FORMATION OF CYCLOBUTANE THYMINE DIMERS PHOTOSENSITIZED BY PYRIDOPSORALENS: A TRIPLET-TRIPLET ENERGY TRANSFER MECHANISM

The 365 nm irradiation of thymine thin films in the presence of pyridopsoralens is shown to induce the formation of cyclobutane thymine dimers, in contrast to other compounds such as 8- and 5-methoxypsoralen. In order to elucidate the mechanism of such a photosensitized reaction, we have determined the energy of the lowest triplet state (T1) of these compounds, using phosphorescence spectroscopy and CNDO/S quantum chemistry calculations. The T1 energy values were found to be significantly higher for pyridopsoralens--up to 0.3 eV--than for 8- and 5-methoxypsoralen (approximately 2.8 eV), which are not able to photoinduce cyclobutane thymine dimers. The determination of the relative efficiency of cyclobutane thymine dimer formation was performed using chromatographic analysis. A good correlation was found between the energy of the T1 state of the psoralen derivatives and the related cyclobutane thymine dimer formation. Moreover, the photosensitized cyclobutane thymine dimer formation appeared to be temperature-dependent. Our results are consistent with a mechanism involving a triplet energy transfer from the pyridopsoralen to thymine.     

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.     

CORRELATION BETWEEN THE TRIPLET PHOTOPHYSICAL PROPERTIES AND THE PHOTOBIOLOGICAL ACTION IN YEAST OF TWO MONOFUNCTIONAL PYRIDOPSORALENS

The photophysical properties of the lowest excited triplet states of pyrido[3,4-c]psoralen (PyPs) and 7-methylpyrido[3,4-c]psoralen (MePyPs) have been investigated by laser flash photolysis, including energy transfer from these triplets to oxygen-generating singlet oxygen. A parallel study of the photosensitization of yeast in vivo by these compounds in the presence and absence of oxygen is also reported. The low triplet and hence singlet oxygen yields, reflected in the lack of an oxygen effect in yeast, suggest that photoadditions to DNA are likely to be the main source of the photosensitized lethal effects induced by these pyridopsoralens in vivo.     

Structure determination of an interstrand-type cis-anti cyclobutane thymine dimer produced in high yield by UVB light in an oligodeoxynucleotide at acidic pH

UVB irradiation of DNA produces photodimers in adjacent DNA bases and on rare occasions in nonadjacent bases. UVB irradiation (312 nm) of d(GTATCATGAGGTGC) gave rise to an unknown DNA photoproduct in approximately 40% yield at acidic pH of about 5. This product has a much shorter retention time in reverse phase HPLC compared to known dipyrimidine photoproducts of this sequence. A large upfield shift of two thymine H6 NMR signals and photoreversion to the parent ODN upon irradiation with 254 nm light indicates that the photoproduct is a cyclobutane thymine dimer. Exonuclease-coupled MS assay establishes that the photodimer forms between T2 and T7, which was confirmed by tandem mass spectrometric MS/MS identification of the endonuclease P1 digestion product pd(T2[A3])=pd(T7[G8]). Acidic hydrolysis of the photoproduct gave a product with the same retention time on reverse phase HPLC and the same MS/MS fragmentation pattern as authentic Thy[ c,a]Thy. 2D NOE NMR data are consistent with a cis-anti cyclobutane dimer between the 3'-sides of T2 and T7 in anti glycosyl conformations that had to have arisen from an interstand type reaction. In addition to pH dependency, the photoproduct yield is highly sequence specific and concentration dependent, indicating that it results from a higher order folded structure. The efficient formation of this interstrand-type photoproduct suggests the existence of a new type of folding motif and the possibility that this type of photoproduct might also form in other folded structures, such as G-quadruplexes and i-motif structures which can be now studied by the methods described.     

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.     

