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Photoreactions of furocoumarins with biomolecules
Journal of Photochemistry and Photobiology, B: Biology, 6 (1990) 197-206
PHOTOREACTIONS BIOMOLECULES*
OF FUROCOUMARINS
197
WITH
JEAN CADET Laboratoires de Chimie, Ddpartement de Recherche Fondamentale, Centre d'Etudes Nuclgaires de Grenoble, 85X, F-38041 Grenoble Cddex (France) PAUL VIGNY Laboratoire de Physique et Chimie Biomoldculaire (CNRS UA 198), Universitd Paris VI, F-75231 Paris Cddex 05 (France) W. ROBERT MIDDEN The Center of Photochemical Sciences, Department of Chemistry, Bowling Green State University, Bowling Green, OH 43403 (U.S.A.) (Received November 24, 1989; accepted December 16, 1989)
Keywords. Furocoumarins, furocoumarin photocycloadducts to DNA, furocoumarin-protein photoadducts, photocycloaddition products of furocoumarin to lipids, photodynamic effects.
Summary Recent aspects of the photoreactions of linear and angular furocoumarins with DNA and related compounds, including [2 + 2 ] cycloaddition to pyrimidine bases, covalent attachment to the osidic moiety of adenine nucleosides and photodynamic effects, are surveyed. Reactions of photoexcited furocoumarins with proteins and unsaturated lipids and the possible biological roles of the resulting adducts are also presented and discussed.
1. Introduction Several furocoumarins including 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP) and 4,5',8-trimethylpsoralen (TMP) are widely used in association with UVA radiation in psoralen plus UVA light (PUVA) therapy of disabling psoriasis, mycosis fungoides, vitiligo and other skin diseases (for a recent review, see ref. 1). The mechanism of the lethal action of photoexcited furocoumarins (initially investigated by Padova's group more than 20 years ago) is still not fully understood despite numerous and *Paper presented at the Meeting on Photomedicine organized by the French Society for Photobiology, Paris, November, 1989.
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198 considerable efforts. However, it is reasonable to assume that the photobiological effects of furocoumarins arise, at least partly, from the [ 2 + 2 ] photocycloaddition of these aromatic planar molecules to thymine residues of cellular DNA following their intercalation within oligonucleotide chains [2]. Several lines of indirect evidence also strongly suggest the involvement of unsaturated lipids and, to a lesser extent, proteins as other important cellular targets [3, 4]. The most recent aspects of the photoreactions of monofunctional and bifunctional furocoumarins with these three classes of biomolecules are surveyed in this paper (for recent reviews, see refs. 5-9). Emphasis is placed on the characterization of the [2 + 2] photocycloadducts of furocoumarins with model compounds and cellular targets. In addition, other photoreactions including photodynamic effects and non-photocycloadditions are critically reviewed.
2. P h o t o r e a c t i o n s o f f u r o c o u m a r i n s w i t h n u c l e i c acids
2.1. [2 + 2] photocycloaddition to p y r i m i d i n e s Recent insights into the photoreactivity of linear furocoumarins (psoralens) and angular furocoumarins (angelicins) with pyrimidine nucleobases and the structural aspects o f the resulting adducts have been gained from various experiments involving isolated DNA and related model compounds. Near-UV photolysis of a dilute solution of bichromophoric molecules in which thymine is linked to 5-MOP by a polymethylene chain of variable length has been observed to give rise to the predominant formation of a cis-anti pyroneside monoadduct [10]. It is worth noting that a similar higher photoreactivity of the 3.4 ethylenic bond of free 5-MOP is observed when reacting with thymidine [11 ]. The two cis-syn diastereoisomers of the furan-side 3-carbethoxypsoralen (3-CPs) monoadducts to 2-deoxycylidine (dCyd) which undergo fast deamination in neutral aqueous solution have been identified as the main photoproducts of the reaction of excited 3-CPs with dCyd in the solid state [12]. The presence of complexed amino acid residues in nucleosome and in chromatin A and B leads to a significant decrease in the photobinding of 5-MOP and 8-MOP to DNA by comparison with that observed for a free double-stranded biopolymer [13]. The photoreaction of various linear and angular pyrrolocoumarin derivatives with tricyclic or tetracyclic structures essentially generates monoadducts involving the pyrone ring. This is explained in terms of a higher delocalization of the ethylenic bond of the pyrrole ring with respect to that of the furan moiety 14]. The cis-anti isomer of the pyrone-side monoadduct of 4'-methylangelicin to thymine is produced together with the cis-syn furan-side monoadduct in isolated DNA [15]. The stereochemistries of the two photoadducts have been unambiguously characterized by specific nuclear Overhauser effects (NOEs) as inferred from 1H nuclear magnetic resonance (NMR) experiments. An unexpected formation of interstrand cross-links occurs on exposure to near-UV light of DNA complexed
199 with tetrahydrobenzo-4,6-dimethylangelicin [16]. The presence of a tetrahydrobenzene ring in the furocoumarin has been suggested to induce pronounced distortion of the DNA duplex, allowing alignment between the photoreactive pyrone 3,4 ethylenic bond and the furan 4',5' ethylenic bond of one pyrimidine base with the 5,6 double bond of an opposite pyrimidine base. The base-catalysed reversal of the cross-link between 4'-hydroxymethyl4,5',8-trimethylpsoralen (HMT) and thymine within the self-complementary oligonucleotide 5'-GGGTACCC-3' exclusively generates a pyrone-side monoadduct through a mechanism involving the opening of the pyrone ring in the initial step of the reaction [17]. The action spectra for the photoreversal of the furan-side and pyrone-side HMT-thymidine monoadducts within the single-stranded deoxyoligonucleotide 5'-GAAGCTACGAGC-3' have been determined [18]. The photocross-linking of the furan-side HMT-thymidine monoadduct of 5'-GAAGCTACGAGC-3' occurs efficiently to the thymine base of the complementary oligonucleotide with a quantum yield of 2 . 4 x 1 0 - 2
