3-(α,α-DIMETHYLALLYL)-PSORALEN: A LINEAR FUROCOUMARIN FORMING MAINLY 4'',5''-MONOFUNCTIONAL ADDUCTS WITH DNA

Abstract— 3-(α,αdimethylallyl)-psoralen, a furocoumarin derivative recently isolated from Hortia arborea . has been studied with regard to its photoreacting (365 nm) capacity with native DNA. This compound photobinds to DNA to a much lower extent than psoralen, forming mainly 4.5'-monofunc-tional adducts, due to the presence in the 3-position of α.α-dimethylallyl group. which exerts a considerable hindrance both for the formation of a molecular complex with native DNA and for the photoreaction of the 3 ,4-double bond. This site of the molecule photoreacts, and, only 2.5% of the bound substance forms cross-linkages. Therefore. 3-(α,α-dimethylallyl).psoralen behaves as a fairly pure mono-functional reagent.     

STUDIES ON THE PHOTO-C4-CYCLO-ADDITION REACTIONS BETWEEN SKIN-PHOTOSENSITIZING FUROCOUMARINS AND NUCLEIC ACIDS*

Abstract— Among the natural or synthetic furocoumarins (psoralens) a group exists which has interesting biological properties, the best known of which is skin-photosensitization. The mechanism of action has remained unclarified for a long time. Furocoumarins lack photooxidative properties; they act by a mechanism that does not require oxygen and are therefore different from photodynamic substances. Photosensitizing furocoumarins when irradiated at 365 nm react with nucleic acids to give a C₄-cyclo-addition to the 5,6-double bond of the pyrimidine bases engaging their 3,4- or 4‵,5‵-double bond. Differences exist in the behaviour of the various furocoumarins; psoralen reacts equally well with native DNA, with denatured DNA and with RNA, whereas bergapten, xanthotoxin and 8-methylpsoralen at room temperature react to a much greater extent with native DNA than with denatured DNA and with RNA. A temperature effect has also been observed. In the case of native DNA an intercalation, occurring in the dark, of furocoumarins between two adjacent base pairs of the double helix is suggested as the first step in the reaction. The photoreaction is not accompanied by breaks in the polynucleotide chain or by conformational modifications of the macromolecule. A parallelism was observed between the order of activity of the substances of this group for photoreaction with native DNA and for skin-photosensitization. Ehrlich ascites tumor cells lose completely their capacity of transmitting the tumor after irradiation in the presence of psoralen, bergapten and xanthotoxin. By hydrolysis of DNA extracted from Ehrlich ascites tumor cells irradiated in the presence of psoralen a photoadduct between psoralen and thymine was isolated.     

ZUM WIRKUNGSMECHANISMUS PHOTODYNAMISCHER FUROCUMARINE PHOTOREAKTION VON PSORALEN-(4-14C) MIT DNS, RNS, HOMOPOLYNUCLEOTIDEN UND NUCLEOSIDEN

Abstract— The photodynamic binding of psoralen to DNA and RNA was shown using different methods. This binding was found to be mainly located either at thymine or uracil. The concentration and pH dependancy was studied. The photoproduct obtained with uridine was isolated paper chromatographically and was split into initial components photochemically.
The results bring an evidence that psoralen with its 3,4-double bond is able to form a cyclic derivative with the C₆-C₆ doble bond of thymine or uracil.     

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.     

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.     

Benzoangelicins: new monofunctional DNA photobinding agents.

4,6-Dimethylbenzoangelicin, obtained by fusing a benzene ring at the furan side of 4,6-dimethylangelicin, was studied in terms of crystal structure and interactions with DNA in both ground and excited states. 4,6-Dimethylbenzoangelicin has a planar structure and forms a molecular complex with DNA, undergoing intercalation inside the double helix. Under UVA irradiation, it photoconjugates covalently with the macromolecule, showing a DNA photobinding rate slightly lower than that of 8-methoxypsoralen, involving however only its 3,4 double bond, i.e. behaving as a pure monofunctional agent. The parameters of dark binding and photobinding were determined, and two C4 cycloadducts with thymine were isolated and characterized.     

4,5'-dimethylangelicin: a new DNA-photobinding monofunctional agent.

