TRIPLET EXCITED STATES OF 4'',5''-PHOTOMONOADDUCTS OF 3- CARBETHOXYPSORALEN and 8-METHOXYPSORALEN WITH DNA NUCLEOSIDES

Abstract— Triplet absorption spectra, triplet extinction coefficient and intersystem crossing for 4',5'-monocycloadducts of 3-carbethoxypsoralen (3-CPs) with thymidine (dThd) and uridine (dUrd) in ethanol have been investigated in order to elucidate whether their triplet state properties could be the limitating step for a further photoreaction of 3-CPs monoadducts with DNA nucleosides. The comparison between the triplet characteristics of 4',5'-monoadducts of 3-CPs and those of 8-methoxypsoralen (8-MOP) shows that the quantum yield is much higher in the case of 3-CPs than for 8-MOP. The monofunctionality of 3-CPs cannot therefore be ascribed to the triplet excited states properties of its monoadducts. It is likely that steric hindrance introduced by the bulky carbethoxy group remains a reasonable explanation.     

REACTIVITY OF EXCITED STATES OF FLAVIN AND 5-DEAZAFLAVIN IN ELECTRON TRANSFER REACTIONS

Abstract— Quenching of the excited states of lumiflavin and 3-methyl-5-deazalumiflavin by methyl-and methoxy-substituted benzenes and naphthalenes in methanol was investigated. The observed difference in the reactivity of acid and neutral lumiflavin triplets is explained thermodynamically by applying the Michaelis cycle, as being due to the higher reduction potential of the acid triplet. In this connection the p K values of lumiflavin triplet (p K ^M= 6.5) and semiquinone (p K ^M= 11.3) have also been determined in methanol. The difference in the reactivity between the singlet and triplet states of lumiflavin is found to be greater as predicted by the difference in excitation energy. The reactivities of the excited states of flavin and 5-deazaflavin differ only slightly in contrast to the marked difference in the ground state reactivities of electron transfer reactions. This is explained in terms of the model of Rehm and Weller. The pH dependence of the electron transfer quenching of 5-deazaflavin triplet was investigated in water, yielding a triplet p K of 2.5. In contrast to the flavin, this triplet p K does not significantly differ from the p K of the 5-deazaflavin ground state. From this, different sites of protonation are deduced for the photoexcited triplet states of flavin and 5-deazaflavin.     

Solvent-Dependent Stabilization of a Charge Transfer State is the Key to Ultrafast Triplet State Formation in an Epigenetic DNA Nucleoside

2’-Deoxy-5-formylcytidine (5fdCyd), a naturally occurring nucleoside found in mammalian DNA and mitochondrial RNA, exhibits important epigenetic functionality in biological processes. Because it efficiently generates triplet excited states, it is an endogenous photosensitizer capable of damaging DNA, but the intersystem crossing (ISC) mechanism responsible for ultrafast triplet state generation is poorly understood. In this study, time-resolved mid-IR spectroscopy and quantum mechanical calculations reveal the distinct ultrafast ISC mechanisms of 5fdCyd in water versus acetonitrile. Our experiment indicates that in water, ISC to triplet states occurs within 1 ps after 285 nm excitation. PCM-TD-DFT computations suggest that this ultrafast ISC is mediated by a singlet state with significant cytosine-to-formyl charge-transfer (CT) character. In contrast, ISC in acetonitrile proceeds via a dark ¹nπ* state with a lifetime of ∼3 ps. CT-induced ISC is not favored in acetonitrile because reaching the minimum of the gateway CT state is hampered by intramolecular hydrogen bonding, which enforces planarity between the aldehyde group and the aromatic group. Our study provides a comprehensive picture of the non-radiative decay of 5fdCyd in solution and new insights into the factors governing ISC in biomolecules. We propose that the intramolecular CT state observed here is a key to the excited-state dynamics of epigenetic nucleosides with modified exocyclic functional groups, paving the way to study their effects in DNA strands.     

Can a Six‐Letter Alphabet Increase the Likelihood of Photochemical Assault to the Genetic Code?