1,2,4-Triazoles—XXXIV: Photodimerization of some 1,2,4-triazo[4,3--]quinoline and 1,2,4-triazolo[3,4-a]isoquinoline derivatives

Irradiation of 1,2,4-triazolo[4,3-a]quinoline and several Me derivatives at 300 nm afforded the cisoid-fused, head-to-tail cyclobutane dimers 7bα,7cα,14bα,14cα - tetrahydro - 1,2,3a,8,9,10a - hexaazadibenzo [c,i]dicyclopenta[a,g]biphenylenes. However, 1,2,4 - triazolo[3,4-a]isoquinolines gave analogous cisoid-fused, head-to-head dimers except for the 5-Me derivative where the head-to-tail dimer was obtained.Codimerization occurred when 1 - methyl - 1,2,4 - triazolo[4,3-a]quinoline and 5 - methyl - 1,2,4 -triazolo[3,4-a]isoquinoline were irradiated at 300 nm, affording a cisoid-fused head-to-tail cyclobutane co-dimer as the single photoproduct. However, irradiation of their 5-Me substituted derivatives at 300 nm afforded the cisoid-fused, head-to-head, cyclobutane codimer and also a minor amount of the dimer derived from 5 - methyl -1,2,4- triazolo[4,3-a] quinoline. Irradiation of equimolar quantities of 2(1H)-quinolinone and 5 - methyl - 1,2,4 -triazolo [4,3-a]quinoline at 300 nm gave the known 2(1H)-quinolinone dimer and a minor amount of a cisoid-fused co-dimer of undetermined configuration.     

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.     

SPECTROSCOPIC PROPERTIES AND PHOTOREACTIVITY WITH DNA OF NEW MONOFUNCTIONAL PYRIDOPSORALENS

Abstract— New psoralen derivatives have been synthesized in order to enhance their affinity towards DNA. The spectral properties (absorption, fluorescence emission, fluorescence quantum yield) and the photostability of pyrido[3,4-c]psoralen are first reported. The drastic changes observed in the solubility and in the fluorescence emission when these compounds are added to native DNA give evidence of the formation of non covalent dark complexes. Upon UV irradiation (365 nm) of the complexes, a photobinding occurs. Heat denaturation and renaturation experiments of modified DNA show that only monoadducts are formed. From the analysis of their fluorescence properties the involvement of the 4', 5' double bond is assumed. The monofunctional character has also been established for psoralens having a fused pyridine ring in the 4', 5' site. On the opposite, a fused tetrahydropyrido group in the 4', 5' site is inefficient to inactivate this reactive site.     

EFFECT OF MOLECULAR STRUCTURE ON THE PHOTOPHYSICAL PROPERTIES, THE PHOTOREACTIVITY WITH DNA AND THE PHOTOBIOLOGICAL ACTIVITY OF MONOFUNCTIONAL PYRIDOPSORALENS

Abstract— 7-Methyl-pyrido[4,3-c]psoralen (2N-MePyPs) has been synthesized in order to investigate the possible effect of the position of the pyridine-nitrogen atom on the photoreactivity towards DNA and the photobiological activity of pyridopsoralens, a new family of psoralen derivatives. In comparison to its isomer, 7-methyl-pyrido[3,4-c]psoralen (MePyPs), 2N-MePyPs shows a 2.5 times lower DNA photobinding capacity. Photobiological experiments with diploid yeast ( Saccharomyces cerevisiae ) reveal that this compound differs strikingly from its isomer MePyPs. It has only a weak antiproliferative potential and, per unit dose, a lower capacity than MePyPs for the induction of nuclear genotoxic effects. With respect to these latter features, 2N-MePyPs resembles the monofunctional furocoumarin 3-CPs.     

