Water-Soluble Cationic Metalloporphyrins: Specific G-Quadruplex

Article pubs.acs.org/IC

Water-Soluble Cationic Metalloporphyrins: Specific G‑QuadruplexStabilizing Ability and Reversible Chirality of Aggregates Induced by AT-Rich DNA Yan-Fang Huo,†,‡ Li-Na Zhu,*,†,§ Ke-Ke Liu,† Li-Na Zhang,† Ran Zhang,† and De-Ming Kong‡,§ †

Department of Chemistry, Tianjin University, Tianjin 300072, People’s Republic of China State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Nankai University, Tianjin 300071, People’s Republic of China § Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071, People’s Republic of China ‡

S Supporting Information *

ABSTRACT: Cu-TMPipPrOPP and Zn-TMPipPrOPP, two new cationic metallo derivatives of TMPipPrOPP (5,10,15,20-tetrakis{4[3-(1-methyl-1-piperidinyl)propoxy]phenyl}porphyrin), a porphyrin with four bulky side chains, were synthesized and characterized. The interactions of the new metalloporphyrins with structurally different DNAs were then compared with those of TMPipPrOPP. The introduction of bulky side chains provides the porphyrin derivatives with excellent binding specificity for G-quadruplex DNA, which is reflected by (1) the significantly different optical responses of TMPipPrOPP toward G-quadruplexes in comparison with single-stranded and duplex DNAs and (2) the ability of the three porphyrin derivatives to effectively stabilize G-quadruplexes, with no or little effect on the stability of duplex DNA. TMPipPrOPP can achieve colorimetric and fluorescent discrimination of G-quadruplexes from single-stranded and duplex DNAs with extraordinary high specificity. Due the presence of metal ions, Cu-TMPipPrOPP and Zn-TMPipPrOPP are deprived of the ability for optical G-quadruplex recognition but show enhanced ability to stabilize G-quadruplexes. In addition, because of the presence of the four cationic side chain substituents, the three porphyrin derivatives can form chiral aggregates via electrostatic interactions along the surface of structurally diverse DNAs. The chirality of aggregates formed by TMPipPrOPP is not changed by the nature of the template DNAs, whereas aggregates formed by Cu-TMPipPrOPP and Zn-TMPipPrOPP in the presence of adenine-thymine (AT) rich duplex DNA show completely inverted chirality in comparison with those formed in the presence of other DNAs. Interestingly, the chirality of the aggregates can be reversibly switched many times by alternating the ratio of Cu-TMPipPrOPP (or Zn-TMPipPrOPP) to AT-rich duplex DNA.

1. INTRODUCTION As the repository of hereditary information and the agents of gene expression, nucleic acids play important roles in a variety of biological processes. They are also important targets for drugs, particularly anticancer drugs.1 Sequence- and/or structure-specific recognition of nucleic acids at regions encoding particular genes is thus important in both biological and biomedical fields. For example, it has been reported that regions with a high content of adenine and thymine residues (AT-rich sequences) are frequently found in eukaryotic promoter regions.2 Some AT-rich gene domains have been validated as important targets for novel anticancer drugs,3 and several AT-specific minor groove-binding agents are currently in phase I clinical trials.3b,4 As well as nucleotide sequence, secondary structure is also an important determinant of the biological functions of nucleic acids at specific gene regions. The double-stranded helix of B© 2017 American Chemical Society

form DNA is the most favored secondary structure under physiological conditions, but increasing numbers of noncanonical nucleic acid secondary structures have been reported over recent decades.5 One of the most important of these is the G-quadruplex structure, which is formed by guanine (G)-rich DNA or RNA sequences.6 Gene sequences with the potential to form G-quadruplex structures are ubiquitous in the human genome and occur particularly in the telomere and promoter regions of some important oncogenes. Increasing evidence suggests that G-quadruplex structures are involved in key cellular processes, including transcription, recombination, and replication. Small-molecule ligands that can specifically target G-quadruplex structures are thus considered to show promise for treating some diseases, especially certain cancers.7 GReceived: February 16, 2017 Published: May 5, 2017 6330

DOI: 10.1021/acs.inorgchem.7b00426 Inorg. Chem. 2017, 56, 6330−6342

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Inorganic Chemistry Scheme 1. Chemical Structures of TMPyP4, TMPipPrOPP, Zn-TMPipPrOPP, and Cu-TMPipPrOPP

TMPipPrOPP and Cu-TMPipPrOPP show unique chiral aggregation behaviors in the presence of AT-rich duplex DNA.

