Giovanni Rodighiero

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.

BIOCHEMICAL AND MEDICAL ASPECTS OF PSORALENS

A few studies have been devoted to the synthesis of new psoralen derivatives. A new route suitable for preparing psoralen and many of its derivatives has been described by Queval and Bisagni[56] and a semi-synthetic route to psoralen, starting from marmesin, by Loutfy and Abu-ShadyC381. Many new synthetic derivatives of psoralen have been prepared by Loutfy et al. [373 for testing their photosensitizing activity on skin and further investigating the relationship between chemical structure and photobiological activity. Some other tricyclic compounds (i.e. pyranocoumarins such as xanthyletin and dimethylallylxanthyletin) having structure analogous to furocoumarins, have been studied as to their possible photosensitizing activity and the ability to photoreact with DNA. These compounds appear to photosensitize biological substrates and able to form -both monoadducts and cross-linkings in double stranded DNA [22].

Mechanism of skin photosensitization by forucoumarins: Photoreactivity of various furocoumarins with native DNA and with ribosomal RNA

Abstract The photoreactions (by irradiation at 365 nm) between several furocoumarins and native DNA or rRNA have been studied. The initial rates of the photoreactions and the quantum yields were determined. Moreover some experiments have been performed in a manner suitable to give data of photoreactivity which could be directly compared with those previously obtained in determining the skin-photosensitizing activity of the same substances by irradiation at 365 nm. The results showing a close correlation between the capacity of the substances to photoreact with native DNA and that to produce skin photosensitization, offer valid evidence for the mechanism of action of furocoumarins on the skin. By contrast, the photoreactions with rRNA seem to be less (or not) connected with skin photosensitization.

SKIN‐PHOTOSENSITIZING FUROCOUMARINS: PHOTOCHEMICAL INTERACTION BETWEEN DNA AND —O14CH3 BERGAPTEN (5‐METHOXY‐PSORALEN)

Abstract— For some years the mechanism of the photosensitizing effects displayed by some furocoumarins on various biological substrates (human and guinea‐pig skin, bacteria cultures, mammalian cells adapted to in vitro growth, viruses) have been studied. Recently it has been pointed out that a photoreaction occurs between the photosensitizing furocoumarins and DNA after irradiation at 3655 Å. By use of a labeled furocoumarin, i.e.—O14CH3 bergapten or 5‐methoxy‐psoralen, this has been confirmed and more extensively studied. During the irradiation a stable combination of the furocoumarin with native DNA takes place with a quantum yield of 5·2 × 10‐3. It is probable that the reactive sites of DNA are the pyrimidine bases. Yeast‐RNA and the same DNA after heat‐denaturation or in the presence of high NaCl concentration photoreact at a much reduced rate. This photoreaction may explain some various biological photosensitizing effects produced by furocoumarins.

Studies on the photoreactions (365 nm) between DNA and some methylpsoralens

Abstract While many methyl derivatives of psoralen are very active as skin-photosensitizers, some others have a small activity or are practically inactive. This difference is not explicable in terms of different capacity of photobinding to DNA. Other factors are important, especially the ability to form cross-linkings in DNA. In fact, as a result of a comparison of a group of methylpsoralen derivatives, a good correlation has been found between the skin-photosensitizing potencies of the various compounds, as evaluated by determining the minimum irradiation time necessary for the production of erythema on guinea pig skin, and the amounts of cross-linking formed in DNA as a function of the time of irradiation.

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.

Photosensitized effects of furocoumarins: the possible role of singlet oxygen

Abstract The capacity of various furocoumarins to generate singlet oxygen in aqueous solution has been determined. The antiproliferative and the skin-photosensitizing activities of the same furocoumarins did not show any correlation with the capacity to generate the singlet oxygen, while these photobiological properties could be correlated with the capacity of furocoumarins to induce photolesions to the DNA.

FLUORIMETRIC DETERMINATION OF 4‘,5’‐CYCLOADDUCTS IN THE DNA‐PSORALEN PHOTOREACTION

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

A comparison between the photoreactivity of some furocoumarins with native DNA and their skin-photosensitizing activity.

È stata determinata la fotoreattività con DNA nativo di un gruppo di furocumarine per irradiazione a 365 nm. I valori ottenuti sono risultati in accordo con quelli di attività fotosensibilizzatrice cutanea posseduti dalle stesse sostanze.

Photochemical interaction between xanthyletine and DNA.

Abstract— Xanthyletine, a dimethyl‐pyranocoumarin having a structural relationship to psoralen, has been studied in connection with its interaction with DNA. In the dark, it forms a weak molecular complex with DNA, which is not of the intercalated type. Under irradiation at 365nm, it is able to bind covalently to DNA, with, however, a much lower rate relative to psoralen. In this photobinding, it behaves as a pure monofunctional reagent, involving only its3,4‐double bond of the α‐pyronic ring.