In 2014, two unnatural nucleosides, d5SICS and dNaM, were shown to selectively base pair and replicate with high fidelity in a modified strain of E. coli, thus effectively expanding its genetic alphabet from four to six letters. More recently, a significant reduction in cell proliferation was reported in cells cultured with d5SICS, and putatively with dNaM, upon exposure to brief periods of near‐visible radiation. The photosensitizing properties of the lowest‐energy excited triplet state of both d5SICS and dNaM were implicated in their cytotoxicity. Importantly, however, the excited‐state mechanisms by which near‐visible excitation populates the triplet states of d5SICS and dNaM are currently unknown. In this study, steady‐state and time‐resolved spectroscopies are combined with quantum‐chemical calculations in order to reveal the excited‐state relaxation mechanisms leading to efficient population of the triplet states in these unnatural nucleosides in solution. It is shown that excitation of d5SICS or dNaM with near‐visible light leads overwhelmingly to ultrafast population of their triplet states on the femtosecond time scale. The results presented in this work lend strong support to the proposal that photoexcitation of these unnatural nucleosides can accelerate oxidatively generated damage to DNA and other biomolecules within the cellular environment.     

THE EFFECT OF MERCURIC IONS ON THE EXCITED STATES OF DNA-INTERCALATED ETHIDIUM BROMIDE

The first excited triplet state of DNA-intercalated ethidium bromide is produced with a quantum yield of 0.010.002 on irradiation at 532 nm. A difference extinction coefficient of 1.50.210³ m² mol^?1 is measured for the triplet state at 380 nm. Mercuric ions quench the first excited singlet state of DNA-intercalated ethidium bromide via induced spin orbit coupling to give an increased yield of ethidium triplet states. The same mercuric ion that quenches the singlet state then quenches the triplet state, via the same mechanism, with a rate constant of ca 3.510³ s^?1. An upper limit for the rate of detachment of Hg²⁺ from its binding site in DNA may be fixed at ca 10³ s^?1.     

A FLASH PHOTOLYSIS STUDY OF THE TRIPLET STATE OF ACRIDINE ORANGE IN BASIC SOLVENTS

Abstract— Photochemical reactions of acridine orange (AO) in basic aqueous and ethanolic solutions were studied using the flash photolysis technique. The absorption spectrum of AO was determined in detail (230–900 nm) and extinction coefficients were obtained. The decay of the triplet state is the result of a first order process, a triplet-triplet annihilation process and a quenching by the dye in the ground state. The main part of the triplet decays to the ground state; however the observation of semi-reduced AO shows that the decay is partly due to chemical reactions.
An efficient reversible reaction is observed on flashing aqueous solutions of AO containing tetramethyl- p -phenylenediamine: semi-reduced AO is formed in high yield by reaction between the triplet dye and the diamine. In addition, irreversible reactions of AO occur; these are shown to be due to the triplet state by the method of triplet energy transfer.     

TRIPLET STATE PROPERTIES OF POLYCYCLIC AROMATIC HYDROCARBON-DNA and PORPHYRIN-DNA COMPLEXES DETERMINED BY OPTICALLY DETECTED ZERO FIELD MAGNETIC RESONANCE

The photoexcited triplet states of the physically bound (intercalated) complexes of benzanthracene, pyrene, and free base porphin with DNA have been examined at 77 and 2 K. Measurements of triplet lifetimes, zero field splittings, and individual triplet sublevel intcrsystem crossing rates were obtained by optically detected zero field magnetic resonance (ODMR) for the benzan-thracene-DNA and free base porphin-DNA complexes. The triplet lifetime was measured for the pyrene-DNA complex. No ODMR was observable in the pyrene-DNA system. Comparison of triplet state data with new and previous results for the benzo(a)pyrene-DN A complex is discussed in an attempt to characterize the interactions experienced by polycyclic aromatic hydrocarbons and porphyrins within the DNA environment.     

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.     

