GENOTOXICITY OF THE NATURAL FUROCHROMONES, KHELLIN AND VISNAGIN AND THE IDENTIFICATION OF A KHELLIN-THYMINE PHOTOADDUCT

Abstract— Longwave UV irradiation of a frozen aqueous solution of khellin and thymine resulted in the formation of a 2+2 photoadduct between the 2,3 double bond of khellin and the 5', 6' double bond of thymine. This behaviour is analogous to that of the furocoumarins and serves as a mechanism to describe the genotoxicity of the furochromones khellin and visnagin. These two compounds are phototoxic towards bacteria and fungi and inhibit mitosis and cause gross chromosomal changes in Chinese hamster ovary (CHO) cells in near UV light. For comparative purposes, 8-methoxypsoralen was also tested and was found to be more active than visnagin which, in turn, was more active than khellin.     

UV IRRADIATION OF NUCLEIC ACIDS: CHARACTERIZATION OF PHOTOPRODUCTS OF THYMIDYLYL-(3'→5')-2'-DEOXY-5-FLUOROURIDINE

Abstract— The acetone-sensitized irradiation using UV-B (ultraviolet light, 280–320 nm; sunlamps) of thymidyl-yl(3'→5')deoxyfluorouridine monophosphate produces two main photoproducts. The distribution of these photo-products is dependent on the pH of the irradiation solution. At pH 6, the cis-syn cyclobutane-type photodimer is the major product, whereas at high pH (8–10) a photoadduct is the major product. These photoproducts have been identified and structurally characterized by H-1 and C-13 NMR spectroscopy. The photoadduct arises from defluorination of the 5-fluorouracil moiety. The structure of the photoadduct maintains the sugar-phosphate backbone of the starting material (d-TpF), and contains a saturated thymine moiety with an added Thy(C6-hydroxyl) and a Thy(C5)-(C5)Ura covalent bond.     

Evidence for [2+2] Photoreaction of α-Methylene-γ-Butyrolactones with Thymine: An Explanation for Chronic Actinic Dermatitis to Sesquiterpene Lactones?

The photoreactivity of isoalantolactone, a natural sesquiterpene lactone, toward thymine was studied. After 313 nm irradiation of a deoxygenated acetone solution of isoalantolactone (2 x 10(-3) M) and thymine (4 x 10(-3) M), two intermolecular [2 + 2] photoadducts, 3 and 4, were isolated with respective yields of 30% and 18%. The structures of these two photoadducts were determined by a combination of NMR experiments. Compound 3 was identified as a cis-syn-exo intermolecular [2 + 2] photoadduct involving the 5,6 double bond of thymine and the exomethylenic double bond of the lactone, while compound 4 was identified as an intermolecular [2 + 2] photoadduct involving the same bonds but with the cis-syn-endo conformation. This high photoreactivity of sesquiterpene lactones toward thymine could be an explanation of the progressive evolution of allergic contact dermatitis toward chronic actinic dermatitis.     

6,4,4'-trimethylangelicin photoadduct formation in DNA: production and characterization of a specific monoclonal antibody

The photochemical reactions of 6,4,4'-trimethylangelicin (TMA) with calf thymus DNA and an octanucleotide containing a single thymine have been characterized. HPLC analyses of enzymatically hydrolyzed TMA-DNA showed that isomeric forms of 4',5'-furan-side monoadducts were the major products. To develop monoclonal antibodies Balb c mice were immunized with the TMA-DNA complexed with methylated bovine serum albumin. The resultant antibodies were characterized by enzyme-linked immunosorbent assays (ELISA). The most sensitive antibody (7E3) has high specificity for TMA-DNA, very low cross-reactivity with DNA modified with either 4',5'-dimethylangelicin or 4'-methylangelicin and no cross-reactivity with non-modified DNA or with DNA modified with either 4'-aminomethyl-4,5',8-trimethylpsoralen or 8-methoxypsoralen. To characterize further this antibody, oligonucleotides containing specific TMA photoadducts were isolated from the photoreaction mixture by polyacrylamide gel electrophoresis and used as competitive inhibitors in the ELISA. Autoradiography of the gel showed an intense band corresponding to the 4',5'-monoadduct and two weaker unidentified bands. Antibody 7E3 reacted only with the 4',5'-monoadduct band as would be expected since this photoadduct was the principal photoadduct in the original antigen.     

