Evaluation of the photosensitizer activity of hydrophobic phosphorus(V)porphyrins using the absorption spectral change of 1-benzyl-1,4-dihydronicotinamide.

Under visible light irradiation, water-insoluble P(V)porphyrins oxidized 1-benzyl-1,4-dihydronicotinamide (BNADH), a model compound for nicotinamide adenine dinucleotide, and diminished the typical absorption of BNADH at around 340 nm. A singlet oxygen quencher, sodium azide, partially inhibited photosensitized BNADH oxidation. This BNADH oxidation photosensitized by P(V)porphyrins in the presence of sodium azide can be explained by electron transfer oxidation from BNADH to the photoexcited P(V)porphyrins. The quantum yields of BNADH oxidation via electron transfer by these P(V)porphyrins were larger than those of a singlet oxygen mechanism. Redox potential measurements supported the electron transfer mechanism from a thermodynamic point of view, and fluorescence lifetime measurement also suggests this mechanism. The process of this electron transfer oxidation involves the radical formation of BNADH and the further reaction of this radical to the oxidized form (cationic form of BNADH). Analysis of the quantum yields of BNADH photooxidation by P(V)porphyrins suggests that the photoinduced electron transfer from BNADH to photoexcited P(V)porphyrins triggers the radical chain reaction of BNADH oxidation. The electron transfer rate coefficient and this efficiency were increased with an increase in the Gibbs energy of electron transfer from tryptophan to photoexcited P(V)porphyrins (-ΔG). However, the BNADH oxidation quantum yield via electron transfer decreased with an increase in the -ΔG of electron transfer. These results suggest that reverse electron transfer inhibits the decomposition of BNAD radicals. This assay using BNADH can be used to evaluate the photosensitizer activity of water-insoluble compounds. These P(V)porphyrins may be used as photosensitizers for photodynamic therapy in a relatively hydrophobic environment in cancer tissues.

Protein Photodamaging Activity and Photocytotoxic Effect of an Axial-Connecting Phosphorus(V)porphyrin Trimer.

An axial-connecting trimer of the porphyrin phosphorus(V) complex was synthesized to evaluate the relaxation process of the photoexcited state and the photosensitizer activity. The photoexcitation energy was localized on the central unit of the phosphorus(V)porphyrin trimer. The photoexcited state of the central unit was relaxed through a process similar to that of the monomer phosphorus(V)porphyrin. The excited state of this axially connected type of phosphorus(V)porphyrin trimer was not deactivated through intramolecular electron transfer. The singlet oxygen generation quantum yield of the trimer was almost the same as that of the monomer. The phosphorus(V)porphyrin, trimer, and monomer bound to human serum albumin and oxidized the tryptophan residue via singlet oxygen generation and electron transfer during visible light irradiation. The photocytotoxicity of these phosphorus(V)porphyrins on two cell lines was examined. The monomer induced photocytotoxicity; however, the trimer did not show cytotoxicity with or without photoirradiation. In summary, the photoexcited state of the trimer was almost the same as that of the monomer, and these phosphorus(V)porphyrins demonstrated a similar protein-photodamaging activity. The difference in association between the photosensitizer molecules and cells is the key factor of phototoxicity by these phosphorus(V)porphyrins.

Photosensitized Protein Damage by DiethyleneglycoxyP(V)tetrakis(p-n-butoxyphenyl)porphyrin Through Electron Transfer: Activity Control Through Self-aggregation and Dissociation.

