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Photoaddition of angelicin to linolenic acid methyl ester
Journal of Photochemistry
and Photobiology,
B: Biology,
2 (1988)
515 - 521
515
Preliminary Note
Photoaddition of angelicin to linolenic acid methyl ester S. CAFFIERI,
A. DAGA, D. VEDALDI and F. DALL’ACQUA
Department of Pharmaceutical Sciences, sulla Chimica de1 Farmaco e dei Prodotti (Italy)
University of Padova, and Centro di Studio Biologicamente Attivi de1 C.N.R., Padova
(Received April 29, 1988; accepted May 27, 1988)
Keywords. Photoaddition, angelicin, unsaturated lipids, UV-A. The photobinding of furocoumarins to DNA was for many years taken to be the mechanism causing the photobiological effects of these drugs [ 11. In fact, their antiproliferative activity is mainly due to this photochemical event [ 21. Other effects, however, such as erythemogenic and pigmentogenic activities, did not prove to be directly connected with the damage induced in DNA [ 3, 4). Thus, different mechanisms were investigated, such as photobinding to proteins [4, 51, hemolytic effects [6], production of active species of oxygen [3, 7, 81 and so on. The photochemical interaction between furocoumarins and unsaturated fatty acids has only recently been recognized [ 9 - 141. Until now, three furocoumarins have been used for these studies, i.e. psoralen, %methoxypsoralen (8-MOP) and 4,8,5’-trimethylpsoralen (TMP). They are all linear furocoumarins, capable of forming cross-links in DNA, and having strong erythemogenic and pigmentogenic activity on the skin under UV-A irradiation [15,16]. This study considers angelicin, an angular furocoumarin, which lacks both cross-linking [ 171 and phototoxic capacity [15]. Angelicin and linolenic acid methyl ester (LAME), both 4 X 10e3 M in ethanol, were irradiated in test tubes with two Philips HPW 125 lamps, emitting mainly at 365 nm, for 2 h (total incident dose about 1.2 X 10” quanta *ml-‘). The photoreacted mixture was then chromatographed by TLC on silica-gel plates developed with ethyl ether-n-hexane-formic acid (25:25: 1 v/v/v). Besides angelicin and some of its photodegradation products, in the plates four new compounds were found (I, II, III, IV) by means of a mineral lamp. Their Rf values were 0.72, 0.66, 0.61 and 0.55 respectively, higher than those of all the other compounds. Each compound was recovered from silica by elution with ethanol. The resulting solution was submitted to UV absorption and fluorescence analysis. The UV absorption spectrum of compound II, which is formed with the highest yield, is shown in Fig. 1, together with those of intact angelicin and loll-1344/88/$3.50
0 Elsevier Sequoia/Printed
in The Netherlands
0.i
0
i
300
400nm
Fig. 1. UV absorption spectra of angelicin (- * -), its cycloadduct with LAME (compound II) () and the cycloadduct between psoralen and LAME (- - -).
the 3,4-cycloadduct between psoralen and LAME [ 141. This spectrum is also similar to those of other cycloadducts of angelicins involving the 3,4 double bond (pyrone-side adducts with thymine [ 181, pyrone-pyrone cyclodimers [ 19; 201). The almost complete disappearance of the absorption band at 300 nm is characteristic of the saturation of the 3,4 double bond of the furocoumarin. On irradiating the ethanol solution of compound II with 254 nm light, the spectrum undergoes modification (Fig. 2). Photocycloreversion at short wavelengths, with re-formation of the parent compounds, is typical of cyclobutane adducts of furocoumarins [21].
517
Fig. 2. UV absorption spectrum of an ethanol solution of compound II before (a) and after irradiation at 254 nm ((b) 5 min; (c) 10 min; (d) 20 min; (e) 40 min).
