Ariel
Adamski‡
a,
Marta A.
Fik‡
a,
Maciej
Kubicki
a,
Zbigniew
Hnatejko
a,
Dorota
Gurda
b,
Agnieszka
Fedoruk-Wyszomirska
b,
Eliza
Wyszko
b,
Dariusz
Kruszka
c,
Zbigniew
Dutkiewicz
c and
Violetta
Patroniak
*a
aFaculty of Chemistry, Adam Mickiewicz University, Umultowska 89B, Poznań, 61-614, Poland. E-mail: violapat@amu.edu.pl
bInstitute of Bioorganic Chemistry, Polish Academy of Science, Noskowskiego 12/14, Poznań, 61-704, Poland
cDepartment of Chemical Technology of Drugs, Poznań University of Medical Sciences, Grunwaldzka 6, Poznań, 60-780, Poland
First published on 26th July 2016
The self-assembly of 6,6′′′′-dimethyl-2,2′;6′,2′′;6′′,2′′′-quaterpyridine L with tetrahedral ions of silver(I) and copper(I) leads to the formation of dinuclear double stranded helicates 1–4. 1H NMR studies confirm the stability of each helicate both in 2% aqueous DMSO solution, which plays the role of a medium in biological studies, and in the cell extract solution. The cytotoxic properties of complexes were examined on two cancerous cell lines: HeLa and T-47D as well as on the non-neoplastic fibroblasts HaCaT by using the MTT assay method. The IC50 values of cisplatin towards cancerous cell lines are rather comparable. However, the trend of cancerous/healthy cell selectivity is not maintained. One may assume a different mechanism of action. Moreover, it is possible to distinguish the cell death pathway triggered by Ag(I) and Cu(I) helicates presented in this study. Flow cytometry, EtBr displacement titrations, CD titrations, DNA melting experiments and DFT calculations were used to characterize the type of interaction and the mechanism of cytotoxic action.
Helicates are a distinct class of metallosupramolecular complexes with fascinating topologies,9 composed of at least one intertwining strand of an organic ligand, which is wrapped around metal ion(s), thus generating a helical axis between metallic knots.10,11 The design of helicates requires the consideration of multiple factors. The most important are the stereoelectronic preferences of the metal ions, ligand topology and its flexibility as well as the variation of the spacing groups that link the coordination domains.12 Tailoring the ligand structure (type, the number and distribution of donor units) to the geometry of the metal ion is a key parameter in the self-assembly of helicates. Therefore, a ligand should be designed so as to possess an individual donor domain able to coordinate a separate metal ion.13 Thus, the formation of helicates is directed by the interplay between the flexibility and rigidity of polydentate ligands.14 All these features are satisfied within the frameworks of polypyridine systems, especially those containing the 2,2′-bipyridine motifs.15 The coordination linkage of bipyridine units with tetragonal metal ions is a well-known supramolecular synthon in the engineering process of helicates.16 In this context, quaterpyridine functions as a bis(bpy) bridging ligand by using two independent bipyridine subunits in the coordination of tetrahedral silver(I) or copper(I) ions. This results in the formation of helical structures.17
The extraordinary architecture and topology of helicates generate numerous applications, especially in terms of medicinal chemistry,18 chiral synthesis,19 asymmetric catalysis20 as well as the generation of optical devices21 and advanced supramolecular materials.22 Double-stranded metal helicates are positively charged in their core23 which results in their ability to bind the DNA helix and consequently prevents the DNA cleavage by restriction enzymes.24 The action of many helical metal–drug complexes is the result of selective molecular recognition of helical structures by enzymes which may lead to their inhibition or formation of metalloenzymes.25 In this context, the properties of helicates are not only the result of their purely structural form, but also are amenable to the nature of the metal ion. Ag(I) ions, excellent templates for the formation of polypyridine helicates, are often used in the fields of medicinal and analytical chemistry. Ag(I) complexes show enhanced bioactivity compared to their simple salts,26 which is related to distinct reversible Ag–N and Ag–O interactions.27 Consequently, Ag(I) compounds find applications in the treatment of epilepsy, nicotine addiction or mental illnesses,28 or as agents in the treatment of burn wounds.29 Replacement of Ag(I) ions with Cu(I) ions in quaterpyridine complexes has no significant effect on their helicity, but substantially modifies the biological properties or provides additional features due to the activity of the Cu(I) complexes in asymmetric reactions.30 Cu(I) ions are known for their high cytotoxicity as a result of oxidative damage (especially against eukaryotic and prokaryotic cells),31 whereas in the form of Cu(I) helicates may be precisely directed to molecular targets into the diseased cells. Moreover, their cytotoxic effect may be decreased and their mechanism of action may be totally different.
