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Aug 31, 2016 - Reiko Nagano,. §. Hiromi Morita,. § ... Nanotechnology Innovation Station, NIMS, 1-2-1 Sengen, Tsukuba 305-0047, Japan. •S Supporti...
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Selective DNA Recognition and Cytotoxicity of Water-Soluble Helical Metallosupramolecular Polymers Utpal Rana,† Chanchal Chakraborty,†,‡ Rakesh K. Pandey,† Md. Delwar Hossain,†,# Reiko Nagano,§ Hiromi Morita,§ Shinya Hattori,§ Takashi Minowa,§ and Masayoshi Higuchi*,† †

Electronic Functional Macromolecules Group, National Institute for Materials Science (NIMS), Tsukuba 305-0044, Japan International Center for Materials Nanoarchitectonics (MANA), NIMS, Tsukuba 305-0044, Japan § Nanotechnology Innovation Station, NIMS, 1-2-1 Sengen, Tsukuba 305-0047, Japan ‡

S Supporting Information *

ABSTRACT: Water-soluble helical Fe(II)-based metallosupramolecular polymers ((P)- and (M)-polyFe) were synthesized by 1:1 complexation of Fe(II) ions and bis(terpyridine)s bearing a (R)- and (S)-BINOL spacer, respectively. The binding affinity to calf thymus DNA (ct-DNA) was investigated by titration measurements. (P)-PolyFe with the same helicity as B-DNA showed 40-fold higher binding activity (Kb = 13.08 × 107 M−1) to ct-DNA than (M)-polyFe. The differences in binding affinity were supported by electrochemical impedance spectroscopy analysis. The charge-transfer resistance (Rct) of (P)-polyFe increased from 2.5 to 3.9 kΩ upon DNA binding, while that of (M)-polyFe was nearly unchanged. These results indicate that ionically strong binding of (P)-polyFe to DNA chains decreased the mobility of ions in the conjugate. Unique rod-like images were obtained by atomic force microscopy measurement of the DNA conjugate with (P)-polyFe, likely because of the rigid binding between DNA chains and the polymer. Differences in polymer chirality lead to significantly different cytotoxicity levels in A549 cells. (P)-PolyFe showed higher binding affinity to B-DNA and much higher cytotoxicity than (M)polyFe. The helicity in metallosupramolecular polymer chains was important not only for chiral recognition of DNA but also for coordination to a biological target in the cellular environment.

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were recently reported by Yeh et al.11 Chiral metal complexes have been used as unique molecular probes for DNA recognition, as reported by Barton et al.12 The recognition of B-DNA by chiral bis(EDTA−distamycin)−fumaramide was reported by Denan et al.13 Chiral (P)-forms of drugs selectively bind to right-handed B-DNA, as reported by Qu et al.14 Here, we report an interesting example of a metallosupramolecular polymer in which two useful properties were combined in a single polymeric compound: the ability of the polymer to serve as a structural template for DNA recognition and a chemotoxic agent that induces apoptosis in cancer cells. Briefly, we synthesized helical metallosupramolecular polymers with opposite helicity (Figure 1) and induced a helical conformation of the polymers, resulting in DNA recognition and cytotoxic activity in human cancer cells.

INTRODUCTION Conjugation of metal complexes and DNA has received attention for their potential in the development of chemotherapeutic agents because these DNA-binding agents can be effectively used as anticancer drugs.1,2 Transition-metal complexes and cationic polymers are considered promising candidates in the field of anticancer drug design because of their high level of design flexibility and ability to bind anionic nucleic acids such as DNA.3−8 However, the low solubility and binding activity to DNA under biologically relevant conditions limit the utility of metal complexes. We recently introduced a new family of water-soluble metallosupramolecular polymers based on bisterpyridine and transition metals that exhibits strong binding activity to DNA under biologically relevant conditions and shows cytotoxicity toward human lung cancer cell lines (NCIH460) by inducing apoptosis.9 Computational analysis indicated that the polymers are conjugated with DNA chains via minor groove binding.10 Our previous study indicated that metallosupramolecular polymers are potential candidates as anticancer drugs because their strong binding to DNA can prevent cancer cell proliferation. Because of the helicity of DNA, chirality and helicity in metal complexes and polymers may enhance anticancer activities. For example, the high binding affinity of peptide nucleic acids with right-handed helix conformations to B-DNA and strong cytotoxicity to cancer cells © 2016 American Chemical Society