Thus, the increase in the absorption at 300 nm (Fig. 2) is an indication that angelicin is restored during this irradiation. Its presence was also confirmed by chromatographic analysis of the photoreverted solution. The shape of the spectrum, lack of fluorescence and behaviour under UV-C light suggest that compound II is a C4-cycloadduct between the 3,4 double bond of angelicin and a double bond of the fatty acid. Figure 3 shows the NMR spectrum of compound II, obtained by means of a Varian FT80A spectrometer. The four signals at 6 = 7.61, 7.22, 7.03
518
1: I
-_(
3 -, -. -Y
520
The capacity of furocoumarins to induce specific lesions in unsaturated fatty acids may be a factor involved in skin phototoxicity. However, angelitin, which causes neither erythema nor pigmentation of the skin [15], is able to photobind effectively to linolenic acid methyl ester, although to a lesser extent than that shown by the strong phototoxic linear furocoumarins (psoralen, &MOP, TMP). Further studies are therefore necessary for a full understanding of the biological significance of this new mechanism.
1 L. Musajo and G. Rodighiero, Mode of photosensitizing action of furocoumarins. In A. C. Giese (ed.), Photophysiology, Vol. VII, Academic Press, New York, 1972, pp. 115 - 147. 2 F. DaIl’Acqua, D. Vedaidi, F. Baccichetti, G. Rodighiero and A. Gennaro, QSAR on furocoumarins, agents for the photochemotherapy of psoriasis. In M. Kuchar (ed.), QSAR in Design of Bioactiue Compounds, Prous, Barcelona, 1984, pp. 87 - 95. 3 M. A. Pathak and P. C. Joshi, Production of active oxygen species (‘02 and 02’) by psoralens and ultraviolet radiation;(320 - 400 nm), Biochim. Biophys. Acta, 798 (1984) 115 - 126. : 4 K. Yoshikawa, N. Mori, S. Sakakihara, N. Mizuno and P.-S. Song, Photoconjugation of 8-methoxypsoralen with proteins, Photochem. Photobiol., 29 (1979) 1127 - 1133. 5 F. Veronese, 0. Schiavon, R. Bevilacqua, F. Bordin and G. Rodighiero, Photoinactivation of enzymes by linear and angular furocoumarins, Photochem. PhotobioZ., 36 (1982) 25 - 30. 6 F. Dall’Acqua, D. Vedaldi, G. Miolo and P. Arslan, Dark and photohemolysis of erythrocytes by furocoumarins, II Congr. of the Eur. Sot. for Photobiology, Padova, Italy, 1987, abstract C-63. I W. Poppe and L. I. Grossweiner, Photodynamic sensitization by 8-methoxypsoralen via singlet oxygen mechanism, Photochem. PhotobioZ., 22 (1975) 217 - 219. 8 D. Vedaldi, F. Dall’Acqua, A. Gennaro and G. Rodighiero, Photosensitized effects of furocoumarins: the possible role of singlet oxygen, 2. Naturforsch., Teil C, 38 (1983) 866 - 869. 9 L. Kittler and G. LSber, Furocoumarins: biophysical investigations on their mode of action, Stud. Biophys., 97 (1983) 61 - 67. 10 L. Kittler and G. Lober, Photoreactions of furocoumarins with membrane constituents. Results with fatty acids and artificial bilayers, Stud. Biophys., 101 (1984) 69 - 72. 11 S. Caffieri, G. Tamborrino and F. Dall’Acqua, Formation of photoadducts between unsaturated fatty acids and furocoumarins, Med. Biol. Environ., 15 (1987) 11 - 14. 12 K. G. Specht, L. Kittler and R. W. Midden, A new biological target of furocoumarins: photochemical formation of covalent adducts with unsaturated fatty acids, Photothem. Photobiol., 47 (1988) 537 - 541. 13 K. G. Specht, P. Bhan, M. R. Chedekel and R. W. Midden, Furocoumarin photosensitized reactions with fatty acids. In G. Moreno, R. H. Pottier and T. G. Truscott (eds.), Photosensitization. Molecular, Cellular and Medical Aspects, NATO ASI Series, Springer, Berlin, 1988, pp. 301 - 303. 14 S. Caffieri, D. Vedaldi, A. Daga and F. Dail’Acqua, Photosensitizing furocoumarins: photocycloaddition to unsaturated fatty acids. In T. B. Fitzpatrick, P. Forlot, M. A. Pathak and F. Urbach (eds.), Psoralens in 1988. Past, Present and Future, John Libbey Eurotext, Montrouge, 1988, in the press. 15 L. Musajo and G. Rodighiero, The skin-photosensitizing furocoumarins, Experientia, 18 (1962) 153 - 162. 16 G. Caporale, L. Musajo, G. Rodighiero and F. Baccichetti, Skin-photosensitizing activity of some methylpsoralens, Experientia, 23 (1967) 985 - 988.