Our previous studies on the coordination properties of dimethylquaterpyridine self-assembled with transition metal ions have resulted in a broad spectrum of architectures, including mono- and poly-nuclear complexes with octahedral metal ions32–34 as well as organometallic35 and helical complexes.36 Simultaneously, our recent studies on the terpyridine complexes of silver(I)37 became the inspiration for the design of new helicates of silver(I) and copper(I) with 6,6′′′′-dimethyl-2,2′;6′,2′′;6′′,2′′′-quaterpyridine L (Fig. 1).
Herein, we would like to present new crystal structures of [Ag2L2(CF3SO3)2] (1), [Ag2L2(PF6)2] (2), [Cu2L2(CF3SO3)2] (3), and [Cu2L2(BF4)2] (4), their synthesis, characterization and luminescence studies. However, the main emphasis in this project is put on the investigation of their cytostatic effect towards HeLa and T-47D cancer cell lines as well as non-neoplastic fibroblast HaCat cells. In addition, we carry out DNA binding experiments, by using circular dichroism (CD), competitive binding fluorescence measurements and molecular docking with DNA as well as flow cytometry. Our study represents the first example of the biological evaluation of the quaterpyrdine complexes of Ag(I) and Cu(I).
The helical form of complexes 1–4 was confirmed by electrospray ionization mass analysis. Each complex shows the presence of two sets of peaks: [M2L2]2+ and [M2L2(X)]+ (where M is the cation of metal and X is a counterion). A detailed analysis of the isotopic distribution of both peaks leads to the conclusion that helical form of complexes 1–4 is rather stable even under harsh conditions of the measurements, whereas the ratio of peaks [M2L2]2+ to [M2L2(X)]+ depends on the applied cone voltage (Fig. S3, ESI†).38
As well, FT-IR spectra are in good agreement with the solid state structures and are described in detail in Fig. S4, ESI†.
Fig. 4 Anisotropic ellipsoid representation of complex 3; ellipsoids are drawn at the 50% probability level; hydrogen atoms are shown as spheres of arbitrary radii. |
In three out of four cases (1, 2 and 4) the complex molecules are C2-symmetrical. In 1 and 4 a twofold axis passes through the two Ag cations (so one ligand molecule is symmetry-independent); in 2 the C2 axis halves both ligand molecules, and it runs through the middle points of the central C–C bonds.
In order to allow for such constrained geometry, the ligand molecules are significantly twisted, the dihedral angle between the planes of terminal rings is as high as 60–70°. Table 1 lists the values of individual dihedral angles. The relatively compact geometry of the complexes is additionally enforced by intramolecular π⋯π interactions (the distance between centroids of approximately parallel rings from different ligand molecules is around 3.6 Å). In the crystal structures the electrostatic interactions are dominant; in the structure of 2 the voids in the crystal structure are filled with electron density, described as disordered water molecules (Fig. 3).
