Helmut Görner

Photochemistry of DNA and related biomolecules: quantum yields and consequences of photoionization.

The reactions of nucleic acids and constituents, which can be induced by laser UV irradiation, are described. Emphasis is placed on the quantum yields of various stable photoproducts of DNA and model compounds upon irradiation at 193, 248, 254 or 266 nm. In particular, those quantum yields and processes are discussed which involve photoionization as the initial step and occur in aqueous solution under well defined conditions, e.g. type of atmosphere. The efficiencies of some photoproducts, with respect to photoionization using irradiation at 193 or 248 nm, are presented. Radical cations of nucleobases are important sources of damage of biological substrates since they can cause lesions other than dimers and adducts, e.g. strand breakage, abasic sites, crosslinks or inactivation of plasmid and chromosomal DNA. While competing photoreactions, such as hydration, dimerization or adduct formation, diminish the selectivity of the photoionization method, a combination with model studies on pyrimidine- and purine-containing constituents of DNA has brought about an enhanced insight into the reaction mechanisms. The knowledge concerning the lethal events in plasmid and cellular DNA has been greatly improved by correlation with the chemical effects obtained by gamma-radiolysis, vacuum-UV (< 190 nm) and low-intensity irradiation at 254 nm.

Photochromism of nitrospiropyrans: effects of structure, solvent and temperature

The thermal and photochemical ring opening and ring closure of the spiropyran/merocyanine couple of three nitro-substituted spiropyrans (6-NO2: 6, 7 and 8-NO2: 8) were studied and the results were compared with those of other 6-NO2 spirobenzopyranindolines (BIPS) (1–5). The photocolouration, which occurs in the triplet manifold throughout, and the photochemical conversion and thermal relaxation of the two observable photomerocyanines into the closed spiropyran (Sp) form (decolouration) were quantified as a function of solvent polarity and temperature. The relaxation time (τt-Sp) at 25°C ranges from 2 s for 5 in methylcyclohexane to 104 s for 7 in ethanol. This large variation in τt-Sp is due to changes in both the activation energy (Ea = 75–105 kJ mol−1, increasing with polarity) and the pre-exponential factor (A = 1012–1015 s−1). The quantum yield of colouration with 308 nm pulses is substantial in solvents of low polarity (Φcol = 0.3–0.8) and decreases (<0.2) with increasing polarity. The observed merocyanine triplet state is rather short-lived (<10 μs) and the precursor of the trans- and a cis-merocyanine. The same intermediates, the triplet state and the cis-isomer, were observed when the trans-merocyanine was excited at 530 nm. The thermal cis → trans isomerization takes place in the μs–ms range and has an activation energy of Ec → t = 30–40 kJ mol−1. The effects of substituents and medium properties are described and the mechanism of photochromism is discussed.

Photo-induced electron transfer from nucleotides to ruthenium-tris-1,4,5,8-tetraazaphenanthrene: model for photosensitized DNA oxidation

Abstract— The luminescence quenching of ruthenium‐tris‐l,4,5,8‐tetraazaphenanthrene [Ru(tap)32+] by nucleotides approaches the diffusion rate only with guanosine‐5′‐monophosphate (GMP), the most reducing nucleotide, and leads to an electron transfer with the production of the monoreduced complex and the oxidized base. The resulting deprotonated GMP(‐H)radical recombines with the monoreduced complex according to a bimolecular equimolar process.

Laser-induced strand break formation in DNA and polynucleotides.

The main objective of this review is to summarize the current knowledge of the chemical steps leading to laser pulse-induced strand breakage in nucleic acids. One of the motivations behind these photoionization studies is to gain a better understanding of the direct effects of high-energy radiation on DNA which leads to strand breaks (SchulteFrohlinde, 1986a, 1986b, 1989). The radiationinduced double-strand breaks are the most serious damage to DNA in cells which, if unrepaired, lead to cell transformation, mutation and cell death (Ward, 1975, 1990; Schulte-Frohlinde, 1987a, 1987b). Strand break (sb) formation induced by UV light is discussed only in so far as it is not incorporated in recent reviews by Nikogosyan and Letokhov (1983); Peak et al. (1987); HClkne (1987); Cadet and Vigny (1990); Nikogosyan (1990). Physical and spectroscopically observable primary processes have been summarized by Nikogosyan and Letokhov (1983) and Nikogosyan (1990). Photosensitized sb formation has been reviewed by numerous authors, see for example: Amagasa (1981); Cadet et al. (1986); Piette and van de Vorst (1987); Kochevar and Dunn (1990). Also not incorporated in the present work is the content of articles edited by Wang (1976) under the title: “Photochemistry and Photobiology of Nucleic Acids” since strand breakage is virtually not discussed in these reviews. Furthermore, most of the earlier articles which do not explicitly contribute to the elucidation of mechanistic steps are also not included. The present article describes mainly the chemical effects from biphotonic excitation of polynucleotides and DNA leading to singleand double-strand breaks (ssb and dsb). In addition some recent results concerning monophotonic generation of sb are discussed. Methods for the determination of sb are reviewed in Section 2. Of special importance is the time dependence of sb formation since this allows conclusions concerning the mechanism to be drawn. In Section 3 photoionization of nucleic acids as initiating process is outlined and the following Sections (4-12) deal with the chemical mechanisms of ssb and dsb initiation of polynucleotides and DNA under a wide range of experimental conditions. 2 . Methods