521 17 F. Dall’Acqua, S. Marciani, L. Ciavatta and G. Rodighiero, Formation of inter-strand cross-linkings in the photoreactions between furocoumarins and DNA, 2. Naturforsch., Teil B, 26 (1971) 561 - 569. 18 F. Dall’Acqua, S. Caffieri, D. Vedaldi, A. Guiotto and P. Rodighiero, Monofunctional 3,4- and 4’,5’-photocycloadducts between 4,5’-dimethylangelicin and thymine, Photothem. Photobiol., 37 (1983) 373 - 379. 19 S. Caffieri and F. Dall’Acqua, Fotostabilitl e fotodimerizzazione della 4,5’-dimetilangelicina, Chim. Oggi, (1985) 29 - 34. 20 S. Caffieri, G. M. J. Beijersbergen van Henegouwen, C. Erkelens, C. de Bruijn and F. Dall’Acqua, Photodimerization of 4,6,4’-trimethylangelicin and 6,5’-dimethylangelicin, J. Photochem. Photobiol., B: Biol., 1 (1987) 213 - 221. 21 C. H. Krauch, D. M. Kramer and A. Wacker, Zum Wirkungsmechanismus photodynamischer Furocumarine. Photoreaktion von Psoralen-(4-14C) mit DNS, RNS, Homopolynucleotiden und Nucleosiden, Photochem. Photobiol., 6 (1967) 341 - 354.
and Photobiology,
B: Biology,
2 (1988)
515 - 521
515
Preliminary Note
Photoaddition of angelicin to linolenic acid methyl ester S. CAFFIERI,
A. DAGA, D. VEDALDI and F. DALL’ACQUA
Department of Pharmaceutical Sciences, sulla Chimica de1 Farmaco e dei Prodotti (Italy)
University of Padova, and Centro di Studio Biologicamente Attivi de1 C.N.R., Padova
(Received April 29, 1988; accepted May 27, 1988)
Keywords. Photoaddition, angelicin, unsaturated lipids, UV-A. The photobinding of furocoumarins to DNA was for many years taken to be the mechanism causing the photobiological effects of these drugs [ 11. In fact, their antiproliferative activity is mainly due to this photochemical event [ 21. Other effects, however, such as erythemogenic and pigmentogenic activities, did not prove to be directly connected with the damage induced in DNA [ 3, 4). Thus, different mechanisms were investigated, such as photobinding to proteins [4, 51, hemolytic effects [6], production of active species of oxygen [3, 7, 81 and so on. The photochemical interaction between furocoumarins and unsaturated fatty acids has only recently been recognized [ 9 - 141. Until now, three furocoumarins have been used for these studies, i.e. psoralen, %methoxypsoralen (8-MOP) and 4,8,5’-trimethylpsoralen (TMP). They are all linear furocoumarins, capable of forming cross-links in DNA, and having strong erythemogenic and pigmentogenic activity on the skin under UV-A irradiation [15,16]. This study considers angelicin, an angular furocoumarin, which lacks both cross-linking [ 171 and phototoxic capacity [15]. Angelicin and linolenic acid methyl ester (LAME), both 4 X 10e3 M in ethanol, were irradiated in test tubes with two Philips HPW 125 lamps, emitting mainly at 365 nm, for 2 h (total incident dose about 1.2 X 10” quanta *ml-‘). The photoreacted mixture was then chromatographed by TLC on silica-gel plates developed with ethyl ether-n-hexane-formic acid (25:25: 1 v/v/v). Besides angelicin and some of its photodegradation products, in the plates four new compounds were found (I, II, III, IV) by means of a mineral lamp. Their Rf values were 0.72, 0.66, 0.61 and 0.55 respectively, higher than those of all the other compounds. Each compound was recovered from silica by elution with ethanol. The resulting solution was submitted to UV absorption and fluorescence analysis. The UV absorption spectrum of compound II, which is formed with the highest yield, is shown in Fig. 1, together with those of intact angelicin and loll-1344/88/$3.50
0 Elsevier Sequoia/Printed
in The Netherlands
0.i
0
i
300
400nm
Fig. 1. UV absorption spectra of angelicin (- * -), its cycloadduct with LAME (compound II) () and the cycloadduct between psoralen and LAME (- - -).