quadruplex probes that can efficiently and specifically recognize G-quadruplex structures are also important for elucidating the biological functions of G-quadruplex-forming regions, following the biological events surrounding G-quadruplex formation and/ or monitoring telomere-targeted anticancer therapy, designing G-quadruplex-specific drugs, and G-quadruplex-based sensors.8 Porphyrins and metalloporphyrins are widely distributed in nature and, in living organisms, participate in essential biological processes, including photosynthesis, dioxygen transport, and storage. In recent years, porphyrin derivatives have also been widely studied as promising ligands for G-quadruplex structures.9 Because of the good size match between the porphin core and the G-quartet planes of G-quadruplexes, some porphyrins are able to induce G-quadruplex formation and stabilize these structures.9a,b,10 The best-known and mostly widely studied example is 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphyrin (TMPyP4, Scheme 1), but this ligand has poor selectivity for G-quadruplex over doublestranded DNAs.11 In previous studies, we have shown that the specificity of G-quadruplex recognition can be improved by introducing bulky side chains at the meso positions of the porphin core.10d,e Insertion of a metal ion into the porphyrin to give a metalloporphyrin might further improve the ability of porphyrin derivatives to induce and stabilize G-quadruplex, since it has been reported that when a metal complex (such as a metalloporphyrin) stacks onto the last G-quartet of the Gquadruplex, the metal ion might be positioned at the center of the G-quartet (i.e., the surface of the G-quadruplex ion channel). Electrostatic interactions between the central metal ion and the carbonyl oxygens of the guanine bases in the ion channel would then provide additional stabilization.12 A number of metalloporphyrin analogues of TMPyP4 have been prepared13 but, because of the poor selectivity of TMPyP4, most of these metalloporphyrins also interact nonspecifically with double-stranded DNAs.13b−e,14 To address this problem, we have now designed, synthesized, and characterized a water-soluble cationic porphyrin with four bulky cationic side chains (5,10,15,20-tetrakis{4-[3-(1-methyl1-piperidinyl)propoxy]phenyl}porphyrin, TMPipPrOPP, Scheme 1), together with the two metalloporphyrin derivatives Zn-TMPipPrOPP and Cu-TMPipPrOPP. Comparison of the interactions of these compounds with single-stranded, duplex, and G-quadruplex DNAs showed that TMPipPrOPP might be a good optical probe for the highly selective recognition of Gquadruplexes versus single-stranded and duplex DNAs. ZnTMPipPrOPP and Cu-TMPipPrOPP, especially Cu-TMPipPrOPP, are better able to stabilize G-quadruplexes than TMPipPrOPP but lose the capacity for specific optical recognition of G-quadruplexes. Unlike TMPipPrOPP, Zn-

2. EXPERIMENTAL SECTION 2.1. Reagents and Instruments. The oligonucleotides (Table S1 in the Supporting Information), which can fold into single-stranded, duplex, and G-quadruplex structures, were purchased from Sangon Biotech. Co. Ltd. (Shanghai, People’s Republic of China). Their concentrations were represented as single-stranded concentrations. The molar extinction coefficient was calculated using a nearestneighbor approximation (http://www.idtdna.com/analyzer/ Applications/OligoAnalyzer), and the calculated molar extinction coefficients of these oligonucleotides are given in Table S1. Calf thymus DNA (CtDNA) was also purchased from Sangon Biotech. Co. Ltd. (Shanghai, People’s Republic of China). Its concentration was represented as the base concentration, which was determined by the absorbance at 260 nm using the molar absorption coefficient of 6600 M−1 cm−1. The CtDNA solution gave a ratio of ultraviolet absorbance at 260 and 280 nm of 1.83:1, which indicates that the DNA was sufficiently free of protein. Na2EDTA (disodium ethylenediaminetetraacetic acid), Tris (tris(hydroxymethyl)aminomethane), K2CO3, N,Ndimethylformamide (DMF), and CH2Cl2 were obtained from Sigma. 5,10,15,20-Tetrakis(4-hydroxyphenyl)porphyrin (THPP) was obtained from TCI Development Co. Ltd. (Shanghai, People’s Republic of China). N-(3-Chloropropyl) piperidine hydrochloride was bought from Huai’an City East Chemical Factory (Jiangsu, People’s Republic of China). DMF was distilled over CaH2 before use. CH2Cl2 and CHCl3 was distilled from CaH2 and stored over molecular sieves. Other chemical reagents were of analytical grade and were used without further purification. Deionized and sterilized water (resistance >18 MΩ/cm) was used throughout the experiments. Nuclear magnetic resonance (NMR) spectra were recorded on a Mercury Vx-300 spectrometer operating for 1H NMR. High-resolution Fourier transform mass spectrometry (FT-MS) was measured on a Varian 7.0T FT-MS mass spectrometer. The infrared spectra of the samples (as KBr pellets) were recorded in the 3500−400 cm−1 region on a BIO-RAD FTS3000 spectrophotometer. Electron paramagnetic resonance (EPR) spectra were recorded with a Bruker EMX-6/1 spectrometer. Inductively coupled plasma mass spectrometry (ICPMS) was performed on an Agilent 7700x instrument, and elemental analysis was carried out on a Vario EL CUBE instrument. 2.2. Synthesis and Characterization of Zn-TMPipPrOPP and Cu-TMPipPrOPP. 2.2.1. Synthesis and Characterization of ZnTPipPrOPP. Free base porphyrins TPipPrOPP and TMPipPrOPP were synthesized according to the procedure reported by us recently.15 Then, a suspension of TPipPrOPP (0.2000 g, 0.1696 mmol) and zinc acetate dihydrate (0.3724 g, 1.696 mmol) in dry CHCl3/MeOH (20 mL) was stirred in the dark at room temperature for 24 h. The mixture was poured into CH2Cl2 (300 mL) and washed with water. The organic layer was evaporated under reduced pressure. The crude residue was redissolved in a CH2Cl2/MeOH mixture and purified using silica gel columns (100−200 mesh) with CH2Cl2/MeOH/TEA (v/v/v 10:0.5:0.01). The second fraction was collected, and the solvent was evaporated. Purple crystals of Zn-TPipPrOPP were obtained in 43% yield (0.0905 g, 0.0729 mmol). 1H NMR (300 MHz, 6331


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Inorganic Chemistry Scheme 2. Synthetic Route of TMPipPrOPP, Zn-TMPipPrOPP, and Cu-TMPipPrOPP

