Article pubs.acs.org/JPCB
Singlet Oxygen Generating Activity of an Electron Donor Connecting Porphyrin Photosensitizer Can Be Controlled by DNA Kazutaka Hirakawa,*,† Yoshinobu Nishimura,‡ Tatsuo Arai,‡ and Shigetoshi Okazaki§ †
Department of Applied Chemistry and Biochemical Engineering, Graduate School of Engineering, Shizuoka University, Johoku 3-5-1, Naka-ku, Hamamatsu, Shizuoka 432-8561, Japan ‡ Department of Chemistry, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8571, Japan § Medical Photonics Research Center, Hamamatsu University School of Medicine, Handayama 1-20-1, Higashi-ku, Hamamatsu, Shizuoka 431-3192, Japan S Supporting Information *
ABSTRACT: To control the activity of singlet oxygen (1O2) generation by photosensitizer through interaction with DNA, the electron- donor-connecting water-soluble porphyrin, meso-(9-anthryl)tris(N-methyl-p-pyridinio)porphyrin (AnTMPyP), was designed and synthesized. Molecular orbital calculation speculated that the photoexcited state of AnTMPyP can be deactivated via intramolecular electron transfer from the anthracene moiety to the porphyrin moiety, forming a charge-transfer (CT) state. The electrostatic interaction between the cationic porphyrin and anionic DNA predicts a rise in the CT state energy, leading to the inhibition of the electron transfer quenching. AnTMPyP showed almost no fluorescence in an aqueous solution, and the fluorescence lifetime was very short (600 nm), were designed and synthesized.27,28 Unfortunately, previous reported porphyrins could not be used at physiological pH27 and low quantum yield of 1 O2 generation (ΦΔ < 0.05).28 In general, the electron donor is easily oxidized by 1O2, leading to self-quenching of the generated 1O2 by the photosensitizer itself. To avoid this selfquenching and recovery of 1O2 generating yields, the molecular design is essentially important. The strategy for improving the 1 O2 generating yield is steric hindrance of the photosensitizer. Steric hindrance should inhibit the interaction between the
INTRODUCTION Control of electron transition including photoinduced electron transfer and energy transfer is an important theme of physical chemistry.1−7 Especially, porphyrins have been extensively studied for this theme.1,3,4,6,7 The porphyrin photosensitizer is an important drug in photodynamic therapy (PDT), which is a less invasive treatment for cancer and some nonmalignant conditions using visible-light irradiation.8−13 An important mechanism of PDT is the oxidation of biomacromolecules by singlet oxygen (1O2), which is generated through energy transfer from the excited photosensitizer to molecular oxygen. Singlet oxygen, the lowest excited state of molecular oxygen,14−16 can oxidize a wide range of organic compounds. DNA is one of the important target biomacromolecules for PDT, and DNA-targeting drugs have been studied.17−19 The activity control of photosensitized 1O2 generation by DNA should be the initial step in achieving tailor-made PDT. We have reported that berberine and palmatine, naturally occurring photosensitizers, can bind to DNA through electrostatic interaction and generate 1O2 only when the DNA−photosensitizer complex is formed.20−22 The interaction changes the redox potentials of these photosensitizers to suppress quenching by intramolecular electron transfer, resulting in the elongation of the lifetime of the photoexcited state and making the energy transfer to molecular oxygen possible.21,22 Generated 1O2 © 2013 American Chemical Society
Received: July 22, 2013 Revised: September 3, 2013 Published: October 4, 2013 13490
dx.doi.org/10.1021/jp4072444 | J. Phys. Chem. B 2013, 117, 13490−13496
The Journal of Physical Chemistry B
Article
Figure 1. Structure of AnTMPyP. The optimized structure and the HOMO of AnTMPyP were obtained by the calculation at the Hartree−Fock/631G* level.
electron-donating moiety and the generated 1O2. The purpose of this study is to confirm the concept to design a porphyrin photosensitizer whose activity can be controlled through interaction with DNA. The electron-donor-connecting porphyrin, meso-(1-anthryl)tris(N-methyl-p-pyridinio)porphyrin (AnTMPyP, Figure 1), was designed and synthesized to investigate the photochemical property in the presence of DNA. The molecular design of photosensitizer based on physical chemistry should be important for development of new drugs.