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RESULTS AND DISCUSSION The chiral ligands ((R)- and (S)-L1) were synthesized by a Suzuki−Miyaura cross-coupling reaction in ∼44% yield (Scheme 1) and characterized by 1H-, 13C-, and 1H−1H COSY NMR and MALDI-MS (Figures S1−17). Initially, Received: May 20, 2016 Revised: August 26, 2016 Published: August 31, 2016 2307

DOI: 10.1021/acs.bioconjchem.6b00255 Bioconjugate Chem. 2016, 27, 2307−2314

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Bioconjugate Chemistry

Figure 2. (a) UV−vis spectral change of (R)-L1 by addition of Fe(II); (b) absorption change at 570 nm as a function of the molar ration of Fe(BF4)2/(R)-L1. Figure 1. Helical metallosupramolecular polymers ((P)- and (M)polyFe) formation by the complexation of (R)-L1 or (S)-L1 with Fe(II) salt.

A chiral structure was expected to form following complexation between the chiral ligand L1 and Fe(II). When Fe(BF4)2 was added to (R)-L1, a new circular dichroism (CD) signal at 570 nm appeared, which was attributed to the MLCT band (Figure 3a). The intensity of the CD signal decreased as Fe(II)

complexation between the (R)-L1 and Fe(II) salt was monitored by UV−vis spectroscopic titration (Figure 2). To a ligand solution in CHCl3−MeOH (1:1), when Fe(BF4)2 was added successively, a new absorbance peak at 570 nm appeared, which was attributed to metal-to-ligand charge transfer (MLCT).15−19 With increasing metal salt concentration, the MLCT peak intensity linearly increased and became saturated at [Fe(II)]/[(R)-L1] ≈ 1.0. Stepwise complexation of L1 and Fe(II) resulted in increased L1 absorbance at 320 nm. The stoichiometric ratio of the complex was determined by plotting the absorbance at 570 nm and the molar ratio of Fe(II) to (R)L1, which revealed 1:1 complexation between L1 and Fe(II) (Figure 2b).20 The absorbance intensity of the MLCT band did not change in the presence of excess metal ions, indicating that the polymer were stable in solution.16

Figure 3. (a) CD spectral changes of the (R)-L1 with the addition of Fe(II); (b) CD spectra of (P)-polyFe (red line) and (M)-polyFe (black line).

Scheme 1. Synthesis of (R)- and (S)-L1

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DOI: 10.1021/acs.bioconjchem.6b00255 Bioconjugate Chem. 2016, 27, 2307−2314