1 | 2 | 3 | 4 | |||
---|---|---|---|---|---|---|
A/B | 11.1(2) | 17.2(2) | 17.2(2) | 11.92(5) | 18.00(8) | 15.6(2) |
B/C | 62.56(9) | 44.7(1) | 45.96(9) | 64.2(1) | 61.8(1) | 54.76(9) |
C/D | 11.7(2) | 17.2(2) | 17.2(2) | 4.3(1) | 7.3(1) | 7.5(2) |
A/D | 73.9(1) | 60.46(9) | 53.88(9) | 69.9(1) | 76.9(1) | 66.89(9) |
Complex | Medium | Absorption λ/nm (ε M−1 cm−1) | Emission (excitation) λ/nm | ΔEabs—em/cm−1 | Φ em |
---|---|---|---|---|---|
1 | ACN | 298.0 (33645) | 356.0 (298) | 5456 | 5.52 × 10−2 |
H2O–DMSO | 301.2 (36740) | 360.8 (301) | 5484 | 2.09 × 10−2 | |
2 | ACN | 298.0 (37340) | 354.8 (298) | 5372 | 5.27 × 10−2 |
H2O–DMSO | 302.2 (36372) | 362.6 (302) | 5512 | 2.42 × 10−2 | |
3 | ACN | 250.5 (32950); 291.5 (35655); 451.0 (4455) | 355.2 (292) | 6152 | 3.78 × 10−2 |
H2O–DMSO | 293.4 (46472); 448.4 (6543) | 339.5; 358.0 (293) | 6150 | 0.61 × 10−2 | |
4 | ACN | 250.5 (36340); 291.5 (39395); 450.5 (5125) | 355.6 (292) | 6184 | 4.24 × 10−2 |
H2O–DMSO | 292.9 (34083); 448.0 (4822) | 339.0; 361.8 (293) | 6502 | 0.25 × 10−2 | |
L | ACN | 250.0 (17215); 290.0 (21030) | 342.6 (290) | 5294 | 8.27 × 10−2 |
H2O–DMSO | 291.8 (23043) | 366.0 (292) | 6948 | 3.70 × 10−2 |
UV-Vis absorption spectra of the Cu(I) quaterpyridine complexes 3 and 4 are composed of two high energy bands and one broad low energy band. The high energy absorption bands peaking at 250.5 and 291.5 nm are associated with ligand-centered transitions. The weak absorption bands of 3 and 4 maximized at 450 and 451 nm, respectively, are caused by a charge transfer mechanism, and can be identified as the metal to ligand charge transfer (MLCT) transitions. Similar assignments have also been made in the related copper(I) pyridine systems.39 The absorption spectra of the ligand and the complexes in 2% aqueous DMSO solution are shown in Fig. 7. Peak wavelengths and their absorption coefficients are given in Table 2.
Fig. 7 Electronic absorption spectra of studied compounds in 2% aqueous DMSO solution at room temperature. |
The absorption maxima are red-shifted when increasing the polarity of the solvent. The Stokes shifts (differences between absorption edges and emission peaks) are lowest in the cases of 1 and 2 and highest for 3 and 4 both in CH3CN and 2% aqueous DMSO solutions. Photoluminescence studies of ligand L and their complexes were carried out at room temperature.
Upon photo-excitation at ∼300 nm solutions of all complexes give a broad emission band with maxima at ∼355 or ∼360 nm (Fig. 9 and 10). In the acetonitrile solution ligand L excited at 290 nm displays luminescence with the emission maximum at 342.6 nm (Fig. 8), which can be presumed that the peak originates from intraligand transition. The intense emissions of all complexes exhibit a red shift compared to that of the corresponding free ligand L. Complexes 1–4 show similar emission spectra. Upon excitation at 292 nm emission is observed in compounds 3 and 4 at ∼355 nm. Compounds 1 and 2 exhibit peaks at 356 nm and 354.8 nm, respectively (λex = 298 nm).
The emission spectra of compounds 1–4 in 2% aqueous DMSO solution present emissions at about 360 nm (λex = 301 and 293 nm, Table 2, Fig. 9).
Fig. 9 Emission spectra of 2% aqueous DMSO solutions of 1–4 complexes and ligand L at room temperature. |
The emission bands are blue-shifted compared to the corresponding ligand L (λem = 366 nm, λex = 292 nm). The red or blue shifted emissions of complexes 1–4, in both solutions, may be ascribed to the intraligand charge transitions modified by the coordination of the Ag(I) and Cu(I) metal ions.40,41
Luminescence quantum yields (Φ) for all compounds (Table 2) were determined relative to a reference luminophore solution of anthracene. The quantum yield is dependent on the medium and the values of emission quantum yields are the highest in acetonitrile solution for all studied compounds. The quantum efficiencies of 1 and 2 are a little higher than those of 3 and 4.