591, 649. Octanol/water partition coefficient (log10 Pow = log10(Co/Cw)) = −0.39. Measurements of Absorption and Fluorescence Spectra. The absorption spectra of AnTMPyP and DNA were measured with a UV−vis spectrophotometer, model UV1650PC (Shimadzu, Kyoto, Japan). The fluorescence spectra of samples were measured with a model F-4500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). The fluorescence quantum yield (Φf) of AnTMPyP was measured with an absolute photoluminescence quantum yield measurement system (C9920-02, Hamamatsu Photonics, Hamamatsu, Japan). Fluorescence Lifetime Measurements. Fluorescence decay was measured using a time-correlated single-photon counting method. 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. The temporal profiles of fluorescence decay were detected by using a microchannel plate photomultiplier (R3809U, Hamamatsu Photonics) equipped with a TCSPC computer board module (SPC630, Becker and Hickl Gmbh, Berlin, Germany). The full-width at halfmaximum (fwhm) of the instrument response function was 51 ps. The values of χ2 and the Durbin−Watson parameters were used to determine the quality of the fit obtained by nonlinear regression. Measurement of Near-Infrared Emission Spectra of 1 O2. The 1O2 formation was directly measured by near-infrared luminescence at around 1270 nm from deactivated 1O2, which corresponds to the 1O2(1Δg)−3O2(3Σg−) transition. The emission from 1O2 was measured using an apparatus based on commercially available apparatuses and was improved for high-sensitivity detection (NIR-PII System, Hamamatsu Photonics). The excitation pulse was obtained using an optical parametric oscillator (OPO) (L5996, Hamamatsu Photonics) excited by an Nd:YAG laser (SureliteI-20, Continuum, CA, USA). The excitation wavelength was 532 nm. Pulse width and intensity were approximately 7 ns and 50 J/pulse, respectively, and the repetition rate was 20 Hz. Emission of 1O2 was monitored using an infrared-gated image intensifier (NIR-PII, Hamamatsu Photonics) after passage through a polychromator (MS257, Oriel Instruments, CT, USA). Measurements started at 1 μs after application of the excitation pulse, and the exposure time was 100 μs. Signals were accumulated by repeated detection (2000 times). Calibration of the wavelength was performed using a spectral calibration lamp (krypton type,
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EXPERIMENTAL SECTION Materials. The synthesized oligonucleotides of the AT sequence (AATT, d(AAAATTTTAAAATTTT)2) and the guanine-containing sequence (AGTC, d(AAGCTTTGCAAAGCTT) 2 ) and tetrakis(N-methyl-p-pyridinio)porphyrin (TMPyP) were purchased from Sigma-Aldrich Co. LLC. (St. Louis, MO, USA). In this paper, these oligonucleotides are described as DNA. The spectroscopic grade water (H2O) was from Dojin Chemicals Co. (Kumamoto, Japan) and used as received. Sodium phosphate buffer (pH 7.6) was from Nakalai Tesque Inc. (Kyoto, Japan). Octanol and dimethyl sulfoxide-d6 were from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Methyl iodide, diethyl ether, and N,N-dimethylformamide were from Kanto Chemical Co., Inc. (Tokyo, Japan). Synthesis of AnTMPyP. AnTMPyP was obtained by the methylation of meso-(1-anthryl)tris(p-pyridyl)porphyrin (AnTPyP). Synthesis of AnTPyP was previously reported.27 To obtain AnTMPyP, the methylation of AnTPyP was carried out according to the literature.29,30 An amount of 3 mg (4.2 μmol) of AnTPyP was methylated in 8 mL of N,N-dimethylformamide with 0.8 mL (80 μmol) of methyl iodide for 3.5 h at room temperature. The solvent and methyl iodide were removed under vacuum. The residue was taken up in N,Ndimethylformamide and precipitated with diethyl ether. The brown powder was washed with diethyl ether and dried, the result being a brown product. 1H NMR (dimethyl sulfoxide-d6, TMS, 300 MHz) δ = −2.74 (s, 2H, central-H), 4.67 (s, 6H, methyl-H), 4.74 (s, 3H, methyl-H), 6.81 (d, J = 8.4 Hz, 2H, anthryl-H), 7.12 (t, J = 9.3 Hz, 2H, anthryl-H), 7.57 (t, J = 6.9 Hz, 2H, anthryl-H), 8.40 (d, J = 4.8 Hz, 2H, βH), 8.48 (d, J = 9.0 Hz, 2H, βH), 8.87 (d, J = 4.8 Hz, 2H, βH), 9.00−9.03 (m, 6H, pyridyl-H), 9.15 (d, J = 5.4 Hz, 4H, βH), 9.24 (s, 1H, anthryl-H), 9.43 (d, J = 6.9 Hz, 4H, pyridyl-H), 9.50 (d, J = 6.6 Hz, 2H, pyridyl-H). FAB-MS: m/z 762 (M+, C52H40N7). UV− vis absorption peaks (λmax/nm) in ethanol: 255, 426, 518, 553, 13491
dx.doi.org/10.1021/jp4072444 | J. Phys. Chem. B 2013, 117, 13490−13496
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Figure 2. Proposed structure of AnTMPyP oxidized by 1O2.