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Bioconjugate Chemistry concentration increased and became saturated at a 1:1 ratio. The induced MLCT band in the CD spectra indicates that an asymmetric field surrounding the optically inactive Fe(II) was generated.21 In the case of (R)- and (S)-L1, addition of Fe(II) revealed an MLCT band at ∼570 nm with opposite sense, demonstrating the formation of oppositely helical polymers. Hence, on the basis of the CD spectrum, it is determined that the chiral (R)- or (S)-L1 ligand bound to Fe(II) to form the corresponding right-handed helix (P)-polyFe or left-handed helix (M)-polyFe, respectively (Figure 3b). These complexation results (Figures 2 and 3) were consistent with those reported by Kimura et al.15 The metallosupramolecular polymers were prepared by the 1:1 complexation of (R)-L1 or (S)-L1 with Fe(OAc)2. Fe(OAc)2 (0.03 mmol) and the ligand (R)- or (S)-L1, (0.03 mmol) were refluxed in argon-saturated dry acetic acid for 24 h.19,22 The reaction mixture was cooled to 25 °C and filtered to remove insoluble residues. The filtrate was transferred to a Petri dish and dried by evaporation. The blue-colored film was collected and further dried under a vacuum for 24 h to obtain (P)- or (M)-polyFe in >90% yields. The molecular weights (Mw) of (P)- and (M)-polyFe were determined using the SEC−Viscometry−RALLS method (sizeexclusion chromatography−viscometry−right-angle light scattering) with poly(ethylene oxide) as a standard (std) in methanol. Molecular weights were found to be 1.07 × 105 and 9.83 × 104 Da, respectively. FTIR and Raman spectroscopic investigations of polymers were carried out at room temperature for structural analysis. In FTIR, antisymmetric (νas) and symmetric (νs) vibrations of the uncoordinated acetate ion were obtained at 1560 and 1414 cm−1, respectively (Figure S18). Typical pyridine vibrations at 734 (ring deformation of pyridine), 792, and 820 cm−1 (C−H deformation vibration of pyridine) were observed in the spectra.23,24 The Raman bands at 1604, 1561, 1531, 1470, 1362, 1288, 1163, and 1017 cm−1 were all characteristics peaks of the terpyridyl unit in a Fe(dπ)− Terpy (π*) MLCT transition (Figure S19).25,26 Studies of metallosupramolecular polymer−DNA interactions are of paramount importance to understand the basic mechanism of tumor inhibition for cancer treatment. UV−vis spectroscopy is an important tool for investigating the interaction between DNA and metal complex by detecting changes in the absorbance and shifts in the wavelength. Electrostatic interactions between (P)- or (M)-polyFe with ctDNA were investigated by UV−vis titration. To a 5% ethanol− aqueous solution of each polymer (5 μM concentration, determined on the basis of the repeat unit), a buffer solution of ct-DNA was added stepwise up to 7.5 μM (per nucleotide of DNA). With increasing ct-DNA concentration, both polymers showed noticeable bathochromic shifts with hypochromism of the MLCT band. The bathochromic shifts for (P)- and (M)polyFe were 3 and 2 nm, with hypochromicity 24% and 11.6%, respectively (at 1:1 D−P; Figure 4a,b). The maximum red shift and highest hypochromicity of (P)-polyFe indicated the strong electrostatic interaction between the metal center of the polymers and phosphate anions of the DNA in the periphery of the DNA helix.12,27 To quantify the extent of the DNA binding, the intrinsic binding constant (Kb) of the two polymers was determined on the basis of the McGhee−von Hipper (MvH) model using eq 1.28 These values were obtained by monitoring the variation in absorbance at the MLCT band of the polymers with increasing concentrations of ct-DNA.

Figure 4. UV−vis spectral changes of polymers with the addition of ctDNA, (a) (P)-polyFe, and (b) (M)-polyFe; nonlinear fitting of the absorption change of (c) (P)-polyFe and (d) (M)-polyFe as a function of DNA concentration.

It is well-known that DNA molecules consist of two biopolymer strands coiled around each other to form a double helix and stable conjugated structure with the same configuration as the chiral molecule with a strong positive center.29 The binding constants (Kb) of (P)- and (M)-polyFe were calculated from the nonlinear fitting curve of the absorption of each polymer as a function of DNA concentration to be 13.08 × 107 and 3.05 × 106 M−1, respectively. (P)-polyFe showed stronger binding affinity than (M)-polyFe to B-DNA because of the right-handed helicity of the polymer chains (Table 1). Nonlinear fitting using the MvH eq (panels c and d Table 1. DNA Binding of Polymers

(P)-polyFe (M)polyFe

hypochromicity (%)

bathochromic shift (nm)

Kb × 106 (M−1)

s

24 11.6

3 2

130.8 3.05

0.51 0.48

of Figure 4) revealed a clear saturation point in the case of the (P)- enantiomer; however, the results for the (M)- enantiomer were less clear. This is because the conjugate structure was stabilized and favorable electrostatic interactions present between same configuration chiral polymer molecule with a positive metal center and phosphate anions of the B-DNA chain.10,30 This was an exciting initial observation because selective binding between the chiral metallosupramolecular polymers with DNA has not been previously reported. When we assumed that the dihedral angle of the BINOL unit was 90°, one pitch of the helical structure in polyFe was estimated by simple calculation (Scheme 2). In this estimation, four Fe(II) ions and four ligands formed one helical pitch. Because the distance between the two BINOLs was determined to be 1.77 nm by simple molecular modeling, the width and pitch length of the helical polymer were calculated to be approximately 1.5 and 3.5 nm, respectively, as shown in panels a and b of Scheme 2a. In contrast, the width and pitch length of B-DNA are known to be approximately 2.0 and 3.4 nm, respectively. The higher binding affinity of (P)-polyFe to ct2309