[19]. The role of DNA sequence on the photobinding reaction of various linear and angular furocoumarins has been investigated recently using the gel sequencing method [20-22]. Thymine appears to be the main target for the photoaddition of bifunctional 8-MOP, 5-MOP and HMT and monofunctional 3-CPs, pyrido[3,4-c]psoralen (PyPs) and angelicin; the thymidylyl-(3'-5')2'deoxyadenosine (5'-TpA) sites are the most reactive with hot spots for photo addition in alternating (AT)n sequences [20, 21 ]. It is worth mentioning that HMT may also significantly photoadd to cytosine with a preference for 2'-deoxycytidylyl-(3'-5')-2'-deoxyadenosene (5'-CpA) and/or 2'-deoxyaden osylyl-(3'-5')-2'-deoxyadenosine (5'-ApC) sites [21]. Attempts have been made to assess the role of furan-side psoralen monoadducts on the secondary structure of isolated DNA using the $1 nuclease hydrolysis assay [23]. 3-CPs and 7-methylpyrido[3,4-c]psoralen (MePyPs) induce the destruction of seven base pairs around the monoadduct, whereas no local denaturation is observed when 7-methylpyrido[4,3-c]psoralen (2NMePyPs) is used as the photosensitizer. The presence of a TMP cross-link in the central TpA site of oligomeric partially double-stranded decadeoxyribonucleotide has no effect on the gel electrophoretic migration of the modified oligonucleotides [24]. This has been rationalized in terms of a lack of significant bending of the duplex at the site of the cross-link in contrast with the conclusions of previous X-ray crystallographic and 1H NMR studies, which indicate a 40o-50 ° bending [25, 26], and recA protein binding assay and electron microscopic analysis [27]. Further studies are required in order to resolve these apparent conflicting results. Significant progress has been accomplished in the quantitative measurement of psoralen pyrimidine photoadducts within cellular DNA. The formation of the two cis-syn diastereoisomers of the 3-CPs furan-side monoadduct to thymidine (3-CPs <5,6>4'5dThd) has been monitored by fluorescence detection in line with high performance liquid chromatography (HPLC) analysis [28].
200 It is worth noting that the two photoadducts exhibit similar kinetics of repair in the DNA of yeast and mammalian cells [29 ]. The furan-side 8-MOP-thymine m o n o a d d u c t has been shown to be the main DNA lesion induced in haploid repair-proficient yeast cells on treatment with 8-MOP and UVA radiation [30]. Furan-side monoadducts to thymidine and 2'-deoxycytidine have been tentatively characterized as the main photoadducts of 6,4,4'-trimethylangelicin to DNA on the basis of HPLC behavior and absorption features [31]. The quantitative meas ur em e nt of the 8-[C3H3]MOP-thymine adduct was achieved by scintillation counting following gel permeation c h r o m a t o g r a p h y of the 0.4 M HC1 DNA hydrolysate. It should also be mentioned that the TMP furanside mo n o ad d u c t s generated in the DNA of human cells are present in a higher yield on 365 nm exposure than following 405 nm irradiation for the same total amount of TMP adducts [32]. Significant improvement in both the specificity and sensitivity of detection of furocoumarin m o n o a d d u c t s and diadducts has been made recently in cellular DNA and skin (for recent reviews, see refs. 33 and 34). Monoclonal antibodies specifically recognize furan-side mo n oadduct s of 8-MOP [34] and 6,4,4'-trimethylangelicin [31] and 8-MOP cross-links in DNA [34].
2.2. Photobinding to the osidic moiety of adenine nucleosides Photoexcited 8-MOP photobinds to 2'-deoxyadenosine on exposure to near-UV light in the solid state. Three isomers involving covalent binding between the 3 or 4 positions of the pyrone ring and the 1' or 5' furanose carbons of the nucleoside have been isolated and characterized [35]. Similar adducts are generated in the photoreaction of 5,7-dimethoxycoumarin with adenosine [36]. 2.3. Other photosensitization reactions which do not involve binding of the f u r o c o u m a r i n s 3-CPs exerts a p r o n o u n c e d photodynamic action on the guanine residue in defined sequence DNA fragments [37]. Photo-oxidized guanine arising from type I and type II mechanisms is p r o d u c e d to the same extent as thymine monoadducts when oxygen is present in the irradiated aqueous DNA solution. In contrast, PyPs and MePyPs are not able to induce any detectable photodynamic effects on haploid yeast cells in agreement with the low efficiency of these c om pounds in the generation of singlet oxygen [38]. It is noteworthy that pyridopsoralens induce the formation of thymine cyclobutane dimer within nucleoside and DNA [39]. The cyclodimerization which occurs in the order of efficiency PyPs > MePyPs > 2N-MePyPs is likely to arise through non-radiative triplet energy transfer from the psoralens to thymine involving excited vibrational levels [40]. 3. P h o t o b i n d i n g
to proteins
There are n u m e r o u s indications that p h o t o e x c i t e d furocoumarin derivatives are able to react efficiently with proteins [4, 41] (for earher studies,
201 see ref. 9). However, m or e definite p r o o f of the o c c u r r e n c e of photobinding of furocoumarin to protein or amino acids awaits the isolation and characterization of such adducts.