DiethyleneglycoxyP(V)tetrakis(p-n-butoxyphenyl)porphyrin (EGP(V)TBPP) forms a self-aggregation in an aqueous solution, and the photoexcited state of this molecule was effectively deactivated. Association with human serum albumin (HSA), a water-soluble protein, causes dissociation of the self-aggregation, resulting in recovery of the photosensitizer activity of EGP(V)TBPP. Under visible light irradiation, EGP(V)TBPP photosensitized HSA oxidation. The photosensitized singlet oxygen-generating activity of EGP(V)TBPP was confirmed by near-infrared emission measurement. A singlet oxygen quencher, sodium azide, partially inhibited the HSA photodamage; however, the quenching effect was estimated to be 57%. Another 43% of the HSA photodamage could be explained by the electron transfer mechanism. The redox potential of EGP(V)TBPP and the calculated Gibbs energy of electron transfer from tryptophan to photoexcited EGP(V)TBPP demonstrated the possibility of HSA oxidation through electron extraction. Fluorescence lifetime measurements of EGP(V)TBPP verified the electron transfer from HSA. The photosensitizer activity of EGP(V)TBPP can be controlled through an association with biomolecules, such as protein, and the electron transfer-mediated biomolecule photooxidation plays an important role in photodynamic therapy under hypoxia.

Phenothiazine Dyes Induce NADH Photooxidation through Electron Transfer: Kinetics and the Effect of Copper Ions.

Phenothiazine dyes, methylene blue, new methylene blue, azure A, and azure B, photosensitized the oxidation of nicotinamide adenine dinucleotide (NADH), an important coenzyme in the living cells, through electron transfer. The reduced forms of these phenothiazine dyes, which were produced through electron extraction from NADH, underwent reoxidation to the original cationic forms, leading to the construction of a photoredox cycle. This reoxidation process was the rate-determining step in the photoredox cycle. The electron extraction from NADH using phenothiazine dyes can trigger the chain reaction of the NADH oxidation. Copper ions enhanced the photoredox cycle through reoxidation of the reduced forms of phenothiazine dyes. New methylene blue demonstrated the highest photooxidative activity in this experiment due to the fast reoxidation process. Electron-transfer-mediated oxidation and the role of endogenous metal ions may be important elements in the photosterilization mechanism.

Fluorescence lifetime probes for detection of RNA degradation.

To investigate RNA degradation in live cells, detection methods that do not require RNA extraction from cells are necessary. In this study, we examined the utility of fluorescence lifetime measurements using a probe attached to the end of an RNA molecule for detecting RNA degradation. We optimized a short fluorescein-labeled RNA sequence whose fluorescence lifetime varied significantly before and after degradation. The selected HHG-fluorescein sequence (H = U, C, or A) is a promising RNA labeling unit (fluorescence lifetime probe) for live cell imaging of RNA degradation.

Photooxidation Activity Control of Dimethylaminophenyl-tris-(N-methyl-4-pridinio)porphyrin by pH.

To control the activity of photodynamic agents by pH, an electron donor-connecting cationic porphyrin, meso-(N',N'-dimethyl-4-aminophenyl)-tris(N-methyl-p-pyridinio)porphyrin (DMATMPyP), was designed and synthesized. The photoexcited state (singlet excited state) of DMATMPyP was deactivated through intramolecular electron transfer under a neutral condition. The pK a of the protonated DMATMPyP was 4.5, and the fluorescence intensity and singlet oxygen-generating activity increased under an acidic condition. Furthermore, the protonation of DMATMPyP enhanced the biomolecule photooxidative activity through electron extraction. Photodamage of human serum albumin (HSA) was observed under a neutral condition because a hydrophobic HSA environment can reverse the deactivation of photoexcited DMATMPyP. However, an HSA-damaging mechanism of DMATMPyP under a neutral condition was explained by singlet oxygen production. Therefore, it is indicated that the protein photodamaging activity of DMATMPyP goes into an OFF state under a neutral hypoxic condition. Under an acidic condition, the HSA photodamaging quantum yield by DMATMPyP through electron extraction could be preserved in the presence of a singlet oxygen quencher. Photooxidation of nicotinamide adenine dinucleotide by DMATMPyP was also enhanced under an acidic condition. This study demonstrated the concept of using pH to control photosensitizer activity via inhibition of the intramolecular electron transfer deactivation and enhancement of the oxidative activity through the electron extraction mechanism. Specifically, biomolecule oxidation through electron extraction may play an important role in photodynamic therapy to treat tumors under a hypoxic condition.

