REVERSIBLE PHOTOCYCLOADDITION OF A 4',5'-DIHYDROPSORALEN DERIVATIVE WITH THYMINE

The photocycloaddition reaction between a 4′,5′-dihydropsoralen derivative and thymine was studied in solution using a synthetic bichromophoric model 8 in which the two rings are associated by a tetramethylene chain. In water this model molecule exhibits intramolecular ring-ring stacking interactions as evidenced by UV and NMR spectroscopies. Irradiation at 365 nm at usual concentrations ( 5.10^?-⁴M) leads exclusively to a regio- and stereo-selective dimerization reaction involving the 3,4 double bonds of the psoralen moities. Extreme dilutions (ca 2.10^?-⁵M) were necessary to observe the intramolecular reaction which results in the exclusive formation of a 3,4 cb-anti adduct. This reaction is completely reversed by irradiation at 254 nm. These results are discussed with regard to the behavior of the homologous models in which the furan part of the psoralen ring is not hydrogenated, These latter compounds also lead exclusively to a 3,4 cis-anti adduct. It appears that saturation of the furan ring increases strongly the quantum yield of the photaddition at 365 nm (0.01 → 0.18) and that the triplet excited state of the 4′,5′-dihydropsoralen is involved in the photoaddition.     

FLUORIMETRIC DETERMINATION OF 4',5'-CYCLOADDUCTS IN THE DNA-PSORALEN PHOTOREACTION

Abstract— 4',5'-Photocycloadducts of psoralen with pyrimidine bases of DNA have been quantitatively determined by fluorescence measurements on the acid hydrolysate of DNA-psoralen photoadducts.     

ADENOSINE-MEDIATED PHOTOREACTION OF 4,5',8-TRIMETHYLPSORALEN WITH ALCOHOLS

Abstract— Irradiation of thin films consisting of 4,5',8-trimethylpsoralen (TMP), adenosine and small amounts of alcohols led to TMP-alcohol photoadducts in addition to TMP-adenosine photoadducts. Four TMP-ethanol and two TMP-methanol adducts have been separated and characterized. Covalent bonds were formed between the 4-carbon of TMP and the α-carbon to the hydroxy group in the alcohols. The TMP-alcohol photoadducts were formed only in the TMP film containing small amounts of alcohol and adenosine. Furthermore, no photoadduct of TMP and ribose was detected upon photolysis of a TMP-ribose film, suggesting that the adenine moiety plays a specific role in the reaction. The interaction of adenosine with psoralens in a dry film may be related to the DNA sequence selectivity observed for the photoreaction of psoralens with DNA.     

TRIPLET EXCITED STATE OF THE 4'5' PHOTOADDUCT OF PSORALEN AND THYMINE

Abstract— The triplet-triplet absorption spectrum of the 4'5' psoralen-thymine mono-adduct has been determined in water and methanol using the technique of laser flash photolysis. The extinction coefficient of the triplet was measured by the energy-transfer method with retinol triplet as standard, and used to determine the singlet → triplet intersystem crossing quantum yield for 353 nm excitation. Reaction rate constants for mono-adduct triplet with thymine and tryptophan were measured in water. Long-lived transient absorptions detected after quenching the mono-adduct triplet with thymine and tryptophan are assigned mainly to the corresponding mono-adduct radical anion, whose spectrum was established in separate pulse radiolysis studies of the mono-adduct in aqueous formate.
The significant singlet → triplet quantum yields found for the mono-adduct might be consistent with the involvement of triplet excited mono-adduct in DNA cross-link formation, as also may be the high reactivity obtained for the triplet with thymine. The initial quenching products observed resulted from a charge-transfer reaction.     

MONOFUNCTIONAL 4',5'-PHOTOCYCLOADDUCT BETWEEN 4,5'-DIMETHYLANGELICIN AND THYMINE

Abstract A new fluorescent compound has been isolated from the products of hydrolysis of DNA irradiated in the presence of 4,5'-dimethylangelicin, a monofunctional photosensitizing furocoumarin. The marked similarity of the UV absorption and fluorescence spectra of this photoproduct to those of 4',5'-dihydro-4,5'-dimethylangelicin, as well as its behaviour on photodissociation (254 nm) yielding thymine and 4,5'-dimethylangelicin in equimolecular amounts, are consistent with a cycloadduct between one molecule of 4,5'-dimethylangelicin and one of thymine, and the C₄-cycloaddition occurs through 4',5'–double bond of the furocoumarin and the 5,6-double bond of the pyrimidine.     