4. P h o t o r e a c t i o n s w i t h lipids In addition to nucleic acids and proteins, psoralens can also form photochemical adducts with unsaturated fatty acids. The first indication of a photoreaction between psoralens and fatty acids was r e p o r t e d by Midden and coworkers in 1983 [42] based on work p e r f o r m e d by Leonhard Kittler while visiting the Midden laboratory in 1982. Confirmation of these results was r e p o r t e d in 1 9 8 4 - 1 9 8 6 [ 4 3 - 4 5 ] . In these studies near-UV illumination of TMP or 8-MOP with the free unsaturated fatty acids or methyl esters (oleic, linoleic, linolenic or arachidonic) in m e t h a n o l - w a t e r solution caused the formation of p h o t o p r o d u c t s that eluted later than the parent fatty acids on an octadecylsilylsilica gel HPLC column. No such p h o t o p r o d u c t s were observed with the completely saturated fatty acid, stearic acid. The later elution suggests that these p h o t o p r o d u c t s are more hydrophobic than the parent fatty acids and therefore are probably not photo-oxidation products. In fact, no peroxides were detected by iodine test and the p h o t o p r o d u c t s were f o r med readily even in the absence of oxygen. The p h o t o p r o d u c t s in the reaction of TMP with oleic acid or its methyl ester have been proved to be covalent adducts by mass spectrometry, double isotope labeling and UV absorbance s p e c t r o s c o p y analyses [ 4 6 - 4 8 ]. Dall'Acqua and coworkers [ 4 9 - 5 1 ] have also recently investigated these reactions. They have r ep o r ted results that indicate the formation of covalent adducts between the angular furocoumarin, angelicin, and other furocoumarins with linolenic acid methyl ester and other unsaturated fatty acids, further confirming the generality of this photoreaction. The molecular structures and stereoconfigurations of the four diastereoisomeric adducts formed in the reaction with oleic acid methyl ester (OAME) have b een established by 1H NOE NMR analysis and molecular mechanics calculations using the pr ogr a m MM2 [53]. Addition of the furocoumarin is observed to occur at the 3,4 bond of the coumarin portion of TMP [52] and addition of psoralen occurs at the 4',5' bond of the furan ring [50]. This is in contrast with the reaction observed with pyrimidines in doublestranded DNA; with this substrate the 4',5' bond of TMP appears to be the first to react [53]. This difference may be due to differences in the relative orientations of the psoralens and substrates, since orientation of the double bonds undergoing cycloaddition plays an important role in determining the stereochemistry of the adducts [54, 55]. It is interesting to note that only four of the eight possible diastereoisomers are detected in the reaction of TMP with OAME. Other diastereoisomers may form, but their yield is apparently much lower than the four main photoadducts. This stereospecificity is most
202 probably due to preferential orientation of the reactants in a ground state non-covalent complex or exciplex intermediate [52, 56]. The quantum yield for the formation of adducts between TMP and OAME is estimated to be about 0.003 [47] in ethanol for 4 mM TMP and 20 mM OAME; however, this quantum yield is only an estimate because of the difficulty in measuring light absorption due to scattering in the somewhat non-homogeneous mixture. The quantum yield is also observed to vary with the relative concentrations of the reagents and with the solvent. While this quantum yield is low c o m p a r e d with the quantum yields for formation of psoralen-DNA adducts, it should be noted that the quantum yields for reactions of free pyrimidines with psoralens in solution are significantly lower [55, 57]. Quantum yields for psoralen adduct formation with nucleic acids are much higher with the polymers than with the free bases or nucleosides due to the important role of association of the ground state reactants [53, 55]. The quantum yield for formation of psoralen-lipid adducts in lipid bilayers, where the psoralen can be intercalated into the lipid bilayer, has not yet been determined and this reaction may be much more efficient. Adduct formation is faster in polar solvents such as ethanol than in non-polar solvents such as benzene. Adduct formation has been observed with all of the c o m m o n unsaturated fatty acids including oleic, linoleic, linolenic and arachidonic acids. It occurs with 8-MOP and TMP, but is significantly slower with the former psoralen. It is interesting to note that, in addition to adduct formation, cis-trans isomerization of the fatty acid is also observed with a quantum yield four times larger than that for the sum of the formation of the four diastereoisomeric adducts [47, 52].
5. C o n c l u d i n g r e m a r k s Although the extent of binding to different cellular c o m p o n e n t s does not solely determine the bioeffects, it is certainly of interest. Recently, Beijersbergen van Henegouwen et al. [4] have r e p o r t e d a m et hod for measuring the binding of psoralens to various fractions of biomolecules in animal tissue. Using this method they measured significant binding of 8-MOP to proteins (57%), lipids (26%) and DNA (17%). Some years earlier, Pathak and Kramer [58] also measured binding of psoralens to protein, RNA and DNA and found greater binding to DNA than the other components; they did not include lipids in these measurements. It has been hypothesized that psoralen-lipid adduct formation may play an important role in the therapeutic benefit of PUVA therapy [9, 44, 59]. If this hypothesis is confirmed, it will open up a new pathway for the improvement of the safety of PUVA therapy for psoriasis by suggesting a new and straightforward strategy for eliminating the carcinogenicity of this treatment. More research is certainly warranted to determine the role of psoralen-lipid, p s o r a l e n - p r o t e i n and psoralen-nucleic acid adducts in the biological effects of these intriguing drugs.
203 Acknowledgments T h i s w o r k w a s s u p p o r t e d b y t h e L i g u e N a t i o n a l e F r a n ~ a i s e c o n t r e le C a n c e r and by the C e n t r e N a t i o n a l de la R e c h e r c h e Scientifique ( I n t e r f a c e Chimie-Biologie).