Molecular mechanics and dynamics of DNA-furocoumarin complexes: effect of the aromatization of the pyrone ring on the intercalation geometry

Results of molecular mechanics and dynamics calculations on intercalation complexes of DNA with various furocoumarins (psoralen, angelicin, 7-methylpyrido[3,4-c]psoralen and 7-methylpyrido[4,3-c]psoralen) and their corresponding aromatized derivatives are presented. These calculations were undertaken with the aim to elucidate the roles of the pyrone and pyridine moieties in the interactions which tend to orient the furocoumarins and pyridopsoralens between DNA base pairs. It appears that the intercalation geometries are very similar for the furocoumarins and related aromatized compounds. Therefore the oxygen and nitrogen atoms of the pyrone and pyridine moieties are not important in the orientation of the drug within the oligonucleotide.     

MUTAGENIC EFFECTS PHOTOINDUCED IN MAMMALIAN CELLS IN VITRO BY TWO MONOFUNCTIONAL PYRIDOPSORALENS

Abstract— The photobiological activity of the two monofunctional pyridopsoralens pyrido (3,4-c) psoralen (PyPs) and 7-methyl pyrido (3,4-c) psoralen (MePyPs) was studied in mammalian cells in vitro taking 8-methoxypsoralen (8-MOP) as a reference compound.
In the presence of 365-nm irradiation (UVA) MePyPs was found to be more effective than 8-MOP in terms of DNA photobinding capacity and inhibition of cell cloning ability in Chinese hamsterV–79 cells. As a function of UVA dose and of the number of total photoadducts induced MePyPs produced a higher frequency of 6-thioguanine resistant mutants than 8-MOP. PyPs showed an intermediate response for cell killing and mutation induction. At equal cytotoxic levels both monofunctional pyridopsoralens exhibited the same mutagenic activity as the Afunctional furocoumarin 8-MOP.
The antiproliferative effect being taken as indicative for an efficient photochemotherapeutic activity against psoriasis, the inhibition of cloning capacity induced by MePyPs plus UVA was studied in parallel on human skin fibroblasts. Such cells were more sensitive to 8-MOP photoadditions thanV–79 cells and even more so to MePyPs photoadditions. Data obtained on the rate of DNA semi conservative synthesis on both cell lines following treatments with the two compounds are in line with these observations.     

STRUCTURE OF 3-CARBETHOXYPSORALEN PHOTOLYSIS PRODUCTS

Abstract— Upon UV-A irradiation(320–400 nm), the photorcaction of psoralens either with a solvent molecule or with a second psoralen molecule competes with the well-known photoaddition of psoralens to DNA. In the present study, a structural assignment of the 3-carbethoxypsoralen (3-CPs) photolysis products is proposed on the basis of their chromatographic (high performance liquid chromatography) and of their spectroscopic (absorption, fluorescence, mass spectrometry and ¹H Nuclear Magnetic Resonance) properties. Four photolysis products have been isolated and identified. The first one results from a water molecule addition on the 4',5' double bond of 3-CPs, the second from an ethanol molecule addition on the 4',5' double bond. Two cyclobutane type dimers of 3-CPs have also been characterized. These results confirm that, in protic solvents, the furan(4',5') double bond of 3-CPs is more photoreactive than the pyrone(3,4) double bond.     

A comparative repair study of thymine- and uracil-photodimers with model compounds and a photolyase repair enzyme

Cyclobutane uridine and thymidine dimers with cis-syn-structure are DNA lesions, which are efficiently repaired in many species by DNA photolyases. The essential step of the repair reaction is a light driven electron transfer from a reduced FAD cofactor (FADH ) to the dimer lesion, which splits spontaneously into the monomers. Repair studies with UV-light damaged DNA revealed significant rate differences for the various dimer lesions. In particular the effect of the almost eclipsed positioned methyl groups at the thymidine cyclobutane dimer moiety on the splitting rates is unknown. In order to investigate the cleavage vulnerability of thymine and uracil cyclobutane photodimers outside the protein environment, two model compounds, containing a thymine or a uracil dimer and a covalently connected flavin, were prepared and comparatively investigated. Cleavage investigations under internal competition conditions revealed, in contrast to all previous findings, faster repair of the sterically less encumbered uracil dimer. Stereoelectronic effects are offered as a possible explanation. Ab initio calculations and X-ray crystal structure data reveal a different cyclobutane ring pucker of the uracil dimer, which leads to a better overlap of the pi*-C(4)-O(4)-orbital with the sigma*-C(5)-C(5')-orbital. Enzymatic studies with a DNA photolyase (A. nidulans) and oligonucleotides, which contain either a uridine or a thymidine dimer analogue, showed comparable repair efficiencies for both dimer lesions. Under internal competition conditions significantly faster repair of uridine dimers is observed.     

