[August 12, 2011] Control of Singlet Oxygen Generation Photosensitized by meso-Anthrylporphyrin through Interaction with DNA [Photochemistry and Photobiology] Tweet (Photochemistry and Photobiology Via Acquire Media NewsEdge) ABSTRACT To control the activity of photosensitized singlet oxygen (1 O2) generation, the electron donor-connecting porphyrin, 5-(9'-anthryl)-10,15,20-tris(p-pyridyl)porphyrin (AnTPyP), was designed and synthesized. AnTPyP became water-soluble by the protonation of the pyridyl moieties in the presence of 5 mM trifluoroacetic acid (pH 2.3). The photoexcited state of the porphyrin ring in an AnTPyP molecule was effectively deactivated by intramolecular electron transfer from the anthracene moiety within 0.04 ns in an aqueous solution. The deactivation was suppressed by the interaction with a DNA strand, resulting in the elongation of the lifetime of the porphyrin excited state and the enhancement of the fluorescence intensity. Furthermore, it was confirmed that the interaction enabled the photoexcited AnTPyP to generate 1 O2. Selective 1 O2 generation by forming a complex with DNA should be the initial step to realize the target selective photodynamic therapy. INTRODUCTION As an anticancer agent, DNA is one of the most important target biomacromolecules, and DNA-targeting drugs have been extensively studied (1,2). Photodynamic therapy (PDT) is a promising treatment for cancer and some nonmalignant conditions using a photosensitizer as a drug and visible-light irradiation (3-5). An important mechanism of PDT is the oxidation of biomacromolecules by singlet oxygen (1 O2), which is generated through energy transfer from the excited photosensitizer to molecular oxygen. A DNA-selective photosensitizer should be developed to improve the treatment effect (6-9). The control of 1 O2 generation by a specific DNA sequence using photosensitizer/quencher/oligonucleotides systems has been studied (6-8). The demonstrated principle is selectively placing the 1 O2 photosensitizer close to a molecule that can quench the excited state of the photosensitizer by using a positioning system that can then be manipulated to change the distance between photosensitizer and the quencher. Furthermore, the pH regulated 1 O2 photosensitizer/quencher/ DNA i-motif system was reported (9). We reported on two photosensitizers, berberine and palmatine, which can easily bind to DNA through electrostatic interaction and generate 1 O2 only when the DNA-photosensitizer complex is formed (10,1 1). The interaction changes their redox potentials and suppresses the quenching by intramolecular electron transfer, resulting in the elongation of the lifetime of the photoexcited state, making the energy transfer to molecular oxygen possible (11). Generated 1 O2 effectively oxidizes every guanine residue of DNA (10). Indeed, it has been reported that the guanine residue is the selective target of 1 O2 (12). The main oxidized product of guanine by 1 O2 in both isolated and cellular DNA is 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxodGuo) (13,14). In the case of the DNA photodamage by berberine and palmatine, 8-oxodGuo was detected (10). Berberine and palmatine can act as a DNA-targeting photosensitizer, and guanines are specifically oxidized through 1 O2 generation. However, these photosensitizers cannot absorb long-wavelength light, which is advantageous for PDT. Thus, on the basis of this controlling mechanism of 1 O2 generation, we designed and synthesized a porphyrinoid photosensitizer, which is important for clinical use because of its high absorptivity for the red region (> 600 nm). Relevantly, the control of photosensitized 1 O2 generation using porphyrinoid molecular systems has been extensively studied (6-9,15-17). The purpose of this study is the development of an electron donor-connecting porphyrin whose electron-accepting