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204 18 Y.-B. Shi, J. Gri_ffith, H. Gamper and J. E. Hearst, Evidence for structural deformation of the DNA helix by a psoralen diadduct but not by a monoadduct, N u c l e i c A c i d s Res., 18 (1988) 8 9 4 5 - 8 9 5 2 . 19 Y.-B. Shi and J. E. Hearst, Wavelength dependence for the photoreactions of DNA-psoralen monoadducts. 1. Photoreversal of monoadducts, B i o c h e m i s t r y , 26 (1987) 3 7 8 6 - 3 7 9 2 . 20 E. Sage and E. Moustacchi, Sequence effects on 8-methoxypsoralen photobinding to defined DNA fragments, B i o c h e m i s t r y , 26 (1987) 3 3 0 7 - 3 3 1 4 . 21 V. Boyer, E. Moustacchi and E. Sage, Sequence specificity in photoreaction of various psoralen derivatives with DNA: role in biological activity, B i o c h e m i s t r y , 27 (1988) 3011-3018. 22 G. Miolo, M. Scarpa, F. Dall'Acqua and F. Zacchello, Determination of base specificity in 6,4,4'-trimethylangelicin photobinding to single-stranded DNA, J. Photochem. Photobiol. B, 3 (1989) 1 2 3 - 1 2 7 . 23 F. Gaboriau, P. Vigny and J. Moron, Secondary structure of DNA modified by monofunctional psoralen derivatives, B i o c h e m i s t r y , 28 (1989) 5 8 0 1 - 5 8 0 7 . 24 W.-P. Zhen, O. Dahl, O. Buchardt and P. E. Nielsen, On the DNA bending by psoralen interstrand crosslinking. A gel electrophoresis study, Photochem. Photobiol., 48 (1988) 643-646. 25 M. T. Tomic, D. E. W e m m e r and S.-H. Kim, Structure of a cross-linked psoralen, Science, 238 (1987) 1 7 2 2 - 1 7 2 5 . 26 D. A. Pearlman, S. R. Holbrook, D. H. Pirkle and S.-H. Kim, Molecular models for DNA damaged by photoreaction, Science, 227 (1985) 1 3 0 4 - 1 3 0 8 . 27 Y.-B. Sift and J. E. Hearst, Wavelength dependence for the photoreactions of DNA-psoralen monoadducts. 2. Photo-cross-linking of monoadducts, B i o c h e m i s t r y , 26 (1987) 3 7 9 2 - 3 7 9 8 . 28 A. Moysan, P. Vigny, M. Dardalhon, D. Averbeck, L. Voituriez and J. Cadet, 3-Carbethoxypsoralen-DNA photolesions: identification and quantitative detection in yeast and mammalian cells of the two c i s - s y n diastereoisomers with thymidine, Photochem. Photobiol., 47 (1988) 803-808. 29 M. Dardalhon, A. Moysan, D. Averbeck, P. Vigny, J. Cadet and L. Voituriez, Repair of the two cis-syn diastereoisomers formed between 3-carbethoxypsoralen and thymidine in yeast cells followed by a chemical method, J. Photochem. Photobiol. B, 2 (1988) 380-304. 30 M. B a n k m a n n and M. Brendel, Molecular dosimetry of 8-MOP+UVA-induced DNA photoproducts in S a c c h a r o r n y c e s cerevisiae: correlations of lesion n u m b e r with genotoxic potential, J. Photochem. Photobiol. B, 4 (1989) 5 7 - 7 4 . 31 G. Miolo, M. Stefanidis, R. M. Santella, F. Dall'Acqua and F. P. Gasparro, 6,4,4'-Tri mcthylangelicin photoadduct formation in DNA: production and characterization of a specific monoclonal antibody, J. Photochem. Photobiol. B, 3 (1989) 1 0 1 - 1 1 2 . 32 D. Papadopoulo, D. Averbeck and E. Moustacchi, High levels of 4,5',8-trimethylpsoralen photoinduced furan-side monoadducts can block cross-link removal in normal h u m a n cells, Photochem. Photobiol., 47 (1988) 3 2 1 - 3 2 6 . 33 F. P. Gasparro, Immunological assay for 8-MOP photoadducts to DNA, J. Photochem. Photobiol. B, 2 (1988) 2 8 6 - 2 8 8 . 34 F. P. Gasparro and R. M. Santella, Immunoassay of DNA damage, Photochem. Photobiol., 48 (1988) 3 2 1 - 3 2 8 . 35 J. Cadet, L. Voituriez, R. Nardin, A. Viari and P. Vigny, Isolation and characterization of 8-MOP adducts to the osidic moiety of 2'-deoxyadenosine, J. Photochem. Photobiol. B, 2 (1988) 3 2 1 - 3 3 9 . 36 T. H. Cho, H. K. Shim and S. C. Shim, Isolation and characterization of the photoadducts of 5,7-dimethoxycoumarin and adenosine, Photochem. Photobiol., 46 (1987) 3 0 5 - 3 0 9 . 37 E. Sage, T. Le Doan, V. Boyer, D. E. Helland, L. Kittler, C. Hdl~ne and E. Moustacchi, Oxidative DNA damage photo-induced by 3-carbethoxypsoralen and other furocoumarins. Mechanisms of photo-oxidation and recognition by repair enzymes, J. Mol. Biol., 20P (1989) 297-314.