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Scheme 2. Estimation of the Width and Pitch of Helical PolyFe When the Dihedral Angle of the BINOL Unit Is 90° (a); Top View (b); and Helicity and Pitch Matching of (P)-PolyFe and B-DNA (c)

DNA suggests strong electrostatic binding resulting from the well-matching of helicity and pitch length between the polymer and ct-DNA (Scheme 2c). Electrochemical impedance spectroscopy (EIS) is an effective technique for bioelectro-analysis such as the monitoring of DNA−metal complex interactions.31 To study the ability of the polymers to bind DNA, we carried out EIS titration by successive addition of DNA into the polymer solution. The impedance titration results are shown in Figure 5. Chargetransfer resistance (Rct) was calculated from the x-axis intercept of the semicircle at high frequencies. The charge-transfer resistance (semicircle diameter) increased with ct-DNA concentration; in the case of (P)-polyFe, resistance increased rapidly with the addition of DNA, while little change was observed in (M)-polyFe. This suggests that the binding of DNA with the former is stronger, which influenced the diffusion of redox polymer species toward the electrode and eventually increased the charge-transfer resistance.31,32 Additionally, at a 1:1 molar ratio, resistance no longer increased, indicating a saturation state, which was in accordance with the UV−vis data shown in Figure 4. Rct values increased from 2.5 to 3.9 kΩ for (P)-polyFe and 2.5 to 2.6 kΩ for (M)-polyFe at the saturation point. This rise in the Rct is consistent with the less-hindered

Figure 5. Nyquist plots measured after successive addition of DNA into solution of (a) (P)-polyFe and (b) (M)-polyFe, (c) impedance variation in both polymers after addition of DNA, and (d) resistance change after addition of equimolar DNA to both polymers.

charge-transfer diffusion upon strong binding between same helical B-DNA and (P)-polyFe. For detailed analysis of the conjugate structure of the DNA and polymers, AFM analysis was performed. B-DNA without 2310

DOI: 10.1021/acs.bioconjchem.6b00255 Bioconjugate Chem. 2016, 27, 2307−2314

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Bioconjugate Chemistry polymer showed well-known chain-like morphology (Figure S20). When B-DNA conjugated with the same helical (P)polyFe, assembled chainlike structures were observed (Figure 6a), while for opposite helical (M)-polyFe and DNA

normal cells (NIH 3T3 cell) under identical conditions. As shown in Figure 8, no significant cytotoxicity was observed up

Figure 6. AFM images of (a) (P)-polyFe−DNA and (b) (M)-polyFe− DNA conjugate structures.

Figure 8. CCK-8 assay of NIH 3T3 cell line in the presence of (P)polyFe.

to 25 μM of (P)-polyFe. At this concentration, 33% of cancer cells (A549) and 91% of normal cells (NIH 3T3) were viable. These results indicate that the polymer is cancer-cell-specific. The cytotoxicity of the two polymers was further tested visually by calcein-AM and propidium iodide (PI) double staining. The staining method is fluorescence-based for the simultaneous detection of live cells (green), dead cells (red), or both with two probes that reflect cellular activities and plasma membrane integrity.35 For (P)-polyFe, we observed a greater number of dead cells (red) than for (M)-polyFe in A549 cells. The percentages of dead and total cells were 36 and 23% for (P)- or (M)-polyFe, respectively (Table 2). Fluorescence

conjugates, disassembled structures were observed (Figure 6b). The diameter of B-DNA is 2.0 nm with a pitch distance of 3.4 nm; the diameter of a single (P)-polyFe chain is ∼1.5 nm with a pitch distance ∼3.5 nm (Scheme-1). On the basis of the AFM images, the average diameter and width of the (P)polyFe-DNA conjugate was determined to be ∼6.5 and ∼28 nm, respectively (Figure S21) because of the conjugation of three polymer chains with one B-DNA chain; both (P)-polyFe and B-DNA showed the same helical with close pitch distances. The AFM images suggest that the strong binding between the same helical DNA and (P)-polyFe formed better conjugate structures.33 Inhibition of DNA replication, which causes cell death, may occur because of the stabilization of the DNA structure with DNA binding molecules when the binding is sufficiently strong.34 To identify metallosupramolecular anticancer drugs, we tested two polymers for their anticancer activities by measuring cytotoxicity in A549 cancer cells (Figure 7).