EXPLORATORY PHOTOCHEMISTRY OF 5-AZIDO-8-ALKOXY–SUBSTITUTED PSORALENS FREE AND BOUND TO DNA

5-Azido-8-alkoxy psoralens were synthesized. Laser flash photolysis (LFP: XeF, 351 nm, 55 mJ, 17 ns) of the azides in acetonitrile or benzene solution produces the triplet nitrene and a small amount of ketenimine. Laser flash photolysis of the azides in methanol or aqueous solution cleanly produces the triplet nitrene. In aqueous solution containing highly polymerized calf thymus DNA, LFP produces a mixture of triplet nitrene and ketenimine corresponding to photolysis of free and bound psoralen, respectively. The two transients decay slowly but at different rates. Assignment of the transient spectra were secured by matrix EPR and UV-visible spectroscopy. The triplet nitrene lifetime is the same in buffer and in the presence and absence of calf thymus DNA. The results explain why psoralen azides are unable to efficiently nick plasmid DNA pBR322 upon UV activation.     

DNA Photosensitization by an “Insider”: Photophysics and Triplet Energy Transfer of 5‐Methyl‐2‐pyrimidone Deoxyribonucleoside

The main chromophore of (6‐4) photoproducts, namely, 5‐methyl‐2‐pyrimidone (Pyo), is an artificial noncanonical nucleobase. This chromophore has recently been reported as a potential photosensitizer that induces triplet damage in thymine DNA. In this study, we investigate the spectroscopic properties of the Pyo unit embedded in DNA by means of explicit solvent molecular‐dynamics simulations coupled to time‐dependent DFT and quantum‐mechanics/molecular‐mechanics techniques. Triplet‐state transfer from the Pyo to the thymine unit was monitored in B‐DNA by probing the propensity of this photoactive pyrimidine analogue to induce a Dexter‐type triplet photosensitization and subsequent DNA damage.     

TRIPLET STATE STUDIES OF FLAVlNS BY ELECTRON PARAMAGNETTC RESONANCE-III

Abstract— A continuation of studies is presented on the excited triplet state of flavins using EPR techniques. Detailed experiments are reported on the triplet state of flavin-mono-nucleotide (FMN) and flavin-adenine-dinucleotide (FAD). Action spectra of triplet yield are explained in terms of the optical absorption for FMN and FAD. Effects of light saturation, concentration quenching and oxygen on the triplet state are discussed. It is suggested that the rate constant k ₃ for the intersystem crossover from the excited singlet to the triplet state is increased by oxygen and quenchers such as KI. Detailed kinetic studies are presented on the formation of the triplet state.     

PHOTOCLEAVAGE OF DNA: IRRADIATION OF QUINONE-CONTAINING REAGENTS CONVERTS SUPERCOILED TO LINEAR DNA

Irradiation (350 nm) of air-saturated solutions of reagents containing an anthraquinone group linked to quaternary alkyl ammonium groups converts supercoiled DNA to circular and to linear DNA. Generation of linear DNA does not occur by accumulation of numerous single-strand cuts but by coincident-site double-strand cleavage of DNA. Irradiation forms the triplet state of the anthraquinone, which reacts either by hydrogen atom abstraction from a sugar of DNA or by electron transfer from a base of the DNA. Subsequent reactions result in chain scission. The quinone is apparently reformed after this sequence and reirradiation leads to double-strand cleavage.     

QUENCHING OF THE TRIPLET STATE OF CHLOROPHYLL a BY ASCORBIC ACID AND ASCORBATE IN ETHANOLIC SOLUTION

Abstract— Ascorbic acid and ascorbate in chlorophyll ethanol solution were found to be fairly efficient quenchers of the chlorophyll triplet state; comparable to the efficiency of ascorbic acid as a quencher in aqueous pyridine solution.
It has been well established that ascorbic acid quenches the triplet state of chlorophyll in aqueous pyridine solution.^(1,2) The bimolecular quenching constant, kQ , is very much less than that for O₂ or quinine.^(3,4)
Information regarding the quenching of the triplet state of chlorophyll by ascorbic acid in ethanolic solution is lacking. There has been some question as to whether ascorbic acid reduces photoexcited chloro-phyll-ethanolic solution because of its high oxidation potential, or because like the ascorbate ion, it acts only as a quencher; both ascorbic acid and ascorbate in high concentrations gave low quantum yields.⁽⁵⁾ The quenching of the triplet state by ascorbic acid and ascorbate was determined by the flash-photolytic method.     