CDCl3): δ 8.76 (s, 8H), 7.89 (d, J = 8.4 Hz, 8H), 7.19 (d, J = 8.5 Hz, 8H), 4.23 (t, J = 6.2 Hz, 8H), 2.68−2.57 (m, 8H), 2.51 (s, 16H), 2.14 (dd, J = 13.9, 7.1 Hz, 8H), 1.67 (dd, J = 10.7, 5.3 Hz, 16H), 1.51 (d, J = 4.4 Hz, 8H). FT-MS: m/z calcd for [C76H88N8O4Zn + H], 1241.6220; found, 1241.6298 [M + H] (Figures S1 and S2 in the Supporting Information). Anal. Calcd for C76H88N8O4Zn: C, 73.44; H, 7.14; N, 9.02. Found: C, 73.05; H, 7.27; N, 9.17. 2.2.2. Synthesis and Characterization of Cu-TPipPrOPP. A suspension of TPipPrOPP (0.2000 g, 0.1696 mmol) and cupric acetate monohydrate (0.6772 g, 3.392 mmol) in dry CH2Cl2/MeOH (40 mL) was stirred at reflux for 12 h. Then, the mixture was poured into CHCl3 (300 mL) and washed with water. The solvent was removed under reduced pressure. The violet residue was dissolved in CHCl3 and purified using silica gel columns (100−200 mesh) with CHCl3/PE/TEA (v/v/v 2.5/1/0.01). The first fraction was collected, and the solvent was evaporated. Purple crystals of Cu-TPipPrOPP were obtained in 73% yield (0.1535 g, 0.1238 mmol). Because CuTPipPrOPP is paramagnetic, it cannot be characterized by NMR. FTMS: m/z calcd for [C76H88N8O4Cu + H], 1240.6225; found, 1240.6302 [M + H] (Figure S3 in the Supporting Information). Anal. Calcd for C76H88N8O4Cu: C, 73.55; H, 7.15; N, 9.03. Found: C, 73.63; H, 7.19; N, 9.07. 2.2.3. Synthesis and Characterization of Zn-TMPipPrOPP-4I and Cu-TMPipPrOPP-4I. To a suspension of Zn-TPipPrOPP (0.0500g, 0.0403 mmol) or Cu-TPipPrOPP (0.0700 g, 0.0565 mmol) in dry CH2Cl2 (20 mL) was added CH3I (10 mL, 0.16 mmol). The mixture was stirred under N2 and heated by using an oil bath at 40 °C for 24 h.

After this mixture was cooled, the solvent was evaporated and the resulting solid was washed with CH2Cl2 and diethyl ether in turn. A red-purple solid of Zn-TMPipPrOPP-4I and red-brown solid of CuTMPipPrOPP-4I were obtained in 50% yield (0.0262 g, 0.0201 mmol) and 70% yield (0.0514 g, 0.0396 mmol), respectively. 1H NMR for ZnTMPipPrOPP-4I (300 MHz, DMSO): δ 8.73 (s, 8H), 7.93 (d, J = 8.4 Hz, 8H), 7.32 (d, J = 8.5 Hz, 8H), 4.32 (d, J = 5.1 Hz, 8H), 3.70−3.56 (m, 8H), 3.44 (t, J = 5.2 Hz,16H), 3.13 (s, 12H), 2.40−2.25 (m, 8H), 1.87 (s, 16H), 1.61 (s, 8H). FT-MS: m/z calcd for [C80H100N8O4Zn4I]/4, 325.1784; found, 325.1812 [M + − 4I]/4; calcd for [C80H100N8O4Cu-4I]/4, 324.9286; found, 324.9311 [M+-4I]/4 (Figures S4−S6 in the Supporting Information). Anal. Calcd for ZnTMPipPrOPP-4I: C, 53.07; H, 5.57; N, 6.19. Found: C, 53.14; H, 5.43; N, 6.15. Calcd for Cu-TMPipPrOPP-4I: C, 53.12; H, 5.57; N, 6.19. Found: C, 53.24; H, 5.86; N, 6.30. ICP-MS: calcd for ZnTMPipPrOPP-4I Zn, 3.61%; found Zn, 3.53%; calcd for CuTMPipPrOPP-4I Cu, 3.51%; found Cu, 3.49%. 2.3. UV−Vis Absorption Spectroscopy. UV−vis absorption spectra were measured on a Cary 60 UV−vis spectrophotometer (Agilent Technologies) with a 1 cm path length micro quartz cell (40 μL, Starna Brand, England). Solutions containing 0−50 μM of the individual DNAs, 10 mM Tris-HCl buffer (pH 7.4), 50 mM KCl, and 1 mM Na2EDTA were prepared. Each solution was heated at 95 °C for 5 min to remove any possible DNA secondary structures and then cooled rapidly to 25 °C and allowed to incubate at 25 °C for 30 min. After overnight incubation at 4 °C, 5 μM of TMPipPrOPP, Zn6332


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Inorganic Chemistry TMPipPrOPP, or Cu-TMPipPrOPP was added and absorption spectra in the range of 350−800 nm were recorded. 2.4. Fluorescence Spectroscopy. Fluorescence spectra were measured on a Shimadzu RF-5301PC spectrofluorimeter. Solutions were prepared as above. When 697 nm was used as the excitation wavelength, the excitation and emission slits were both set at 3 nm. When other excitation wavelengths were used, the excitation and emission slits were both set at 5 nm. 2.5. Melting Temperature (T1/2) Detection of G-Quadruplex and Duplex DNA. Melting temperature (T1/2) detection of Gquadruplex or duplex DNA was carried out on a Cary-60 UV−vis spectrophotometer equipped with a single-cell Peltier temperature control accessory. The DNA (5 μM) solutions were prepared in 10 mM Tris-HCl buffer (pH 7.4) containing 50 mM KCl and 1 mM Na2EDTA. The solutions were heated at 95 °C for 5 min and then cooled rapidly to 25 °C and allowed to incubate at 25 °C for 30 min. After overnight incubation at 4 °C, 0 or 5 μM of TMPipPrOPP, ZnTMPipPrOPP, or Cu-TMPipPrOPP was added. After a sufficient mixing, the absorption signal at 295 nm (G-quadruplex) or 260 nm (duplex DNA) (400 nm as control wavelength) was recorded at ∼10 °C. When the absorption signal became constant, the temperature was increased in steps of 1 °C, and the absorption signal was recorded at each temperature until the signal no longer changed. At each temperature, the mixture was left to equilibrate for 1 min before the absorption signal was recorded. 2.6. Induced Circular Dichroism (CD) Spectra of DNATemplated Chiral Aggregates. A 3 mL mixture was prepared in 10 mM Tris-HCl buffer (pH 7.4) containing 50 mM KCl, 1 mM Na2EDTA, and 3 μM individual DNA oligonculeotides. Each mixture was heated at 95 °C for 5 min to remove any possible aggregates and then cooled rapidly to 25 °C and incubated at 25 °C for 30 min. After overnight incubation at 4 °C, 5 μM of TMPipPrOPP, ZnTMPipPrOPP, or Cu-TMPipPrOPP was added. A CD spectrum of the mixture was recorded between 220 and 600 nm in 1 cm path length cuvette on a Jasco J-715 spectropolarimeter. Spectra were averaged from three scans, which were recorded at 100 nm/min with a response time of 1 s and a bandwidth of 0.5 nm.

