Metal Bacteriochlorins Which Act as Dual Singlet ... - ACS Publications

The same photosensitizer can also act as an efficient singlet oxygen generator. Thus, the same zinc bacteriochlorin can function as a sensitizer with ...
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J. Phys. Chem. B 2008, 112, 2738-2746

Metal Bacteriochlorins Which Act as Dual Singlet Oxygen and Superoxide Generators Shunichi Fukuzumi,*,† Kei Ohkubo,† Xiang Zheng,‡ Yihui Chen,‡ Ravindra K. Pandey,*,‡,§ Riqiang Zhan,| and Karl M. Kadish*,| Department of Material and Life Science, Graduate School of Engineering, Osaka UniVersity, SORST, Japan Science and Technology Agency (JST), Suita, Osaka 565-0871, Japan, Chemistry DiVision, Photodynamic Therapy Center, Roswell Park Cancer Institute (RPCI), Buffalo, New York 14263, Department of Nuclear Medicine, Roswell Park Cancer Institute, Buffalo, New York 14263, Department of Chemistry, UniVersity of Houston, Houston, Texas 77204-5003 ReceiVed: August 20, 2007; In Final Form: NoVember 29, 2007

A series of stable free-base, ZnII and PdII bacteriochlorins containing a fused six- or five-member diketo- or imide ring have been synthesized as good candidates for photodynamic therapy sensitizers, and their electrochemical, photophysical, and photochemical properties were examined. Photoexcitation of the palladium bacteriochlorin affords the triplet excited state without fluorescence emission, resulting in formation of singlet oxygen with a high quantum yield due to the heavy atom effect of palladium. Electrochemical studies revealed that the zinc bacteriochlorin has the smallest HOMO-LUMO gap of the investigated compounds, and this value is significantly lower than the triplet excited-state energy of the compound in benzonitrile. Such a small HOMO-LUMO gap of the zinc bacteriochlorin enables intermolecular photoinduced electron transfer from the triplet excited state to the ground state to produce both the radical cation and the radical anion. The radical anion thus produced can transfer an electron to molecular oxygen to produce superoxide anion which was detected by electron spin resonance. The same photosensitizer can also act as an efficient singlet oxygen generator. Thus, the same zinc bacteriochlorin can function as a sensitizer with a dual role in that it produces both singlet oxygen and superoxide anion in an aprotic solvent (benzonitrile).

Introduction Porphyrin-based photosensitizers have been the subject of enormous interest due to their potential use in photodynamic therapy (PDT).1-9 A rational design of photosensitizers is generally aimed toward finding compounds with high singlet oxygen quantum yields and long wavelength absorptions between 700 and 900 nm. The later point is important because light below 700 nm is scattered by tissue and that above 900 nm (near-infrared region) is absorbed by water.10 It is now wellestablished that both absorption and scattering of light by tissue increase as the wavelength decreases.1-9 To date, Photofrin (a hematoporphyrin-based compound) is the only photosensitizer that has been approved by health organizations around the world for the treatment of a variety of cancers. Unfortunately, there remain several major drawbacks associated with Photofrin, the most important of which are its complex chemical nature, its weak absorption in the long-wavelength region (630 nm) of the spectrum, and its skin phototoxicity. It has been shown that the incorporation of different metal ions into a porphyrin core makes a significant difference in the compound’s photophysical properties, including photobleaching and its pharmacokinetic/pharmacodynamic characteristics.2 A palladium(II) bacteriochlorin complex has been shown to be an efficient candidate for PDT sensitizers.11-13 We reported * To whom correspondence may be addressed: fukuzumi@ chem.eng.osaka-u.ac.jp; [email protected]; [email protected]. † Department of Material and Life Science, Graduate School of Engineering, Osaka University. ‡ Chemistry Division, Photodynamic Therapy Center, Roswell Park Cancer Institute. § Department of Nuclear Medicine, Roswell Park Cancer Institute. | Department of Chemistry, University of Houston.

earlier that replacement of the five-membered exocyclic ring in naturally occurring bacteriochlorophyll-a with a sixmembered fused imide ring system (named bacteriopurpurinimide) or the introduction of an additional keto group at the position 132 will both make a significant difference in the stability of these molecules.14 We also demonstrated that freebase bacteriopurpurinimides exhibit strong absorptions close to 800 nm and have singlet oxygen yields in the range of 4550%.15 It is generally believed that the primary biological effect of PDT is caused by singlet oxygen molecules (type II mechanism)16 formed via energy transfer from photosensitizers to O2 in tumor and endothelial cells of the tumor tissue.4,17,18 It has recently been suggested that a reactive oxygen species other than singlet oxygen, for example, superoxide anion, is formed by electron transfer from the triplet excited-state of Pdbacteriopheophorbide to O2 in aqueous media but not in organic solvents where singlet oxygen almost exclusively forms.13 It was suggested that photosensitizers with reductants (e.g., ascorbic acid) afforded radical anions of reductants, which are oxidized further by oxygen with superoxide formation, increasing the type I free radical route and hence photodynamic efficiency on the whole.19-21 However, there has been no report on the formation of superoxide anion by photosensitizers alone for PDT in organic solvents. We report herein the synthesis, photophysical and photochemical properties of a series of stable metal bacteriochlorins containing a fused six- or five-membered diketo or imide ring (Figure 1). The most interesting finding in this study is the occurrence of an intermolecular photoinduced electron transfer from the triplet excited state of the photosensitizer to its ground