The cytostatic effect is expressed as the value of half maximal inhibitory concentration (IC50) and presented in Table 3. The cytotoxic effect of helicates 1–4 is visible against both cancer cell lines, HeLa and T-47D. Interestingly, the IC50 parameters are lower in comparison to that of cisplatin.44 In all cases a dose-dependent antiproliferative activity may be observed.
In general, one is able to observe an almost two times greater cytotoxic effect of Ag(I) complexes (overall IC50 = 23.7 μM) than Cu(I) compounds (overall IC50 = 49.2 μM) towards all three cell lines. In particular, complex 2 seems to exhibit the most prominent cytotoxic effect (IC50 value oscillates around 14 μM), while complex 3 is the least active (average IC50 = 50 μM) (Fig. 10). The differences among complexes comprising the same metal ions may be related to the counterions used in the syntheses, since they may have an impact on the permeability through cell membranes.
Fig. 10 Comparison of IC50 between HeLa, T-47D and HaCat cell lines. The vertical bars indicate 95% confidence interval. |
Referring to our previous study on the cytotoxicity of 2,2′:6′,2′′-terpyridine (tpy) complexes of Ag(I) it is worth noting that the cell growth-inhibitory properties of quaterpyridine (qpy) complexes are slightly weaker towards T-47D and HeLa cells. The comparison of Ag(I) complexes with triflate anions [Ag2(tpy)2](OTf)2 and [Ag2(qtp)2](OTf)2 (complex 1) indicates 3–4 times higher cytotoxicity of the tpy complex towards HeLa and T-47D cells.36 It may be attributed to the increased size of cations of qpy complexes which may make the permeability through membranes more difficult. 2,2′-Bipyridine, 2,2′:6′,2′′-terpyridine and 1,10-phenanthroline ligands are often used as ancillary ligands for the construction of bioactive complexes due to their electronic properties and chemical inertness (in terms of the formation of covalent bondings and stability).45,46
Detailed spectroscopic DNA binding studies and cell cycle analysis were performed to gain further insight into the mechanistic aspects of action of Ag(I) and Cu(I) complexes presented in this work.
An interesting tendency for presented helicates could be observed. In fact, the analysis of cell death induction by complexes 1–4 showed that 3 and 4 tend to trigger apoptosis of HeLa and T-47D cells (around 40–50% of the cell population) rather than necrosis (±1.3%) (Fig. 11–13). On the other hand, in cases of 1 and 2 the trend is reversed, which is especially visible for complex 2 (at 8 μM concentration) where almost 70% of cells undergo necrosis. One may assume that the in vivo acceptable redox potential of Cu ions allows them to interfere with the cell cycle. The activity of Cu complexes turns out to be in accordance with previously reported studies.47
Fig. 12 The graphical representation of the distribution of the types of HeLa cell death. Inset: The amount of dead cells in total. |
Fig. 13 The graphical representation of the distribution of the types of T-47D cell death. Inset: The amount of dead cells in total. |
Fig. 14 The highest scored poses of 3 docked into d(CCCCGGGG) DNA (a) in the major groove and (b) in the minor groove. |
Fig. 15 The highest scored poses of 4 docked into d(CCCCGGGG) DNA (a) in the major groove and (b) in the minor groove. |
It was found that complex 3, in its preferred orientations, binds slightly stronger to the minor groove than to the major groove. The corresponding CDOCKER interaction energies are −75.7 and −68.2 kcal mol−1. For complex 4 the highest scored poses have similar values of CDOCKER interaction energies both in major and minor grooves, which are −74.1 and −73.3 kcal mol−1, respectively.
The analysis of short contacts between the complexes and DNA shows that cationic helicates 3 and 4 may bind to DNA through non-covalent van der Waals interactions and electrostatic interactions with negatively charged DNA's sugar-phosphate backbone. In the major groove the complexes form specific hydrophobic interactions with aromatic rings of nucleobases, e.g. π–π T-shaped, π–alkyl and π–hydrogen bond donor (Fig. 16).