Controlled Photodynamic Action of Axial Fluorinated DiethoxyP(V)tetrakis(p-methoxyphenyl)porphyrin through Self-Aggregation.

DiethoxyP(V)tetrakis(p-methoxyphenyl)porphyrin (EtP(V)TMPP) and its fluorinated derivative (FEtP(V)TMPP) were synthesized to examine their photodynamic action. These P(V)porphyrins were aggregated in an aqueous solution, resulting in the suppression of their photodynamic activity. In the presence of human serum albumin (HSA), a water-soluble protein, the aggregation states were resolved and formed a binding complex between P(V)porphyrin and HSA. These P(V)porphyrins photosensitized the oxidation of the tryptophan residue of HSA under the irradiation of long-wavelength visible light (>630 nm). This protein photodamage was explained by the electron transfer from tryptophan to the photoexcited state of P(V)porphyrins and singlet oxygen generation. The axial fluorination reduced the redox potential of the one-electron reduction of P(V)porphyrin and increased the electron transfer rate constant. However, this axial fluorination decreased the binding constant with HSA, and the quantum yield of photosensitized HSA damage through electron transfer was decreased. The photocytotoxicity of these P(V)porphyrins to HaCaT cells was also confirmed, and FEtP(V)TMPP demonstrated stronger phototoxicity than EtP(V)TMPP. In summary, a self-aggregation of porphyrin photosensitizers and resolving by targeting biomacromolecules may be used to target selective photodynamic action. The redox potential and an association with a targeting biomolecule are the important factors of the electron transfer-mediated mechanism, which is advantageous under hypoxic tumor conditions.

Sequence selective photoinduced electron transfer of a pyrene-porphyrin dyad to DNA.

The binding modes of a pyrene-porphyrin dyad, (1-pyrenyl)-tris(N-methyl-p-pyridino)porphyrin (PyTMpyP), to various DNAs (calf thymus DNA (Ct-DNA), poly[d(G-C)2], and poly[d(A-T)2]) have been investigated using circular dichroism and linear dichroism measurements. Based on the polarization spectroscopic results, it can be shown that the pyrenyl and porphryin planes are skewed to a large extent for PyTMPyP in an aqueous environment and in the binding site of poly[d(G-C)2]. In this complex, a photoinduced electron transfer (PET) process between the pyrenyl and porphyrin moieties occurs. On the other hand, PET was not observed in the PyTMPyP-poly[d(A-T)2] complex, whereas the fluorescence intensity of TMPyP was enhanced. The molecular planes of the pyrene and porphyrin moieties are almost parallel in the poly[d(A-T)2] and Ct-DNA adducts. Moreover, the generation of 1O2 species occurs only for the PyTMPyP-Ct-DNA and PyTMPyP-poly[d(A-T)2] complexes. We discuss the photophysical properties of PyTMPyP which are attributed to the binding patterns and the sequence of DNA bases.

Kinetics of Spontaneous Bimetallization between Silver and Noble Metal Nanoparticles.

A physical mixture of polymer-protected Ag nanoparticles and Rh, Pd, or Pt nanoparticles spontaneously forms Ag-core bimetallic nanoparticles. The formed nanoparticles were smaller than the parent Ag nanoparticles. In the initial process of this reaction, the surface plasmon absorption of Ag nanoparticles diminished and then almost ceased within one hour. Within several minutes, the decrease in Ag surface plasmon absorption could be analyzed by second-order reaction. This reaction was accelerated with an increase of temperature and the energy gap in the Fermi level between Ag and the other metals. The activation energy (Ea ) of this reaction could be determined. An electron transfer reaction from Ag to other metal nanoparticles was proposed as the initial interaction between these metal nanoparticles because the Fermi level of Ag is relatively high, and the electron transfer is possible in terms of energy. The Marcus plot between the rate constant and the driving force, roughly estimated from the work function of metals, and the observed Ea values reasonably explained the proposed electron transfer mechanism.