10.1021/jp0766757 CCC: $40.75 © 2008 American Chemical Society Published on Web 02/07/2008

Metal Bacteriochlorins

Figure 1. Chemical structures of investigated bacteriochlorins.

state to produce both the radical cation and radical anion and this phenomenon only occurs under conditions where the HOMO-LUMO gap is smaller than the triplet energy. The radical anion thus produced can transfer an electron to molecular oxygen to produce superoxide anion. The same photosensitzer can also in parallel act as a singlet oxygen generator, and this dual role of the sensitizer, described for the first time herein, should provide important new insights into the further development of new compounds for effective use in the area of PDT. Experimental Section Instrumentation. Melting points were determined in a hot plate melting point apparatus and are uncorrected. 1H NMR spectra were recorded in CDCl3 at 400 MHz with TMS as an internal standard. E. Merck silica gel and E. Merck precoated TLC plates, silica gel F254, were used for column and preparative thin layer chromatography, respectively. The electronic absorption spectra were taken with a Varian Gary 50 Bio UV-visible spectrophotometer using dichloromethane as a solvent. Synthesis. The synthetic procedure is summarized in Scheme 1. The purity of products is confirmed by 1H NMR (see Supporting Information). Zn(II) Complex of 3-Acetyl-bacteriopurpurin-18-N-hexylimide Methyl Ester, 1(Zn). 3-Acetyl-bacteriopurpurin-18-Nhexylimide methyl ester (50 mg) was prepared from methyl bacteriopheophorbide-a following a published procedure14 and dissolved in 40 mL of anhydrous N,N-dimethylformide (DMF). Anhydrous zinc acetate (600 mg) was then added, and the mixture was heated at 140 °C for 0.5 h under an inert atmosphere. The solvent was evaporated and the residue chromatographed over a silica column eluting with MeOH/ CH2Cl2 (5% v/v). The appropriate fractions were combined. The residue obtained after removing the solvent was crystallized from CH2Cl2/n-hexane as dark red crystals in a 70% yield (38 mg). mp 170-172 °C. UV-vis λmax (, M-1 cm-1 in CH2Cl2): 831 (6.2 × 104), 699 (1.01 × 104), 563 (1.42 × 104), 425 (5.22 × 104) and 360 nm (5.81 × 104). 1H NMR (400 MHz, CDCl3) δ (ppm): 8.58 (two singlet overlapped, 2H, 5-H and 10-H), 8.35 (s, 1H, 20-H), 5.12 (m, 1H, 17-H), 4.21(m, 1H, -NCHH(CH2)4CH3), 4.06 (m, 3H, 1H, -NCHH(CH2)4CH3, 1H for 7-H, 1H for 18-H), 3.95 (m, 1H, 8-H), 3.50 (s, 3H,12-CH3), 3.26(s, 3H, -CH2CH2COOCH3), 3.00 (s, 3H, 2-CH3), 2.51 (s, 3H, CH3CO-), 2.30 (m, 2H, -CH2CH2COOCH3), 2.00 (m, 4H, 2H for 8-CH2CH3, 2H for -NCH2CH2(CH2)3CH3), 1.80 (2H, for -CH2CH2COOCH3), 1.65 (d, J ) 6.84 Hz, 3H, 7-CH3), 1.53 (d, J ) 6.88 Hz, 3H, 18-CH3), 1.38-1.15 (m, 9H, 6H for -NCH2CH2CH2CH2CH2CH3, 3H for 8-CH2CH3), 0.87 (t, J ) 7.58 Hz, 3H, N(CH2)5CH3). HRMS for C40H47O5N5Zn: calcd, 741.2868; found, 741.2869. Cd(II) Complex of 3-Acetyl-bacteriopurpurin-18-N-hexylimide Methyl Ester, 1(Cd). 3-Acetyl-bacteriopurpurin-18-N-