Interaction between thymine dimer and flavin-adenine dinucleotide: a DFT and direct ab initio molecular dynamics study

The interaction between the fully reduced flavin-adenine dinucleotide (FADH (-)) and thymine dimer (T) 2 has been investigated by means of density functional theory (DFT) calculations. The charges of FADH (-) and (T) 2 were calculated to be -0.9 and -0.1, respectively, at the ground state. By photoirradiation, an electron transfer occurred from FADH (-) to (T) 2 at the first excited state. Next, the reaction dynamics of electron capture of (T) 2 have been investigated by means of the direct ab initio molecular dynamics (MD) method (HF/3-21G(d) and B3LYP/6-31G(d) levels) in order to elucidate the mechanism of the repair process of thymine dimer caused by the photoenzyme. The thymine dimer has two C-C single bonds between thymine rings (C 5-C 5' and C 6-C 6' bonds) at the neutral state, which is expressed by (T) 2. After the electron capture of (T) 2, the C 5-C 5' bond was gradually elongated and then it was preferentially broken. The time scale of the C-C bond breaking and formation of the intermediate with a single bond (T) 2 (-) was estimated to be 100-150 fs. The present calculations confirmed that the repair reaction of thymine dimer takes place efficiently via an electron-transfer process from the FADH (-) enzyme.     

2'-chloro-2',3'-dideoxy-3'-fluoro-d-ribonucleosides: synthesis,stereospecificity, some chemical transformations,and conformational analysis

The synthesis of methyl 5-O-benzoyl-2-chloro-2,3-dideoxy-3-fluoro-beta-d-ribofuranoside (5) and its use as a glycosylating agent for persilylated thymine, N(6)-benzoyladenine, and N(4)-benzoylcytosine are described (Scheme 1). The 2'-chloro-2',3'-dideoxy-3'-fluoro-d-ribonucleosides 10-12 synthesized were transformed to 2',3'-dideoxy-3'-fluoro-alpha- and -beta-d-erythro-pentofuranoside nucleosides of thymine (13a,b), adenine (14a,b), and cytidine (15a,b) by treatment with tributyltin hydride in the presence of alpha,alpha'-azobisisobutyronitrile (Scheme 2). Treatment of 2'-chloro-2',3'-dideoxy-3'-fluoro-d-ribonucleosides with 1 M MeONa/MeOH under reflux for 1-5 h afforded 2',3'-didehydro-2',3'-dideoxy-2'-chloro-d-pentofuranosyl nucleosides as the principal products (47-81%) of the reaction, along with recovered starting nucleoside (11-33%) (Scheme 3). Easy HF elimination was also observed in the case of the 2'-azido-2',3'-dideoxy-3'-fluoro-beta-d-ribofuranosides of thymine (17) and adenine (20) (Scheme 3). The role of conformational peculiarities of 2'-chloro-2',3'-dideoxy-3'-fluoro-d-ribonucleosides as well as of 17 and 20 in the observed exclusive elimination of HF is discussed. The conformational analysis of a rather broad palette of 2,3-dideoxy-3-fluoro-2-(X-substituted)-d-ribofuranosides was performed with the aid of the PSEUROT (version 6.3) program, using (i) the recently reparametrized Karplus-type relation (Chattopadhyaya and co-workers. J. Org. Chem. 1998, 63, 4967) and (ii) empirical bond angle correction terms suggested by us. The predictive power of the Brunck and Weinhold model (J. Am. Chem. Soc. 1979, 101, 1700) of the gauche effect between atoms and groups as a conformational driving force acting upon the pentofuranose ring is explored. Their model invokes maximum antiperiplanar sigma <--> sigma stabilization when the donating bond is the least polar one and the acceptor orbital is at the most polarized bond and is found at least as satisfactory, and in various specific cases more so than, as rationalizations on the basis of the preference of the gauche vs the trans conformation of two vicinal electronegative substituents (Wolfe. Acc. Chem. Res. 1972, 5, 102).     

PHOTO REACTION OF 4,5'',8-TRIMETHYLPSORALEN WITH ADENOSINE

The near-UV induced photoreaction of 4,5',8-trimethylpsoralen (TMP) with adenosine was investigated in a dry film state. Four major photoadducts were isolated and purified by reverse-phase liquid chromatography. The structures of the photoproducts were elucidated on the basis of spectroscopic methods, including UV, FT-IR, mass spectrometry (FAB and EI methods) and 1H-NMR analysis. These photoproducts were characterized to be TMP-adenosine 1:1 adducts, which resulted from the covalent bond formation between the carbon C(4) of TMP and ribose 1' or 5' carbon of adenosine. Of the photoadducts, one photoadduct (V) was the major product, reflecting some selectivity in the photoreaction of TMP with adenosine in the solid state.     