COMPLEXES OF SPIROPYRAN-DERIVED MEROCYANINES WITH METAL IONS : RELAXATION KINETICS, PHOTOCHEMISTRY AND SOLVENT EFFECTS

Abstract The complexation of spiropyran-derived merocyanines having a 6-nitro group in the pyran moiety (6-NO 2 -BIPSs) or no nitro group and certain transition or rare-earth metal ions (M n + , n =2, 3) were studied by steady-state and time-resolved techniques in acetonitrile, butyronitrile, acetone and dioxane at room temperature. The overall kinetics of complex formation are governed by equilibria between the spiropyran (Sp), trans-merocyanine (trans) and complex (trans-M n + ) forms. For this two-step system two relaxation times are experimentally accessible which refer to fast ( τ I ) and slow ( τ II ) relaxation kinetics. Thermal or photochemical perturbation of the equilibria leads to the same τ II value for a given 6-NO 2 -BIPS and metal ion in defined concentrations. With increasing [M n + ], τ II strongly increases and finally approaches the time needed for the thermal Sp→trans conversion. Water in small concentration ( 2 -BIPSs are weak fluorescence and mainly radiationless decay of the excited singlet state, whereas intersystem crossing plays a role mainly for Pb II and intramolecular energy transfer mainly for Co II or Ni II . Photochemical reactions are trans→cis photoisomerization via the triplet state (Pb II ) and photosubstitution of the trans isomer by a solvent as another ligand via the singlet state (with spiropyran ring closure), e.g. for Mn II or Zn II .

Complexes of spiropyran-derived merocyanines with metal ions Thermally activated and light-induced processes

The properties of complexes of five spiropyran-derived merocyanines with MgII, PbII, transition and rare-earth metal ions (Mn+, n = 2, 3) have been studied in acetone by steady-state and time-resolved techniques. Within nanoseconds, excitation of the spiropyran (Sp) form of 1′,3′,3′-trimethyl-6-nitro-8-methoxy-spiro[2H-1-benzopyran-2,2′-indoline] (3) and derivatives leads to atriplet state of the zwitterionic merocyanine form, which converts into a mixture of trans- and cis-isomers. Eventually, bidentatemetal complexes (trans-Mn+) were observed, with rate constants for 3 from 3 × 107 (with NiII) to 1.2 × 109 d mol−1 s−1 (with PbII). This reaction also occurs with similar rate constants for 1 (where only unidentate complexes are formed) and 2 (derivatives without the methoxy group). For 5, where the methyl group on N(1′) is replaced by a phenyl group, the yield of complex is small. The kinetics were studied for equilibria of the Sp form with both trans-merocyanine and trans-Mn+, being perturbed either thermally or photochemically. Upon excitation of complexes with 3 and 4 [where the methyl group on N(1′) is replaced by C2H4OH], triplet formation, trans → cis photoisomerization (into cis-Mn+) and photosubstitution (into the Sp form) were observed. The yields depend on the nature of the metal ion; they are negligible for CoII, NiII or CuII, substantial for CdII or ZnII and largest for PbII. The lifetime of the observed cis-Mn+ complex is limited by interaction with the metal ion and water.

CIS-TRANS PHOTOISOMERIZATION OF 4-NITROSTILBENES

Abstract Results on cis-trans photoisomerization of 4-nitrostilbenes obtained in recent years are summarized and discussed. Mechanisms for the direct trans → cis and cis → trans photoisomerization are presented. The trans → cis isomerization occurs via a triplet state as intermediate whereas in the cis → trans isomerization the main route bypasses the triplet state. The triplet state shows a configurational equilibrium between the planar trans (tr3) and more twisted forms (p3 or c3). The equilibrium is mainly on the trans side. The influence of substitution, temperature, viscosity, solvent, and addition of quenchers on quantum yields of isomerization, fluorescence studies, and spectroscopic as well as kinetic laser flash photolysis results support the above conclusions.