J. Phys. Chem. B, Vol. 112, No. 9, 2008 2739 hexylimide methyl ester (50 mg) was dissolved in 40 mL of anhydrous N,N-dimethylformide (DMF), and anhydrous cadmium acetate (600 mg) was added. Under argon flow, the mixture was heated at 140 °C for 0.5 h. The solvent was evaporated and the residue chromatographed over a silica column eluting with MeOH/CH2Cl2 (10% v/v). The appropriate fractions were combined. The residue obtained after removing the solvents was recrystallized from CH2Cl2/n-hexane as dark red crystals in 40% yield (24 mg). mp >300 °C. UV-vis λmax (, M-1 cm-1 in CH2Cl2): 822 (5.62 × 104), 577 (1.48 × 104), 423 (4.25 × 104) and 367 nm (5.87 × 104). 1H NMR (400 MHz, CDCl3) δ (ppm): 8.45 (s, 1H, 5-H), 8.27 (two singlet overlapped, 2H, 10-H and 20-H), 4.85 (m,1H, 17-H), 4.06 (m, 5H, 2H for -NCH2(CH2)4CH3, 1H for 7-H, 1H for 18-H, 1H for 8-H), 3.51 (s, 3H,12-CH3), 3.25 (s, 3H, CH2CH2COOCH3), 3.02 (s, 3H, 2-CH3), 2.48 (s, 3H, CH3CO-), 2.31 (m, 2H, -CH2CH2COOCH3), 2.08 (m, 4H, 2H for 8-CH2CH3, 2H for -NCH2CH2(CH2)3CH3), 1.70 (m,5H, 2H for -CH2CH2COOCH3), 3H for 7-CH3), 1.50 (d, J ) 6.88 Hz, 3H, 18-CH3), 1.30-1.10 (m, 9H, 6H for -NCH2CH2CH2CH2CH2CH3, 3H for 8-CH2CH3), 0.85 (t, J ) 7.58 Hz, 3H, N(CH2)5CH3). HRMS for C40H47O5N5Cd: calcd, 791.2612; found, 791.2624. Pd(II) Complex of 3-Acetyl-bacteriopurpurin-18-N-hexylimide Methyl Ester, 1(Pd). The bacteriopurpurinimide cadmium complex (50 mg) was dissolved in 40 mL of anhydrous acetone, and anhydrous palladium chloride (600 mg) was added. Under argon flow, the mixture was refluxed for 0.5 h. The solvent was removed and the residue chromatographed over a silica column, eluting with acetone/CH2Cl2 (1% v/v). The corresponding fractions of 1(Pd) were combined. The residue obtained after removing the solvents was crystallized from CH2Cl2/n-hexane as dark red crystals in 50% yield (25 mg). mp 210-212 °C. UV-vis λmax (, M-1 cm-1 in CH2Cl2): 815 (5.79 × 104), 677 (0.42 × 104), 539 (0.91 × 104), 419 (2.88 × 104), and 345 nm (3.59 × 104). 1H NMR (400 MHz, CDCl3) δ (ppm): 9.27 (s, 1H, 5-H), 8.65 (s, 1H, 10-H), 8.55 (s, 1H, 20H), 5.45 (m, 1H, 17-H), 4.40 (m, 4H, 2H for -NCH2(CH2)4CH3, 1H for 7-H, 1H for 18-H), 4.05 (m, 1H, 8-H), 3.60 (two overlapped singlets, altogether 6H, 3H for 12-CH3, 3H for -CH2CH2COOCH3), 3.45 (s, 3H, 2-CH3), 3.05 (s,3H, CH3CO-), 2.65 (m, 2H, -CH2CH2COOCH3), 2.35 (m, 4H, 2H for 8-CH2CH3, 2H for -NCH2CH2(CH2)3CH3), 2.00 (2H, for -CH2CH2COOCH3), 1.90 (d, J ) 6.84 Hz, 3H, 7-CH3), 1.80 (d, J ) 6.88 Hz, 3H, 18-CH3), 1.50-1.20 (m, 9H, 6H for -NCH2CH2CH2CH2CH2CH3, 3H for 8-CH2CH3), 0.90 (t, J ) 7.58 Hz, 3H, N(CH2)5CH3). HRMS for C40H47O5N5Pd: calcd, 783.2606; found, 783.2629. Bacteriopyropheophorbide-a Methyl Ester. Bacteriopheophorbide-a methyl ester (315 mg) was obtained from Rb. sphaeroides by following the literature procedure.14 It was then dissolved in collidine (20 mL) and refluxed (about 150 °C) under nitrogen for 2 h. Collidine was evaporated under vacuum, and hexane was added. The precipitate was filtered and washed with hexane. The product was chromatographed on a silica column (3% acetone in CH2Cl2) to afford 180 mg (63% yield) of bacteriopyropheophorbide-a. UV-vis λmax (, M-1 cm-1 in CH2Cl2): 755 (10.7 × 104), 532 (3.21 × 104), and 361 nm (7.76 × 104). 1H NMR (CDCl3): 8.99, 8.48, and 8.43 (each s, 1H, 5-H, 10-H and 20-H); 5.10 (m, 1H, 17-H); 4.93 (m, 1H, 7-H); 4.30 (m, 2H, 8-H and 18-H); 4.14 and 4.04 (m, 2H, 132-H); 3.62 (s, 3H, 17-CO2CH3); 3.50 (s, 3H, 3-CH3); 3.45 (s, 3H, 12-CH3); 3.17 (s, 3H, 2-CH3); 2.59 (m, 2H, 172CH2); 2.32 and 2.25 (each m, 3H, 81-CH2 and 171-CH2); 2.10