Photosensitized Protein-Damaging Activity, Cytotoxicity, and Antitumor Effects of P(V)porphyrins Using Long-Wavelength Visible Light through Electron Transfer.

Photodynamic therapy (PDT) is a less-invasive treatment for cancer through the administration of less-toxic porphyrins and visible-light irradiation. Photosensitized damage of biomacromolecules through singlet oxygen (1O2) generation induces cancer cell death. However, a large quantity of porphyrin photosensitizer is required, and the treatment effect is restricted under a hypoxic cellular condition. Here we report the phototoxic activity of P(V)porphyrins: dichloroP(V)tetrakis(4-methoxyphenyl)porphyrin (CLP(V)TMPP), dimethoxyP(V)tetrakis(4-methoxyphenyl)porphyrin (MEP(V)TMPP), and diethyleneglycoxyP(V)tetrakis(4-methoxyphenyl)porphyrin (EGP(V)TMPP). These P(V)porphyrins damaged the tryptophan residue of human serum albumin (HSA) under the irradiation of long-wavelength visible light (>630 nm). This protein photodamage was barely inhibited by sodium azide, a quencher of 1O2. Fluorescence lifetimes of P(V)porphyrins with or without HSA and their redox potentials supported the electron-transfer-mediated oxidation of protein. The photocytotoxicity of these P(V)porphyrins to HeLa cells was also demonstrated. CLP(V)TMPP did not exhibit photocytotoxicity to HaCaT, a cultured human skin cell, and MEP(V)TMPP and EGP(V)TMPP did; however, cellular DNA damage was barely observed. In addition, a significant PDT effect of these P(V) porphyrins on a mouse tumor model comparable with the traditional photosensitizer was also demonstrated. These findings suggest the cancer selectivity of these P(V)porphyrins and lower carcinogenic risk to normal cells. Electron-transfer-mediated oxidation of biomacromolecules by P(V)porphyrins using long-wavelength visible light should be advantageous for PDT of hypoxic tumor.

Mechanism of oxidative DNA damage induced by metabolites of carcinogenic naphthalene.

Naphthalene is a carcinogenic polycyclic aromatic hydrocarbon, to which humans are exposed as an air pollutant. Naphthalene is metabolized in humans to reactive intermediates such as 1,2-hydroxynaphthalene (1,2-NQH2), 1,4-NQH2, 1,2-naphthoquinone (1,2-NQ), and 1,4-NQ. We examined oxidative DNA damage by these naphthalene metabolites using 32P-labeled DNA fragments from human cancer-relevant genes. 1,2-NQH2 and 1,4-NQH2 induced DNA damage in the presence of Cu(II). The DNA-damaging activity of 1,2-NQH2 was significantly increased in the presence of the reduced form of nicotinamide adenine dinucleotide (NADH), whereas that of 1,4-NQH2 was not. In the presence of NADH, 1,2-NQ induced Cu(II)-dependent DNA damage, whereas 1,4-NQ did not. The calculated energy of the lowest unoccupied molecular orbital (LUMO), which corresponds to the reduction potential, was estimated to be -0.67 eV for 1,2-NQ and -0.75 eV for 1,4-NQ. These results suggest that 1,2-NQ was reduced more easily than 1,4-NQ. Furthermore, 1,2-NQH2, 1,4-NQH2, and 1,2-NQ plus NADH formed 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) as an oxidative DNA marker. Catalase and bathocuproine inhibited DNA damage, suggesting that H2O2 and Cu(I) were involved. These results indicate that NQH2s are oxidized to the corresponding NQs via semiquinone radicals, and that H2O2 and Cu(I) are generated during oxidation. 1,2-NQ is reduced by NADH to form the redox cycle, resulting in enhanced DNA damage. The formation of the corresponding semiquinone radicals was supported by an electron paramagnetic resonance (EPR) study. In conclusion, the redox cycle of 1,2-NQ/1,2-NQH2 may play a more important role in the carcinogenicity of naphthalene than that of 1,4-NQ/1,4-NQH2.