ability and activity of the photosensitized 1 O2 generation can be controlled through interaction with DNA. MATERIALS AND METHODS Materials. As DNA molecules, the synthesized 16-mer oligonucleotides (AATT: d(AAAATTTTAAAATTTT)2 and AGTC: d(AAG CTTTGCAaAGCTT)2, Sigma Chemical Co., St. Louis, MO) were used. Trifluoroacetic acid (TFA) and distilled water were purchased from Wako Pure Chemical Industries (Osaka, Japan). Deuterium oxide (D2 O) was from Across Organics (Morris Plains, NJ). The spectroscopic grade solvents of dichloromethane and water were from Dojin Chemicals Co. (Kumamoto, Japan) and used as received. 5,10,15,20-Tetrakis(4-pyridyl)porphyrin (TPyP) was from Aldrich Chemical Co., Inc. (Milwaukee, WI). 5,10,l5,20-Tetrakis(4-sulfophenyl)porphyrin (TPPS) was from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). Synthesis of AnTPyP. 5-(9'-anthryl)-10,15,20-tris(p-pyridyl)porphyrin (AnTPyP, Fig. 1) was synthesized by the following procedure based on the literature (18-20). In 8 mL of propionic acid (WAKO Pure Chemical Industries), 206 mg of 9-anthraldehyde (Aldrich Chem. Co., Inc.) and 288 µL of 4-pyridinecarboxaldehyde (Wako Pure Chemical Industries) were dissolved and 2.0 mL of pyrrole (WAKO Pure Chemical Industries) was added to the reflux of the mixture for 1 h. After this reaction, sodium acetate trihydrate (1.6 mg) was dissolved in water (16 mL) and added to the reaction mixture and stirred to bring the pH to approximately 3.0. To allow the porphyrin to settle, the mixture was left overnight. The resulting purple precipitate was collected by Büchner filtration and washed with N,N-dimethylformamide and methanol. The crude product was purified three times by column chromatography on silica gel with an eluent of chloroform-methanol (98/2, vol/vol), resulting in a pure product with a 0.8% (4 mg) yield. 1 H-NMR (CDCl3, TMS) d -2.60 (s, 2H, central-H), 7.00-7.02 (m, 4H, 4',8'-anthryl-H (2H) and 2,8-ßH (2H)), 7.44-7.48 (m, 2H, 3,7-ßH), 8.15 (d, 4H, JH-H = 3.0 Hz, 10,20-o-pyridyl-H), 8.19 (d, 2H, JH-H = 4.3 Hz, 15-m-pyridyl-H), 8.30 (d, 2H, JH-H = 10.8 Hz, 1,5-anthrylH), 8.33 (d, 2H, JH-H = 5.0 Hz, 12,18-ßH), 8.64 (d, 2H, JH-H = 5.0 Hz, 13,17-ßH), 8.89 (t, 2H, JH-H = 5.1 Hz, 2,3,6,7-anthryl-H), 8.96 (s, 1H, 10-anthryl-H), 9.08 (d, 2H, JH-H = 4.3 Hz, 15-o-pyridyl-H). FAB-MS: m/z 718 (M+). UV-Vis absorption peaks (?max nm-1) in dichloromethane: 258, 419, 514, 548, 588, 645. Measurement of absorption and fluorescence spectra. Absorption spectra of the synthesized porphyrin and the samples were measured with a UV-Vis spectrophotometer UV-1650PC (Shimadzu, Kyoto, Japan). Fluorescence spectra of the samples were measured with an F-4500 spectrophotometer (Hitachi, Tokyo, Japan). Fluorescence lifetime measurement. Fluorescence decay measurements were performed by using a time-correlated single-photon counting method (21). Laser excitation at 410 nm was achieved by using a diode laser (LDH-P-C-410; PicoQuant, Berlin, Germany) with a power control unit (PDL 800-B; PicoQuant) in a repetition rate of 2.5 MHz. Temporal profiles of fluorescence decay were detected by using a microchannel plate photomultiplier (R3809U; Hamamatsu Photonics, Shizuoka, Japan) equipped with a TCSPC computer board module (SPC630; Becker and Hickl, Berlin, Germany). Full-width at half-maximum (FWHM) of the instrument response function was 51 ps. The values of ?2 and the Durbin-Watson parameters were used to determine the quality of the fit obtained by nonlinear regression. Detection of near-infrared luminescence from 1 O2. The 1 O2 generation was directly measured by the near-infrared luminescence around 1270 nm, which corresponds to the 1 O2 (1 ?g) - 3 O2 (3 Sg-) transition. The sample was D2 O solution of 5 µM AnTPyP, with or without DNA. A direct detection system, which consists of an Nd:YAG laser (THG/355 nm, 30 