route), in which the neutral porphyrin TPipPrOPP is reacted with metal salts to provide the metalloporphyrin ZnTPipPrOPP or Cu-TPipPrOPP. This reaction occurs readily, and since the products, Zn-TPipPrOPP and Cu-TPipPrOPP, are soluble in CH2Cl2 or CHCl3 and the metal salts are soluble in water, the metal salts can be easily removed by partitioning the reaction mixture between CH2Cl2 and water. ZnTPipPrOPP and Cu-TPipPrOPP can then be separated from TPipPrOPP by silica gel chromatography. Using this synthetic route, pure crystals of Zn-TPipPrOPP and Cu-TPipPrOPP were obtained in 43% and 73% yields, respectively. Fourier transform mass spectroscopy (FT-MS) of Zn-TPipPrOPP and Cu-TPipPrOPP and proton nuclear magnetic resonance (1H NMR) spectroscopy of Cu-TPipPrOPP (Figures S1−S3 in the Supporting Information) confirmed the formation and purity of Zn-TPipPrOPP and Cu-TPipPrOPP. The cationic metalloporphyrins Zn-TMPipPrOPP and Cu-TMPipPrOPP were then obtained by N-methylation of Zn-TPipPrOPP or CuTPipPrOPP. The tetraiodide salts of Zn-TPipPrOPP and CuTPipPrOPP were obtained in 50% and 70% yields, respectively. This strategy thus allowed preparation of the cationic metalloporphyrins in satisfactory yield and with relatively simple purification procedures. The new cationic metalloporphyrins Zn-TMPipPrOPP and Cu-TMPipPrOPP were characterized by FT-MS, infrared (IR) spectroscopy, 1H NMR spectroscopy, and electron paramagnetic resonance (EPR) spectroscopy (Figures S4−S7 in the Supporting Information). The FT-MS spectra of ZnTMPipPrOPP and Cu-TMPipPrOPP in methanol showed molecular ions at 325.1812 and 324.9311, respectively, which are in good agreement with the calculated exact masses of 325.1784 for Zn-TMPipPrOPP and 324.9286 for CuTMPipPrOPP. Since infrared spectra can provide important structural information for coordination compounds, the infrared spectra of Cu-TMPipPrOPP, Zn-TMPipPrOPP, and the free base TMPipPrOPP were compared. The IR spectra of the three compounds displayed similar characteristics, with strong bands appearing mainly between 700 and 1600 cm−1 (Figure 1). The spectrum of the free base TMPipPrOPP shows two characteristic bands at 3313 and 966 cm−1, which are associated with the N−H stretching vibration and the in-plane bending vibration of the pyrrole rings. These two bands disappeared in the spectra of both Zn-TMPipPrOPP and CuTMPipPrOPP because of deprotonation and metalation of the pyrrole rings. This observation supports the coordination of

3. RESULTS AND DISCUSSION 3.1. Synthesis of the Cationic Metalloporphyrins of Zn-TMPipPrOPP and Cu-TMPipPrOPP. The synthetic route to the cationic porphyrin TMPipPrOPP and the cationic metalloporphyrins Zn-TMPipPrOPP and Cu-TMPipPrOPP is shown in Scheme 2. The preparation of TMPipPrOPP has been reported by us recently.15 Briefly, meso-tetrakis(4hydroxylphenyl)porphyrin (THPP) and N-(3-chloropropyl)piperidine hydrochloride were used to synthesize the neutral porphyrin precursor TPipPrOPP. The cationic porphyrin TMPipPrOPP was then prepared by N-alkylation of TPipPrOPP in the presence of CH3I and K2CO3. Theoretically, there are three possible ways to obtain the cationic metalloporphyrins. One way would be to metallize THPP first and then use the metallo derivatives as precursors (Scheme 2, red route). The reaction between the metallo derivatives of THPP and N-(3-chloropropyl)piperidine hydrochloride could, however, generate several byproducts, such as mono-, bi-, and trisubstituted metalloporphyrins, leading to difficult purification steps and low yields. The second way would be to react TMPipPrOPP with metal salts. This is the most direct route to cationic metalloporphyrins (Scheme 2, blue route), but TMPipPrOPP and the product metalloporphyrins have similar polarities and solubilities, making it difficult to separate the product from the starting materials by silica gel chromatography or recrystallization. The reaction between the cationic ligand and cationic metal ions is also somewhat difficult, and the reaction efficiency is relatively low. These problems can be overcome by using a third synthetic route (Scheme 2, green

Figure 1. IR spectra of TMPipPrOPP, Cu-TMPipPrOPP, and ZnTMPipPrOPP. 6333


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Figure 2. 1H NMR of Zn-TMPipPrOPP-4I and TMPipPrOPP-4I.