Photosensitized oxidation of nicotinamide adenine dinucleotide by diethoxyphosphorus(V)tetraphenylporphyrin and its fluorinated derivative: Possibility of chain reaction.

Water-soluble porphyrins, diethoxyphosphorus(V)tetraphenylporphyrin (EtP(V)TPP) and its fluorinated analogue (FEtP(V)TPP), decreased the typical absorption around 340nm of nicotinamide adenine dinucleotide (NADH) under visible light irradiation, indicating oxidative decomposition. A singlet oxygen quencher, sodium azide, and a triplet quencher, potassium iodide, slightly inhibited photosensitized NADH oxidation. However, these inhibitory effects were very small. Furthermore, the fluorescence lifetime of these P(V)porphyrins was decreased by NADH, suggesting the contribution of electron transfer to the singlet excited (S1) state of P(V)porphyrin. The redox potential measurement supports the electron transfer-mediated oxidation of NADH. The quantum yields of NADH photodecomposition by P(V)porphyrins could be estimated from the kinetic data and the effect of these quenchers on NADH oxidation. The obtained values suggest that the electron accepting by the S1 states of P(V)porphyrins triggers a chain reaction of NADH oxidation. This photosensitized reaction may play an important role in the photocytotoxicity of P(V)porphyrins. The axial ligand fluorination of P(V)porphyrins improved electron accepting ability. However, fluorination slightly suppressed static interaction with NADH, resulting in decreased oxidation quantum yield.

Relationship between the photoinduced electron transfer and binding modes of a pyrene-porphyrin dyad to DNA.

The binding modes of a pyrene-porphyrin dyad, (1-pyrenyl)-tris(N-methyl-p-pyridino)porphyrin (PyTMpyP), to DNA and its photophysical properties have been investigated using various spectroscopic techniques. The circular dichroism (CD) spectrum of PyTMpyP bound to DNA (PyTMpyP-DNA) showed one negative and two positive bands in the Soret region. The CD signal in the pyrene absorption region was positive. The shape of the CD spectrum does not support an intercalative binding mode of TMpyP, which would typically afford a negative CD band in the absence of the pyrene moiety. Linear dichroism (LD) experiments revealed a very small signal in the Soret region, which also challenges the intercalation of TMpyP into DNA. Upon excitation of the pyrene moiety, the emission intensity of porphyrin in aqueous solution was quenched due to a photoinduced electron transfer (PET) process between the pyrenyl and porphyrin moieties. On the other hand, the emission of porphyrin was markedly enhanced upon binding to DNA, as the PET process from the excited pyrene moiety to TMpyP was suppressed when bound to DNA. The PET process occurs in the timescale of 65 ps, and could be detected by femtosecond transient absorption spectroscopic methods. Two fluorescence decay times were observed for PyTMpyP in aqueous solution (0.78 and 4.8 ns). Both decay times increased upon binding to DNA owing to environment and/or conformational changes in PyTMpyP. The driving force (ΔG) of the PET process was evaluated under conditions of minor and major groove binding. The PET process and photophysical properties of the PyTMpyP dyad were concluded to be influenced by the binding mode.

Photosensitized enzyme deactivation and protein oxidation by axial-substituted phosphorus(V) tetraphenylporphyrins.