the 3,4-cycloadduct between psoralen and LAME [ 141. This spectrum is also similar to those of other cycloadducts of angelicins involving the 3,4 double bond (pyrone-side adducts with thymine [ 181, pyrone-pyrone cyclodimers [ 19; 201). The almost complete disappearance of the absorption band at 300 nm is characteristic of the saturation of the 3,4 double bond of the furocoumarin. On irradiating the ethanol solution of compound II with 254 nm light, the spectrum undergoes modification (Fig. 2). Photocycloreversion at short wavelengths, with re-formation of the parent compounds, is typical of cyclobutane adducts of furocoumarins [21].
517
Fig. 2. UV absorption spectrum of an ethanol solution of compound II before (a) and after irradiation at 254 nm ((b) 5 min; (c) 10 min; (d) 20 min; (e) 40 min).
Thus, the increase in the absorption at 300 nm (Fig. 2) is an indication that angelicin is restored during this irradiation. Its presence was also confirmed by chromatographic analysis of the photoreverted solution. The shape of the spectrum, lack of fluorescence and behaviour under UV-C light suggest that compound II is a C4-cycloadduct between the 3,4 double bond of angelicin and a double bond of the fatty acid. Figure 3 shows the NMR spectrum of compound II, obtained by means of a Varian FT80A spectrometer. The four signals at 6 = 7.61, 7.22, 7.03
518
1: I
-_(
3 -, -. -Y
520
The capacity of furocoumarins to induce specific lesions in unsaturated fatty acids may be a factor involved in skin phototoxicity. However, angelitin, which causes neither erythema nor pigmentation of the skin [15], is able to photobind effectively to linolenic acid methyl ester, although to a lesser extent than that shown by the strong phototoxic linear furocoumarins (psoralen, &MOP, TMP). Further studies are therefore necessary for a full understanding of the biological significance of this new mechanism.
1 L. Musajo and G. Rodighiero, Mode of photosensitizing action of furocoumarins. In A. C. Giese (ed.), Photophysiology, Vol. VII, Academic Press, New York, 1972, pp. 115 - 147. 2 F. DaIl’Acqua, D. Vedaidi, F. Baccichetti, G. Rodighiero and A. Gennaro, QSAR on furocoumarins, agents for the photochemotherapy of psoriasis. In M. Kuchar (ed.), QSAR in Design of Bioactiue Compounds, Prous, Barcelona, 1984, pp. 87 - 95. 3 M. A. Pathak and P. C. Joshi, Production of active oxygen species (‘02 and 02’) by psoralens and ultraviolet radiation;(320 - 400 nm), Biochim. Biophys. Acta, 798 (1984) 115 - 126. : 4 K. Yoshikawa, N. Mori, S. Sakakihara, N. Mizuno and P.-S. Song, Photoconjugation of 8-methoxypsoralen with proteins, Photochem. Photobiol., 29 (1979) 1127 - 1133. 5 F. Veronese, 0. Schiavon, R. Bevilacqua, F. Bordin and G. Rodighiero, Photoinactivation of enzymes by linear and angular furocoumarins, Photochem. PhotobioZ., 36 (1982) 25 - 30. 