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SCHEME 1

(m, 1H, 171-CH2); 1.81 (d, J ) 6.44 Hz, 3H, 18-CH3); 1.77 (d, J ) 6.07 Hz, 3H, 7-CH3); 1.12 (t, J ) 7.17 Hz, 3H, 82-CH3); 0.35 and -1.03 (each s, 1H, 2NH). 132-Oxo-bacteriopyropheophorbide-a Methyl Ester, 2(H2). Bacteriopyropheophorbide-a methyl ester (50 mg) was dissolved in THF (10 mL), and a suspension of LiOH (80 mg) in water (1 mL) was added to the solution. The reaction mixture was exposed to air and vigorously stirred for 24 h at room temperature, after which 70 mL of a then 1% (v/v) AcOH water solution was added. The product was extracted with a CH2Cl2/ THF mixture (1:1). The combined extracts were washed with water and dried over sodium sulfate, and the solvent was evaporated. The intermediate product, mainly containing the corresponding carboxylic acid, was dissolved in a CH2Cl2/THF mixture (1:1) and treated with excess diazomethane. The conversion of carboxylic acid to the corresponding methyl ester was monitored by thin-layer chromatography. A few drops of acetic acid were then added to decompose the excess diazomethane. The reaction mixture was washed with water and the solvent evaporated under vacuum. The residue was purified by silica column chromatography (5% f 8% acetone in dichloromethane) to afford 20 mg (39% yield) of 2(H2). UV-vis λmax (, M-1 cm-1 in CHCl3): 771 (9.80 × 104), 388 (9.79 × 104), and 348 nm (8.05 × 104). 1H NMR (CDCl3): 9.53, 8.99 and 8.92 (each s, 1H, 5-H, 10-H, and 20-H); 5.02 (m, 1H, 7-H); 4.53 (m, 2H, 8-H and 17-H); 4.29 (m, 1H, 18-H); 3.69 (s, 3H, 17-CO2CH3); 3.65 (s, 3H, 3-CH3); 3.59 (s, 3H, 12-CH3); 3.24 (s, 3H, 2-CH3); 2.72 (m, 1H, 172-CH2); 2.65 (m, 171-CH2); 2.34 and 2.32 (each m, 2H, 81-CH2); 2.17 (m, 1H, 171-CH2); 1.90 (d, 7.01 Hz, 3H, 18-CH3); 1.81 (d, J ) 7.17 Hz, 3H, 7-CH3);

1.15 (t, J ) 7.49 Hz, 3H, 82-CH3); -0.39 and -2.11 (each s, 1H, 2NH). HRMS for C34H36N4O5: calcd, 580.2685; found, 580.2686. Pd(II) Complex of 132-Oxo-bacteriopyropheophorbide-a Methyl Ester, 2(Pd). L-Ascorbic acid 6-palmitate (120 mg) was dissolved in MeOH (40 mL) and degassed. Compound 2(H2) (40 mg) and palladium acetate (40 mg) were dissolved in degassed CHCl3 (34 mL) and added to the methanolic solution. The mixture was stirred under N2 for 1 h and the solvent then evaporated under vacuum. The residue was purified by preparative plate (5% acetone in CHCl3) and was obtained in 83% yield (39 mg). UV-vis λmax (, M-1 cm-1 in CHCl3): 770 (8.87 × 104), 384 (3.23 × 104), and 320 nm (2.89 × 104). 1H NMR (400 MHz, CDCl3) δ (ppm): 9.52, 8.89, and 8.82 (each s, 1H, 5-H, 10-H, and 20-H); 5.02 (m, 1H, 7-H); 4.59 (m, 1H, 17-H); 4.53 (m, 1H, 8-H); 4.26 (m, 1H, 18-H); 3.57 (s, 3H, 12-CH3); 3.55 (s, 6H, 3-CH3 and 17-CO2CH3); 3.12 (s, 3H, 2-CH3); 2.54 (m, 2H, 172-CH2); 2.33 (m, 3H, 81-CH2 and 171-CH2); 2.17 (m, 1H, 171-CH2); 1.84 (d, J ) 7.17 Hz, 3H, 7-CH3); 1.72 (d, J ) 6.66 Hz, 3H, 18-CH3); 1.10 (t, J ) 8.07 Hz, 3H, 82-CH3). HRMS for C34H34N4O5Pd: calcd, 684.1564; found, 684.1566. Zn(II) Complex of 132-Oxo-bacteriopyropheophorbide-a Methyl Ester, 2(Zn). Compound 2(H2) was dissolved in 30 mL of DMF. Oven-dried Zn(OAc)2 (300 mg) was added. The reaction mixture was heated to 130 °C for 1 h, and the solvent was evaporated under vacuum. The residue was purified by preparative plate (3% MeOH in CH2Cl2) to afford 21 mg (95% yield) of 2(Zn). UV-vis (, M-1 cm-1 in CH2Cl2): 780 (5.71 × 104), 392 (3.96 × 104), and 338 nm (2.86 × 104). 1H NMR (400 MHz, CDCl3) δ (ppm): 9.16, 8.95, and 8.78 (each s, 1H,

Metal Bacteriochlorins

J. Phys. Chem. B, Vol. 112, No. 9, 2008 2741

Figure 2. (a) UV-vis-NIR and (b) fluorescence spectra of 1(Pd) in PhCN.