The activity for photodynamic therapy of water-soluble cationic porphyrins, tetraphenylporphyrin P(V) complexes, was investigated. Bis(cyclohexylmethoxy)P(V)tetraphenylporphyrin (DCHMP(V)TPP), dichloroP(V)tetraphenylporphyrin (Cl2P(V)TPP), and dimethoxyP(V)tetraphenylporphyrin (DMP(V)TPP) could cause the photosensitized deactivation of tyrosinase. The tryptophan residue of human serum albumin (HSA) and several kinds of amino acids could be damaged by these P(V)porphyrins under visible light irradiation. The photosensitized damage of these biomolecules was inhibited by sodium azide, a singlet oxygen (1O2) quencher, and enhanced in deuterium oxide, suggesting the contribution of 1O2. However, an excess amount of sodium azide did not completely inhibit the photosensitized damage. In addition, the redox potential measurements demonstrated the possibility of electron transfer from tryptophan and tyrosine to photoexcited P(V)porphyrins. These results suggest that electron transfer-mediated oxidation of amino acids contributes to the photosensitized protein and amino acid damage by these P(V)porphyrins. Specifically, Cl2P(V)TPP showed the highest photodamaging activity in the P(V)porphyrins used in this study. Oxidized products of amino acids by photoexcited P(V)porphyrins were analyzed with a liquid chromatography-mass spectrometer. Because of the hypoxic condition of a tumor, photodynamic therapy through a 1O2-mediated mechanism should be restricted, and the electron transfer-mediated mechanism may improve the photodynamic effect. In the cases of these P(V)porphyrins, redox potential is the most important factor for photosensitized protein and amino acid oxidation through photoinduced electron transfer.

The molecular mechanism of photochemical internalization of cell penetrating peptide-cargo-photosensitizer conjugates.

In many drug delivery strategies, an inefficient transfer of macromolecules such as proteins and nucleic acids to the cytosol often occurs because of their endosomal entrapment. One of the methods to overcome this problem is photochemical internalization, which is achieved using a photosensitizer and light to facilitate the endosomal escape of the macromolecule. In this study, we examined the molecular mechanism of photochemical internalization of cell penetrating peptide-cargo (macromolecule)-photosensitizer conjugates. We measured the photophysical properties of eight dyes (photosensitizer candidates) and determined the respective endosomal escape efficiencies using these dyes. Correlation plots between these factors indicated that the photogenerated (1)O2 molecules from photosensitizers were highly related to the endosomal escape efficiencies. The contribution of (1)O2 was confirmed using (1)O2 quenchers. In addition, time-lapse fluorescence imaging showed that the photoinduced endosomal escape occurred at a few seconds to a few minutes after irradiation (much longer than (1)O2 lifetime), and that the pH increased in the endosome prior to the endosomal escape of the macromolecule.

Relaxation Process of Photoexcited meso-Naphthylporphyrins while Interacting with DNA and Singlet Oxygen Generation.

Electron donor-connecting cationic porphyrins meso-(1-naphthyl)-tris(N-methyl-p-pyridinio)porphyrin (1-NapTMPyP) and meso-(2-naphthyl)-tris(N-methyl-p-pyridinio)porphyrin (2-NapTMPyP) were designed and synthesized. DFT calculations speculate that the photoexcited states of 1- and 2-NapTMPyPs can be deactivated via intramolecular electron transfer from the naphthyl moiety to the porphyrin moiety. However, the quenching effect through the intramolecular electron transfer is insufficient, possibly due to the orthogonal position of the electron donor and the porphyrin ring and the relatively small driving force: Gibbs energies are 0.11 and 0.07 eV for 1- and 2-NapTMPyPs, respectively. It was speculated that more than 0.3 eV of the driving force is required to realize effective electron transfer in similar electron-donor connecting porphyrin systems. These porphyrins aggregated around the DNA strand, accelerating the deactivation of their excited singlet state and decreasing their photosensitized singlet oxygen-generating activities. In the presence of a sufficiently large concentration of DNA, these porphyrins can bind to a DNA strand stably, leading to an increased fluorescence quantum yield and lifetime. Singlet oxygen generation was also suppressed by the aggregation of porphyrins around DNA. Although the quantum yield of singlet oxygen generation was recovered in the presence of sufficient DNA, the singlet oxygen generated by DNA-binding porphyrins was significantly smaller than that without DNA. These results suggest that DNA-binding drugs limit the generation of photosensitized singlet oxygen by quenching the DNA strand.

Determination of Singlet Oxygen and Electron Transfer Mediated Mechanisms of Photosensitized Protein Damage by Phosphorus(V)porphyrins.

