Theoretical and experimental analysis of the luminescence signal of singlet oxygen for different photosensitizers

After the generation by different photosensitizers, the direct detection of singlet oxygen is performed by measuring its luminescence at 1270 nm. Using an infrared sensitive photomultiplier, the complete rise and decay time of singlet oxygen luminescence is measured at different concentrations of a photosensitizer, quencher, or oxygen. This allows the extraction of important information about the photosensitized generation of singlet oxygen and its decay, in particular at different oxygen concentrations. Based on theoretical considerations all important relaxation rates and rate constants were determined for the triplet T(1) states of the photosensitizers and for singlet oxygen. In particular, depending on the oxygen or quencher concentration, the rise or the decay time of the luminescence signal exhibit different meanings regarding the lifetime of singlet oxygen or triplet T(1)-state. To compare with theory, singlet oxygen was generated by nine different photosensitizers dissolved in either H2O, D2O or EtOD. When using H2O as solvent, the decaying part of the luminescence signal is frequently not the lifetime of singlet oxygen, in particular at low oxygen concentration. Since cells show low oxygen concentrations, this must have an impact when looking at singlet oxygen detection in vitro or in vivo.     

Enabling Photo-Crosslinking and Photo-Sensitizing Properties for Synthetic Fluorescent Protein Chromophores

Synthetic fluorescent protein chromophores have been reported for their singlet state fluorescence properties and applications in bioimaging, but rarely for the triplet state chemistries. Herein, we enabled their photo-sensitizing and photo-crosslinking properties through rational modulations. Extension of molecular conjugation and introduction of heavy atoms promoted the generation of reactive oxygen species. Unlike other photosensitizers, these chromophores selectively photo-crosslinked aggregated proteins and uncovered the interactome profiles. We also exemplified their general applications in chromophore-assisted light inactivation, photodynamic therapy and photo induced polymerization. Theoretical calculation, pathway analysis and transient absorption spectroscopy provided mechanistic insights for this triplet state chemistry. Overall, this work expands the function and application of synthetic fluorescent protein chromophores by enabling their triplet excited state properties.     

Quantum yields of triplet and O2(1 delta g) formation of 4-thiouridine in water and acetonitrile

The quantum yield of triplet formation, phi T, and that of the photosensitized formation of singlet molecular oxygen, phi delta, were determined for a rare nucleoside, 4-thiouridine (4t-Urd), in water and in acetonitrile, using singlet molecular oxygen phosphorescence, laser-induced optoacoustics and time-resolved thermal lensing. These yields, phi T and phi delta, the latter in aerated solutions, were found to be, respectively, in water: 0.67 +/- 0.17 and 0.18 +/- 0.04 and in acetonitrile: 0.61 +/- 0.15 and 0.50 +/- 0.20. The fraction of the 4t-Urd triplet molecules quenched by oxygen leading to singlet molecular oxygen, S delta, was calculated to be between 0.7 and unity in both solvents, this value being indicative of a pi pi*character for the lowest triplet state of 4t-Urd.     

KINETIC PROPERTIES OF THE TRIPLET STATES OF METHYLENE BLUE AND OTHER PHOTOSENSITIZING DYES

Abstract— The long-lived (> 1 μsec) transients formed in the flash excitation of the representative photosensitizers methylene blue, eosin Y and pyrene have been investigated and various criteria have been used to distinguish between triplet state intermediates and chemical intermediates. Previous assignments of the triplet transients of methylene blue appeared less secure in view of the photochemical reactivity of this dye and its lack of phosphorescence. Earlier assignments of monomeric and dimeric triplet transients of methylene blue are substantiated, however, by the observations that the rate constant for quenching by oxygen is approximately 1/9^th diffusion controlled and the formation rates are commensurate with singlet decay rates and by the observation of triplet-triplet annihilation. Additional evidence in support of monomer triplet assignments for methylene blue and eosin Y is provided by the effect of heavy atom quenchers Cs⁺, Hg²⁺ and T1⁺ on decay rates. Due to chemical reactivity, quenching by I⁻appears less suitable as a diagnostic test for triplet state intermediates. The effect of N₃⁻, which is known to quench singlet oxygen molecules and to alter the course of photosensitized oxidations, on the triplet decay of methylene blue, eosin Y and pyrene is also investigated.     

