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.     

THE EFFECT OF PSORALENS AND ANGELICINS ON PROTEINS IN THE PRESENCE OF UV-A IRRADIATION

Abstract— The photobinding to proteins of furocoumarins with linear and angular structure (psoralens and angelicins) has been found to occur at relatively high fluences of UV-A irradiation (66.5 kJm²). The extent of photobinding between serum albumin and the investigated furocoumarins (psoralen, 8-methylpsoralen, 8-methoxypsoralen, angelicin and 4,5'-dimethylangelicin) varies largely with the furocoumarin structure and is correlated with the extent of photodegradation of the same furocoumarins when irradiated alone in aqueous solution. On the other hand, for each furocoumarin, the extent of photobinding varies considerably with different proteins.     

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.     

C4-CYCLODIMERS OF PSORALEN ENGAGING THE 4'',5''- DOUBLE BOND

Abstract— By irradiating psoralen (the parent linear furocoumarin) in thin solid film two new fluorescent photodimers. compounds I and II, have been isolated and characterized. Unlike previously isolated dimers of furocoumarins which showed a 'pyrone-pyrone' structure involving in the C₄-cycloaddition the 3,4-double bond of the furocoumarinic moiety, the new dimers show respectively a 'furan-furan' and a 'furan-pyrone' structure, involving therefore the 4',5'-double bond. By analogy with the photocycloaddition reactions between furocoumarins and pyrimidine bases of DNA, in this case too the 4',5'-double bond of the furocoumarin can be involved. The experimental conditions, however, in which irradiation is carried out play an important role.     

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.     

4,4'-DIMETHYLANGELICIN, A MONOFUNCTIONAL FUROCOUMARIN SHOWING HIGH PHOTOSENSITIZING ACTIVITY

Abstract— 4,4'-Dimethylangelicin, a monofunctional furocoumarin, showed a high photosensitizing activity on various biological substrates. The lethal effect on Escherichia coli B₄₈, a wild type strain, and the extent of DNA synthesis inhibition in Ehrlich ascites tumor cells, were not only much more pronounced than that observed with the previously studied monofunctional 4,5'-dimethylangelicin, but even higher than with bifunctional psoralen, 4,4'-Dimethylangelicin, contrary to the other methylangelicin, proved to be phototoxic on guinea pig skin and showed an evident mutagenic effect in E. coli WP₂, but to a much lower extent than psoralen.     

MONOFUNCTIONAL ANGULAR FUROCOUMARINS: SEQUENCE SPECIFICITY IN DNA HOTOBINDING OF 6,4,4''-TRIMETHYLANGELICIN and OTHER ANGELICINS

The sequence specificity in the photoreaction (365 nm) of 6,4,4'-trimethylangelicin (TMA) with DNA fragments of the lac I gene of Escherichia coli was studied by using DNA sequencing methodology. In order to map the sites of TMA photoaddition, we took advantage of the (3'-5') exonuclease activity associated with T4 DNA polymerase, which is blocked by bulky adducts, such as furocoumarin photoadducts. A quantitative analysis of the sites of photoaddition is reported. TMA was demonstrated to photoreact with thymine and, to a lower extent, to cytosine. AT-rich sequences and TTT sites in a GC context are the most reactive sites towards TMA whereas TA, AT, CA, AC sites are weaker sites with similar reactivity. Cytosines in alternated CG sequences are also targets of TMA photobinding. We observed a less pronounced sequence specificity of TMA than that of other psoralen derivatives already studied (Sage and Moustacchi, 1987; Boyer et al., 1988). A comparison with other furocoumarins 4,4'-dimethylangelicin (4,4'-DMA), 4'-methylangelicin (4'-MA), angelicin, 4,5',8-trimethylpsoralen (TMP) and 8-methoxypsoralen (8-MOP) is also reported. The role of flanking sequence and consequently of the local conformation at the various sites of photoaddition is discussed. A preferential orientation of the TMA molecule during the intercalation in the dark is suggested. Hot alkali treatment of TMA-modified DNA did not reveal any DNA strand breakage due to photooxidized bases.     

Dark and Photochemical Interactions of Dimethyltetrahydrobenzoangelicin with DNA

A study of dark interaction and photoreaction between 4,6-dimethyltetrahydrobenzoangelicin (THBA) and DNA is described. 4,6-Dimethyltetrahydrobenzoangelicin is a furocoumarin derivative in which 4'and 5'carbons are linked by a four-methylene bridge. In spite of the bulky aliphatic ring, THBA forms a complex with DNA in the dark and, on UVA irradiation, reacts with pyrimidine bases of DNA yielding monoadducts only involving its furan side double bond. Two main photoproducts form: they derive from a C₄-cycloaddition to thymine and cytosine, respectively, and account for 56% and 39% of the total photoreaction yield. Both show cis-syn configuration. Two other isomers, one with thymine and one with cytosine, formed with so much lower yield ( ca 3 and 1%, respectively) that their structure could not be assigned. Furthermore, in spite of its angular structure, THBA induces a small number of crosslinks in DNA.     

RESEARCH NOTE: PHOTOREACTION OF 4', 5'-DIHYDROPSORALEN WITH THYMINE

Abstract— The photocycloaddition reaction of 4',5'-dihydropsoralen with thymine was carried out in solution and in the frozen state. A major photoadduct was isolated and characterized by elemental analysis and physical methods. The photoadduct was proven to be the 1:1 C₄-cycloaddition product, an analogue of furocoumarin-DNA biadduct, with the stereochemistry of anti -head-to-head formed through 2+2 addition reaction between the pyrone double bond of 4', 5'-dihydropsoralenand 5,6-double bond of thymine.     