205 38 J. C. Ronfard-Haret, D. Averbeck, R. V. Bensasson, E. Bisagni, E. J. Land and J. Moron, Correlation between the triplet photophysical properties and the photobiological action in yeast of two monofunctional pyridopsoralens, Photochem. Photobiol., 45 (1987) 235-239. 39 A. Moysan, J. Cadet, E. Moustacchi, E. Sage, A. Viari, P. Vigny and L. Voituriez, Pyridopsoralens are able to photosensitize the formation of DNA cyclobutyl dimers, 3rd Congress of the European Society f o r Photobiology, Budapest, Hungary, August 27-September 2, 1989, Book of Abstracts, p. 181. 40 R. Coastalat, J. Blais, J.-P. Ballini, A. Moysan, J. Cadet, O. Chalvet and P. Vigny, Formation of cyclobutane thymine dimers photosensitized by pyridopsoralens: a triplet-triplet energy transfer mechanism, Photochem. Photobiol., in the press. 41 S. Frederiksen, P. E. Nielsen and P. E. Hoyer, Lysosomes: a possible target for psoralen photodamage, J. Photochem. Photobiol. 13, 3 (1989) 437-447. 42 L. Kittler, W. R. Midden and S. Y. Wang, Photoreactions of lipids sensitized by furocoumarins, Photochem. Photobiol., 37s (1983) 16s. 43 L. Kittler and G. L6ber, Photoreactions of furocoumarins with m e m b r a n e constituents, Stud. Biophys., 101 (1984) 6 9 - 7 2 . 44 L. Kittler, K. G. Specht and W. R. Midden, Furocottmarins form covalent adducts with fatty acids. Implications for the design of noncareinogenie psoralens, Photochem. Photobiol., 43s (1986) 17s. 45 L. Kittler, W. R. Midden and S. Y. Wang. Interactions of furocoumarins with subunits of cell constituents. Photoreaction of fatty acids and aromatic amino acids with trimethylpsoralen (TMP) and 8-methoxypsoralen (8-MOP), Stud. Biophys., 114 (1986) 1 3 9 - 1 4 8 . 46 K. G. Specht, P. Bhan, M. R. Chedekel and W. R. Midden, The chemical structures of the trimethylpsoralen--oleie acid methyl ester adducts, Photochem. Photobiol., 45s (1987) 51s. 47 K. G. Specht, L. Kittler and W. R. Midden, A new biological target of furocoumarins: photochemical formation of covalent adducts with unsaturated fatty acids, Photochem. Photobiol., 47 (1988) 5 3 7 - 5 4 1 . 48 K. G. Specht, P. Bhan, M. R. Chedekel and W. R. Midden, Furocoumarin photosensitized reactions with fatty acids, in G. Moreno, R. H. Pottier and T. G. Truscott (eds.), Photosensitisation, Springer, Berlin, 1988, pp. 3 0 1 - 3 0 3 . 49 S. Cattieri, G. Tamborrino and F. DaU'Acqua, Formation of photoadducts between unsaturated fatty acids and furocoumarins, Med. BioL Environ., 15 (1987) 11-14. 50 S. Caffieri, A. Daga, D. Vedaldi and F. Dall'Acqua, Spectroscopic studies on the C4photocycloadducts between psoralen and unsaturated fatty acid methyl esters, Photomed PhotobioL, 10 (1988) 1 1 1 - 1 2 2 . 51 S. CalIieri, A. Daga, D. Vedaldi and F. Dall'Acqua, Photoaddition of angelicin to linolenic acid methyl ester, J. Photochem. Photobiol. B, Biol., 2 (1988) 5 1 5 - 5 2 1 . 52 K.G. Specht, W. R. Midden and M. R. Chedekel, Photocyeloaddition of 4,5',8-trimethylpsoralen and oleic acid methyl ester: product structures and reaction mechanisms, J. Org. Chem., 54 (1989) 4 1 2 5 - 4 1 3 4 . 53 D. Kanne, K. Straub, J. E. Hearst and H. Rapoport, Isolation and characterization of pyrimidine-psoralen-pyrimidine photodiadducts from DNA, J. Am. Chem. Soc., 104 (1982) 6754--6764. 54 K. Straub, D. Kanne, J. E. Hearst and H. Rapoport, Isolation and characterization of pyrimidine-psoralen photoadducts from DNA, J. Am. Cherm Soc., 103 (1981) 2 3 4 7 - 2 3 5 5 . 55 G. D. Cimino, H. B. Gamper, S. T. Isaacs and J. E. Hearst, Psoralens as photoactive probes of nucleic acid structure and function: organic chemistry, photochemistry and biochemistry, Annu. Rev. Biochem., 54 (1985) 1 1 5 1 - 1 1 9 3 . 56 N. J. Turro, Modern Molecular Photochemistry, Benjamin/Cummings, Menlo Park, CA, 1978. 57 J. C. Ronfard-Haret, D. Averbeck, R. V. Bensasson, E. Bisagni and E. J. Land, Some properties of the triplet excited state of the photosensitizing furocoumarin: 3-carbethoxypsoralen, Photochem. Photobiol., 35 (1982) 4 7 9 - 4 8 9 .
206 58 M. A. Pathak and D. M. Kramer, Photosensitization of skin in vivo by furocoumarins (psoralens), Biochim. Biophys. Acta, 105 (1969) 197-206. 59 W. R. Midden and J. E. Klaunig, Testing the antipsoriasis potential of psoralen-lipid photoproducts, lOth Int. Congress of Photobiology, Jerusalem, Israel, 1088 Book of Abstracts, p. 25.