Figure 3. Absorption spectra of (a) free TMPipPrOPP, Zn-TMPipPrOPP, and Cu-TMPipPrOPP and (b−d) their spectral changes induced by different DNAs. The insert in (a) shows the aqueous solution photograph of the three porphyrin derivatives. Conditions: [TMPipPrOPP] = [CuTMPipPrOPP] = [Zn-TMPipPrOPP] = 5 μM; [Hum24] = [C-MYC] = [AT] = [ssDNA1] = 20 μM (strand concentration); [CtDNA] = 960 μM (base concentration). The absorption spectra in the presence of other DNAs can be found in Figure S8 in the Supporting Information.

disappears since the hydrogen atoms of the N−H bonds are replaced by the zinc ion. Coordination of the Cu(II) ion to the nitrogen atoms in the porphin core is evidenced by the EPR spectrum. The EPR spectrum of Cu-TMPipPrOPP in ethanol solution at room temperature (Figure S7 in the Supporting Information) shows typical EPR signals attributable to the one unpaired electron in the outer layer d orbital of copper(II) in the complex. The EPR

both nitrogen atoms of the pyrrole rings to the metal and further confirms the formation of the metal complexes.16 The 1H NMR spectra of TMPipPrOPP and Zn-TMPipPrOPP in DMSO-d6 are shown in Figure 2. For the free base porphyrin TMPipPrOPP, the characteristic chemical shift of the pyrrole NH in the porphyrin core is located at −2.87 ppm.17 After formation of the Zn complex, the peak at −2.87 ppm 6334


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Figure 4. (a) Fluorescence spectra of free TMPipPrOPP, Zn-TMPipPrOPP, Cu-TMPipPrOPP and (b, c) their spectral changes induced by different DNAs. (d) Ratio of the fluorescence intensity at high wavelength to that at low wavelength. The excitation wavelengths for TMPipPrOPP, ZnTMPipPrOPP, and Cu-TMPipPrOPP were 422, 426, and 417 nm, respectively. Conditions: [TMPipPrOPP] = [Cu-TMPipPrOPP] = [ZnTMPipPrOPP] = 5 μM; [Hum24] = [C-MYC] = [AT] = [ssDNA1] = 20 μM (strand concentration); [CtDNA] = 960 μM (base concentration). The fluorescence spectra in the presence of other DNAs can be found in Figure S12 in the Supporting Information. Since no fluorescence is observed, the spectra of Cu-TMPipPrOPP in the presence of DNA are not given.

parameters (g∥ = 2.30, g⊥ = 2.05, g0 = 2.13, g∥ > g⊥) indicate that the Cu(II) ion is coordinated to the nitrogen atoms in the porphin core with a slightly distorted square planar geometry.18 3.2. Colorimetric Responses of TMPipPrOPP, ZnTMPipPrOPP, and Cu-TMPipPrOPP to Different DNAs. First, the electronic absorption spectra of the three porphyrin derivatives were compared. The absorption spectrum of free TMPipPrOPP (black line) has a strong Soret band centered at 422 nm and four weak Q bands centered at 521, 559, 596, and 650 nm (Figure 3). In comparison with the spectrum of TMPipPrOPP, the Soret band in the spectrum of ZnTMPipPrOPP shows a red shift of ∼4 nm and that in the spectrum of Cu-TMPipPrOPP shows a blue shift of ∼5 nm. Because of the increased molecular symmetry, the spectra of both Zn-TMPipPrOPP and Cu-TMPipPrOPP show only two Q bands. The two Q-bands of Zn-TMPipPrOPP, which are centered at 563 and 606 nm, have comparable absorption intensities. For Cu-TMPipPrOPP, however, the Q band at 542 nm is much stronger than that at 580 nm. The relatively strong absorption at 542 nm imparts a noticeable red color to an aqueous solution of Cu-TMPipPrOPP, in comparison with solutions of TMPipPrOPP and Zn-TMPipPrOPP (Figure 3a, insert). The effects of different DNAs (Table S1 in the Supporting Information), including single-stranded, duplex, and Gquadruplex DNAs, on the absorption spectra of the three porphyrin derivatives were then investigated. TMPipPrOPP gave absorption signal responses to G-quadruplexes significantly different from those to single-stranded and duplex DNAs. Addition of G-quadruplexes (Hum24, C-MYC, KRAS, and Oxy28) caused obvious hypochromicity of the Soret band

(422 nm), accompanied by the emergence of two new bands at 454 and 697 nm (Figure 3b and Figure S8 in the Supporting Information). Under the same conditions, neither singlestranded DNAs (ssDNA1 and ssDNA2) nor duplex DNAs (AT, GC, LD, and CtDNA) caused such marked spectral changes. Only a slight red shift (∼5 nm) and different levels of hyperchromicity were observed for the Soret band and, more importantly, no new absorption bands emerged, implying that TMPipPrOPP can specifically discriminate G-quadruplexes from single-stranded and duplex DNAs. This finding is consistent with our earlier hypothesis that the introduction of bulky side-chain substituents can improve the specificity of Gquadruplex recognition by porphyrin derivatives, since the presence of these large side chains increases the steric hindrance for the intercalation of porphyrin derivatives into the base planes or grooves of duplex DNAs.10d,e Disappointingly, electronic absorption spectroscopy demonstrated that Cu-TMPipPrOPP and Zn-TMPipPrOPP cannot optically discriminate G-quadruplexes from single-stranded and duplex DNAs, since spectral signal changes induced by structurally different DNAs were almost indistinguishable. Although slight red shifts (7 nm for Cu-TMPipPrOPP and 6 nm for ZnTMPipPrOPP) and different degrees of hyperchromicity were observed for the Soret band, no new bands emerged. These experiments demonstrate that TMPipPrOPP can be used as a specific colorimetric probe for G-quadruplex DNA but that CuTMPipPrOPP and Zn-TMPipPrOPP cannot. Such a conclusion was confirmed by absorption titration spectra recorded by fixing the porphyrin concentration but varying the DNA concentration (Figures S9−S11 in the Supporting Information). 6335


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Figure 5. (a−c) Melting curves of G-quadruplexes (Hum24 and C-MYC) and duplex (ssDNA2/ssDNA2c) in the absence or presence of the individual porphyrin derivatives. (d) Melting temperature changes of the tested DNAs induced by the porphyrin derivatives.