SOME PROPERTIES OF THE TRIPLET EXCITED STATE OF THE PHOTOSENSITIZING FUROCOUMARIN: 3-CARBETHOXYPSORALEN

Abstract— 3-Carbethoxypsoralen (3-CPs) has been tested in the photochemotherapy of psoriasis. It only forms monoadducts with DNA and is being considered as a non-carcinogenic alternative to 8-MOP which itself forms DNA crosslinks that arc difficult to repair. Using laser flash photolysis or pulse radiolysis, the triplet state of 3-CPs, a possible intermediate in photosensitization, has been generated in several solvents: ethanol, water and benzene. The triplet lifetime, extinction coefficient and quantum efficiency of formation have been measured. Triplet reactivities towards (i) the solvents used, (ii) 3-CPs, (iii) oxygen, (iv) tryptophan and (v) tyrosine, leading, respectively, to photoadditions with water, ethanol and 3-CPs, to ¹O₂, semioxidized tryptophan and semioxidized tyrosine, (vi) thymine and (vii) uracil have been investigated. The dark binding of 3-CPs to DNA has been studied by comparing the reactivity of e_aq^- with free 3-CPs, free DNA and the 3-CPs DNA complex. Some photophysical and photochemical properties of 4',5'di-hydro-3-carbethoxypsoralen(DH–3-CPs), model of the main fluorescent photo-product of 3-CPs, have also been investigated. Biological consequences of the photochemical properties of 3-CPs andDH–3-CPs have been studied in a cellular system (haploid yeast).     

DNA‐backbone radio resistivity induced by spin blockade effect

Coherent control of OH‐free radicals interacting with the spin‐triplet state of a DNA molecule is investigated. A model Hamiltonian for molecular spin singlet‐triplet resonance is developed. We illustrate that the spin‐triplet state in DNA molecules can be efficiently populated, as the spin‐injection rate can be tuned to be orders of magnitudes greater than the decay rate due to small spin‐orbit coupling in organic molecules. Owing to the nano‐second life‐time of OH free radicals, a non‐equilibrium free energy barrier induced by the injected spin triplet state that lasts approximately longer than one‐micro second in room temperature can efficiently block the initial Hydrogen abstraction and DNA damage. For a direct demonstration of the spin‐blockade effect, a molecular simulation based on an ab‐initio Car‐Parrinello molecular dynamics is deployed. © 2010 Wiley Periodicals, Inc. J Comput Chem 2010     

Photochemical properties of meso-substituted thiacarbocyanine dyes in solutions and in complexes with DNA

The processes of cis-trans photoisomerization and thermal back isomerization as well as the effect of DNA on the spectral and kinetic characteristics of the triplet state of a number of meso-substituted thiacarbocyanine dyes: 3,3’-diethyl-9-methoxythiacarbocyanine iodide (K1), 3,3’,9-triethylthiacarbocyanine iodide (K2), 3,3’-diethyl-9-methylthiacarbocyanine iodide (K3), and 3,3’-diethyl-9-chlorothiacarbocyanine perchlorate (K4), were studied by the flash photolysis method. Upon flash photoexcitation, the processes of trans-cis and cis-trans photoisomerization were observed for dye K1; the data on the structure of the absorption bands of the photoisomers were obtained. Complexation with DNA leads to an increase in the quantum yield of the triplet state of the dyes, which is explained by growing rigidity of the bound molecules. In the presence of DNA, triplet state deactivation follows the two-exponential law, thus showing that the dyes form complexes of two different types. The processes of quenching of the dye triplet state by oxygen were studied in solutions and in complexes with DNA. The rate constants for oxygen quenching of the triplet state of the dyes in complexes with DNA were found to be much lower than the values expected for the diffusion-controlled reactions (with allowance for the spin statistical factor, $k_{qO_2 } < 1/9k_{dif} $ ), which is explained by the steric factor of the complexation.     