Fig. 16 Specific interactions of complex 3 with d(CCCCGGGG) DNA in the major groove (π–π T-shaped in magenta, π–alkyl in light green and π–hydrogen bond donor in light gray). |
van der Waals and electrostatic interactions of the ligands' pyridine rings and Cu(I) with the DNA phosphate groups are responsible for binding in the minor groove (Fig. 17).
Fig. 17 Specific interactions of complex 4 with d(CCCCGGGG) DNA in the minor groove (π–anion in orange, attractive charge interaction in red). |
Therefore, the CD spectra of the d(CCCCGGGG) helix were recorded in the absence and presence of increasing concentrations of all complexes in the phosphate buffer at pH = 7.25 in the molar ratios of 1, 2 and 4.
All complexes generate some structural changes in the B-DNA ribbon (Fig. 18). The positive band at 260 nm slightly decreases in cases of complexes 2–4. This suggests that no significant perturbations in the DNA structure occur and most probably it is related to binding in the grooves and stabilizing the helix. These results may be nicely supported by the molecular docking studies for compounds 3 and 4, which revealed that mentioned molecules are able to bind in the grooves or interact electrostatically with the surface of the B-DNA helix.
On the other hand, prominent changes may be observed for complex 1. The negative band at 240 nm is hypsochromically shifted for about 10 nm and the positive band at 260 nm decreases upon addition of the complex. It may be concluded as a distortion of the B-DNA caused by another kind of interactions with the complex resulting in attaching tightly to DNA. Evidence supporting this theory might be two additional negative bands visible at ca. 280 and 330 nm which may be generated due to the connection of a further chromophore to the helix.52
According to the experimental results one may assume similar behavior of complexes 2–4 in the presence of DNA in terms of the interaction nature. The changes are indicative of a non-intercalative mode of binding of these complexes and offer support to their groove binding nature. Only complex 1 behaves differently and no straightforward correlations with performed theoretical calculations may be done.
Fig. 19 Emission spectra of EB bound to CT DNA in the presence of increasing amounts of complexes 1–4 (a–d, respectively). |
In general, intercalators and groove binders stabilize the helix and increase the Tm values. These kinds of interactions stabilize the second order structure of the DNA helix. Helicates presented in our study slightly increase the Tm values (2.6–6 °C) of the 12-mer (Table 4). It may support the concept concerning groove binding and may suggest that the binding phenomenon is of moderate strength.54
Compound | Complex 1 | Complex 2 | Complex 3 | Complex 4 |
---|---|---|---|---|
T m [°C] | 65.9 | 65.1 | 62.7 | 66.1 |
In general, 1H NMR reference spectra of free helicates performed in D2O as well as in CDCl3 show a set of seven signals corresponding to 36 protons of two ligand strands wrapped around metal ions in a helical complex. In the aromatic region of spectra 1–4 one of the most downfield doublet turns out to be strongly upfield, below the shifts of both triplets, as compared to the ligands' NMR spectrum, which is characteristic of the helical arrangement of ligand molecules in the complex.
1H NMR reference spectra of free helicates overlap with the spectra of helicates after incubation in the HeLa cell extract. The signals in the spectra of free helicates were found to be slightly shifted by 0.02 ppm as compared to the spectra of helicates incubated with the cell extract which is likely due to the presence of deionized water in studied samples (Fig. 20). The spectra of incubated helicates 1–4 performed in D2O do not reveal significant changes, which suggests the stability of complexes and resistance against the action of enzymes in the cell extract.
Fig. 20 An aromatic region of NMR spectra of ligand L and its helicate 4 before and after incubation with the HeLa cell extract. |
Proton NMR studies of organic layers obtained by the extraction of incubated helicates with CDCl3 were aimed to reveal the presence of a de-coordinated ligand (L is very soluble in deuterated chloroform). The proton NMR spectra of organic layers do not reveal even trace amounts of ligand, which is in agreement with previous studies.