Hz; Tempest-30, New Wave Research, OR) as an excitation light source (355 nm, intensity: 280 mW cm-2), a quartz cuvette as an irradiation cell, a spectroscope and a near-infrared gated multichannel detector ICCD camera (NIR-II; Hamamatsu Photonics) has been built (22,23). Gate time and accumulation time are 5-50 µs after laser pulse and 128 s (total: 36 J cm-2), respectively. The time profile of 1 O2 emission. The sample was 2 mL of D2 O solution in a cell (1 cm × 1 cm × 4.5 cm) containing 5 µM AnTPyP, 50 µM-bp DNA (AATT or AGTC) and 5 mM TFA. The excitation light source was the second harmonic (532 nm) of a pulsed Nd:YAG laser (5 ns, 10 Hz; Continuum Minilite-II, Photonic Solutions, Edinburgh, UK). The laser light was passed through a set of dielectric multilayer film mirrors to eliminate stray light and irradiate from 45° direction of the surface of the quartz cell. The emission from the front surface of the sample cell was collected with a set of quartz lenses, passed through a cold mirror (CLDM-50S; Sigma Koki, Tokyo, Japan) separated by a Bosch-Lomb Shimadzu monochromator, and then introduced into a photomultiplier (R5509-41; Hamamatsu Photonics), which was cooled to 200 K with liquid nitrogen. The signal from the photomultiplier was amplified by 75 with an amplifier (SR-455; Stanford Research Systems, CA) and then counted with a scaler/averager (SR430; Stanford Research Systems). By changing wavelength, the luminescence intensity showed a maximum at 1270 nm, confirming the detection of phosphorescence of 1 O2. To record the time profile of 1 O2 emission, the signal obtained at 1270 nm was accumulated for 25,000 scans with a bin width of 320 ns. Molecular orbital calculation for design of photosensitizer. The equilibrium geometry of porphyrin and its molecular orbital (MO) energy were estimated from the ab initio MO calculation at the Hartree-Fock/6-31G* level utilizing the Spartan 08' (Wavefunction Inc., CA). RESULTS AND DISCUSSION Figure 1 shows the molecular structure of the synthesized photosensitizer, AnTPyP, in which the anthracene moiety directly connects to the porphyrin ring as an electron donor. The MO calculation was performed at the Hartree-Fock 6-31G* level to predict the equilibrium geometry and the photophysical property of the porphyrinoid photosensitizer. The optimized structure of AnTPyP indicated the steric rotational hindrance of the anthracene moiety around the meso-position of the porphyrin, which keeps the two p-electronic systems nearly orthogonal to each other (Fig. 1). The MO calculation showed that the highest occupied MO (HOMO) of the protonated AnTPyP locates on the anthracene moiety (Fig. 2), suggesting that the S1 excitation of the porphyrin ring corresponds to the electron transition from the HOMO-1 to the lowest unoccupied MO. Consequently, the photoexcited state of the protonated form of AnTPyP should be deactivated via intramolecular electron transfer from the anthracene moiety to the porphyrin moiety, forming a charge transfer (CT) state (Fig. 3). The electrostatic interaction with anionic DNA and the hydrophobic environment of the DNA strand should raise the CT state energy, leading to the recovery of the photochemical activity, as in the case reported previously (10,11). From the CPK model, AnTPyP is considered to bind to the DNA major groove. A similar binding form of another cationic porphyrin has been reported (24). The speculated geometry of the DNA-AnTPyP complex is shown in Fig. 4. The UV-Vis absorption spectrum of AnTPyP was redshifted by the addition of TFA in a dichloromethane solution (Fig. 5). The acid titration curve demonstrated that the pKa is 2,9.1 Since the central nitrogen of the nonsubstituted reference porphyrin, TPyP, is protonated at about pH = 1 (25), the observed pKa corresponds to the protonation to the pyridyl moiety of AnTPyP. AnTPyP could then be