Photoreduction of 9,10-Anthraquinone Derivatives: Transient Spectroscopy and Effects of Alcohols and Amines on Reactivity in Solution¶

The photoreduction of 9,10‐anthraquinone (AQ), the 2‐methyl, 2‐ethyl, 2,3‐dimethyl, 1,4‐difluoro, 1‐chloro and 1,8‐dichloro derivatives as well as 1,4,4a,9a‐tetrahydroanthraquinone, 1,2‐benzanthraquinone and 6,13‐pentacenequinone in nonaqueous solution at room temperature was studied by time‐resolved UV–visible spectroscopy. Upon 308 nm excitation of AQ the triplet state reacts with alcohols and triethylamine (TEA). The rate constant of triplet quenching by amines is close to the diffusion‐controlled limit. The semiquinone radical ·QH/Q·− is the main intermediate, and the half‐life of the second‐order decay kinetics depends significantly on the donor and the medium. Photoinduced charge separation after electron transfer from amines to the triplet state of AQ in acetonitrile and the subsequent charge recombination or neutralization also were measured by transient conductivity. The maximum quantum yield, λirr= 254 nm, of photoconversion into the strongly fluorescing 9,10‐dihydroxyanthracenes is close to unity. The fluorescence with maximum at 460–480 nm and a lifetime of 20–30 ns disappears as a result of a complete recovery into AQ, when the dihydroxyanthracenes are exposed to oxygen. The mechanisms of photoreduction of parent AQ in acetonitrile by 2‐propanol and in benzene and acetonitrile by TEA are discussed. The effects of AQ follow essentially the same pattern. The various functions of oxygen, e.g. (1) quenching of the triplet state; (2) quenching of the semiquinone radical, thereby forming HO2·/O2·− radicals; and (3) trapping of the dihydroxyanthracenes are outlined.

Photoprocesses in spiropyrans and their merocyanine isomers: Effects of temperature and viscosity

Abstract The spectroscopic properties of five spiro[2H-1-benzopyran-2,2′-indoline] compounds containing a nitro group in the 6-position of the benzopyran moiety (1–5) were studied as a function of temperature in media of different polarity, e.g., methylcyclohexane, 2-methyltetrahydrofuran (MTHF), glycerol triacetate, and ethanol. UV excitation of a spiropyran at ambient temperature leads to a triplet state of the merocyanine (3t∗) prior to population of the trans and cis isomers. The yields of formation of 3t∗ and the merocyanine ground states decrease in a similar manner on decreasing the temperature. In highly viscous media, where the photochemical ring opening is suppressed, phosphorescence and the T-T absorption of the spiropyran could be detected: e.g., λTT = 451 nm, τT = 0.2 s for 3 (R-1′: CH3, R-8: OCH3) in MTHF at temperatures below 100 K. Upon excitation at 530 nm photosomerization into a less stable cis isomer occurs. The quantum yield is reduced on decreasing the temperature, whereas the yields of fluorescence and population of the 3t∗ state increase in a similar manner. A semilogarithmic plot of the inverse triplet life-time vs 1 T shows two linear sections, indicating rotation about the CC bond in fluid solution and a strong increase in triplet life-time due to the hindrance of rotation at very high viscosities. The 3t∗ state, which is an intermediate in the photocolouration is not directly involved in trans→cis photoisomerization.

Photoinduced electron transfer between styrylanthracenes and electron donors and acceptors in acetonitrile

The decay pathways of the lowest singlet and triplet excited states (1trans* and 3trans*) of trans-n-styrylanthracenes (n-StA, where n= 1, 2 or 9 on the anthracene) have been studied in acetonitrile at room temperature. Fluorescence lifetimes (τF) and quantum yields (ϕF), as well as the yield and spectral and kinetic properties of the lowest triplet state of the three StAs, were determined by steady-state and transient techniques. The formation and the decay of the respective StA radical cations (trans˙+) were observed by laser flash photolysis; the yield of photoionization is ca. 0.07 on 353 nm excitation and is enhanced by fluorescence quenching with 1,4-dicyanobenzene. The formation and decay of the StA radical anions (trans˙–) in the presence of diethylaniline (DEA) is concluded from transient conductivity (Δκ) and optical results. DEA significantly enhances the yield of trans˙– and the initial amplitude of Δκ and correspondingly quenches ϕF. The bimolecular interaction between 1trans* and 4-bromodimethylaniline enhances the triplet population.