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Oriel Instruments). The values of ΦΔ were estimated from a comparison of the 1O2 emission intensities by AnTMPyP (2.5 μM) in a 2.0 cm3 solution of sodium phosphate buffer (pH 7.6) and methylene blue (ΦΔ = 0.52 in H2O).31 The time profile of 1 O2 emission was also measured with the above system and multichannel scale (SR430, Stanford Research Systems, CA, USA) with NIR PMT (R5509-42, Hamamatsu Photonics). Calculations. The structure of AnTMPyP was optimized by molecular orbital (MO) calculations at the Hartree−Fock/631G* level utilizing the Spartan 10′ (Wavefunction Inc., CA, USA). The structure of DNA-binding AnTMPyP was predicted by molecular mechanics calculation using Spartan 10′. Analysis of Association between AnTMPyP and DNA. The apparent association constant between AnTMPyP and DNA (Kapp) was calculated using the following equation:32,33
RESULTS AND DISCUSSION Calculated Structure and Energy of AnTMPyP. The structure of AnTMPyP was optimized by the MO calculation at the Hartree−Fock/6-31G* level (Figure 1). This result indicated the steric rotational hindrance of the anthracene moiety around the meso-position of the porphyrin, which keeps the two π-electronic systems nearly orthogonal to each other. This calculation showed that the S1 excitation of the porphyrin ring is assigned to the electronic transition from the next HOMO to the LUMO, and the HOMO is located on the anthracene moiety (Figure 1). Consequently, it is speculated that the photoexcited state of AnTMPyP can be deactivated via intramolecular electron transfer from the anthracene moiety to the porphyrin moiety, forming a charge-transfer (CT) state. The electrostatic interaction between the cationic porphyrin and anionic DNA predicts a rise in the CT state energy, leading to the recovery of photochemical activity. In addition, steric hindrance between the hydrogen atom at the β-position of the porphyrin ring and anthracene could be predicted. Steric hindrance by the hydrogen atom may prevent self-oxidation of the anthracene moiety by 1O2. Because an electron donating group is easily oxidized by 1O2, the connection of an electron donor tends to decrease the apparent yield of 1O2 generation. Singlet oxygen should oxidize anthracene at the 9′- and 10′-position through the Diels− Alder reaction (Figure 2). However, the oxidation of the anthracene moiety directly connecting at the meso-position of
[DNA] [DNA] 1 = + Abs − Abs0 Abs b − Abs0 K app(Abs b − Abs0 ) (1)
where [DNA] is the base-pair concentration of DNA and Abs, Abs0, and Absb correspond to the observed absorbance of AnTMPyP with DNA, the absorbance without DNA, and the absorbance of DNA-binding AnTMPyP, respectively. In the plot of [DNA]/(Abs − Abs0) versus [DNA], Kapp is given by the ratio of the slope to the intercept. 13492
dx.doi.org/10.1021/jp4072444 | J. Phys. Chem. B 2013, 117, 13490−13496
The Journal of Physical Chemistry B
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the porphyrin should be difficult because of steric hindrance, resulting in recovery of the 1O2 yield. Interaction between AnTMPyP and DNA. The UV−vis absorption spectrum of AnTMPyP was red-shifted by the addition of DNA (Figure 3), indicating the binding interaction
Figure 5. Fluorescence spectra of AnTMPyP. The sample solution contained 2.5 μM AnTMPyP with or without DNA in a 10 mM sodium phosphate buffer (pH 7.6). Excitation was at 532 nm. No fluorescence was observed by photoexcitation of DNA solution in the absence of porphyrin. The effect of two-photon absorption by DNA could be excluded. Figure 3. Absorption spectra of AnTMPyP in the absence or presence of DNA. The sample solution contained 2.5 μM AnTMPyP with or without 100 μM bp DNA in a 10 mM sodium phosphate buffer (pH 7.6).
Table 1. Fluorescence Lifetime and Quantum Yield of AnTMPyP and TMPyPa
of AnTMPyP with the DNA strand. It has been reported that TMPyP, a reference porphyrin of AnTMPyP, binds to the minor groove of DNA.34−36 The CPK model and the molecular mechanics calculation also support this binding interaction between AnTMPyP and the DNA minor groove (Figure 4).
porphyrin
DNA
AnTMPyP
without AATT AGTC without AATT AGTC
TMPyP
τf, ns (fraction) 0.04 (0.94) 3.6 (0.12) 2.8 (0.21) 5.1 (1.00) 12.0 (1.00) 2.4 (0.29)
6.3 (0.06) 10.4 (0.88) 10.6 (0.79)
Φf