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.     

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.     

Computational study of thymine dimer radical anion splitting in the self-repair process of duplex DNA

Formation of the thymine dimer is one of the most important types of photochemical damage in DNA, responsible for several biological pathologies. Though specifically designed proteins (photolyases) can efficiently repair this type of damage in living cells, an autocatalytic activity of the DNA itself was recently discovered, allowing for a self-repair mechanism. In this paper, we provide the first molecular dynamics study of the splitting of thymine dimer radical anions, using a quantum mechanical/molecular mechanics (QM/MM) approach based on density functional theory (DFT) to describe the quantum region. A set of seven statistically representative molecular dynamics trajectories is analyzed. Our calculations predict an asynchronously concerted process in which C5-C5' bond breaking is barrierless while C6-C6' bond breaking is characterized by a small free energy barrier. An upper bound of 2.5 kcal/mol for this barrier is estimated. Moreover, the molecular dynamics study and the low free energy barrier involved in C6-C6' bond breaking characterize the full process as being an ultrafast reaction.     

A NOVEL PHOTOADDUCT OF 4,5'',8-TRIMETHYLPSORALEN AND ADENOSINE

A novel photoadduct of 4,5',8-trimethylpsoralen (TMP) and adenosine was isolated and purified by reverse-phase liquid chromatography. The structure of the photoproduct was determined by various spectral methods and found to be a TMP-adenosine 1:1 adduct resulting from the covalent bond formation between the carbon C(4) of TMP and ribose 4'-carbon of adenosine.     

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.     

PHOTOALLERGIC RESPONSES TO CHEMICALS

Abstract— –Photochemical and immunologic knowledge about photoallergy to chemicals is briefly summarized. Studies in in vitro systems have demonstrated that photoallergic compounds can covalently bond to proteins through a photochemical reaction. The immunologic nature of the photoallergic response is based mainly on clinical observations, induction of photoallergy in man and in guinea-pigs and on results of in vitro immunologic tests.
Studies of the photoreactions of the photoallergic compound, 3,3',4',5-tetrachlorosalicylanilide (TCSA), with proteins are discussed. TCSA noncovalently bonds to human serum albumin prior to irradiation. Prior interaction is essential for formation of a photoaddition product indicating that a short-lived reactive species derived from TCSA is involved in the photoaddition and limiting the number of skin proteins which can participate in antigen formation. By fragmentation of the TCSA-albumin photoadduct with CNBr, it was determined that TCSA can bond to at least three sites on the albumin molecule. TCSA also can sensitize the photooxidation of histidine in albumin.     

Photosensitization of DNA of defined sequence by furochromones, khellin and visnagin

The sequence specificity in the in vitro DNA photobinding of khellin and visnagin, two naturally occurring furochromones proposed for chemotherapy of vitiligo, was investigated by using DNA sequencing methodology. The 3'-5' exonuclease associated with the T4 DNA polymerase served as a tool for determining photoadducts distribution on DNA fragments of the lac I gene of Escherichia coli. The photoadduct distribution of psoralen is also studied for comparison. Upon UVA irradiation, visnagin mainly forms monoadducts with thymine and to a lower extent with cytosine. Alternating (A-T)n sequences are hot spots for visnagin photoaddition. This is a property shared with furocoumarins. TTT sites are also quite reactive to visnagin, as they are to methylated angelicins. In contrast, with psoralen derivatives, there is no preferential photobinding in 5'-TpA sites, and 5'-ApT sites react as well. Furthermore, many sites such as T in the GC context, and C in any context, react, although weakly. The visnagin photoadduct distribution resembles very much the photoadduct distribution of methylated angelicins as described by Miolo et al. The photoreaction of these two series of compounds is less sequence dependent than the photobinding of psoralen derivatives as described by Sage and Moustacchi and by Boyer et al. The sequence specificity in khellin-DNA photobinding is the same as for visnagin, even though it forms much fewer photoadducts. The absence of photo-oxidation of DNA after treatment with visnagin or khellin plus UVA suggests that furochromones do not present any photodynamic effect on DNA.     