water-soluble without the protonation of the central nitrogen at pH ranging from 1 to 2.9, which could be realized with an aqueous solution containing 1-10 mM TFA. In the present study, 5 mM TFA of an aqueous solution was used to protonate the pyridyl moiety of AnTPyP (pH 2.3). In the presence of 5 mM TFA, the hyperchromic effect of absorption spectra of DNA (26% for AATT and 25% for AGTC at 260 nm) was observed, suggesting the partial denaturation of the DNA duplex. The fluorescence spectra and the time profile of the fluorescence intensity are shown in Fig. 6A,B, respectively. The protonated AnTPyP showed almost no fluorescence in an aqueous solution, and the fluorescence lifetime (tf) was markedly short. It is notable, however, that, in the presence of DNA, the fluorescence intensity increased and the lifetime became long. This observation is in contrast to the findings for TPyP, as listed in Table 1. TPyP shows a relatively large fluorescence quantum yield (Ff) and a longer tf in the absence of DNA. The extremely short lifetime for free AnTPyP stems from an effective quenching of the photoexcited state by the intramolecular electron transfer from the anthracene moiety to the porphyrin moiety to form the CT state. Owing to the positive charge in the protonated porphyrin moiety, AnTPyP can interact with anionic DNA. The interaction between AnTPyP and DNA was confirmed by the change in the UVV is absorption spectra, where the maximum wavelength of the Soret band was redshifted from 420 nm to 428 nm (Fig. 7). The electron-accepting ability of the porphyrin moiety should be decreased by the electrostatic interaction with anionic DNA, as in the case of previously reported photosensitizers (10,11). in the case of palmatine, a cationic photosensitizer, the interaction with DNA decreases the free energy (-?G) for the electron transfer by about 0.12 eV (26). In the present case, the decrease of this energy could be estimated to be in 0.10.37 eV.2 The roughly estimated -?G of the intramolecular electron transfer in the photoexcited AnTPyP without DNA is about 0.12 eV,3 suggesting that the control of the electron transfer by the interaction with DNA is possible from an energetic point of view. Furthermore, the hydrophobic environment of DNA is also unfavorable for the CT state. Thus, the intramolecular electron transfer process was suppressed, and the tf of the porphyrin ring elongated (Fig. 6, Table 1). The apparent value of Ff reached a plateau at 50 µM-bp, where all AnTPyP molecules interact with DNA. With the GCcontaining sequence of DNA, the tf value of AnTPyP was slightly smaller than that of the AT-only sequence. Since guanine has the lowest oxidation potential in the nucleobases (31), the electron transfer from guanine to the photoexcited porphyrin ring takes place more easily, and then the tf of AnTPyP possibly decreases. To evaluate the 1 O2 generation activity of the photosensitizer, the near-infrared emission was measured. The nearinfrared emission at around 1,270 nm, which is assigned to the radiative deactivation of 1 O2 to its ground state, was clearly observed during the photoexcitation of AnTPyP in the presence of DNA, whereas very weak emission was observed in the absence of DNA. The near-infrared emission was effectively diminished by sodium azide (NaN3), a physical quencher of 1 O2. Thus, Fig. 8 demonstrates that the photosensitized 1 O2 generation by AnTPyP became possible through the interaction with DNA. The apparent quantum yield of the 1 O2 generation (F?) with AnTPyP was estimated to be 0.75 and 0.52 for AnTPyPAATT and AnTPyP-AGTC, respectively, in comparison with the 1 O2 emission intensity for methylene blue in D2 O (F? = 0.52) (34). These values indicate that apparently 31% of 1 O2 generation was decreased by the guanines of DNA. The comparison of the values of Ff (Table 1) suggests