PHOTOREACTIONS BIOMOLECULES*
OF FUROCOUMARINS
197
WITH
JEAN CADET Laboratoires de Chimie, Ddpartement de Recherche Fondamentale, Centre d'Etudes Nuclgaires de Grenoble, 85X, F-38041 Grenoble Cddex (France) PAUL VIGNY Laboratoire de Physique et Chimie Biomoldculaire (CNRS UA 198), Universitd Paris VI, F-75231 Paris Cddex 05 (France) W. ROBERT MIDDEN The Center of Photochemical Sciences, Department of Chemistry, Bowling Green State University, Bowling Green, OH 43403 (U.S.A.) (Received November 24, 1989; accepted December 16, 1989)
Keywords. Furocoumarins, furocoumarin photocycloadducts to DNA, furocoumarin-protein photoadducts, photocycloaddition products of furocoumarin to lipids, photodynamic effects.
Summary Recent aspects of the photoreactions of linear and angular furocoumarins with DNA and related compounds, including [2 + 2 ] cycloaddition to pyrimidine bases, covalent attachment to the osidic moiety of adenine nucleosides and photodynamic effects, are surveyed. Reactions of photoexcited furocoumarins with proteins and unsaturated lipids and the possible biological roles of the resulting adducts are also presented and discussed.
1. Introduction Several furocoumarins including 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP) and 4,5',8-trimethylpsoralen (TMP) are widely used in association with UVA radiation in psoralen plus UVA light (PUVA) therapy of disabling psoriasis, mycosis fungoides, vitiligo and other skin diseases (for a recent review, see ref. 1). The mechanism of the lethal action of photoexcited furocoumarins (initially investigated by Padova's group more than 20 years ago) is still not fully understood despite numerous and *Paper presented at the Meeting on Photomedicine organized by the French Society for Photobiology, Paris, November, 1989.
1011-1344/90/$3.50
© Elsevier Sequoia/Printed in The Netherlands
198 considerable efforts. However, it is reasonable to assume that the photobiological effects of furocoumarins arise, at least partly, from the [ 2 + 2 ] photocycloaddition of these aromatic planar molecules to thymine residues of cellular DNA following their intercalation within oligonucleotide chains [2]. Several lines of indirect evidence also strongly suggest the involvement of unsaturated lipids and, to a lesser extent, proteins as other important cellular targets [3, 4]. The most recent aspects of the photoreactions of monofunctional and bifunctional furocoumarins with these three classes of biomolecules are surveyed in this paper (for recent reviews, see refs. 5-9). Emphasis is placed on the characterization of the [2 + 2] photocycloadducts of furocoumarins with model compounds and cellular targets. In addition, other photoreactions including photodynamic effects and non-photocycloadditions are critically reviewed.
2. P h o t o r e a c t i o n s o f f u r o c o u m a r i n s w i t h n u c l e i c acids
2.1. [2 + 2] photocycloaddition to p y r i m i d i n e s Recent insights into the photoreactivity of linear furocoumarins (psoralens) and angular furocoumarins (angelicins) with pyrimidine nucleobases and the structural aspects o f the resulting adducts have been gained from various experiments involving isolated DNA and related model compounds. Near-UV photolysis of a dilute solution of bichromophoric molecules in which thymine is linked to 5-MOP by a polymethylene chain of variable length has been observed to give rise to the predominant formation of a cis-anti pyroneside monoadduct [10]. It is worth noting that a similar higher photoreactivity of the 3.4 ethylenic bond of free 5-MOP is observed when reacting with thymidine [11 ]. The two cis-syn diastereoisomers of the furan-side 3-carbethoxypsoralen (3-CPs) monoadducts to 2-deoxycylidine (dCyd) which undergo fast deamination in neutral aqueous solution have been identified as the main photoproducts of the reaction of excited 3-CPs with dCyd in the solid state [12]. The presence of complexed amino acid residues in nucleosome and in chromatin A and B leads to a significant decrease in the photobinding of 5-MOP and 8-MOP to DNA by comparison with that observed for a free double-stranded biopolymer [13]. The photoreaction of various linear and angular pyrrolocoumarin derivatives with tricyclic or tetracyclic structures essentially generates monoadducts involving the pyrone ring. This is explained in terms of a higher delocalization of the ethylenic bond of the pyrrole ring with respect to that of the furan moiety 14]. The cis-anti isomer of the pyrone-side monoadduct of 4'-methylangelicin to thymine is produced together with the cis-syn furan-side monoadduct in isolated DNA [15]. The stereochemistries of the two photoadducts have been unambiguously characterized by specific nuclear Overhauser effects (NOEs) as inferred from 1H nuclear magnetic resonance (NMR) experiments. An unexpected formation of interstrand cross-links occurs on exposure to near-UV light of DNA complexed
199 with tetrahydrobenzo-4,6-dimethylangelicin [16]. The presence of a tetrahydrobenzene ring in the furocoumarin has been suggested to induce pronounced distortion of the DNA duplex, allowing alignment between the photoreactive pyrone 3,4 ethylenic bond and the furan 4',5' ethylenic bond of one pyrimidine base with the 5,6 double bond of an opposite pyrimidine base. The base-catalysed reversal of the cross-link between 4'-hydroxymethyl4,5',8-trimethylpsoralen (HMT) and thymine within the self-complementary oligonucleotide 5'-GGGTACCC-3' exclusively generates a pyrone-side monoadduct through a mechanism involving the opening of the pyrone ring in the initial step of the reaction [17]. The action spectra for the photoreversal of the furan-side and pyrone-side HMT-thymidine monoadducts within the single-stranded deoxyoligonucleotide 5'-GAAGCTACGAGC-3' have been determined [18]. The photocross-linking of the furan-side HMT-thymidine monoadduct of 5'-GAAGCTACGAGC-3' occurs efficiently to the thymine base of the complementary oligonucleotide with a quantum yield of 2 . 4 x 1 0 - 2