3,4 AND 4'',5''-PHOTOCYCLOADDUCTS BETWEEN 4''-METHYLANGELICIN AND THYMINE FROM DNA

Abstract— Two C₄-cycloadducts between 4'-methylangelicin and thymine were isolated from the photo-reaction (365 nm) between this furocoumarin and DNA. Their capacity to undergo photoreversion at 254 nm and their spectroscopic and NMR data allowed us to assign furan- and pyrone-side structures, respectively, to these photocompounds. The former adduct has a cis-syn configuration; the latter. which was also isolated from a photoreaction between the furocoumarin and thymine in water-methanol solution, was assigned a cis-anti structure.     

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.     

PHOTOINACTIVATION OF ENZYMES BY LINEAR AND ANGULAR FUROCOUMARINS

Abstract Furocoumarins with linear (psoralen, 8-methylpsoralen, 8-methoxypsoralen and 3-carbethox-ypsoralen) and angular molecular structures (angelicin and 4,5'-dimethylangelicin) were found to inactivate enzymes to different extents through UV-A irradiation. Moreover, enzymes with different structures (glutamate dehydrogenase, lysozyme, 6-phosphogluconate dehydrogenase, enolase, thermoly-sine and ribonuclease) are inactivated to different extents by the same furocoumarin. UV-A irradiation produces both covalent incorporation of the furocoumarins into the protein molecule and photodegra-dation of amino acid residues; the latter phenomenon seems to be mainly responsible for the photoinactivation process. A close correlation was found between the capacity of the furocoumarins to photoinactivate enzymes and their capacity to modify free amino acids.
A study of the effects of quenchers of various forms of activated oxygen on the photoinactivation of glutamate dehydrogenase, used as a model enzyme, and psoralen and 8-methylpsoralen as a reference for furocoumarins, showed that singlet oxygen is the species most involved in the photoinactivation process.     

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.     

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).     

Pyrrolocoumarin derivatives: DNA-binding properties

The spectroscopic and DNA-binding properties of a number of pyrrolocoumarin derivatives, including linear tricyclic, angular tricyclic, linear tetracyclic and angular tetracyclic compounds were investigated. The compounds we examined form non-covalent complexes with duplex DNA, probably of the intercalation type. The binding constants are comparable with the constants found for the furocoumarin analogues. Although for some of the compounds the photoreactivity with DNA is comparable with that of 8-MOP, pyrrolocoumarins behave as monofunctional reagents. This fact is explained in terms of an increased delocalization of the 4',5' double bond in the pyrrole moiety. Denaturation-renaturation experiments and HPLC analysis of the photoadducts confirm that pyrrolocoumarins are essentially monofunctional DNA-photobinding agents.     

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.     

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.     

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.     

1,4-shifts in a dinuclear Ni(I) biarylyl complex: a mechanistic study of C-H bond activation by monovalent nickel

The known aryne complex (PEt3)2Ni(eta2-C6H2-4,5-F2) (1a) reacts with a catalytic amount of Br2Ni(PEt3)2 over 1% Na/Hg to afford the dinuclear Ni(I) biarylyl complex [(PEt3)2Ni]2(mu-eta1:eta1-3,4-F2C6H2-3',4'-F2C6H2) (2a), which results from a combination of C-C bond formation and C-H bond rearrangement. The dinuclear benzyne [(PEt3)2Ni]2(mu-eta2:eta2-C6H2-4,5-F2) (3) was obtained by the reaction of 1a with a stoichiometric amount of Br2Ni(PEt3)2 over excess 1% Na/Hg, and 3 was found to catalyze the conversion of 1a to 2a. The reaction of 1a with B(C6F5)3 produced the trinuclear complex (PEt3)3Ni3(mu3:eta1:eta1:eta2-4,5-F2C6H2)(mu3:eta1:eta1:eta2-4,5-F2C6H2-4',5'-F2C6H2) (6). The addition of PEt3 to 6 produced 1 equiv of 1a and 1 equiv of [(PEt3)2Ni]2(mu-eta1:eta1-4,5-F2C6H2-4',5'-F2C6H2) (7a). Both 6 and 7a were identified as intermediates in the conversion of 1a to 2a. The analogue [(PEt3)(PMe3)Ni]2(mu-eta1:eta1-4,5-F2C6H2-4',5'-F2C6H2) (7b) was prepared by the addition of PMe3 to 6 and was structurally characterized. NMR spectroscopic evidence identified the additional asymmetric biarylyl [(PEt3)2Ni]2(mu-eta1:eta1-4,5-F2C6H2-3',4'-F2C6H2) (8a) during the conversion of 1a to 2a. The initial observation of 2 equiv of 8a for every equivalent of 2a produced from solutions of 7a suggests that 8a and 2a are formed from a common intermediate. A crossover labeling experiment shows that the C-H bond rearrangement steps in the conversion of 1a to 2a occur with the intermolecular scrambling of hydrogen and deuterium labels. The evidence collected suggests that Ni(I) complexes are capable of activating aromatic C-H bonds.     

Photoaddition of 4,6-dimethyltetrahydrobenzoangelicin to thymine in DNA: X-ray studies and experiments with model oligonucleotides

The crystal structures of 4,6-dimethyltetrahydrobenzoangelicin (THBA), a furocoumarin analog, and of its furan-side cis-syn cycloadduct with thymine formed in the photoreaction with DNA, have been determined. The crystal structure of the latter compound contained only one enantiomeric form corresponding to the addition to a 5'-XpT site. Contrary to most psoralen derivatives studied, THBA showed higher photoreactivity toward synthetic oligonucleotides containing that sequence than toward those with the 5'-TpX sequence.