that almost 10% of the S1 state of AnTPyP was decreased by the guanine residues. Another decreased part of 1 O2 generation (almost 21%) should be the deactivation of 1 O2 by guanines. Guanine is the only base that significantly reacts with 1 O2 (35,36). The reported rate constant of the chemical quenching of 1 O2 by guanine is relatively large (1.7 × 107 M-1 S-1) (37), whereas the total quenching rate constant and that of the chemical reaction by adenine are 4.1 × 10s M-1 S-l and 8 × 103 M-1 S-1, respectively (38). Therefore, the chemical quenching of 1 O2 by guanines may be the predominant process of the 1 O2 deactivation by DNA. In the case of AnTPyP, 1 O2 is necessarily generated in the vicinity of DNA. Some parts of generated 1 O2 are deactivated by guanines, and the other parts of 1 O2 diffuse into the bulk solution, as can be observed by the spectroscopic method. On the other hand, the oligonucleotides used in this study hardly affected the lifetime of 1 O2,6 suggesting that the quenching of 1 O2 by the DNA strand without the bindingphotosensitizer should be negligible. Although the guaninecontaining DNA strand prevents the 1 O2 generation, the estimated F? is comparable to that of other water-soluble porphyrins (39). The superior characteristic of this sensitizer is that the interaction with DNA can control the 1 O2 generation activity, which can be realized by increasing the energy level of the CT state of AnTPyP and suppressing the electron transfer quenching by the interaction with DNA. CONCLUSION The singlet excited state of AnTPyP is effectively quenched through intramolecular electron transfer from the anthracene moiety. The interaction with DNA suppresses the intramolecular electron transfer, resulting in the elongation of the lifetime of the photoexcited state. This elongation enhances the intersystem crossing and makes the photoenergy transfer to molecular oxygen possible. The activity control of porphyrinoid photosensitizers for PDT through an interaction with DNA should provide a possible increase in the selectivity for targeting DNA. Although AnTPyP can act at pH 2-3, which is not a normal physiological pH, this study demonstrated the activity control of an electron donor-connecting porphyrin by DNA. This designing concept may be available to develop the DNA-selective porphyrinoid photosensitizer. Acknowledgements-This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of the Japanese Government and Takahashi Industrial and Economic Research Foundation. 1 The pKa value of AnTPyP was estimated from the acid titration curve, i.e. the relationship between the absorbance of AnTPyP and the TFA concentration, under an assumption that the pH of solution is equal to -log10 [TFA]. 4 The quenching rate coefficient (kq) of Tt state can be expressed as follows: 1/tT = kq [O2] + k0, where the oxygen concentrations ([O2]) under air- and oxygen-saturated conditions at 25°C were 260 and 1,140 µM, respectively, and k0 is the deactivation rate constant in the absence of oxygen. 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Kazutaka Hirakawa*1, Tom Hirano2, Yoshinobu Nishimura3, Tatsuo Arai3 and Yoshio Nosaka4 1 Department of Basic Engineering (Chemistry), Faculty of Engineering, Shizuoka University, Shizuoka, Japan 2 Photon Medical Research Center, Hamamatsu University School of Medicine, Shizuoka, Japan 3 Department of Chemistry, University of Tsukuba, Ibaraki, Japan 4 Department of Materials Science and Technology, Nagaoka University of Technology, Niigata, Japan Received 14 January 2011, accepted 30 March 2011, DOI: 10.1111/j.1751-1097.2011.00929.x * Corresponding author email: [email protected] (Kazutaka Hirakawa) © 2011 The Authors Photochemistry and Photobiology © 2011 The American Society of Photobiology 0031-8655/11 (c) 2011 American Society for Photobiology [ Back To TMCnet.com's Homepage ]