PHOTOREACTIVITY OF 5-GERANOXYPSORALEN AND LACK OF PHOTOREACTION WITH DNA

5-Geranoxypsoralen, commonly called bergamottin, a major furocoumarin contained in bergamot oil, is reported in vitro as a highly photoreactive psoralen. In ethanol, it exhibits quite a high triplet state quantum yield (approximately 0.37). The triplet state is involved in subsequent photochemistry which depends on the initial concentration and on the presence of oxygen. In contrast to most psoralens, absorption and fluorescence data suggest that 5-geranoxypsoralen does not interact with DNA in the dark. No UVA-induced interstrand cross-links in DNA were shown.     

MONOFUNCTIONAL3,4-and4',5'-PHOTOCYCLOADDUCTS BETWEEN 4,5'-DIMETHYLANGELICIN and THYMINE

By irradiating (365 nm) an aqueous liquid solution of 4,5'-dimethylangelicin. a monofunctional photosensitizing furocoumarin, in the presence of an excess of thymine, two new compounds, I and II, have been obtained; they do not show fluorescence when observed with Wood's light. The nuclear magnetic resonance data, the marked similarity of UV absorption and fluorescence spectra of these compounds with those of synthetic 3.4-dihydro-4,5'-dimethylangelicin and their capacity to undergo photodissociation (254 nm) yielding the starting thymine and 4,5'-dimethylangelicin in equimolecular amounts, are consistent with C₄-cycloadducts between the 3,4-double bond of the furocoumarin and 5,6-double bond of thymine. Nuclear magnetic resonance data indicate for I and II a head-to-head and a head-to-tail structure, respectively. When irradiation is carried out in the frozen state, two adducts. III and IV, fluorescent at Wood's light, have been obtained other than the two above-mentioned compounds I and II. Compounds III and IV have been identified as 4'.5'-fluorescent adducts between the 4',5'-double bond of the furocoumarin and the 5.6-double bond of thymine; one of them (III) is identical to that formed in the photoreaction between DNA and 4,5'-dimethylangelicin; for this last compound a cis head-to-head structure has been suggested.     

PHOTOREACTION OF 5-METHOXYPSORALEN WITH THYMIDINE and THE THYMINE MOIETY OF ISOLATED and Saccharomyces cerevisiae DNA. CHARACTERIZATION and MEASUREMENT OF THE TWO cis-syn FURAN-SIDE MONOCYCLOADDUCTS

The photoreaction of the furan-side moiety of 5-methoxypsoralen (5-MOP) with thymidine used as a DNA model compound was investigated in the dry state. Under these conditions, two main fluorescent photoadducts were formed and isolated by HPLC. The two modified nucleosides were characterized as the two cis-syn diastereoisomers of furan-side monoadducts of 5-MOP to thymidine on the basis of spectroscopic measurements including UV, fluorescence, ¹H-NMR and circular dichroism analysis. The identification and quantification of the latter photoproducts within naked DNA exposed to photoexcited 5-MOP were achieved by enzymatic digestion completed by HPLC separation and fluorescence detection. Similarly, the two cis-syn furan-side monoadducts were found to be formed in the DNA of Saccharomyces cerevisiae cells after incubation with 5-MOP and subsequent exposure to 365 nm at an incident dose of 38.4 kJ m^?2. Under these conditions, the rate of induction of two diastereoisomeric photoadducts was as low as one modification per 10⁶ and 2 × 10⁵ bases, respectively.     

PHOTOREACTION OF MONOFUNCTIONAL 3-CARBETHOXYPSORALEN WITH DNA: IDENTIFICATION AND CONFORMATIONAL STUDY OF THE PREDOMINANT cis-syn FURAN SIDE MONOADDUCT TO THYMINE

The main DNA-3-CPs furan side adduct has been isolated following acidic hydrolysis of photomodified DNA and subsequent reversed-phase high performance liquid chromatography purification. This photoadduct has been identified as a thymine-3-CPs C₄-cycloadduct Thy<^{5 4'}_{6 5'}, >3-CPs based on its optical spectroscopic features and of its plasma desorption mass spectrometric characteristics. Moreover, its reversed-phase high performance liquid chromatography retention time is in agreement with a cis-syn stereochemistry as compared to the chromatographic properties of the cis-syn model adduct Thy< ^{5 4'}_{6 5'} >3-CPs previously identified. Further support for the cis-syn stereoconfiguration assignment was provided by fluorescence quenching experiments using iodide and silver ions as external and internal quenchers respectively. These data strongly indicate that the Thy< ^{5 4'}_{6 5'} >3-CPs photoadduct is located inside the DNA helix in agreement with its cis-syn stereochemistry.     