Characterization of the triplet state of tris(8-hydroxyquinoline)aluminium(III) in benzene solution

The lowest triplet state of tris(8-hydroxyquinoline)aluminium(III) (Alq3) has been prepared by pulse radiolysis/energy transfer from appropriate donors in benzene solutions and has an absorption maximum around 510 nm with a lifetime of about 50 mus. It is quenched by molecular oxygen, leading to singlet oxygen formation. From flash photolysis and singlet oxygen formation measurements, a quantum yield of triplet formation of 0.24 was determined for direct photolysis of the complex. A value of 2.10 +/- 0.10 eV was determined for the energy of the lowest triplet state by energy transfer studies and was confirmed by phosphorescence measurements on Alq3, either in the heavy atom solvent ethyl iodide or photosensitized by benzophenone in benzene. Dexter (exchange) energy transfer was observed from triplet Alq3 to platinum(II) octaethylporphyrin.     

Imidazole functionalized magnesium phthalocyanine photosensitizer: modified photophysics, singlet oxygen generation and photooxidation mechanism

Magnesium phthalocyanine (MgPc) was covalently attached by four imidazole units to form a novel photosensitizer (PS). The photophysical processes within the dyad PS were explored by steady state and time-resolved fluorescence as well as laser flash photolysis. Although the imidazole units caused a 50% decrease in fluorescence quantum yield and a remarkable shortening of fluorescence lifetime of the MgPc moiety, the triplet yield (Φ(T)) is higher and the triplet lifetime becomes longer. The transient absorption bands for MgPc(?-) were observed, indicating the occurrence of intramolecular photoinduced electron transfer (PET) from imidazole subunits to the lowest excited singlet state (S(1)) of the MgPc moiety. The kinetic and thermodynamic analysis also supports the involvement of PET in S(1) deactivation. The quantum efficiency of photosensitized oxidation of diphenylisobenzofuran (DPBF) by the PS is 0.52. This value is much higher than Φ(T) (0.26), since DPBF is photo-oxidized not only by singlet oxygen (type II reaction, 54%) but also by superoxide anion radical (type I reaction, 46%). The result suggests that the mechanism of photosensitized oxidation could be changed upon the conjugation of a PS to biological molecules, so that the importance of type I reaction is enhanced.     

Triplet state spectroscopic studies on some 5,10,15,20-tetrakis(methoxyphenyl)porphyrins

Extensive triplet state spectroscopic investigations were carried out with a series of 5,10,15,20-tetrakis(methoxyphenyl)porphyrins. Triplet absorption spectra, triplet lifetime, triplet quantum yield and quantum yield for singlet oxygen production were determined with different absorption and emission techniques, using the frequency-doubled beam of a Nd:YAG laser. It has been found that these synthetic porphyrins are effective photosensitizers which can be used as model compounds to investigate the theoretical and instrumental aspects of PDT.     

Two-step photoactivation of hematoporphyrin by excimer-pumped dye-laser pulses

The selective excitation of high lying singlet or triplet states of hematoporphyrin has been achieved using high peak-power nanosecond pulses generated by excimer-pumped dye lasers. The interaction involves two steps: a pulse at 630 nm raises the molecules to the S1 state and a second one, at 481 nm, further excites them either to a higher singlet state if shed simultaneously or to a triplet state higher in energy than T1 if it arrives delayed with respect to the pulse at 630 nm by a time interval longer than the S1 lifetime. Photodegradation of L-tryptophan (100 microM in 30vol.%methanol-70vol.% buffer, pH 7.4) sensitized by 21 microM hematoporphyrin is reported. While a pure type-II mechanism, which obeys the time-intensity reciprocity law up to peak-intensity values of about 20 MW cm-2, is photosensitized by pulses at 630 nm, strong non-linearities are found for pulsed irradiation at both 630 nm and 481 nm, i.e. when the sensitizer is pumped to high lying singlet states and when it is pumped to high lying triplet states. The dependence of the subsequent reactions on the presence of oxygen and their competition with the photodynamic action has been investigated; in particular, a pathway was observed in which an electron was photoejected from a hematoporphyrin high energy triplet, showing maximum efficiency when the pulses were delayed by 16.4 ns.     

Mechanisms of photoinitiated cleavage of DNA by 1,8-naphthalimide derivatives.