SIMULTANEOUS ESTABLISHMENT OF MONOCLONAL ANTIBODIES SPECIFIC FOR EITHER CYCLOBUTANE PYRIMIDINE DIMER OR (6-4)PHOTOPRODUCT FROM THE SAME MOUSE IMMUNIZED WITH ULTRAVIOLET-IRRADIATED DNA

Six new monoclonal antibodies (TDM-2, TDM-3, 64M-2, 64M-3, 64M-4 and 64M-5) specific for ultraviolet (UV) induced DNA damage have been established. In the antibody characterization experiments, two TDM antibodies were found to show a dose-dependent binding to UV-irradiated DNA (UV-DNA), decrease of binding to UV-DNA after cyclobutane pyrimidine dimer photoreactivation, binding to DNA containing cyclobutane thymine dimers, and unchanged binding to UV-DNA after photoisomerization of (6-4)photoproducts to Dewar photoproducts. These results indicated that the epitope of TDM monoclonal antibodies was the cyclobutane pyrimidine dimer in DNA. On the other hand, four 64M antibodies were found to show a dose-dependent binding to UV-DNA, unchanged binding to UV-DNA after cyclobutane pyrimidine dimer photoreactivation, undetectable binding to DNA containing thymine dimers, and decrease of binding to UV-DNA after photoisomerization of (6-4)photoproducts. These results indicated that the epitope of 64M antibodies was the (6-4)photoproduct in DNA. This is the first report of the simultaneous establishment of monoclonal antibodies against the two different types of photolesions from the same mouse. By using these monoclonal antibodies, we have succeeded in measuring both cyclobutane pyrimidine dimers and (6-4)photoproducts in the DNA from human primary cells irradiated with physiological UV doses.     

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.     

A pyrazine-based fluorescence-enhancing ligand with a high selectivity for thymine in AP site-containing DNA duplexes

A fluorescent pyrazine derivative, 3,5-diamino-6-chloro-2-pyrazine carbonitrile (DCPC), is presented as a promising light-up ligand for single-nucleotide polymorphisms (SNPs) typing. In solutions buffered to pH 7.0 (I = 0.11 M, at 5 degrees C), DCPC can bind to thymine selectively over other nucleobases opposite an abasic site in DNA duplexes (5'-GTGTG CGTTG ANA TGGAC GCAGA-3'/3'-CACAC GCAAC TXT ACCTG CGTCT-5', X = abasic site, N = target nucleotide) with a dissociation constant of 2.6 microM. The binding of DCPC is accompanied by a significant enhancement of its fluorescence (lambda(max), 412 nm), and the response is highly selective to thymine base. These binding and sensing properties allow a clear detection of thymine-related mutations present in polymerase chain reaction (PCR) amplification products.     

Condensed isoquinolines. 37.* Heterocyclization using 3-(arylamino)- and 3-(hetarylamino)isoquinolin-1(2H)-ones