TABLE 1: Photophysical and Electrochemical Properties of Metal Bacteriochlorins in PhCN at 298 K UV-vis-NIR λmax, nm 1(Zn) 1(Pd) 2(H2) 2(Zn) 2(Pd) a

368, 431, 578, 708, 764, 838 345, 421, 540, 681, 738, 819 350, 390, 471, 526, 698, 770 341, 394, 494, 547, 719, 788 321, 387, 476, 524, 704, 771

fluorescence λmax, nm, (τ, ns) 850 (1.36) 834 (0.89) 785 (1.55) 798 (1.34) 830 (0.58)

TsT λmax, nm, (τ, µs)

kEN, M-1 s-1

Eox,a V

Ered,a V

530, 620 (100) 440, 580 (300) 410, 600 (100) 410, 600 (170) 440, 580 (360)

4.2 × 6.9 × 108 1.6 × 109 1.4 × 109 1.8 × 109

0.45 0.66 0.81 0.55 0.76

-0.81 -0.67 -0.67 -0.82 -0.76

107

1

Eox*,a V

-1.02 -0.84 -0.78 -1.01 -0.79

1

Ered*,a V

Φ(1O2)b

0.66 0.83 0.92 0.74 0.79

0.58 0.94 0.58 0.64 0.99

vs SCE. b Excitation wavelength at 532 nm in C6D6.

5-H, 10-H, and 20-H); 4.85 (m, 1H, 7-H); 4.48 (m, 1H, 17-H); 4.38 (m, 2H, 8-H and 18-H); 3.51 (s, 3H, 12-CH3); 3.50 (s, 3H, 3-CH3); 3.20 (s, 3H, 17-CO2CH3); 2.91 (s, 3H, 2-CH3); 2.51-2.26 (m, total 6H, 172-CH2, 81-CH2, and 171-CH2); 1.96 (d, J ) 5.96 Hz, 3H, 7-CH3); 1.91 (d, 8.98 Hz, 3H, 18-CH3); 1.16 (t, J ) 7.07 Hz, 3H, 82-CH3). HRMS for C34H34N4O5Zn: calcd, 642.1820; found, 642.1819. Photophysical Measurements. Absorption spectra were recorded on a Hewlett-Packard 8453A diode array spectrophotometer. Time-resolved fluorescence and phosphorescence spectra were measured by a Photon Technology International GL-3300 with a Photon Technology International GL-302, nitrogen laser/pumped dye laser system, equipped with a fourchannel digital delay/pulse generator (Stanford Research System Inc. DG535) and a motor driver (Photon Technology International MD-5020). Excitation wavelengths were from 538 to 551 nm using coumarin 540A (Photon Technology International, Canada) as a dye. Fluorescence lifetimes were determined by a two-exponential curve fit using a microcomputer. Nanosecond transient absorption measurements were carried out using a Nd: YAG laser (Continuum, SLII-10, 4-6 ns full width at halfmaximum) at 355 nm with the power of 10 mJ as an excitation source. Photoinduced events were monitored by using a continuous Xe lamp (150 W) and an InGaAs-PIN photodiode (Hamamatsu 2949) as a probe light and a detector, respectively. The output from the photodiode and a photomultiplier tube was recorded with a digitizing oscilloscope (Tektronix, TDS3032, 300 MHz). Transient spectra were recorded using fresh solutions in each laser excitation. All experiments were performed at 298 K. For the 1O2 phosphorescence measurements, an O2-saturated C6D6 solution containing the sample in a quartz cell (optical path length 10 mm) was excited at 532 nm using a Cosmo System LVU-200S spectrometer. A photomultiplier (Hamamatsu Photonics, R5509-72) was used to detect emission in the nearinfrared region (band path 1 mm).