3.3. Fluorescence Responses of TMPipPrOPP, ZnTMPipPrOPP, and Cu-TMPipPrOPP to Different DNAs. We next investigated whether or not the different porphyrin derivatives can be used as G-quadruplex-specific fluorescent probes. When excited at their individual maximal absorption wavelengths, both TMPipPrOPP and Zn-TMPipPrOPP gave two emission peaks. Compared with the corresponding peaks for TMPipPrOPP, the two emission peaks of Zn-TMPipPrOPP show blue-shifts of approximately 43 and 56 nm and the fluorescence intensity at the lower wavelength decreases markedly (Figure 4). Cu-TMPipPrOPP emits no fluorescence at all, perhaps because of fluorescence quenching by Cu(II), where the outer layer d orbital has one unpaired electron. TMPipPrOPP gave markedly different fluorescence responses to G-quadruplexes, in comparison with single-stranded or duplex DNAs. For free TMPipPrOPP, the intensity of the peak centered at 720 nm was much lower than that of the peak centered at 657 nm and the fluorescence ratio was lower than 1. Addition of G-quadruplexes led to a significant decrease in the fluorescence intensity at 657 nm, accompanied by a slight increase in that at 720 nm (Figure 4b and Figure S12 in the Supporting Information). When the concentration of Gquadruplex is sufficiently high, the fluorescence intensity at 720 nm will be higher than that at 657 nm and the fluorescence intensity ratio becomes larger than 1 (Figure 4d and Figure S12c). Single-stranded and duplex DNAs do not cause such marked spectral changes; the fluorescence intensity at 720 nm is always lower than that at 657 nm, and the ratio is always much lower than 1. These results suggest that changes in the fluorescence signals of TMPipPrOPP could be used to discriminate G-quadruplexes from single-stranded and duplex DNAs. Zn-TMPipPrOPP, on the other hand, cannot discriminate between structurally different DNAs, since neither

G-quadruplexes nor single-stranded and duplex DNAs cause obvious changes in the fluorescence spectrum of ZnTMPipPrOPP (Figure 4c and Figure S12 in the Supporting Information). The fluorescence intensity at higher wavelength is always lower than that at lower wavelength. Free CuTMPipPrOPP emits no fluorescence, and fluorescence cannot be activated by any DNAs. These data suggest that only TMPipPrOPP has the potential to discriminate G-quadruplexes from single-stranded and duplex DNAs by changes in fluorescence signals. Since Gquadruplexes induce two new absorption bands (centered at 454 and 697 nm) in the absorption spectrum of TMPipPrOPP but single-stranded and duplex DNAs do not, much better Gquadruplex-probing efficiency can be obtained by using 454 or 697 nm as the excitation wavelength (Figures S13 and S14 in the Supporting Information). Overall, it can be concluded that TMPipPrOPP would be a useful optical probe for colorimetric and fluorescent discrimination of G-quadruplex from singlestranded and duplex DNAs but that Cu-TMPipPrOPP and ZnTMPipPrOPP would not. 3.4. Stabilization of G-Quadruplex and Duplex DNAs by Porphyrin Derivatives. The abilities of the three porphyrin derivatives to stabilize G-quadruplex or duplex DNA were compared by investigating their effects on the melting temperatures (T1/2) of the two different DNA structures. Because the T1/2 values of intramolecular duplexes are too high to be determined, an intermolecular duplex (ssDNA2/ssDNA2c, Table S1 in the Supporting Information) was used as the model duplex DNA. As expected, TMPipPrOPP effectively and specifically stabilized G-quadruplexes, reflected by increases of ∼8 °C in T1/2 for Hum24 and C-MYC, but had no almost effect on duplex DNA (Figure 5). This further demonstrates the excellent recognition specificity 6336


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Figure 6. (a, c, e) Induced CD spectra and (b, d, f) CD signal differences of TMPipPrOPP, Zn-TMPipPrOPP, and Cu-TMPipPrOPP in the presence of different DNAs. Δθ = θlow wavelength − θhigh wavelength. Conditions: [TMPipPrOPP] = [Zn-TMPipPrOPP] = [Cu-TMPipPrOPP] = 5 μM. The concentrations of all DNAs are 3 μM. The induced CD spectra in the presence of other DNAs can be found in Figure S15 in the Supporting Information.

those of G-quadruplex. The markedly different abilities of CuTMPipPrOPP and Zn-TMPipPrOPP, especially Cu-TMPipPrOPP, to stabilize G-quadruplex and duplex DNA suggest that these two metalloporphyrins also have high specificity for binding to G-quadruplex DNA. These results allow us to conclude that metallization of TMPipPrOPP to give CuTMPipPrOPP or Zn-TMPipPrOPP abolishes the ability to optically discriminate G-quadruplexes from single-stranded and duplex DNAs but confers enhanced ability to stabilize Gquadruplex, while maintaining relatively high specificity against duplex DNA. 3.5. DNA-Templated Chiral Aggregation of TMPipPrOPP, Zn-TMPipPrOPP, and Cu-TMPipPrOPP. To further investigate the interactions between porphyrin derivatives and DNAs, circular dichroism (CD) spectra of the three porphyrin derivatives in the presence of different DNAs were compared. The three porphyrin derivatives themselves possess no chiral

of TMPipPrOPP for G-quadruplex over duplex DNA. At the same concentration, Cu-TMPipPrOPP and Zn-TMPipPrOPP, especially Cu-TMPipPrOPP, were even more effective at stabilizing G-quadruplex than TMPipPrOPP, perhaps because of an additional contribution from electrostatic interactions between the central metal ion of the metalloporphyrin and carbonyl oxygens in the G-quadruplex ion channel. The Gquadruplex-stabilizing ability of Cu-TMPipPrOPP was obviously higher than that of Zn-TMPipPrOPP. This might be caused by the effect of the axial ligand. Zn-TMPipPrOPP might have an axially coordinated water molecule, but CuTMPipPrOPP does not. The presence of the axial ligand would hinder the end stacking of Zn-TMPipPrOPP on Gquadruplexes to some degree. Different from TMPipPrOPP that stabilized only G-quadruplex DNA, Cu-TMPipPrOPP and Zn-TMPipPrOPP also slightly increased the T1/2 value of duplex DNA, but the T1/2 value changes were much lower than 6337