Using water-soluble 1,8-naphthalimide derivatives, the mechanisms of photosensitized DNA damage have been elucidated. Specifically, a comparison of rate constants for the photoinduced relaxation of supercoiled to circular DNA, as a function of dissolved halide, oxygen and naphthalimide concentration, has been carried out. The singlet excited states of the naphthalimide derivatives were quenched by chloride, bromide and iodide. In all cases the quenching products were naphthalimide triplet states, produced by induced intersystem crossing within the collision complex. Similarly, the halides were found to quench the triplet excited state of the 1,8-naphthalimide derivatives by an electron transfer mechanism. Bimolecular rate constants were < 10(5) M-1 s-1 for quenching by bromide and chloride. As expected from thermodynamic considerations quenching by iodide was 6.7 x 10(9) and 8.8 x 10(9) M-1 s-1 for the two 1,8-naphthalimide derivatives employed. At sufficiently high ground-state concentration self-quenching of the naphthalimide triplet excited state also occurs. The photosensitized conversion of supercoiled to circular DNA is fastest when self-quenching reactions are favored. The results suggest that, in the case of 1,8-naphthalimide derivatives, radicals derived from quenching of the triplet state by ground-state chromophores are more effective in cleaving DNA than reactive oxygen species or radicals derived from halogen atoms.     

One- and two-photon photosensitized singlet oxygen production: characterization of aromatic ketones as sensitizer standards

Singlet molecular oxygen, O2(a1Deltag), can be efficiently produced in a photosensitized process using either one- or two-photon irradiation. The aromatic ketone 1-phenalenone (PN) is an established one-photon singlet oxygen sensitizer with many desirable attributes for use as a standard. In the present work, photophysical properties of two other aromatic ketones, pyrene-1,6-dione (PD) and benzo[cd]pyren-5-one (BP), are reported and compared to those of PN. Both PD and BP sensitize the production of singlet oxygen with near unit quantum efficiency in a nonpolar (toluene) and a polar (acetonitrile) solvent. With their more extensive pi networks, the one-photon absorption spectra for PD and BP extend out to longer wavelengths than that for PN, thus providing increased flexibility for sensitizer excitation over the range approximately 300-520 nm. Moreover, PD and BP have much larger two-photon absorption cross sections than PN over the range 655-840 nm which, in turn, results in amounts of singlet oxygen that are readily detected in optical experiments. One- and two-photon absorption spectra of PD and BP obtained using high-level calculations model the salient features of the experimental data well. In particular, the ramifications of molecular symmetry are clearly reflected in both the experimental and calculated spectra. The use of PD and BP as standards for both the one- and two-photon photosensitized production of singlet oxygen is expected to facilitate the development of new sensitizers for application in singlet-oxygen-based imaging experiments.     

The microenvironment of DNA switches the activity of singlet oxygen generation photosensitized by berberine and palmatine

The effect of the interaction between DNA and the photosensitizer on photosensitized singlet oxygen (1O2) generation was investigated using DNA-binding alkaloids, berberine and palmatine. These photosensitizers were bound to DNA by electrostatic force. Near-infrared luminescence measurement demonstrated that the photoexcited alkaloids can generate 1O2 only when the photosensitizers are bound to DNA. A fluorescence decay study showed significant enhancement of the lifetime of their photoexcited state with the DNA binding. A calculation study suggested that the electrostatic interaction with DNA inhibits the quenching of the photoexcited state of these alkaloids via intramolecular electron transfer, leading to the prolongation of the lifetime of their excited state. This effect should enhance their intersystem crossing and the yield of energy transfer to molecular oxygen. The results show that the electrostatic interaction with DNA significantly affects the 1O2 generation activity of a photosensitizer. In addition, this interaction may be applied to the control and the design of photosensitizers for medical applications such as photodynamic therapy.     