CHARACTERIZATION OF PHOTOCYCLOADDITION PRODUCTS FROM REACTION BETWEEN THYMIDINE and THE MONOFUNCTIONAL 3-CARBETHOXYPSORALEN

The photochemical reaction of 3-carbethoxypsoralen. a monofunctional furocoumarin. with thymidine was investigated as a model system for its photoaddition to DNA. Near UV irradiation (320 nm > λ > 400 nm) of a mixture of thymidine and 3-carbethoxypsoralen as a dry film gave rise to two main nucleoside diastereoisomers which were isolated by reverse phase high performance liquid chromatography. The structure of these products was assigned on the basis of UV absorption, fluorescence.'H-NMR and plasma desorption mass spectra analysis. The results are consistent with 1:1 C, cycloadducts involving the 5,6 double bond of thymine and the 4', 5'double bond of 3-carbethoxypsoralen. These two cycloadducts of cis-syn stereoconfiguration show opposite circular dichroism suggesting a diastereoisomeric relationship.     

Nucleophilic addition of benzenethiol to 1',2'-unsaturated nucleosides: 1'-C-phenylthio-2'-deoxynucleosides as anomeric radical precursors

The addition reaction of benzenethiol to the glycal portion of 1',2'-unsaturated uridine proceeds efficiently in the presence of Et(3)N. The mechanism involves nucleophilic attack of thiolate at the anomeric position in the rate-determining step, wherein conjugation between the nucleobase and the glycal portion is crucial. The derivative having a methyl group either at the 2'- or 6-position did not undergo this addition reaction, due to their sterically prohibited coplanarity. The 1',2'-unsaturated derivatives of thymine and adenine can also be used as substrates for this addition reaction. It was also shown that the resulting 1'-C-phenylthio-2'-deoxynucleosides serve as precursors for radical-mediated C-C bond formation at the anomeric position.     

Expanding the horizon of the thymine isostere biochemistry: unique cyclobutane dimers formed by photoreaction between a thymine and a toluene residue in the dinucleotide framework

Substituted toluenyl groups are considered as close isosteres of the thymine residue. They can be recognized by DNA polymerases as if they were thymine. These toluene derivatives are generally inert toward radical additions, including the [2+2] photo-cycloadditions, due to the stable structure of the aromatic ring and are usually used as solvents for radical reactions. Surprisingly, after incorporating toluene into the dinucleotide framework, we found that the UV excited thymine residue readily dimerizes with the toluenyl moiety through a [2+2] photo-addition reaction. Furthermore, the reaction site on the toluenyl moiety is not the C5=C6 bond, as commonly observed in cyclobutane pyrimidine dimers, but the C4=C5 or C3=C4 instead. Such a reaction pattern suggests that in the stacked structure, it is one of these bonds, not the C5=C6, that is close to the thymine C5=C6 bond. A similar structural feature is found in DNA duplex with a thymine replaced by a 2,4-difluorotoluene. Our results argue that although the substituted toluenyl moieties closely mimic the size and shape of the thymine residue, their more hydrophobic nature determines that they stack on DNA bases differently from the natural thymine residue and likely cause local conformational changes in duplex DNA.     

Product and pulse radiolysis studies on radical-ion splitting of N(1)-C(5')-linked dimer hydrates of 5-substituted uracils by one-electron reduction in anoxic aqueous solution