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Figure 7. AT concentration-dependent induced CD spectral changes of (a) TMPipPrOPP, (b, c) Cu-TMPipPrOPP and (d, e) Zn-TMPipPrOPP. In (b) and (c), the AT concentration is changed by 0−3 and 3−10 μM, respectively. In (d) and (e), the AT concentration is changed by 0−2.4 and 2.4−10 μM, respectively. Conditions: [TMPipPrOPP] = [Zn-TMPipPrOPP] = [Cu-TMPipPrOPP] = 5 μM. The inserts in (a), (b), and (d) show the AT concentration dependent CD signal change at low and high wavelengths, respectively.

center and show no CD signals. When these species are bound to chiral templates (e.g., DNAs), however, induced CD signals are found in the Soret region. Induced CD spectroscopy is now a widely used tool for the analysis of binding modes between porphyrins and DNAs. It has been reported that intercalation of porphyrins in DNAs might induce a negative CD band, whereas groove-binding might induce a positive CD band.19a When a porphyrin binds at the outside of the DNA strand, a bisignate induced CD band might be generated. It has also been reported that the stacking of porphyrins on the ends of G-quadruplexes can result in the emergence of bisignate induced CD bands.19b,c Bisignate- and trisignate-induced CD bands were observed in the spectra of all three porphyrin derivatives in the presence of structurally different DNAs (Figure 6 and Figure S15 in the Supporting Information), indicating that all of the derivatives can bind at the outside of single-stranded, duplex, and G-

quadruplex DNAs. Since each porphyrin derivative has four positively charged side chains and the DNA strand contains many negatively charged phosphate groups, electrostatic interactions inevitably occur between them. The electrostatic interactions between the positively charged porphyrin derivatives and the negatively charged DNAs can shield the electrostatic repulsion between two porphyrin units, enabling them to form chiral aggregates along the DNA strands. π−π interactions between neighboring porphin rings can further stabilize the aggregates, allowing them to remain stable even under conditions of increasing ionic strength (Figure S16 in the Supporting Information). A strong excitation coupling occurs between neighboring porphyrin units in the aggregates, leading to the splitting of excited state energy and the splitting of the Soret band.20 The induced CD signal intensities of ZnTMPipPrOPP were typically lower than those of Cu6338


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Figure 8. (a) Cu-TMPipPrOPP or (b) Zn-TMPipPrOPP concentration-dependent changes in induced CD spectra of the mixtures containing 5 μM Cu-TMPipPrOPP (or Zn-TMPipPrOPP) and 7 μM AT. The inserts show the CD signal changes at low and high wavelengths as a function of total Cu-TMPipPrOPP (or Zn-TMPipPrOPP) concentration.

show much higher effects on AT than other DNAs (Figure S17 in the Supporting Information). The CD band of B-form double-helical AT that can be clearly observed in the presence of TMPipPrOPP nearly disappears in the presence of CuTMPipPrOPP and Zn-TMPipPrOPP, thus suggesting that the structure of AT might be disturbed by these two metalloporphyrins. The effects of changes in DNA concentration on CD spectra were examined to investigate the aggregation of porphyrin derivatives along the surface of the DNA strand. With increased DNA concentration, a monotonous change in the CD signal (CD signal at low and high wavelength increases and decreases, respectively, with DNA concentration) was observed for the three porphyrin derivatives, except for Cu-TMPipPrOPP and Zn-TMPipPrOPP in the presence of AT (Figure 7 and Figure S18 in the Supporting Information). Interestingly, in the presence of AT, Cu-TMPipPrOPP showed a positive induced CD band at ∼427 nm and a continuous increase in signal intensity, concomitant with a marked blue shift in wavelength, was observed with increasing AT concentration. When the AT concentration exceeded 3 μM, the CD signal intensity began to decrease as the AT concentration increased. When the AT concentration reached 4.2 μM, the positive induced CD band changed to a negative induced CD band, and the CD signal intensity continued to decrease with increasing AT concentration. The effects of AT on the induced CD signal at 433 nm were completely different, and the presence of AT induced a negative CD signal. With increasing concentrations of AT, the signal intensity initially decreased and then increased. As the signal intensity increased, the negative CD band changed to a positive band, indicating that the chirality of the aggregates had been inverted. Zn-TMPipPrOPP showed induced CD signal changes similar to those of Cu-TMPipPrOPP, except that the signal intensities were lower than those of Cu-TMPipPrOPP signals. On the other hand, when Cu-TMPipPrOPP (or ZnTMPipPrOPP) was added to a Cu-TMPipPrOPP/AT (or ZnTMPipPrOPP/AT) mixture containing excess AT, complete inversion of the induced CD was observed at both wavelengths (Figure 8). These results indicate that the chirality of the aggregates can be reversibly switched by adjusting the ratio of metalloporphyrin to AT. To further demonstrate this metalloporphyrin- and ATcontrolled reversal of chirality, metalloporphyrin and AT were added alternately to a metalloporphyrin/AT mixture and changes in the CD spectra were monitored. Alternate addition