The evaluation of the biological activity of the resulting compounds was carried out with a focus on their cytotoxic properties. Their biological potential was investigated via MTT assay towards HeLa, T-47D and HaCaT cell lines. It was further investigated by flow cytometry and one was able to observe the DNA dependent mechanism of cytotoxic action. Moreover, it allowed one to observe that Ag(I) complexes induce the necrosis of cells while Cu(I) complexes lead to apoptosis. We find that it is due to the redox properties of metal ions chosen for this study. The redox potential of Cu ions is acceptable in vivo so that it may have an impact on the bioavailability of the complexes. It needs to be mentioned that the preferred form is Cu(I) for presented helicates which we showed in NMR studies with the cell extract.
Since the mechanism of action of helicates is most likely related to their interaction with DNA comprehensive studies of its mode were performed. The results obtained by the circular dichroism (CD) technique, competitive binding fluorescence measurements, as well as DNA melting allowed one to anticipate the groove binding mode. This was further supported by molecular docking studies.
Taking into account the reversible redox nature of Cu(I) helicates the radical activity of these complexes in the cells may become an interesting topic for future studies in our research group.
ESI-MS mass spectra were determined using a Waters Micromass ZQ spectrometer in acetonitrile. The samples were run in positive-ion and negative-ion modes. The concentrations of the compounds were about 10−4 mol dm−3. Sample solutions were introduced into the mass spectrometer source using a syringe pump with a flow rate of 40 μL min−1 with a capillary voltage of +3 kV and a desolvation temperature of 300 °C. The source temperature was 120 °C. Cone voltage (Vc) was set to 30 V to allow the transmission of ions without fragmentation processes. Scanning was performed from m/z = 100 to 1500 for 6 s, and 10 scans were summed to obtain the final spectrum. 1H NMR spectroscopic data were run on a Varian Gemini 400 MHz spectrometer and were calibrated against the residual protonated solvent signals (DMSO-d6: 2.51 (1H)) with chemical shifts represented in ppm. Microanalyses were performed using a Perkin Elmer 2400 CHN microanalyser. FT-IR spectra were obtained using a Perkin Elmer 580 spectrophotometer and peak positions are reported in cm−1. Optical density was measured using an Eppendorf BioPhotometer. All electronic absorption spectra were recorded using a Shimadzu UVPC 2001 spectrophotometer, between 220 and 800 nm, in 10 × 10 mm quartz cells using solutions ∼2 × 10−5 M with respect to the metal ions. Luminescence characterization was performed on a Hitachi F-7000 fluorescence spectrophotometer equipped with a xenon lamp (150 W) which acts as the light source for steady state measurements. For accuracy of data, emission spectra were corrected for the instrumental response. All measurements were carried out under the same experimental conditions. Emission quantum yields were determined by comparison of the integrated emission intensity with that of anthracene56 under identical conditions (exciting wavelengths, optical density, apparatus parameters).
1H NMR (D2O: DMSO-d6, 2% solution, 400 MHz): δ (ppm) = 8.11 (d, 4H, J = 8.01 Hz), 8.03 (t, 4H, J = 7.85 Hz), 7.94 (d, 4H, J = 7.69 Hz), 7.84 (d, 8H, J = 4.33 Hz), 7.26 (t, 4H, J = 4.33 Hz), 1.81 (s, 12H). ESI-MS(+) (%): 445 (100) [AgL]+, 1042 (5) [Ag2L2(CF3SO3)]+. Anal calc. for [Ag2(C22H18N4)2](CF3SO3)2 (1) (1190.68): C, 46.40; H, 3.05; N, 9.41; found: C, 46.37; H, 3.04; N, 9.41%. IR (KBr, cm−1): 3573, 3522, 3079, 2916, 1600, 1591, 1569, 1469, 1448, 1386, 1277, 1269, 1226, 1161, 1107, 1032, 1010, 793, 750, 745, 653, 638, 573, 518.