Oxidation of 2'-deoxyguanosine 5'-monophosphate photoinduced by pterin: type I versus type II mechanism

UV-A radiation (320-400 nm) induces damage to the DNA molecule and its components through different photosensitized reactions. Among these processes, photosensitized oxidations may occur through electron transfer or hydrogen abstraction (type I) and/or the production of singlet molecular oxygen ((1)O2) (type II). Pterins, heterocyclic compounds widespread in biological systems, participate in relevant biological processes and are able to act as photosensitizers. We have investigated the photosensitized oxidation of 2'-deoxyguanosine 5'-monophosphate (dGMP) by pterin (PT) in aqueous solution under UV-A irrradiation. Kinetic analysis was employed to evaluate the participation of both types of mechanism under different pH conditions. The rate constant of (1)O2 total quenching (k(t)) by dGMP was determined by steady-state analysis of the (1)O2 NIR luminescence, whereas the rate constant of the chemical reaction between (1)O2 and dGMP (k(r)) was evaluated from kinetic analysis of concentration profiles obtained by HPLC. The results show that the oxidation of dGMP photosensitized by PT occurs through two competing mechanisms that contribute in different proportions depending on the pH. The dominant mechanism in alkaline media involves the reaction of dGMP with (1)O2 produced by energy transfer from the PT triplet state to molecular oxygen (type II). In contrast, under acidic pH conditions, where PT and the guanine moiety of dGMP are not ionized, the main pathway for dGMP oxidation involves an initial electron transfer between dGMP and the PT triplet state (type I mechanism). The biological implications of the results obtained are also discussed.     

有机分子三重激发态的调控与应用

对近期有机分子三重激发态调控的研究进展进行了总结评述。控制分子的三重激发态性质,可以制备多种具有新颖性质的分子,如用于可激活光动力治疗(PDT)的光敏剂、磷光分子探针与生物标识试剂,以及可控的三重态湮灭上转换等。但目前对三重态控制方面的研究相对较少,其中的规律也很不明确。近期有文献陆续报道了使用超分子方法和共价修饰法进行的三重态调控,利用的光物理过程有单重态能量转移、三重态能量转移、电子转移等等。现有研究结果表明,三重态的调控规律与单重态的调控规律有所不同,例如:发色团的单重激发态(荧光)往往可以被光诱导电子转移(PET)所猝灭,但是在多个例子中已发现,相同发色团的三重态并不能被PET所猝灭。本文总结的研究结果及所作的分析,将对该领域的分子结构设计及后续研究起到一定的促进作用。     

Tuning the singlet-triplet energy gap: a unique approach to efficient photosensitizers with aggregation-induced emission (AIE) characteristics

The efficiency of the intersystem crossing process can be improved by reducing the energy gap between the singlet and triplet excited states (ΔE _ST), which offers the opportunity to improve the yield of the triplet excited state. Herein, we demonstrate that modulation of the excited states is also an effective strategy to regulate the singlet oxygen generation of photosensitizers. Based on our previous studies that photosensitizers with aggregation-induced emission characteristics (AIE) showed enhanced fluorescence and efficient singlet oxygen production in the aggregated state, a series of AIE fluorogens such as TPDC, TPPDC and PPDC were synthesized, which showed ΔE _ST values of 0.48, 0.35 and 0.27 eV, respectively. A detailed study revealed that PPDC exhibited the highest singlet oxygen efficiency (0.89) as nanoaggregates, while TPDC exhibited the lowest efficiency (0.28), inversely correlated with their ΔE _ST values. Due to their similar optical properties, TPDC and PPDC were further encapsulated into nanoparticles (NPs). Subsequent surface modification with cell penetrating peptide (TAT) yielded TAT–TPDC NPs and TAT–PPDC NPs. As a result of the stronger singlet oxygen generation, TAT–PPDC NPs showed enhanced cancer cell ablation as compared to TAT–TPDC NPs. Fine-tuning of the singlet-triplet energy gap is thus proven to be an effective new strategy to generate efficient photosensitizers for photodynamic therapy.     

金属酞菁激发态与氧的相互作用

比较了几种金属酞菁光敏产生单重态氧和超氧负离子的能力,结果表明它们产生¹O₂的能力与中心金属的电子结构有关,取决于三重态寿命和量子产率。顺序如下:Zn>Ga>Cu>H₂>Al>Co。产生O₂^·-的能力不仅与三重态寿命和量子产率有关,也与激发能和氧化还原电位有关。其顺序如下:Ga>Al>Cu>Zn。还研究了酪氨酸与镓酞菁激发态相互作用,酪氨酸猝灭镓酞菁荧光。在除氧条件光激发下,酪氨酸猝灭镓酞菁的激发三重态发生电子转移,检测到GaTSPc^-在560nm处的瞬态吸收,在氧的存在下进一步反应生成O₂^·-。     

Metal bacteriochlorins which act as dual singlet oxygen and superoxide generators

A series of stable free-base, Zn(II) and Pd(II) 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).     