[19]. The role of DNA sequence on the photobinding reaction of various linear and angular furocoumarins has been investigated recently using the gel sequencing method [20-22]. Thymine appears to be the main target for the photoaddition of bifunctional 8-MOP, 5-MOP and HMT and monofunctional 3-CPs, pyrido[3,4-c]psoralen (PyPs) and angelicin; the thymidylyl-(3'-5')2'deoxyadenosine (5'-TpA) sites are the most reactive with hot spots for photo addition in alternating (AT)n sequences [20, 21 ]. It is worth mentioning that HMT may also significantly photoadd to cytosine with a preference for 2'-deoxycytidylyl-(3'-5')-2'-deoxyadenosene (5'-CpA) and/or 2'-deoxyaden osylyl-(3'-5')-2'-deoxyadenosine (5'-ApC) sites [21]. Attempts have been made to assess the role of furan-side psoralen monoadducts on the secondary structure of isolated DNA using the $1 nuclease hydrolysis assay [23]. 3-CPs and 7-methylpyrido[3,4-c]psoralen (MePyPs) induce the destruction of seven base pairs around the monoadduct, whereas no local denaturation is observed when 7-methylpyrido[4,3-c]psoralen (2NMePyPs) is used as the photosensitizer. The presence of a TMP cross-link in the central TpA site of oligomeric partially double-stranded decadeoxyribonucleotide has no effect on the gel electrophoretic migration of the modified oligonucleotides [24]. This has been rationalized in terms of a lack of significant bending of the duplex at the site of the cross-link in contrast with the conclusions of previous X-ray crystallographic and 1H NMR studies, which indicate a 40o-50 ° bending [25, 26], and recA protein binding assay and electron microscopic analysis [27]. Further studies are required in order to resolve these apparent conflicting results. Significant progress has been accomplished in the quantitative measurement of psoralen pyrimidine photoadducts within cellular DNA. The formation of the two cis-syn diastereoisomers of the 3-CPs furan-side monoadduct to thymidine (3-CPs <5,6>4'5dThd) has been monitored by fluorescence detection in line with high performance liquid chromatography (HPLC) analysis [28].
200 It is worth noting that the two photoadducts exhibit similar kinetics of repair in the DNA of yeast and mammalian cells [29 ]. The furan-side 8-MOP-thymine m o n o a d d u c t has been shown to be the main DNA lesion induced in haploid repair-proficient yeast cells on treatment with 8-MOP and UVA radiation [30]. Furan-side monoadducts to thymidine and 2'-deoxycytidine have been tentatively characterized as the main photoadducts of 6,4,4'-trimethylangelicin to DNA on the basis of HPLC behavior and absorption features [31]. The quantitative meas ur em e nt of the 8-[C3H3]MOP-thymine adduct was achieved by scintillation counting following gel permeation c h r o m a t o g r a p h y of the 0.4 M HC1 DNA hydrolysate. It should also be mentioned that the TMP furanside mo n o ad d u c t s generated in the DNA of human cells are present in a higher yield on 365 nm exposure than following 405 nm irradiation for the same total amount of TMP adducts [32]. Significant improvement in both the specificity and sensitivity of detection of furocoumarin m o n o a d d u c t s and diadducts has been made recently in cellular DNA and skin (for recent reviews, see refs. 33 and 34). Monoclonal antibodies specifically recognize furan-side mo n oadduct s of 8-MOP [34] and 6,4,4'-trimethylangelicin [31] and 8-MOP cross-links in DNA [34].
2.2. Photobinding to the osidic moiety of adenine nucleosides Photoexcited 8-MOP photobinds to 2'-deoxyadenosine on exposure to near-UV light in the solid state. Three isomers involving covalent binding between the 3 or 4 positions of the pyrone ring and the 1' or 5' furanose carbons of the nucleoside have been isolated and characterized [35]. Similar adducts are generated in the photoreaction of 5,7-dimethoxycoumarin with adenosine [36]. 2.3. Other photosensitization reactions which do not involve binding of the f u r o c o u m a r i n s 3-CPs exerts a p r o n o u n c e d photodynamic action on the guanine residue in defined sequence DNA fragments [37]. Photo-oxidized guanine arising from type I and type II mechanisms is p r o d u c e d to the same extent as thymine monoadducts when oxygen is present in the irradiated aqueous DNA solution. In contrast, PyPs and MePyPs are not able to induce any detectable photodynamic effects on haploid yeast cells in agreement with the low efficiency of these c om pounds in the generation of singlet oxygen [38]. It is noteworthy that pyridopsoralens induce the formation of thymine cyclobutane dimer within nucleoside and DNA [39]. The cyclodimerization which occurs in the order of efficiency PyPs > MePyPs > 2N-MePyPs is likely to arise through non-radiative triplet energy transfer from the psoralens to thymine involving excited vibrational levels [40]. 3. P h o t o b i n d i n g
to proteins
There are n u m e r o u s indications that p h o t o e x c i t e d furocoumarin derivatives are able to react efficiently with proteins [4, 41] (for earher studies,
201 see ref. 9). However, m or e definite p r o o f of the o c c u r r e n c e of photobinding of furocoumarin to protein or amino acids awaits the isolation and characterization of such adducts.