4',5'-SUBSTITUTED METHYLANGELICINS: PHOTOCYCLOADDUCTS WITH PYRIMIDINE BASES OF DNA

The isolation and characterization of photocycloadducts with pyrimidine bases from DNA samples irradiated (365 nm) in the presence of four 4',5'-substituted methylangelicins was performed. All these furocoumarins yielded mainly the cis-syn furan-side cycloadduct with thymine. For 4',5'-dimethyl-, 5,4',5'-trimethyl- and 6,4',5'-trimethylangelicin this adduct was accompanied by two pyrone-side adducts ( cis-syn and cis-anti ), whereas the 4,4',5'-trimethyl derivative gave the furan-side adduct with cytosine.
The characterization of the regio- and stereochemistry of the adducts was accomplished by ¹H NOE (nuclear Overhauser effect) and ¹H-¹³C HMBC (heteronuclear multiple-bond connectivity) spectroscopies.
The formation of different cycloadducts in DNA by the various derivatives highlights the role of the methyl groups in determining the regio- and stereochemistry of the cycloaddition.     

MECHANISTIC STUDIES OF THE PHOTOREACTION OF FLAVIN WITH N, N-DIMETHYLPROPARGYLAMINE

Abstract Both photoexcited free flavin and protein-bound flavin react with N, N-dimethylpropargyla-mine (DMPA) to yield dihydroflavin-adducts which are not reoxidizable by oxygen. The mechanism of the photoreaction was reinvestigated by flash photolysis and continuous illumination experiments. We suggest that a mechanism occurs in which a flavosemiquinone and a DMPA radical result in the first photoinduced reaction. The flavin-DMPA adducts are then formed by combinations of the two radicals.     

PHOTOREACTION OF 8-METHOXYPSORALEN WITH THYMIDINE

Abstract— The photoreaction of 8-methoxypsoralen (8-MOP) with thymidine in solid film state yielded two 4', 5'-monoadducts (a pair of diastereomers) and three 3,4-monoadducts. The stereochemistry of two 4', 5'-monoadducts was found to be cis-syn and trans-syn and one 3,4-monoadduct was cis-anti. In addition to these monoadducts, 3,4-, 4', 5'-biadducts were also formed during the reaction, but the isolation of each isomer of these adducts was not successful; however, the formation of these biadducts was confirmed by UV, IR, TLC and photosplitting experiments.     

METHYLALLOPSORALEN-THYMINE 3,4- and 4'',5''-MONOADDUCTS FORMED IN THE PHOTOREACTION WITH DNA

Abstract— Two new allopsoralens, i.e. 4,7.5'-trimethylallopsoralen and 4,7,4'-trimethylallopsoralen have been irradiated (365 nm) in the presence of DNA. The DNA so treated was hydrolyzed and among the products of its hydrolysis new 3,4- and 4',5'-monocycloadducts between the two furocoumarins and thymine have been isolated. The monoadducts have been characterized on the basis of their spectroscopic properties, of their capacity to undergo photoreversion forming the parent compounds and of the NMR data. A cis-syn conformation has been suggested for both 3,4- and both 4',5'-monoadducts.     

INTERACTIONS AND PHOTOREACTIVITY OF 5-ALKOXYPSORALENS WITH THYMINE. A MODEL APPROACH

Abstract— In order to investigate the interactions and the photoreactions in solution between the thymine (thy) and the psoralen (Pso) rings, we have prepared model compounds Thy-(CH₂)_n-Pso in which two aromatic chromophores Thy and Pso are linked by flexible polymethylene chains of varying length (CH₂)_n. Two series of compounds were examined and compared as models for the two important drugs 5-methoxypsoralen and 8-methoxypsoralen. Results concerning the 5-alkoxypsoralen series are reported here. In water, these model molecules exhibit intramolecular ring-ring stacking interactions as indicated by hypochromism in the UV and by shielding of the protons in ¹H NMR spectroscopy. These interactions disappear in organic solvents. The photochemical properties of the models were examined in relation with their ground state interaction properties. Irradiation at 365 nm carried out at the usual concentrations (10^-2-10^-3 M) leads exclusively to a stereoselective dimerization involving the psoralen moieties of the models at the 3,4 double bonds. However, when operating at exceedingly low concentrations (2 × 10^-5 M ), the psoralen photodimerization is avoided and a highly regio and stereo-selective psoralen thymine photoaddition is observed involving the 3,4 double bond of psoralen leading to the cis adduct. The same reaction occurs for all models under study being independent of the length of the (CH₂)_n polymethylene linking chain, n = 2 to 6, 12 and of the solvent used. This is unambiguous proof for the highest intrinsic photoreactivity of the 3,4 vs the 4',5' double bond in 5-alkoxy psoralen.     