TMPipPrOPP, perhaps because of the presence of one water molecule axially coordinated to the central Zn(II),21 which hinders close contact between neighboring Zn-TMPipPrOPP molecules. The excellent optical discrimination of TMPipPrOPP toward G-quadruplexes versus single-stranded and duplex DNAs, together with the different stabilizing abilities of the three porphyrin derivatives toward G-quadruplex and duplex DNAs, mean that there must be end-stacking interactions between the porphyrin derivatives and Gquadruplexes in addition to the outside binding interactions. In this binding mode, the porphin core stacks onto the terminal G-quartet(s) by π−π stacking interactions. Meanwhile, the four cationic side chains interact with the negatively charged phosphate backbones of the loops or grooves of the Gquadruplex via electrostatic interactions. The synergy between π−π stacking and electrostatic interactions gives the Gquadruplex enhanced stability. Two binding modes (end stacking and outside binding) thus occur between the porphyrin derivatives and G-quadruplexes. The end-stacking mode gives the porphyrin derivatives good G-quadruplexstabilizing abilities and leads to the obvious changes in the optical properties of TMPipPrOPP. Only outside binding occurs between the porphyrin derivatives and other structurally different DNAs, and this binding mode has no or little effect on the optical properties of the porphyrin or on DNA stability. It is interesting to note that Cu-TMPipPrOPP and ZnTMPipPrOPP show inverted induced CD signals in the presence of AT-rich duplex DNA (AT), in comparison with those in the presence of other DNAs. In the Soret region, CuTMPipPrOPP and Zn-TMPipPrOPP gave a positive induced CD band at low wavelength (∼421−427 nm) and a negative induced CD band at high wavelength (∼433−439 nm) in the presence of AT (Figure 6). On the other hand, a negative band was observed at low wavelength and a positive band was observed at high wavelength for TMPipPrOPP in the presence of all DNAs tested and for Cu-TMPipPrOPP and ZnTMPipPrOPP in the presence of all DNAs except AT. These results suggest that aggregates of Cu-TMPipPrOPP and ZnTMPipPrOPP on an AT template have inverted chirality in comparison with aggregates formed on other DNA templates. Using Δθ (the difference in CD signal between low and high wavelengths), AT can be easily discriminated from other DNAs. By comparing the effects of the three porphyrin derivatives on the DNA CD spectra in the range of 220−300 nm, one can find that Cu-TMPipPrOPP and Zn-TMPipPrOPP 6339


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Figure 9. Reversible changes in (a, c) induced CD spectra and (b, d) CD peak intensities. Conditions: 5 μM Cu-TMPipPrOPP (or ZnTMPipPrOPP) (1, 3, 5, 7, 9) and 3 μM AT (2, 4, 6, 8) were alternately added in the mixture of 5 μM Cu-TMPipPrOPP (or Zn-TMPipPrOPP) and 3 μM AT.

comparison with other aggregates formed on other DNA templates, and the chirality can be reversibly switched many times by adjusting the ratio of the concentration of CuTMPipPrOPP (or Zn-TMPipPrOPP) to AT. Such an interesting finding might provide useful information for further elucidation of chiral aggregation on DNA templates.

of Cu-TMPipPrOPP (or Zn-TMPipPrOPP) and AT resulted in repeated reversal of the induced CD peaks (Figure 9), indicating that the chirality of the aggregates can be reversibly switched many times. To the best of our knowledge, this is the first example of reversible inversion of chiral aggregation triggered by changes in concentration of the DNA template.

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4. CONCLUSIONS In summary, three porphyrin derivatives, TMPipPrOPP, CuTMPipPrOPP, and Zn-TMPipPrOPP, each with four bulky cationic side chains, were synthesized and characterized and their interactions with structurally different DNAs were compared. The four bulky side chains confer perfect selectivity for binding to G-quadruplex rather than single-stranded or duplex DNAs. This is illustrated by the fact that the porphyrin derivatives can efficiently stabilize G-quadruplexes but have no or little effect on the stability of duplex DNA. The different stabilizing abilities toward G-quadruplex and duplex DNA suggest that the porphyrin derivatives adopt different binding modes with G-quadruplex and duplex DNA. End stacking onto the terminal G-quartets of G-quadruplex DNA gives TMPipPrOPP significantly different optical responses toward G-quadruplexes versus single-stranded and duplex DNA and makes colorimetric and fluorescent recognition of G-quadruplexes possible. In addition to end stacking on Gquadruplexes, the presence of cationic side chains means that the three porphyrin derivatives can form chiral aggregates via electrostatic interactions at the outside of all types of DNA. Interestingly, aggregates of Cu-TMPipPrOPP and Zn-TMPipPrOPP formed on AT templates show inverted chirality in

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.7b00426. DNA oligonucleotides used, structural characterization of Cu-TMPipPrOPP and Zn-TMPipPrOPP, colorimetric and fluorescent responses of TMPipPrOPP, Zn-TMPipPrOPP, and Cu-TMPipPrOPP to different DNAs, absorption and fluorescence titration spectra of TMPipPrOPP, and CD studies on DNA-templated chiral aggregation of TMPipPrOPP, Zn-TMPipPrOPP, and Cu-TMPipPrOPP (PDF)

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AUTHOR INFORMATION

Corresponding Author

*L-N.Z.: tel, +86-22-27403475; fax, +86-22-27403475; e-mail, [email protected]. ORCID

Li-Na Zhu: 0000-0002-6396-1592 Notes

The authors declare no competing financial interest. 6340


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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (No. 21371130), the Natural Science Foundation of Tianjin (Nos. 15JCYBJC48300, 16JCYBJC19900), and the Innovation Fund of Tianjin University.

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Water-Soluble Cationic Metalloporphyrins: Specific G-Quadruplex

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