Electrochemical and Spectroelectrochemical Measurements. Cyclic voltammetry (CV) measurements were performed at 298 K on an EG&G model 173 potentiostat coupled with an EG&G model 175 universal programmer in deaerated benzonitrile (PhCN) solution containing 0.10 M TBAP as a supporting electrolyte. A three-electrode system was utilized and consisted of a glassy carbon working electrode, a platinum wire counter electrode, and a saturated calomel reference electrode (SCE). The reference electrode was separated from the bulk of the solution by a fritted-glass bridge filled with the solvent/ supporting electrolyte mixture. Thin-layer spectroelectrochemical measurements of the one-electron oxidized and one-electron reduced bacteriochlorin derivatives were carried out using an optically transparent platinum thin-layer working electrode and a Hewlett-Packard model 8453 diode array spectrophotometer coupled with an EG&G model 173 universal programmer. The complete experimental procedure is as described earlier in the literature.15 Electron Spin Resonance (ESR) Measurements. ESR measurements were performed in benzonitrile (PhCN) with a JEOL X-band spectrometer (JES-RE1XE) and recorded under nonsaturating microwave power conditions. The magnitude of modulation was chosen to optimize the resolution and signalto-noise (S/N) ratio of the observed spectra. The g values were calibrated with a Mn2+ marker. Results and Discussion Absorption and Emission. The bacteriochlorins investigated in this study exhibit near-infrared (NIR) region absorptions in a range 770-838 nm. A typical absorption spectrum is shown in Figure 2a for bacteriochlorin 1(Pd), which has a very strong Qy band (a1u f egx transition) at 819 nm, a Qx band (a2u f egy transition) at 540 nm, and a split Soret band at 345 and 421 nm in benzonitrile (PhCN). The molar absorptivity () of 1(Pd) at 819 nm was determined as 52 000 M-1 cm-1. Such a long wavelength Qy band is highly promising for the compound’s

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Figure 3. Cyclic voltammograms of selected compounds: (a) 1(Pd), (b) 1(Zn), (c) 2(Zn), and (d) 2(Pd) in PhCN containing 0.1 M TBAP at 298 K: sweep rate, 0.1 V s-1.

potential use in photodynamic therapy. The absorption maxima of each examined bacteriochlorin are listed in Table 1. A typical fluorescence spectrum of the compounds (e.g., 1(Pd) is shown in Figure 2b. The fluorescence decay of 1(Pd) is fitted well by a single-exponential line with a lifetime of 0.89 ns. The data for this compound and the other bacteriochlorin derivatives are summarized in Table 1. Fluorescence lifetimes of palladium bacteriochlorins are significantly shortened as compared with values of the related zinc and free-base bacteriochlorins, and this is because the intersystem crossing process is facilitated by a heavy atom effect of palladium. Redox Potentials. A variety of bacteriochlorins and metalated or metal-free bacteriochlorophyll-a derivatives have been investigated as to their redox properties in nonaqueous media.15,22-25 Like the related porphyrins, the bacteriochlorins can be oxidized at the conjugated macrocycle, giving π-radical cations and dications, or reduced, giving π-radical anions and dianions. These reactions are in almost all cases reversible, with the HOMO-LUMO gap being smaller than in the case of related porphyrins due primarily to the easier oxidations resulting from destabilization of the HOMO. The half-wave potentials for oxidation and reduction of the bacteriochlorins are also dependent upon the electron-donating or electron-withdrawing properties of substituents on the macrocycle and the type of central metal ion, and this is seen in the current study by the cyclic voltammograms presented in Figure 3 for Zn and Pd derivatives with the two different macrocycles, The first one-electron oxidation and first two one-electron reductions in a deaerated PhCN solution at 298 K are reversible for all four compounds, and the measured one-electron oxidation and one-electron reduction potentials of these processes (Eox, Ered) are listed in Table 1 together with those of the singlet excited states. The singlet excited-state energies

Figure 4. UV-vis spectral changes of 1(Zn) during (a) first oxidation and (b) first reduction in PhCN containing 0.1 M TBAP at the applied potentials (Eapp) of 0.65 and -0.90 V, respectively.

(E(S)) are determined as the average of the absorption and emission energies.26 The E(S) energies lie between 1.47 and 1.59 eV, being the lowest ever reported among this class of compounds. Radical Cations and Anions. The one-electron oxidation and one-electron reduction potentials in Table 1 were used for selection of an appropriate potential to generate the singly oxidized and singly reduced forms of each compound in a thinlayer cell. Either electron abstraction or electron addition leads to a complete loss of intensity for the long wavelength band of the compound, and this is illustrated in Figure 4 for the case of 1(Zn) where the decrease in intensity of the 838 nm band is accompanied by the appearance of new absorption bands at 922 and 970 nm during controlled potential oxidation or reduction at 0.65 and -0.90 V, respectively. Similar types of spectral changes are also observed during the electrochemical oneelectron oxidation and reduction of 2(Zn) (parts a and b of Figure 5, respectively) and those of 2(Pd) (parts a and b of Figure 6, respectively). In each case, formation of the radical cations and anions of metal bacteriochlorins is well-characterized by the appearance of new near-IR bands at 922 and 970 nm during controlled potential oxidation or reduction at 0.65 and -0.90 V, respectively. Similar types of spectral changes are also observed during the electrochemical one-electron oxidation or reduction of 2(Zn) as shown in Figure 5 or 2(Pd) as shown in Figure 6. In each case, formation of the radical cations and anions of metal bacteriochlorins is well-characterized by ap-

Metal Bacteriochlorins

J. Phys. Chem. B, Vol. 112, No. 9, 2008 2743 TABLE 2: UV-Visible Spectral Data of Singly Oxidized Bacteriochlorin Compounds in PhCN Containing 0.1 M TBAP λmax, nm ( × 10-4, M-1 cm-1) cpd

metal

1a 1

2H Pd Zn 2H Pd Zn

2

a

Figure 5. UV-vis spectral changes of 2(Zn) during (a) first oxidation and (b) first reduction in PhCN containing 0.1 M TBAP at the applied potentials (Eapp) of 0.70 and -1.00 V, respectively.