1H NMR (D2O: DMSO-d6; 2% solution, 400 MHz): δ (ppm) = 8.13 (d, 4H, J = 8.03 Hz), 8.05 (t, 4H, J = 7.83 Hz), 7.87 (d, 4H, J = 7.69 Hz), 7.55 (d, 8H, J = 4.61 Hz), 7.28 (t, 4H, J = 4.29 Hz), 1.83 (s, 12H). ESI-MS(+) (%): 445 (100) [AgL]+, 1037 (3) [Ag2L2(PF6)]+. Anal calc. for [Ag2(C22H18N4)2](PF6)2 (2) (1182.48): C, 44.69; H, 3.07; N, 9.48; found:C, 44.68; H, 3.08; N, 9.46%. IR (KBr, cm−1): 3650, 6413, 3081, 2920, 2004, 1928, 1824, 1731, 1632, 1593, 1572, 1469, 1447, 1387, 1303, 1253, 1191, 1176, 1134, 1110, 1004, 850, 792, 745, 653, 638, 558, 502, 483.
1H NMR (D2O: DMSO-d6; 2% solution, 400 MHz): δ (ppm) = 7.95 (m, 8H), 7.85 (t, 4H, J = 7.76 Hz), 7.73 (t, 4H, J = 7.84 Hz), 7.63 (d, 4H, J = 7.48 Hz), 7.24 (d, 4H, J = 7.48 Hz), 1.80 (s, 12H). ESI-MS(+) (%): 402 (100) [CuL]+, 953 (8) [Cu2L2(CF3SO3)]+. Anal calc. for [Cu2(C22H18N4)2](CF3SO3)2 (3) (1102.04): C, 50.13; H, 3.29; N, 10.17; found: C, 50.10; H, 3.29; N, 10.16%. IR (KBr, cm−1): 3424, 3069, 2921, 2006, 1915, 1830, 1742, 1605, 1595, 1565, 1472, 1446, 1388, 1380, 1257, 1248, 1183, 1175, 1063, 1013, 918, 831, 824, 788, 752, 734, 654, 640, 533, 522, 489.
1H NMR (D2O: DMSO-d6; 2% solution, 400 MHz): δ (ppm) = 7.93 (m, 8H), 7.84 (t, 4H, J = 7.76 Hz), 7.73 (t, 4H, J = 7.85 Hz), 7.62 (d, 4H, J = 7.48 Hz), 7.25 (d, 4H, J = 7.48 Hz), 1.80 (s, 12H). ESI-MS(+) (%): 402 (100) [CuL]+, 990 (6) [Cu2L2(BF4)]+. Anal calc. for [Cu2(C22H18N4)2](BF4)2 (4) (977.51): C, 54.06; H, 3.71; N, 11.46; found: C, 54.04; H, 3.70; N, 11.45%. IR (KBr, cm−1): 3569, 3475, 3076, 3036, 2963, 2918, 2292, 1984, 1902, 1824, 1734, 1606, 1595, 1571, 1473, 1449, 1389, 1278, 1264, 1223, 1186, 1156, 1106, 1090, 1030, 1013, 792, 749, 655, 637, 573, 554, 518.
In 1 the CF3SO3 anion is disordered over two sites (s.o.f.'s refined at 0.705(6)/0.295(6)). In 2 in turn the PF6 anion is also disordered (0.684(6)/0.316(6)); moreover, relatively large diffused residual electron density was interpreted for disordered solvent molecules and the SQUEEZE procedure was applied. Also in 3, one of the CF3SO3 anions is disordered (0.503(5)/0.497(5)). In all the three structures the rigid-bond (RIGU) and similarity (SIMU) restraints have been applied. Finally, in 4 a solvent, methanol, molecule was found to be disordered across the center of symmetry.
Table 5 lists the relevant experimental data and refinement details. Crystallographic data (excluding structural factors) for the structural analysis have been deposited with the Cambridge Crystallographic Data Centre, CCDC 1060683–1060686 (1–4).