RELATIVE REACTIVITIES OF THE EXCITED STATES OF FUROCOUMARINS FOR [2+2] PHOTOCYCLOADDITION REACTION WITH TETRAMETHYLETHYLENE

Abstract— The Stern-Volmer constants for fluorescence quenching by tetramethylethylene decrease in the order DMC ≫ DHP > F-2 > 8-MOP. The same order was observed for the quantum yields of [2+2] cycloaddition reaction with tetramethylethylene on direct irradiation. In [2+2] photocycloaddition of F-2 with tetramethylethylene in ethanol, the ratio of quantum yields deduced from singlet and triplet states of F-2; φ₃⁰/φ₁⁰, is about 5. The excited triplet state is the reactive state for the [2+2] photocycloaddition of F-2 with tetramethylethylene in solution but the excited singlet state of F-2 becomes very important in biological conditions.     

TRIPLET STATE STUDIES OF FLAVINS BY ELECTRON PARAMAGNETIC RESONANCE—I

Abstract— The triplet state of flavin derivatives and d-amino acid oxidase was observed by electron paramagnetic resonance at 77°K.
Flavin triplets (Δ m =± 2) originate from the isoalloxazine ring and are resonant at 1560 guass.The half-life of the FMN triplet in 1 N HCl is 15 nisec.This life-time is prolonged indirectly by the presence of paramagnetic species, such as oxygen or free radicals.
The flavin triplet state is pH dependent.In neutral solution the nlaximum triplet yield is obtained and the longest life-time is observed.The triplet state is affected by intra-and inter-molecular complex formation, FAD is partially quenched by indirectly substituted adenine.Tryptophan quenches completely the FMN triplet.The FAD triplet of d-amino acid oxidase is enhanced but the life-time is shortened relative to a pure FAD solution.     

PHOTOSENSITIZATION OF SINGLE-STRAND BREAKS IN pBR322 DNA BY ROSE BENGAL

Rose bengal photosensitized the formation of frank single-strand breaks (SSBs) in double-stranded, supercoiled pBR322 DNA as measured by neutral agarose electrophoresis. The yield of SSBs followed first order kinetics with respect to light fluence and dye concentration. The efficiency of cleavage was more than 20 times greater in an argon atmosphere than in an oxygen atmosphere. The quantum yield in an air atmosphere was 1.7 (+/- 0.3) X 10(-8). Sodium azide quenched the cleavage more efficiently in an oxygen atmosphere than when the oxygen concentration was reduced. Isopropanol and mannitol were poor quenchers; ribose-5-phosphate and guanosine-5'-monophosphate did not quench the cleavage. Substituting D2O for H2O increased the yield of SSBs in both oxygen and oxygen-depleted atmospheres. The results are consistent with initiation of cleavage by reaction of the triplet state of rose bengal (or a radical derived from it) with DNA. In the presence of oxygen, an additional mechanism is introduced.     

DETECTION OF PORPHYRIN EXCITED STATES IN THE INTACT BOVINE LENS

Previous steady state and time resolved spectroscopic studies on porphyrins have shown that the triplet lifetimes of those sensitizers that bind to lens proteins are lengthened by several orders of magnitude. Presented here is an extension of this experiment to measure these transients in an intact bovine lens. As demonstrated by steady state fluorescence spectroscopy and flash photolysis, mesotetra (p-sulfonatophenyl)porphyrin (TPPS) binds to lens proteins. In air-saturated aqueous solution, TPPS has a triplet lifetime of 2 microseconds. In an intact bovine lens the triplet state decayed via biexponential kinetics with lifetimes of 0.16 and 1.6 microseconds. In addition to a lengthening of the lifetime there was a red shift in the triplet transient spectra of 10-20 nm of the porphyrin in the intact lenses.