Figure 6. UV-vis spectral changes of 2(Pd) during (a) first oxidation and (b) first reduction in PhCN containing 0.1 M TBAP at the applied potentials (Eapp) of 1.00 and -0.90 V, respectively.

pearance of new near-IR bands and their absorption maxima and molar absorptivities are summarized in Tables 2 and 3. Energy Transfer Leading to Formation of Singlet Oxygen. The triplet-triplet transient absorption band observed at 600 nm in the laser flash photolysis measurements of 2(Pd) is shown

Soret band 355 (2.49) 385 (2.54) 360 (3.96) 401 (4.99) 397 (2.98) 404 (3.81)

409 (3.28) 423 (4.06) 432 (3.60) 430 (5.00) 438 (2.39) 445 (2.89)

visible and NIR band 681 (1.42) 701 (0.42) 667 (0.62) 681 (0.32) 685 (0.50)

891 (0.51) 897 (0.59) 920 (0.75) 836 (0.66) 870 (0.79) 878 (0.81)

Taken from ref 15.

Figure 7. T-T absorption spectrum of 2(Pd) obtained by the laser flash photolysis in deaerated PhCN at 5.6 µs after laser excitation (562 nm) at 298 K.

in Figure 7. The T-T absorption decay obeys first-order kinetics (Figure 8a), and this indicates that there is no contribution from the triplet-triplet annihilation under the present experimental conditions. The triplet lifetime was determined as 360 µs. The decay of the T-T absorption of 2(Pd) in air-saturated PhCN (Figure 8b) is enhanced significantly as compared with what is observed in deaerated PhCN (Figure 8a). In the former case, the decay obeys first-order kinetics and the decay rate constant increases with increasing oxygen concentration due to the energy transfer to oxygen. The energy transfer rate constant (kEN) was determined as 1.8 × 109 M-1 s-1 from the dependence of the decay rate constant on oxygen concentration and is close to the diffusion-limited value in PhCN (3.4 × 109 M-1 s-1).27 Irradiation of an oxygen-saturated benzene solution of 2(Pd) results in formation of singlet oxygen which was detected by the 1O2 phosphorescence at 1270 nm.28 The quantum yields (Φ) of 1O2 generation for each compound were determined from the phosphorescence intensity, in reference to the intensity of C60 (Φ ) 0.96).29 The measured quantum yields of singlet oxygen [Φ(1O2)] are listed in Table 1 and are largest for the palladium complexes, being 0.94 and 0.99 for 1(Pd) and 2(Pd), respectively, as compared to 0.58-0.64 for the other three compounds. Thus, more efficient formation of singlet oxygen from palladium bacteriochlorins as compared to the other compounds results from the faster intersystem crossing due to a heavy atom effect of palladium. Intermolecular Photoinduced Electron Transfer Leading to Formation of Superoxide Anion. In contrast to the case of 2(Pd) in Figure 7, the laser photoexcitation of 1(Zn) results in formation of not only the triplet excited state but also the radical cation and radical anion, which are observed in Figure 9. The

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Fukuzumi et al.

TABLE 3: UV-Visible Spectral Data of Singly Reduced Bacteriochlorin Compounds in PhCN Containing 0.1 M TBAP λmax, nm ( × 10-4, M-1 cm-1) cpd

metal

1a 1

2H Pd Zn 2H Pd Zn

2

a

Soret band 339 (2.25) 333 (2.29) 341 (2.16)b 329 (2.33) 327 (2.68) 333 (3.05)

400 (3.06) 354 (2.30) 375 (2.44) 371 (3.50) 420 (1.57) 423 (2.57)

visible and NIR band 608 (1.60) 619 (1.79) 623 (1.54) 587 (0.95) 593 (1.39) 600 (1.01)

737 (0.64) 667 (1.56) 731 (0.92) 755 (1.03) 720 (0.55) 736 (0.54)

980 (0.16) 1018 (1.31) 970 (0.30) 856 (0.52) 893 (0.10) 892 (0.33)b

902 (0.87) 966 (1.07) 958 (0.88)

Taken from ref 15. b Shoulder peak.

Figure 8. Decay curves of transient absorbance at 610 nm of 2(Pd) (a) in the absence of O2 and (b) in air-saturated PhCN ([O2] ) 1.7 × 10-3 M) at 298 K.