Abstract— 4,5′-Dimethylangelicin is a new angular furocoumarin showing interesting photochemical and photosensitizing properties. In the dark it forms a complex with native DNA having higher values of the binding parameters than angelicin; by irradiation at 365 nm it is able to photobind with DNA several times faster than angelicin and in about the same degree as psoralen, without forming crosslinkages. It therefore behaves as a pure monofunctional reagent. The same high photobinding capacity with DNA is shown also in vivo in Ehrlich ascites tumor cells and bacterial E. coli cells. 4,5′-Dimethylangelicin is very active in inhibiting the DNA and RNA syntheses in Ehrlich ascites tumor cells alter irradiation at 365 nm, much more so than angelicin and in the same degree as psoralen. It is also more active than angelicin in decreasing the tumor transmitting capacity of the same cells and the colony-forming ability of E. coli cells, although to a lesser degree than psoralen. It is ineffective in producing erythema on guinea-pig skin. The involvement of repair processes of DNA in these photosensitized effects is discussed.     

PHOTOBINDING OF PSORALENS TO BACTERIAL MACROMOLECULES IN SITU AND INDUCTION OF GENETIC EFFECTS IN A BACTERIAL TEST SYSTEM. EFFECTS OF SINGLET OXYGEN DIAGNOSTIC AIDS D2O AND DABCO

The photobinding of radiolabeled psoralen and 8-methoxypsoralen (8-MOP) to biological macromolecules under conditions that affect the lifetime of singlet oxygen (¹O₂) is reported. These conditions are: increase of ¹O₂ lifetime in D₂O and ¹O₂ quenching with DABCO. The photobinding to calf thymus DNA was studied in vitro and the covalent photobinding to DNA and other biological macromolecules (RNA, proteins) was also studied in intact bacteria. The results of the DNA photobinding experiments have been related to the induction of genetic damage in a bacterial test system. In addition, laser flash photolysis has been used to measure the effect of D₂O and DABCO on the psoralen and 8-MOP triplet lifetimes. In general D₂O increases the triplet lifetimes and DABCO quenches the triplet states with the probable formation of radicals. The results suggest that the covalent photobinding of 8-MOP to various biological macromolecules in situ is a basis for cell damage occurring at various cellular targets. Analysis of the results of the mutagenicity test suggests that in the presence of D₂O the mechanism of induction of genetic lesions is not changed and therefore largely seems to be independent of singlet oxygen.     

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.     

The polarography of coumarins in alcoholic-aqueous media. I

Conclusions 1. The natural coumarin derivatives psoralen and angelicin are reduced at the DME from the adsorbed state at the mercury-solution boundary at the position of the 3,4-double bond of the -pyrone ring. The latter circumstance confirms the ethylenic nature of this bond.2. The existence of the processes of the protonation of the coumarin molecules in acid media and of the opening of the lactone ring in alkaline media has been shown experimentally.3. The investigations performed show that the plarographic method can be used in the study of the physiochemical properties of the natural coumarins and for their quantitative analysis.Khimiya Prirodnykh Soedinenii, Vol. 5, No. 4, pp. 216–222, 1969     

PHOTOBINDING OF PSORALEN AND 8-METHOXYPSORALEN TO CALF THYMUS DNA

Abstract— The photobinding of psoralen and 8-methoxypsoralen to calf thymus DNA induced by 365 nm radiation has been measured for different concentrations of the furocoumarin and nucleotides. The results are consistent with the assumption that dark complexing of the furocoumarin to DNA is a pre-condition for photobinding, but do not exclude the possibility that the free furocoumarin participates in the reactions. The analysis with 'large target' diffusion theory indicates that photobinding should be inefficient for the free excited singlet state and competitive with reactions of the dark-complexed sensitizer for the free triplet state. The analysis indicates also that the diffusive reactions of singlet oxygen generated by the free furocoumarin can compete with photoadduct formation.     

THE PHOTOREACTION BETWEEN FUROCOUMARINS AND VARIOUS DNA WITH DIFFERENT BASE COMPOSITIONS

Abstract— The photoreactivity of psoralen and 8-methylpsoralen towards various samples of DNA having different base-pair compositions has been studied. The total photobinding capacity shown by the two furocoumarins increases by increasing the content of A-T, confirming the data previously obtained using synthetic polynucleotides.
The amount of monofunctional fluorescent 4',5'-cycloadducts and of the cross-linkages formed during the photoreaction is not parallel to the content of A-T, but is highest when the A-T content is between 50 and 60%, while it decreases as the content of A-T is lowered or increased. The possible binding site for the formation of 4',5'-monofunctional cycloadducts as well as of cross-linkages is discussed.     