Table 2. Live and Dead Cell Assay of the Polymers

(P)polyFe (M)polyFe

average no. of live cells (L)

average no. of dead cells (D)

D/total no. of cells (L + D), %

112

64

36

114

34

23

microscopic analysis suggested that the right-handed helical polymer has a greater potential to induce the death of A549 cancer cells (Figure 9). Passive diffusion through the cellular lipid bilayer is considered to be the dominant process involved in polymer uptake, similar to small molecules, and the initiation of apoptosis may explain the cell death.36 The apoptosis mechanism was investigated by FACS using Annexin-V FITC− propidium iodide double staining of A549 cancer cell using 25 μM (P)-polyFe. In this assay, ∼ 6.65% of cells were in early apoptosis, and 17.43% were in late apoptosis (Figure 10). These results indicate that the initiation of apoptosis cause cell death.

Figure 7. CCK-8 assay of A549 cells in the presence of polymers (a) (P)-polyFe and (b) (M)-polyFe.

For this, (P)-polyFe or (M)-polyFe was incubated with A549 cells for 24 h, and cell viability was determined using the CCK8 assay. Both polymers reduced A549 cell survival in a concentration-dependent manner. A significant effect was observed for (P)-polyFe. Cellular survival reached 33% or 64% for (P)-polyFe or (M)-polyFe at 25 μM, respectively. The right-handed helical polymer showed higher cytotoxicity than the left-handed polymer, indicating that the same helical polymer as B-DNA has a greater effect on cell inhibition.34 The exact mechanisms of their accumulation in cells remains unclear, but our study provides a foundation the design of anticancer agent. To determine the specificity of our polymers toward cancer cell, we tested the cytotoxicity of (P)-polyFe in

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CONCLUSIONS In conclusion, we synthesized the opposite helical chiral metallosupramolecular polymers (P)- or (M)-polyFe by 1:1 complexation between the corresponding chiral ligand and Fe(II). Their DNA binding to B-DNA and in vitro cytotoxicity to cancer cells were analyzed by UV−vis and EIS. Right-handed helical polymers showed stronger binding properties and higher cytotoxicity to cancer cells than left-handed polymers. (P)polyFe recognized double-helical B-DNA under physiologically relevant conditions. Strong binding activity and higher 2311

DOI: 10.1021/acs.bioconjchem.6b00255 Bioconjugate Chem. 2016, 27, 2307−2314

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Figure 9. Calcein-AM and propidium iodide (PI) double staining assay of A549 cells (a) without polymer, (b) in the presence of (P)-polyFe, and (c) and (M)-polyFe.