The mechanism of photosensitized protein damage byphosphorus(V) tetraphenylporphyrin derivatives (P(V)TPPs) wasquantitatively clarified. P(V)TPPs bound to human serum albumin(HSA), a water-soluble protein, and damaged its tryptophan residueduring photoirradiation. P(V)TPPs photosensitized singlet oxygen ((1)O(2))generation, and the contribution of (1)O(2) to HSA damage was confirmedby the inhibitory effect of sodium azide, a (1)O(2) quencher. However,sodium azide could not completely inhibit HSA damage, suggesting thecontribution of an electron transfer mechanism to HSA damage. Thedecrement in the fluorescence lifetime of P(V)TPPs by HSA supportedthe electron transfer mechanism. The contribution of these processes could be determined by the kinetic analysis of the effect ofsodium azide on the photosensitized protein damage by P(V)TPPs.

Nile blue can photosensitize DNA damage through electron transfer.

The mechanism of DNA damage photosensitized by Nile blue (NB) was studied using (32)P-5'-end-labeled DNA fragments. NB bound to the DNA strand was possibly intercalated through an electrostatic interaction. Photoirradiated NB caused DNA cleavage at guanine residues when the DNA fragments were treated with piperidine. Consecutive guanines, the underlined G in 5'-GG and 5'-GGG, were selectively damaged through photoinduced electron transfer. The fluorescence lifetime of NB was decreased by guanine-containing DNA sequence, supporting this mechanism. Single guanines were also slightly damaged by photoexcited NB, and DNA photodamage by NB was slightly enhanced in D2O. These results suggest that the singlet oxygen mechanism also partly contributes to DNA photodamage by NB. DNA damage photosensitized by NB via electron transfer may be an important mechanism in medicinal applications of photosensitizers, such as photodynamic therapy in low oxygen.

Effect of DNA microenvironment on photosensitized reaction of watersoluble cationic porphyrins.

Endogenous and exogenous photosensitizers induce DNA damage, leading to carcinogenesis. Further, DNA is an important target biomacromolecule of photodynamic therapy (PDT) for cancer. Since the solar-induced DNA damage and PDT reaction occur in a complex biological environment, the interaction between biomolecule and photosensitizer is important. In this study, we examined the effect of a DNA microenvironment on the photosensitized reaction by watersoluble porphyrin derivatives, tetrakis(N-methyl-p-pyridinio)porphyrin (H(2)TMPyP) and its zinc complex (ZnTMPyP). In the presence of a sufficient concentration of DNA, H(2)TMPyP mainly intercalates to calf thymus DNA, whereas ZnTMPyP binds into a DNA groove. An electrostatic interaction with DNA raises the redox potential of the binding porphyrins. This effect suppressed the photoinduced electron transfer from an electron donor to the DNA-binding porphyrins, whereas the electron transfer from the porphyrins to the electron acceptor was enhanced. In the case of hydrophobic electron acceptors, static complexes with porphyrins were formed, making rapid electron transfer possible. Since the interaction with DNA cleaved this complex, the electron transfer rate was decreased in the presence of DNA. The microenvironment of a DNA strand may assist or inhibit its oxidative damage by photoinduced electron transfer through an electrostatic interaction with binding photosensitizers and the steric effect.

Singlet oxygen generating activity of an electron donor connecting porphyrin photosensitizer can be controlled by DNA.

To control the activity of singlet oxygen ((1)O2) 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 (<0.04 ns). Furthermore, this porphyrin did not demonstrate (1)O2 generating activity under photoirradiation. The fluorescence intensity and lifetime of AnTMPyP were markedly increased in the presence of DNA. The photosensitized (1)O2 generation by this porphyrin was also enhanced through interaction with DNA. The estimated quantum yield of (1)O2 generation by AnTMPyP interacting with DNA without guanine sequence was 0.22. The molecular design to control the photosensitized (1)O2 generation is possible based on the regulation of electron transition and steric hindrance of photosensitizing molecule.