4. P h o t o r e a c t i o n s w i t h lipids In addition to nucleic acids and proteins, psoralens can also form photochemical adducts with unsaturated fatty acids. The first indication of a photoreaction between psoralens and fatty acids was r e p o r t e d by Midden and coworkers in 1983 [42] based on work p e r f o r m e d by Leonhard Kittler while visiting the Midden laboratory in 1982. Confirmation of these results was r e p o r t e d in 1 9 8 4 - 1 9 8 6 [ 4 3 - 4 5 ] . In these studies near-UV illumination of TMP or 8-MOP with the free unsaturated fatty acids or methyl esters (oleic, linoleic, linolenic or arachidonic) in m e t h a n o l - w a t e r solution caused the formation of p h o t o p r o d u c t s that eluted later than the parent fatty acids on an octadecylsilylsilica gel HPLC column. No such p h o t o p r o d u c t s were observed with the completely saturated fatty acid, stearic acid. The later elution suggests that these p h o t o p r o d u c t s are more hydrophobic than the parent fatty acids and therefore are probably not photo-oxidation products. In fact, no peroxides were detected by iodine test and the p h o t o p r o d u c t s were f o r med readily even in the absence of oxygen. The p h o t o p r o d u c t s in the reaction of TMP with oleic acid or its methyl ester have been proved to be covalent adducts by mass spectrometry, double isotope labeling and UV absorbance s p e c t r o s c o p y analyses [ 4 6 - 4 8 ]. Dall'Acqua and coworkers [ 4 9 - 5 1 ] have also recently investigated these reactions. They have r ep o r ted results that indicate the formation of covalent adducts between the angular furocoumarin, angelicin, and other furocoumarins with linolenic acid methyl ester and other unsaturated fatty acids, further confirming the generality of this photoreaction. The molecular structures and stereoconfigurations of the four diastereoisomeric adducts formed in the reaction with oleic acid methyl ester (OAME) have b een established by 1H NOE NMR analysis and molecular mechanics calculations using the pr ogr a m MM2 [53]. Addition of the furocoumarin is observed to occur at the 3,4 bond of the coumarin portion of TMP [52] and addition of psoralen occurs at the 4',5' bond of the furan ring [50]. This is in contrast with the reaction observed with pyrimidines in doublestranded DNA; with this substrate the 4',5' bond of TMP appears to be the first to react [53]. This difference may be due to differences in the relative orientations of the psoralens and substrates, since orientation of the double bonds undergoing cycloaddition plays an important role in determining the stereochemistry of the adducts [54, 55]. It is interesting to note that only four of the eight possible diastereoisomers are detected in the reaction of TMP with OAME. Other diastereoisomers may form, but their yield is apparently much lower than the four main photoadducts. This stereospecificity is most
202 probably due to preferential orientation of the reactants in a ground state non-covalent complex or exciplex intermediate [52, 56]. The quantum yield for the formation of adducts between TMP and OAME is estimated to be about 0.003 [47] in ethanol for 4 mM TMP and 20 mM OAME; however, this quantum yield is only an estimate because of the difficulty in measuring light absorption due to scattering in the somewhat non-homogeneous mixture. The quantum yield is also observed to vary with the relative concentrations of the reagents and with the solvent. While this quantum yield is low c o m p a r e d with the quantum yields for formation of psoralen-DNA adducts, it should be noted that the quantum yields for reactions of free pyrimidines with psoralens in solution are significantly lower [55, 57]. Quantum yields for psoralen adduct formation with nucleic acids are much higher with the polymers than with the free bases or nucleosides due to the important role of association of the ground state reactants [53, 55]. The quantum yield for formation of psoralen-lipid adducts in lipid bilayers, where the psoralen can be intercalated into the lipid bilayer, has not yet been determined and this reaction may be much more efficient. Adduct formation is faster in polar solvents such as ethanol than in non-polar solvents such as benzene. Adduct formation has been observed with all of the c o m m o n unsaturated fatty acids including oleic, linoleic, linolenic and arachidonic acids. It occurs with 8-MOP and TMP, but is significantly slower with the former psoralen. It is interesting to note that, in addition to adduct formation, cis-trans isomerization of the fatty acid is also observed with a quantum yield four times larger than that for the sum of the formation of the four diastereoisomeric adducts [47, 52].
5. C o n c l u d i n g r e m a r k s Although the extent of binding to different cellular c o m p o n e n t s does not solely determine the bioeffects, it is certainly of interest. Recently, Beijersbergen van Henegouwen et al. [4] have r e p o r t e d a m et hod for measuring the binding of psoralens to various fractions of biomolecules in animal tissue. Using this method they measured significant binding of 8-MOP to proteins (57%), lipids (26%) and DNA (17%). Some years earlier, Pathak and Kramer [58] also measured binding of psoralens to protein, RNA and DNA and found greater binding to DNA than the other components; they did not include lipids in these measurements. It has been hypothesized that psoralen-lipid adduct formation may play an important role in the therapeutic benefit of PUVA therapy [9, 44, 59]. If this hypothesis is confirmed, it will open up a new pathway for the improvement of the safety of PUVA therapy for psoriasis by suggesting a new and straightforward strategy for eliminating the carcinogenicity of this treatment. More research is certainly warranted to determine the role of psoralen-lipid, p s o r a l e n - p r o t e i n and psoralen-nucleic acid adducts in the biological effects of these intriguing drugs.
203 Acknowledgments T h i s w o r k w a s s u p p o r t e d b y t h e L i g u e N a t i o n a l e F r a n ~ a i s e c o n t r e le C a n c e r and by the C e n t r e N a t i o n a l de la R e c h e r c h e Scientifique ( I n t e r f a c e Chimie-Biologie).
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