Compound | 1 | 2 | 3 | 4 |
---|---|---|---|---|
Formula | C44H36Ag2N82+·2(CF3SO3)− | C44H36Ag2N82+·2(PF6)− | C44H36Cu2N82+·2(CF3SO3)− | C44H36Cu2N82+·2(BF4)−·2(C7H8)·CH3OH |
Formula weight | 1190.69/c | 1182.49 | 1102.03 | 1193.81 |
Crystal system | Monoclinic | Monoclinic | Monoclinic | Orthorhombic |
Space group | I2/a | I2/a | C2/c | Pbcn |
a (Å) | 16.0589(3) | 12.5733(4) | 23.7716(18) | 14.1499(10) |
b (Å) | 15.5077(3) | 23.2638(6) | 13.2745(8) | 16.6539(10) |
c (Å) | 18.1719(7) | 16.2128(5) | 31.455(3) | 22.4487(13) |
β (°) | 95.270(2) | 96.624(3) | 117.247(11) | 90 |
V (Å3) | 4506.3(2) | 4710.6(2) | 8824.3(14) | 5290.1(6) |
Z | 4 | 4 | 8 | 4 |
D x (g cm−3) | 1.76 | 1.67 | 1.66 | 1.50 |
F(000) | 2384 | 2352 | 4480 | 2456 |
μ (mm−1) | 8.58 | 0.99 | 1.15 | 0.88 |
Θ range (0) | 3.75–70.0 | 2.98–28.07 | 2.90–25.0 | 3.26–25.0 |
Reflections: | ||||
Collected | 9002 | 9658 | 29719 | 12631 |
Unique (Rint) | 4067(0.036) | 4852(0.012) | 7769(0.072) | 4655(0.025) |
With I > 2σ(I) | 3936 | 4287 | 5417 | 3616 |
R(F) [I > 2σ(I)] | 0.053 | 0.032 | 0.052 | 0.054 |
wR(F2) [I > 2σ(I)] | 0.147 | 0.090 | 0.104 | 0.157 |
R(F) [all data] | 0.054 | 0.037 | 0.087 | 0.076 |
wR(F2) [all data] | 0.148 | 0.093 | 0.117 | 0.27 |
Goodness of fit | 1.28 | 1.10 | 0.99 | 1.08 |
Max/min Δρ (e Å−3) | 1.89/−1.31 | 1.07/−0.68 | 0.86/−0.51 | 0.70/−0.53 |
The optimized structures of complexes 3 and 4 were docked into the major and minor grooves of the DNA octamer. Docking studies were performed using the CDOCKER61 procedure implemented in Discovery Studio 4.1. The binding site sphere was centered in the major or minor grooves and the radius of the sphere was set to a value of 14 Å. Partial charges for the receptor and ligands were assigned according to Momany-Rone and MMFF94 charging rules, respectively. Cu(I) ions in helicates were treated as cations with the formal and partial charge equal to +1. CDOCKER uses molecular dynamics to generate ligand conformations, but during docking this step was skipped because the only rotatable bonds in complexes 3 and 4 are those connecting methyl groups with pyridine rings. For every docking run the starting conformation of each ligand was placed in the binding sphere in ten initial orientations by random rotations and translations. Finally, poses were minimized with full potential, the complete CHARMm force-field expression and up to ten poses were saved for each ligand after docking.
Silver complexes were not docked due to the lack of appropriate parameters in the MMFF94 forcefield for Ag.
For the CD experiments 10 μM DNA d(CCCCGGGG) samples were dissolved in 150 mM NaCl, 10 mM sodium phosphate and 0.1 mM EDTA (pH 7.25). 10, 20 or 40 μM complexes were added 5 min before measurements and the samples were annealed by heating at 70 °C for 5 min and then slowly cooled down to room temperature.
The HeLa cell extract corresponding to 1.6 × 10−7 cells in 1 mL of deionized water was prepared according to the literature.63 Helicate (1 mM) was added to the HeLa cell extract (100 μL) and then the resulting suspension was incubated at 37 °C for 24 h. In this manner, two separate samples were performed for each complex. After incubation time, the first sample was extracted with CDCl3 (450 μL) and the organic layer was studied by 1H NMR. To the second sample D2O was added (350 μL) and then the resulting solution was studied by proton NMR. Furthermore, proton NMR reference spectra of free helicates were performed in D2O and CDCl3 as well. The NMR data were obtained by collection of 900 scans, which turns out to be sufficient to obtain good signal-to-noise ratios.
Footnotes |
† Electronic supplementary information (ESI) available. CCDC 1060683–1060686. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c5nj03601a |
‡ Ariel Adamski and Marta A. Fik contributed equally to the experimental results given in this work. |
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2016 |