PHOTOREACTION OF PSORALEN DERIVATIVES WITH STRUCTURALLY ORGANIZED DNA

Abstract— The DNA-photobinding process of a number of psoralen derivatives has been investigated using different nucleic acid structures, such as double helical DNA, nucleosomes, and chromatin under various ionic strength conditions. Important differences were observed using "free" vs organised DNA as the target macromolecule, which are related to conformational, stereochemical, ionic and competition effects. The furocoumarin chemical nature also plays a role; in particular, charged compounds are shown to be more reactive than uncharged analogues with free DNA at low salt concentration, whereas a levelling off in photobinding efficiency occurs on increasing ionic strength and nucleic acid complexity. These results allow an explanation for the photobiological effects of the examined psoralens.     

A5–4 PYRIMIDINE-PYRIMIDONE PHOTOPRODUCT PRODUCED FROM MIXTURES OF THYMINE AND 4-THIOURIDINE IRRADIATED WITH 334 nm LIGHT

The nucleoside 4-thiouridine, present in some bacterial tRNA species, is known to be a chromophore and a target for near-UV light-induced growth delay and also mediates both photoprotection and near-UV cell killing in various bacterial strains. To investigate the photoreaction of 4-thiouridine with DNA or its precursors, we irradiated aqueous mixtures of thymine and 4-thiouridine with 334 nm light and then separated photoproducts using two or more stages of reversed-phase high performance liquid chromatography. The two equally abundant major photoproducts were analyzed by UV absorbance spectrophotometry, fast-atom bombardment and electron-impact mass spectrometry, and ¹H- and ¹³C-NMR spectroscopy, and have been identified as two diastereomers of 6-hydroxy-5-[1-(β-D-erythro-pentofuranosyl)-4′-pyrimidin-2′-one]dihydrothymine (o⁶hThy[5-4]Pdo), of molecular weight = 370.32. These two diastereomers, although stable at room temperature or below, are interconvertible by heating (90d?C for 5 min) in aqueous solution. The possible biological significance of this photoproduct is discussed, and an application as a crosslinker for oligonucleotides to selectively block replication is suggested.     

PHOTOCHEMISTRY OF 4-THIOURIDINE AND THYMINE

Abstract— When thymine is irradiated in aqueous solution with monochromatic 334-nm UV radiation in the presence of 4-thiouridine a photoproduct of thymine is formed, as shown by thin-layer chromatography and autoradiography. The quantum yield for the formation of thymine photoproduct (θ=0.017) is greater than that for cytosine photoproduct formation (θ= 0.0015). The identity of the photoproduct is not known: one possibility is the formation of an adduct between the sensitizer and the base yielding a pyrimidine-pyrimidone type of photoproduct.     

TRIPLET EXCITED STATE OF COUMARIN AND 4'5' DIHYDROPSORALEN: REACTION WITH NUCLEIC ACID BASES AND AMINO ACIDS

Abstract—The triplet-triplet absorption spectra of coumarin, 5.7 dimethoxycoumarin and the furocoumarin 4'5' dihydropsoralen. a model for 4'5' psoralen-pyrimidine mono adducts, have been determined by the techniques of pulse radiolysis and laser flash photolysis. The extinction coefficients of the triplet transitions have been measured and used to determine the singlet → triplet intersystem crossing quantum yields for 347 nm excitation in water. Reaction rate constants for coumarin and 4'5' dihydropsoralen triplets with various pyrimidine and purine nucleic acid bases, and amino acids, have been measured. Long-lived transient absorptions detected after quenching coumarin and 4'5' dihydropsoralen triplets with tryptophan are assigned to mixtures of the corresponding coumarin radical anion and the tryptophan radical cation. The spectra of the radical anions of coumarin and 4'5' dihydropsoralen were established using pulse radiolysis of the coumarins in aqueous formate. It is suggested that coumarins and furocoumarin triplets are quenched by nucleic acid bases and amino acids via a chargetransfer mechanism.