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.     

Photoactivated Psoralens Elicit Defense Genes and Phytoalexin Production in the Pea Plant

In the pea plant ( Pisum sativum ), compounds that intercalate into DNA induce the production of ∼20 major proteins similar to the pattern induced during nonhost disease resistance to the bean fungal pathogen, Fusarium solani f.sp. phaseoli . The pea phytoalexin, pisatin, as well as RNA homologous to several disease-resistance response (DRR) genes accumulate following treatment with these compounds. Psoralen was chosen to characterize this interaction further because it intercalates into DNA and, following irradiation with 365 nm UV light (UV₃₆₅), forms covalent bonds with pyrimidines on either or both strands of DNA. This produces monoadducts or cross-links, respectively. Dose experiments showed that 60 μg/mL 4'-aminomethyl-4,5',8-trimethylpsoralen followed by 18 J/cm² UV₃₆₅ was sufficient to produce an accumulation of pisatin similar to that produced in response to the fungus. Under these inducing conditions, there was an average of 0.19 adducts per kb of pea genomic DNA. The accumulation of pisatin and the RNA of several DRR genes by psoralen required photoactivation, which suggests that covalent binding to DNA was necessary for induction. As the promoters of several putative fungal-induced pea genes contain long stretches of d(AT)_n, which is the preferred psoralen photobinding site, restriction fragments spanning DRR genes were examined after in vivo psoralen treatment. The rate of crosslinking was compared between fungal-induced and noninduced genes using a modified Southern blot analysis. Implications of the induction of the DRR due to psoralen binding are discussed.     

SPECTROSCOPIC PROPERTIES OF PSORALEN DERIVATIVES SUBSTITUTED BY CARBETHOXY GROUPS AT THE 3,4 AND/OR 4,5' REACTION SITE

Abstract— The newly synthesized linear psoralen derivative 3-carbethoxypsoralen has been shown recently to behave as a monofunctional derivative and has attracted some interest in the psoriasis treatment. In a first attempt to understand, by the fluorescence technique, the molecular mechanism by which it interacts with DNA, a spectroscopic study of the molecule was undertaken. The fluorescence of 3-carbethoxypsoralen at room temperature resembles that of 8-methoxypsoralen with a ten times higher quantum yield. 365 nm irradiation of dilute solutions of 3-carbethoxypsoralen rapidly leads to the formation of two types of highly fluorescent photoproducts. Type 1 photoproducts (λ_em^max= 425 nm, λ_exc^max= 360 nm) have been identified as the result of the addition of a solvent molecule to the 4,5' reaction site of the molecule. Their fluorescence intensity is a hundred times higher for 3-carbethoxypsoralen than for 8-methoxypsoralen. They become negligible when the 4',5' reaction site carries also a carbethoxy group. Type 2 photoproducts exhibit a somewhat different emission (λ_em^{max =} 443 nm, λ_exc^max= 413 nm). They are probably the result of an opening of the furocoumarin molecule. The implications of the peculiar photochemical properties of 3-carbethoxypsoralen are discussed in view of its biological activity. In addition, the use of fluorescence in monitoring the photobinding of psoralens to DNA is also discussed     

Synthetic models related to dna-intercalating molecules a study of the ring-ring stacking interactions between 8-alkoxypsoralen and thymine.

As a contribution to the problem of the intercalation and photobinding of psoralens to DNA, model compounds were prepared in which the psoralen ring is linked to thymine by a polymethylene bridge. The ring-ring intramolecular interaction in the models was assessed by hypochromism measurement in the UV.     

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.     

The difference between the reactivities of azomethine bonds in metamitron in electrochemical and chemical reductions

Electrochemical reduction of a herbicide metamitron (4-amino-3-mefhyl-6-phenyl-1,2,4-triazin-5(4H)-one) in buffered aqueous solutions occurs on the protonated 1,6-carbon-nitrogen double bond. The 2,3-double bond in the resulting 1,6-dihydrometamitron is electrochemically reduced at considerably more negative potentials. In reaction with borohydride the 2,3-double bond is reduced first. Reduction of the 1,6-double bond is very little influenced by replacing the 2,3-double bond by the -NHCH- grouping in 2,3-dihydrometamitron.     

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.