6 F. Dall’Acqua, D. Vedaldi, G. Miolo and P. Arslan, Dark and photohemolysis of erythrocytes by furocoumarins, II Congr. of the Eur. Sot. for Photobiology, Padova, Italy, 1987, abstract C-63. I W. Poppe and L. I. Grossweiner, Photodynamic sensitization by 8-methoxypsoralen via singlet oxygen mechanism, Photochem. PhotobioZ., 22 (1975) 217 - 219. 8 D. Vedaldi, F. Dall’Acqua, A. Gennaro and G. Rodighiero, Photosensitized effects of furocoumarins: the possible role of singlet oxygen, 2. Naturforsch., Teil C, 38 (1983) 866 - 869. 9 L. Kittler and G. LSber, Furocoumarins: biophysical investigations on their mode of action, Stud. Biophys., 97 (1983) 61 - 67. 10 L. Kittler and G. Lober, Photoreactions of furocoumarins with membrane constituents. Results with fatty acids and artificial bilayers, Stud. Biophys., 101 (1984) 69 - 72. 11 S. Caffieri, G. Tamborrino and F. Dall’Acqua, Formation of photoadducts between unsaturated fatty acids and furocoumarins, Med. Biol. Environ., 15 (1987) 11 - 14. 12 K. G. Specht, L. Kittler and R. W. Midden, A new biological target of furocoumarins: photochemical formation of covalent adducts with unsaturated fatty acids, Photothem. Photobiol., 47 (1988) 537 - 541. 13 K. G. Specht, P. Bhan, M. R. Chedekel and R. W. Midden, Furocoumarin photosensitized reactions with fatty acids. In G. Moreno, R. H. Pottier and T. G. Truscott (eds.), Photosensitization. Molecular, Cellular and Medical Aspects, NATO ASI Series, Springer, Berlin, 1988, pp. 301 - 303. 14 S. Caffieri, D. Vedaldi, A. Daga and F. Dail’Acqua, Photosensitizing furocoumarins: photocycloaddition to unsaturated fatty acids. In T. B. Fitzpatrick, P. Forlot, M. A. Pathak and F. Urbach (eds.), Psoralens in 1988. Past, Present and Future, John Libbey Eurotext, Montrouge, 1988, in the press. 15 L. Musajo and G. Rodighiero, The skin-photosensitizing furocoumarins, Experientia, 18 (1962) 153 - 162. 16 G. Caporale, L. Musajo, G. Rodighiero and F. Baccichetti, Skin-photosensitizing activity of some methylpsoralens, Experientia, 23 (1967) 985 - 988.
521 17 F. Dall’Acqua, S. Marciani, L. Ciavatta and G. Rodighiero, Formation of inter-strand cross-linkings in the photoreactions between furocoumarins and DNA, 2. Naturforsch., Teil B, 26 (1971) 561 - 569. 18 F. Dall’Acqua, S. Caffieri, D. Vedaldi, A. Guiotto and P. Rodighiero, Monofunctional 3,4- and 4’,5’-photocycloadducts between 4,5’-dimethylangelicin and thymine, Photothem. Photobiol., 37 (1983) 373 - 379. 19 S. Caffieri and F. Dall’Acqua, Fotostabilitl e fotodimerizzazione della 4,5’-dimetilangelicina, Chim. Oggi, (1985) 29 - 34. 20 S. Caffieri, G. M. J. Beijersbergen van Henegouwen, C. Erkelens, C. de Bruijn and F. Dall’Acqua, Photodimerization of 4,6,4’-trimethylangelicin and 6,5’-dimethylangelicin, J. Photochem. Photobiol., B: Biol., 1 (1987) 213 - 221. 21 C. H. Krauch, D. M. Kramer and A. Wacker, Zum Wirkungsmechanismus photodynamischer Furocumarine. Photoreaktion von Psoralen-(4-14C) mit DNS, RNS, Homopolynucleotiden und Nucleosiden, Photochem. Photobiol., 6 (1967) 341 - 354.