Figure 9. Transient absorption spectra of 1(Zn) (1.2 × 10-4 M) in deaerated PhCN taken at 1.4 (B) and 200 µs (O) after laser excitation at 578 nm.

absorption bands at 920 and 970 nm in the near-IR region of this figure agree with the superposition of the absorption bands of the radical cations and anions in Figure 4. The formation of radical cations and anions results from intermolecular photoinduced electron transfer from the triplet excited state of 1(Zn) to its ground state, which becomes possible due to the fact that the HOMO-LUMO gap of this compound (1.26 eV) is smaller than the energy of the triplet excited state (1.4 eV). This sharply contrasts with the case of 2(Pd) whose HOMO-LUMO gap (1.52 eV) is larger than the triplet excited-state energy of 1.4 eV.15,30 In such a case, the photoinduced electron transfer of 1(Zn) becomes energetically possible, whereas that of 2(Pd) is not. The decay of the absorbance at 620 nm due to the radical anion of 1(Zn) is rather slow (Figure 10a) as compared to the

decay of the triplet excited-state of 2(Pd) (Figure 8a), and this is because the decay in the first case corresponds to intermolecular back electron transfer from the radical anion of 1(Zn) to the radical cation, which is controlled by diffusion. Indeed the decay obeys second-order kinetics rather than first-order kinetics and the second-order rate constant was determined as 2.0 × 109 M-1 s-1, a value which agrees with the reported diffusion rate constant in PhCN.27 The decay of the absorbance at 620 nm due to the radical anion of 1(Zn) becomes much faster in aerated PhCN, as shown in Figure 10b. In contrast to the case of 2(Pd), this decay consists of two parts: the first is a fast decay comparable to the decay in the case of 2(Pd) and then a subsequent slow decay. Because the absorption band due to the triplet is overlapped with that due to the radical anion of 1(Zn), the fast decay component may result from an energy transfer from the triplet excited state of 1(Zn) to O2, whereas the subsequent slow decay is ascribed to an electron transfer from the radical anion of 1(Zn) to O2. The latter electron-transfer process should lead to the formation of superoxide anion (O2•-), and this was confirmed by the ESR measurements (vide infra). Photoirradiation of a deaerated PhCN solution of 1(Zn) results in formation of both the radical cation and radical anion of 1(Zn) via electron transfer from the triplet excited state of 1(Zn) to its ground state and this can be detected by ESR as shown in Figure 11a. The ESR signals of the radical cation and anion, produced by chemical oxidation and reduction, have the same g value (2.003), and thus the observed singlet isotropic ESR spectrum with g ) 2.003 is a superposition of two signals due both to the radical cation and radical anion. When the photoirradiation is carried out in an aerated PhCN solution, a g| component of O2•- is observed at 2.070 as shown in Figure 11b. This observed g| value is smaller than value of free O2•-

Metal Bacteriochlorins

J. Phys. Chem. B, Vol. 112, No. 9, 2008 2745

Figure 10. Decay curves of transient absorbance at 620 nm for 1(Zn) (a) in the absence of O2 and (b) in air-saturated PhCN ([O2] ) 1.7 × 10-3 M) at 298 K.

anion of 1(Zn), which is produced by intermolecular photoinduced electron transfer from the triplet excited state of 1(Zn) to its ground state. The HOMO-LUMO gap is smaller than the triplet energy, and this is responsible for the occurrence of the intermolecular photoinduced electron transfer. The fact that metal bacteriochlorins with small HOMO-LUMO gaps are capable of producing both singlet oxygen and superoxide anion should provide a strategy for the synthesis of new compounds to be examined in future PDT studies. Acknowledgment. This work was supported by a Grantin-Aid (Nos. 19205019 and 19750034) from the Ministry of Education, Culture, Sports, Science and Technology, Japan, the Robert A. Welch Foundation (K.M.K., Grant E-680), NIH (CA 55791), and the shared resources of the Roswell Park Cancer Center Support Grant (P30CA16056). Mass spectrometry analyses were performed at the Mass Spectrometry Facility, Michigan State University, East Lansing, MI. Supporting Information Available: 1H NMR data in CDCl3. This material is available free of charge via the Internet at http://pubs.acs.org. Figure 11. ESR spectra of 1(Zn) (2.0 × 10-3 M) in (a) Ar- and (b) O2-saturated glassy PhCN under photoirradiation with a high-pressure mercury lamp at 273 K and immediately cooled down to 77 K.

(2.102), consistent with O2•- forming a complex with Zn2+ ion.31-33 This indicates that O2•- is formed by electron transfer from the radical anion of 1(Zn) to O2 followed by the subsequent binding of the generated superoxide ion with Zn2+ in 1(Zn).34 The O2•-, which is in the protonation equilibrium with HO2•, is known to lead to the DNA cleavage.35-38 Conclusions The photochemical and electrochemical properties of six new free-base and metalated bacteriochlorins are characterized using laser flash photolysis, fluorescence spectroscopy, spectroelectrochemistry and cyclic voltammetry. Among the compounds studied, the palladium bacteriochlorin 2(Pd) exhibits the most promising characteristics as a PDT agent; it has a strong NIR band, a high quantum yield of singlet oxygen formation, and a low excitation energy. The zinc bacteriochlorin 1(Zn) affords a lower quantum yield of singlet oxygen formation, but an additional pathway operates to produce superoxide anion. This pathway is via electron-transfer reduction of O2 by the radical

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