wire counter electrode, and glassy carbon electrode of 3.0 mm as working electrodes (ALS, Tokyo, Japan). Impedance measurements were recorded in 5 mM Tris−HCl and 50 mM NaCl buffer (2:1 buffer−DMF) with scanning frequencies between 10 MHz and 100 Hz applying a 10 mV AC voltage. EIS recordings were performed in 50 μM polymer solution in buffer by successive addition of ct-DNA. After DNA was added and the solution incubated for 10 min, the interaction between DNA and polymer was measured until the signal remained stable for two consecutive measurements. Randel’s equivalent circuit was used as a model for impedance of the electrochemical interface, where the double-layer capacitance was replaced with a constant-phase element for better estimation of the electrochemical double layer of the real system. The morphology of the DNA and conjugate structure were measured by AFM (Model SPI 3800 N, Seiko Instruments, Chiba, Japan). A conjugate solution of ct-DNA and polymer (50 mM) was drop-cast onto a piece of mica pretreated with 1 M MgSO4 solution and rinsed with water and dried prior to measurement. Tapping mode was used to acquire the images under ambient conditions. Cytotoxicity assay reagents, CellCounting Kit-8 (CCK-8), and calcein-AM and propidium iodide (PI) double staining kits were purchased from Dojindo (Kumamoto, Japan). A microplate reader (MTP-880, Corona Electric Co., Ltd., Hitachinaka, Japan) was used for the cytotoxicity assay with CCK-8. Inverted light and fluorescence microscopes (DMIL, Leica Microsystems, Wetzlar, Germany) were used to observe cells stained with the calcein-AM and PI double staining kit. Annexin-V buffer and Annexin-V (10 μg/ mL) were purchased from SONY (Tokyo, Japan). A SONY SP6800 cell analyzer (FACS) was used for apoptosis analysis. Synthesis of (R)-1. To a solution of 6,6′-dibromo-1,1′-bi-2naphthol ((R)-BINOL) (0.20 g, 0.4 mmol) in acetone (10 mL), 1-bromodohexane (0.29 g, 1.8 mmol) and K2CO3 (0.25 g, 1.8 mmol) were added. The mixture was refluxed for 24 h. After the solvent was evaporated, water (100 mL) was added to the mixture and organic compounds were extracted with ethyl acetate. The combined organic portions were washed with water and brine three times and dried over MgSO4 , concentrated, and purified by flash-column chromatography on silica gel (hexane−EtOAc = 8:2) to give (R)-1 as a viscous liquid (0.25 g, 90%). 1 H NMR (300 MHz, CDCl3, 298 K, δ (ppm)): 8.00 (d, 2H, J = 1.8 Hz), 7.84 (d, 2H, J = 8.8 Hz), 7.42 (d, 2H, J = 8.8 Hz), 7.28 (dd, 2H, J = 8.8, 2.2 Hz), 7.00 (d, 2H, J = 8.8 Hz), 3.97 (m, 4H), 1.52 (m, 8H), 1.04 (m, 8H), 0.78 (m, 6H); 13C NMR (75 MHz, CDCl3): 154.79, 132.59, 130.21, 129.73, 129.42, 128.36, 127.12, 120.09, 117.23, 116.43, 69.56, 31.26, 29.25, 25.30, 22.44, 13.87; MALDI-MS: [M + H], 613; HRMS (m/z): [M + Na] found 635.10; requires 635.13.

Figure 10. FACS analysis of A549 cells in the presence of 25 μM (P)polyFe by Annexin-V FITC−PI double staining.

cytotoxicity of (P)-polyFe resulted from the strong electrostatic binding between favorable same helical DNA structures.

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EXPERIMENTAL PROCEDURES Materials and Instruments. 6,6′-Dibromo-1,1-bi-2-naphthol (R- and S-) (97%), 1-bromo hexane, PdCl2(PPh3)2, K2CO3, 4′-(4-bromophenyl)-2,2′:6′,2″-terpyridine, and iron(II) acetate [Fe(OAc)2, > 99.99%] were purchased from TCI (Tokyo, Japan) and Aldrich (St. Louis, MO) and were directly used without further purification. 4′-(4-(4,4,5,5-Tetramethyl1,3,2-dioxaboryl) phenyl)-2,2′:6′,2″-terpyridine was prepared according to our previously reported procedure. 1H- and 13C NMR spectra were recorded at 300 and 75 MHz, respectively, on a JEOL AL 300/BZ instrument (Tokyo, Japan). Mass spectra (MS) were measured using a Shimadzu/Kratos time-offlight mass spectrometer (Kyoto, Japan). High-resolution mass spectra (HRMS) were measured using a Shimadzu LCMS-ITTOF spectrometer. UV−vis spectra were obtained using a Shimadzu UV-2550 UV−visible spectrophotometer. IR spectra were measured on a Shimadzu FTIR 8400S Fourier transform infrared spectrophotometer with KBr pellets. Raman spectroscopy was measured using RAMAN plus instrument (Nanophoton Corporation, Osaka, Japan) with a 785 nm laser. Circular dichroic spectra were recorded with a J-820 spectro polarimeter (JASCO, Oklahoma City, OK). Calf thymus DNA (10 mg/mL, 200−500 bp) was purchased from TREVIGEN (Gaithersburg, MD), and the concentration of ct-DNA per nucleotide was calculated from the known extinction coefficient of 6600 M−1 at 260 nm. The A549 human lung cancer cell line was purchased from the RIKEN Bio-Resource Centre. VersaSTAT 4 (Princeton Applied Research, Oak Ridge, TN) was used to perform impedance measurements in a threeelectrode cell with a Ag−AgCl reference electrode, platinum 2312