Steady-state gamma-radiolysis, pulse radiolysis, and cyclic voltammetry have been performed to identify the mechanism by which N(1)-C(5')-linked homodimer hydrates [1-(6'-hydroxy-5',6'-dihydrothymin-5'-yl)thymine (2a) and [1-(5'-fluoro-6'-hydroxy-5',6'-dihydrouracil-5'-yl)-5-fluorouracil (2b)], N(1)-C(6')-linked dimer hydrate [1-(5'-hydroxy-5',6'-dihydrothymin-6'-yl)thymine (3a)], and N(1)-C(5')-linked heterodimer hydrate [1-(6'-hydroxy-5',6'-dihydrothymin-5'-yl)-5-fluorouracil (2ba)] undergo radiolytic reductive splitting to regenerate the parent monomers in anoxic aqueous solution. Radiolytic reductions of the thymine homodimer hydrates 2a and 3a by hydrated electrons (e(aq)-) regenerated the parent thymine (1a) almost quantitatively, while the 5-fluorouracil homodimer hydrates cis-2b and trans-2b afforded 1-(uracil-5'-yl)-5-fluorouracil efficiently along with a small amount of the parent 5-fluorouracil (1b). In contrast to 2b, the heterodimer hydrate analogue 2ba with noneliminating 5'-methyl substituent releases 5-fluorouracil 1b almost quantitatively in the radiolytic reduction. The pulse radiolysis studies suggested that the electron adducts are produced primarily at the thymine and 5-fluorouracil structural unit in the dimer hydrates 2a,b, respectively, in which the resulting dimer hydrate radical anion of 2b (2b*-) was more stable than that of 2a (2a*-). As characterized by pulse radiolysis and cyclic voltammetry, the 5-fluorouracil homodimer hydrate 2b bearing F-substituent at C(5') undergoes one-electron reduction to eliminate exclusively fluoride ion along with the formation of dimer hydrate C(5') radical (2b(-F)*) with oxidizing property. The formation of a possible dimer hydrate radical intermediate 2b(-F)* was also supported by the effect of amines as the reducing additives on the yields of 1b and 4b in the radiolytic reduction of 2b.     

FLUORESCENCE QUANTUM YIELD DETERMINATION OF PYRIMIDINE (6-4) PYRIMIDONE PHOTOADDUCTS

Abstract An extensive study of the fluorescence characteristics of pyrimidine (6-4) pyrimidone photoadducts, a major class of far-UV-induced DNA lesions, was carried out on dinucleoside monophosphate (6-4) photoadducts, including thymidylyl-(3'→ 5')-thymidine (TpT), 2'-deoxycytidylyl-(3'-5')-thymidine, thymidylyl-(3'→ 5')-2'-deoxy-cytidine, 2'-deoxyuridylyl-(3'→ 5')-thymidine, 5-methyl-2'-deoxycytidylyl-(3'-5')-thymidine (6-4) photoadducts and the corresponding base (6-4) photoadducts, 6-4'-(5'-methylpyrimidin-2'-one) thymine (TT), 5-hydroxy-6-4'-(5'-methylpyrimidin-2'-one)-5,6-dihydrothymine (CT), 5-amino-6-4'-(pyrimidin-2'-one)-5,6-dihydrothymine (UC) obtained by mild acidic hydrolysis of the former derivatives. The fluorescence quantum yield (Φ_F) of these compounds was found to depend on one hand, on the nature of the two bases involved and the base substituent and, on the other hand, on the presence of the phosphate group. The hydrolysis of the phosphodiester bond was shown to enhance Φ_F, the larger effect being observed in the case of the thymine-thymine photoadducts with a seven-fold increase of the Φ_F value in the case of TT as compared to TpT (0.21 and 0.03, respectively). These results are discussed in terms of structural considerations.     

The possibility of the decomposition of 2'-deoxyribose moiety of thymidine induced by the low energy electron attachment

To evaluate the possibility of the decomposition of 2-deoxyribose moiety of thymidine induced by low energy electrons (LEE) attachment, the transition states and the energy barriers of the bond breaking processes of the ribose of the nucleoside have been studied theoretically by applying the density functional theory with the double zeta basis sets (DZP++). The energy barriers for the breakage of the C-C bonds (C(1')-C(2'), C(2')-C(3'), C(3')-C(4'), and C(4')-C(5')) of the ribose group of the radical anion of thymidine are found to be high (ca. 42-57 kcal/mol). The total energies of the C-C bond-broken products are significantly higher than that of the radical anion dT(*-). The decomposition of dT(*-) through the C-C bond rupture is unlikely to take place. The rupture of the C(1')-O(4') bond of dT(*-) needs an activation energy as low as 10.4 kcal/mol. However, the reversed reaction (C(1')-O(4') bond formation) needs the activation energy low as 0.3 kcal/mol. Therefore, the intermediate product LM1(C1')-(O4') is unlikely to be stable and the C(1')-O(4') bond-broken is not favored. The activation energy of the C(4')-O(4') bond rupture process amounts to 20.5 kcal/mol. The total energy of the C(4')-O(4') bond broken product is about 6.5 kcal/mol lower than that of the reactant dT(*-). The subsequent N1-glycosidic bond breaking process is found to have a very low energy barrier. Therefore, the LEE-induced base release through the C(4')-O(4') bond rupture might be a possible pathway.