The reaction of 3-NHR-isoquinolin-1(2H)-ones (R = Ar) with aromatic aldehydes in the presence of Me3SiCl or in acetic acid leads to the formation of derivatives of dibenzo[b,f][1, 8]naphthyridin-5(6H)- one and benzo[f]isoquino[3,4-b][1, 8]naphthyridine-5,9(6H,7H)-dione. The reaction for R = Het in the presence of Me3SiCl gives derivatives of 5H-pyrido[1',2':1,2]pyrimido[4,5-c]isoquinolin-5-one, benzo[f]isoquinoline[3,4-b][1,8]naphthyridine-5,9[6H,7H]-dione, and derivatives of new heterocyclic systems, 5H-pyrazino[1',2':1,2]pyrimido[4,5-c]isoquinolin-5-one, 5H-[1,3]thiazolo[3',2':1,2]pyrimido- [4,5-c]isoquinolin-5-one, 5-H-benzo[f]pyrazolo[3,4-b][1,8]naphthyridin-5-one, and isoquino[3,4-b]- [1,5]naphthyridin-5(6H)-one. The effect of the structure of substituent R and nature of the substituent in the benzaldehydes on the structure of the reaction products was studied.     

酶蛋白直接修复嘧啶二聚体机理的模型研究

去辅基的DNA光解酶在280 nm光辐照下, 能高效修复底物嘧啶二聚体(Φ＝0.56). 为了模拟酶蛋白的这一修复过程, 合成了色氨酸(Trp)和/或酪氨酸(Tyr)与胸腺嘧啶二聚体(D)共价连接的化合物, 作为酶-底物复合物的模型, 研究了它们在295 nm光照射下氨基酸残基光敏化二聚体裂解的性质, 测定了二聚体裂解量子产率(Φ), 获得一些新的结果并对其进行了分析.     

THE PHOTOREACTIONS OF 8-METHOXYPSORALEN WITH TRYPTOPHAN AND LENS PROTEINS*

Abstract— We have previously demonstrated that 8-methoxypsoralen (8-MOP) can be found in the lenses of rats injected (i.p.) with this drug, and that its presence can lead to a photosensitized enhancement of lenticular fluorescence. The cutaneous photosensitizing properties of psoralens are thought to be mediated via their excited triplet states, resulting in photoaddition cyclobutane products between pyri-midine bases and 8-MOP. We have now investigated the possibility that similar types of photoadducts could be generated between 8-MOP and the aromatic amino acid residues in lens proteins. Our experiments involved in vitro irradiation (at 360 nm) of aqueous solutions of 0.1 mM 8-MOP plus purified alpha, beta, or gamma crystallins from calf or normal human (under 20 years of age) lenses. UV absorption and fluorescence emission spectra were measured before and after radiation, and aliquots from all experiments were frozen and kept in the dark for subsequent phosphorescence and EPR spectroscopy. Similar experiments were performed with irradiated aqueous solutions of tryptophan or thymine plus 8-MOP. All controls consisted of solutions kept in the dark. NMR spectra demonstrated that the hydrogen atoms at the 3,4 and 4',5' positions of the 8-MOP molecule were lost following irradiation, suggesting that these two sites were involved in the photoproduct formed between tryptophan and 8-MOP. These studies strongly suggest that 8-MOP is capable of forming photoaddition products with tryptophan and with lens proteins as well as DNA in vivo, resulting in its permanent retention within the ocular lens.     

A FLOW LINEAR DICHROISM STUDY OF THE ORIENTATION OF 4'',5''-PSORALEN-DNA PHOTOADDUCTS

Abstract— The flow linear dichroism properties of covalent adducts derived from the photochemical binding of various psoralen derivatives to salmon sperm DNA were investigated. The psoralens studied include bifunctional derivatives (8-methoxypsoralen,5-methoxypsoralen, tetrahydropyrido [3,4: 4',5'] psoralen) and monofunctional derivatives (pyrido [3,4-c] psoralen, 7-methylpyrido [3,4-c] psoralen, 3-carbethoxypsoralen). The orientation of the psoralen moieties (furan-side monoadducts) relative to the orientation of the DNA bases was determined. All of the furan-side monoadducts are characterized by a similar orientation, with mean angles between the psoralen moiety and the normals of the planes of the DNA bases ranging between 70° and values close—but not equal—to 90°. The results are consistent with a pseudo-intercalative adduct geometry, most probably involving stacking interactions with the DNA bases.