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Article

Bioconjugate Chemistry Synthesis of (S)-1. Using the same procedure as that for (R)-1, we used (S)-BINOL rather than (R)-BINOL, and 0.27 g (92%) (S)-1 was obtained as a viscous liquid. 1 H NMR (300 MHz, CDCl3, 298 K, δ (ppm)): 8.00 (s, 2H), 7.86 (d, 2H, J = 8.8 Hz), 7.46 (d, 2H, J = 8.8 Hz), 7.34 (dd, 2H, J = 8.8, 2.2 Hz), 7.11 (d, 2H, J = 8.8 Hz), 4.05 (m, 4H), 1.48 (m, 8 H), 1.11 (m, 8 H), 0.85 (m, 6H); 13C NMR (75 MHz, CDCl3): 154.71, 132.52, 130.14, 129.71, 129.39, 128.34, 127.08, 119.95, 117.18, 116.32, 69.45, 31.24, 29.20, 25.28, 22.44, 13.90; MALDI-MS: [M + H], 613; HRMS (m/z): [M + Na] found, 635.10; C32H36Br2O2Na requires 635.13. Synthesis of (R)-L1. (R)-1 (0.20 g, 0.3 mmol), terpyridineboronate ester (0.31 g, 0.7 mmol), potassium carbonate (0.55 g, 4.0 mmol), and PdCl2(PPh3)2 (0.02 g, 10 mol %) were placed in a two-neck round-bottom flask. Dry DMSO (20 mL) was added, and the reaction mixture was heated at 80 °C under a nitrogen atmosphere for 24 h. After the resulting mixture was cooled at room temperature, the catalyst was removed by filtration. The filtrate was extracted with chloroform and washed with H2O and brine. The organic layer was dried over MgSO4 and purified by alumina column chromatography and preparative HPLC to give (R)-L1 (0.15 g, 44%) as a light yellow solid. 1 H NMR (300 MHz, CDCl3, 298 K, δ (ppm)): 8.81 (s, 4H), 8.74 (d, 4H, J = 7.9 Hz), 8.69 (d, 4H, J = 7.9 Hz), 8.15 (s, 2H), 8.17 (d, 2H, J = 8.2 Hz), 8.06 (d, 4H, J = 7.6 Hz), 7.85 (bs, 8H) 7.59 (d, 2H, J = 1.5 Hz, 7.2), 7.49 (d, 2H, J = 9.0 Hz), 7.34 (d, 6H), 4.00 (s, 4H), 1.76 (q, 4H), 1.00 (m, 12H), 0.75 (t, 6H); 13 C NMR (75 MHz, CDCl3): 156.29, 155.93, 154.91, 149.78, 149.12, 141.90, 136.79, 135.20, 133.65, 129.55, 129.46, 127.65, 127.56, 126.19, 125.81, 125.53, 123.75, 121.35, 120.42, 118.67, 116.21, 69.71, 31.31, 29.36, 25.33, 22.44, 13.88; MALDI-MS: [M + H], 1069; HRMS (m/z): [M + H] found, 1069.51; C74H64N6O2 requires 1069.51. Synthesis of (S)-L1. (S)-L1 was obtained at 0.14 g (40%) from (S)-1 using the same procedure applied for (R)-L1. 1 H NMR (300 MHz, CDCl3, 298 K, δ(ppm)): 8.81 (s, 4H), 8.75 (d, 4H, J = 7.9 Hz), 8.70 (d, 4H, J = 7.9 Hz), 8.17 (s, 2H), 8.06 (d, 2H, J = 8.2 Hz), 8.04 (d, 4H, J = 7.6 Hz), 7.91 (d, 4H, J = 7.6 Hz), 7.88 (d, 4H, J = 8.2 Hz) 7.60 (q, 2H, J = 1.5 Hz, 7.2), 7.49 (d, 2H, J = 9.0 Hz), 7.39 (d, 6H), 4.07 (m, 4H), 1.50 (q, 4H), 1.01 (m, 12H), 0.76 (t, 6H); 13C NMR (75 MHz, CDCl3): 156.31, 155.96, 154.94, 149.80, 149.13, 141.93, 136.82, 135.22, 133.67, 129.50, 127.60, 126.21, 125.83, 125.55, 123.77, 121.35, 120.44, 118.65, 116.23, 69.72, 31.31, 29.37, 25.34, 22.44, 13.89; MALDI-MS: [M + H] 1069; HRMS (m/z): [M + H] found, 1069.51; C74H64N6O2 requires 1069.51. DNA Binding. The binding constants (Kb) of the polymers to ct-DNA were determined by UV−vis. spectral titration experiments were conducted at room temperature. Dilution of the polymers at the end of each titration was negligible. The Kb was calculated by fitting the changes in the absorption of the polymer as a function of ct-DNA concentration using eq 1. εa − εb b − (b2 − 2Kb2C t[DNA]t /s)1/2 = εb − εf 2KbC t