Isomerization of dewar benzenes involving electron donor-acceptor exciplexes

Valence photoisomerization of hexamethyl (Dewar benzene) (HMDB) is sensitized by aromatic singlet photosensitizers 1,4-dicyanobenzene, 1-cyanonaphthalene, 9-cyanoanthracene, and 9,10-dicyanoanthracene with a limiting quantum efficiency of 1.0 in cyclohexane solvent. Quenching of the fluorescence of the aromatic sensitizers leads to exciplex emission which is identical to that obtained by quenching with the isomer, hexamethylbenzene (HMB). The emission is identified as HMB exciplex emission on the basis of relative lifetime and dual quenching experiments. The relative yield of HMDB-derived (“adiabatic”) emission is 20–50% depending on the excitation energy of the HMB exciplex product. Neither biacetyl singlet or triplet nor 1-cyanonaphthalene triplet photosensitization is successful in bringing about isomerization of HMDB. Dimethyl 1,4,5,6-tetramethylbicy-clo[2.20]hexa-2,5-diene-2, 3-dicarboxylate undergoes valence isomerization on quenching electron donor fluorophores, with a quantum efficiency of 0.2. The aromatic valence isomer is not produced in an excited state in this case. Factors which govern the efficiency of adiabatic and diabatic isomerization of the Dewar benzenes are discussed, including sensitizer redox properties, configuration, and multiplicity, the excitation energy and binding characteristics of exciplexes, and the Dewar benzene substituent pattern.     

具有强可见光吸收的富勒烯-Bodipy衍生物作为光催化剂在硫醚光催化氧化中的应用

以氟硼吡咯染料（Bodipy）做为光吸收天线,富勒烯作为分子内自旋转换单元,分别利用前者的强吸光以及后者的高效系间窜越的优点,制备了吸收波长灵活可调、无重原子、具有强可见光吸收能力、长寿命三重激发态（92.1 μs）的二元（Dyad）、三元化合物（Triad）做为有机三重态光敏剂;其中Triad具有宽谱带可见光吸收能力,提高了光敏剂参与的分子间能量转移或电子转移过程的效率。与传统的Ru（Ⅱ）配合物等三重态光敏剂相比,光催化硫醚氧化的反应时间大大缩短。通过吉布斯自由能的计算、活性氧物种的捕获实验、电子顺磁共振等方法,证明了在光催化氧化硫醚的反应过程中,同时存在超氧负离子自由基和单线态氧两个活性物种,从而加快了光催化氧化反应速率。该研究结果将对新型有机三重态光敏剂的分子结构设计以及在光催化有机合成反应中的应用起到一定的促进作用。     

Photosensitized generation of singlet oxygen

This work gives an overview of what is currently known about the mechanisms of the photosensitized production of singlet oxygen. Quenching of pi pi* excited triplet states by O2 proceeds via internal conversion of excited encounter complexes and exciplexes of sensitizer and O2. Both deactivation channels lead with different efficiencies to singlet oxygen generation. The balance between the deactivation channels depends on the triplet-state energy and oxidation potential of the sensitizer, and on the solvent polarity. A model has been developed that reproduces rate constants and efficiencies of the competing processes quantitatively. Sensitization by excited singlet states is much more complex and hence only qualitative rules could be elaborated, despite serious efforts of many groups. However, the most important deactivation paths of fluorescence quenching by O2 are again directed by excess energies and charge-transfer interactions similar to triplet-state quenching by O2. Finally, two recent developments in photosensitization of singlet oxygen are reviewed: Two-photon sensitizers with particular application potential for photodynamic therapy and fluorescence imaging of biological samples and singlet oxygen sensitization by nanocrystalline porous silicon, a material with very different photophysics compared to molecular sensitizers.     

Micro-organizational control of photochemical oxidations: Rose bengal and derivatives (XV)

we define a microheterogeneous photo-oxidation to be a photosensitized oxidation reaction whose efficiency is enhanced beyond that of diffusion control by the covalent bonding of a sensitizer to a ligand. The ligand is responsible for enhancing the local concentration of a specific substrate susceptible to reaction with an excited state derived from the proximate sensitizer. We illustrate the principle with several applications in singlet oxygen processes.