and bound polymer molar extinction coefficients, respectively. The value of εb was determined from the plateau of DNA titration, at which point addition of DNA did not result in any further changes to the absorption spectrum. Cell Viability. A549 or NIH 3T3 cell lines were grown in DMEM medium (pH 7.4) supplemented with 10% (v/v) FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin (10% FBSDMEM) and grown in the dark at 37 °C in a 5% CO2 humidified environment. Cells were seeded into 96 well tissue culture plates at a density of approximately 5000 cells per well for CCK-8 and into 24 well tissue culture plates at a density of approximately 15 000 cells/cm2 for calcein-AM and PI double staining. The polymers (P)- or (M)-polyFe were dissolved in 20% ethanol aqueous solution and then diluted to 0, 5, 10, 15, 20, and 25 μM in 1% ethanol and 99% of 10% FBS-DMEM for the CCK-8 assay. The old medium was removed from the cell culture, and medium containing polymers was added before measurement. The cytotoxicity assay with CCK-8 was performed according to the manufacturer’s protocol. Briefly, after 24 h of incubating the cells with 0, 5, 10, 15, 20, and 25 μM of (P)- or (M)-polyFe in 96 well plates, 10 μL of CCK-8 solution was added to each well. After an additional 2 h of incubation, the absorbance at 450 nm (reference 630 nm) was measured using a microplate reader. A higher absorbance value corresponded to higher cell proliferation, as the water-soluble formazan dye was formed via cellular respiration. Calcein-AM and PI Assay. After the cells were incubated for 24 h with 25 μM of (P)- or (M)-polyFe in 24 well plates, the cells were washed twice with sterile PBS, and 500 μL of PBS containing calcein-AM (2 μM) and PI (4 μM)) were added. Plates were incubated for 15 min before fluorescence imaging with a microscope. Apoptosis Analysis. After the cells were incubated for 24 h with 25 μM of (P)-polyFe in 24 well plates, the cells were washed twice with sterile PBS, and then 100 μL of 2% BSA containing trypsin was added and incubated for 5 min. The cells were collected in 1.5 mL centrifuge tubes and centrifuged for 3 min (300g). The medium was removed, and 100 μL of staining solution was added to the cells and mixed gently. Next, the cell suspension was kept in the dark at room temperature for 15 min. Finally, 400 μL of 1× binding buffer was added, and the cells were analyzed by FACS. The results were analyzed using SONY SP6800 Software.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00255. Figures showing characterizations of ligands by 1H, 13C, 1 H−1H COSY NMR, FTIR, and Raman spectroscopy techniques; an AFM image of ct-DNA, and a height procile of ct-DNA−(P)-polyFe conjugates. (PDF)

(1)

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Here, b = 1 + KbCt + Kb[DNA]t/2s, Kb is the binding constant between the polymer complex and DNA, Ct represents the total polymer concentration in repeat units, [DNA]t is the DNA concentration per nucleotide, “s” is the size of the binding site per nucleotide, and εa, εf, and εb represent the apparent, free,

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*Tel and fax: +81-29-860-4721; e-mail: HIGUCHI. [email protected]. 2313

DOI: 10.1021/acs.bioconjchem.6b00255 Bioconjugate Chem. 2016, 27, 2307−2314

Article

Bioconjugate Chemistry Present Address

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(M.D.H.) Department of Chemistry, Faculty of Science, Jagannath University, Dhaka-1100, Bangladesh. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

This research was funded by the JST-CREST project.

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DOI: 10.1021/acs.bioconjchem.6b00255 Bioconjugate Chem. 2016, 27, 2307−2314