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.     

Photochemical and photobiological properties of new bispsoralen derivatives (Bis[PsCn]PIP, n = 4, 6, 8)

Bispsoralen derivatives possessing two psoralens and one piperazine molecule, 1,4-bis[n'-(8-psoralenoxy) alkyl] piperazine (Bis[PsCn]PIP, n = 4, 6, 8), show high water solubility, efficient intercalation into DNA and good photocrosslinking efficiency of DNA. Bis(PsC4)PIP shows high lethality on bacteriophage T7 and can effectively inhibit the amplification of DNA by stopping the polymerase chain reactions in a short period of irradiation time.     

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 its 3 ,4-double bond of the α-pyronic ring.
3-(α,α-dimethyl-allyl)-xanthyletine, studied for a comparison, showed only a very low photoreactivity with DNA for covalent addition; this is attributed to the presence of a bulky group at position 3 , which prevents almost completely the photoreaction of the 3 ,4-double bond.
Because of the low capacity of photobinding with DNA and the inability to form cross-links, the photobiological effects of xanthyletine are accordingly reduced: Inhibition of DNA and RNA synthesis in Ehrlich ascites tumor cells, killing of E. coli bacterial cells, inactivation of T₂ bacteriophage have been observed. By contrast, it was inactive in producing erythema on guinea pig skin.     

KINETICS OF SIMIAN VIRUS 40 AND LAMBDA INACTIVATION BY PHOTOADDITION OF PSORALEN DERIVATIVES

Abstract The kinetics of psora/en photoinactivation of two distinct DNA viruses, bacteriophage λ and the papovavirus SV40 were investigated. When λ is treated with near ultraviolet light (UVA, 320-400 nm) and 4,5',8-trimethylpsoralen (TMP) at 1 μg/m/, the phage is rapidly inactivated. The survival curve exhibits a distinct shoulder indicating second or higher-order kinetics. SV40, on the other hand, is much more resistant to psoralen photoinactivation and the survival curve is linear, reflecting first order or'pseudo-first order'kinetics. Two TMP derivatives with increased solubility in aqueous solutions, 4'-aminomethyl-TMP and 4'-hydroxymethyl-TMP, were similarly tested. In both virus systems, TMP was much more effective. In experiments designed to examine the role of psoralen cross-link formation in virus inactivation, treated samples were irradiated a second time in the absence of drug. Since reirradiation causes a decline in λ infectivity as great as that observed in continuously irradiated samples, cross-links are implicated as the primary lethal event. In the case of SV40, the results of such a protocol suggest that both monoadducts and cross-links may be lethal or that monoadduct formation may be rate-limiting.     

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.     

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.     

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.     

SYNTHESIS AND PHOTOBIOLOGICAL PROPERTIES OF 4-HYDROXYMETHYL-4'-METHYLPSORALEN DERIVATIVES

The synthesis and the photobiological activity of two new hydroxymethyl derivatives of psoralen namely 4-hydroxymethyl-4'-methyl- and 4-hydroxymethyl-4'-methyl-8-rnethoxypsoralen are described. Both compounds exhibited efficient photobinding to DNA and RNA. The DNA-photobinding process was investigated using different nucleic acid structures such as double-helical DNA, ribosomal RNA, bacterial DNA and DNA organized in the nucleosomal arrangement. The test derivatives were able to induce cross-links to a similar extent as 8-methoxypsoralen (8-MOP), used as a reference photochemotherapeutic drug. In contrast to 8-MOP, they produced relatively high levels of ^lO₂. Most photobiological effects (DNA synthesis inhibition, T₂ phage sensitization, inhibition of tumor transmitting capacity) showed a good correlation with the extent of covalent photoaddition. On the other hand, the new 4-hydroxymethylpsoralens were unable to induce skin erythema, in striking contrast with 8-MOP. Thus, neither cross-linking of the nucleic acid nor ¹O₂ production were coupled with skin phototoxicity in this class of compounds. The new derivatives appear to represent an important beginning to development of new active photochemotherapeutic agents devoid of undesired phototoxic side effects.     

ESTIMATION OF AN INDEX OF HYDROPHOBICITY OF DNA INTERIOR USING 5-METHOXYPSORALEN AS A FLUORESCENT PROBE

The index of hydrophobicity of DNA interior was estimated by measuring fluorescence spectra of psoralen derivatives associated with DNA. The environment around 5-MOP associated with DNA was as hydrophobic (D_k = 34) as methanol, suggesting that the molecules reside at the space between the base-pairs in B-form DNA. This is also true for 8-MOP. Thus, planar and aromatic molecules of 5- and 8-MOP are more stable in the interior of DNA than in aqueous medium due to hydrophobic affinity.     

Psoralen derivatives and longwave ultraviolet irradiation are active in vitro against human melanoma cell line

Cutaneous malignant melanoma is a very serious form of skin cancer that arises from melanocytes. Currently there is no effective treatment for metastatic melanoma so intense clinical trials are evaluating new drugs for this human malignancy. Psoralens are a group of compounds that bind to DNA in rapidly dividing cells and with ultraviolet light in the A band (UVA) cause DNA crosslinking, thereby preventing cellular division. They are used in the treatment of psoriasis and cutaneous T-cell lymphoma among other skin and blood diseases. We have investigated the cytotoxic potential of three psoralen derivatives plus UVA exposure (PUVA) on a established cell line of human melanoma. Cells were treated with different concentrations of 8-methoxypsoralen (8-MOP), 4,5',8-trimethylpsoralen (TMP) and 7-methylpyridopsoralen (MPP), for 1 h and after exposure to UVA light (0.3 J/cm(2)) were allowed to recover over a 24-72 h period. Viability was assessed by the microculture 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. Cisplatin, one of the most important drugs in the chemotherapy of melanoma, was included for comparative studies. All the psoralen derivatives tested were markedly cytotoxic in a dose and post-exposure-time dependent manner. The IC(50) values for 72 h of post-exposure time were as follows: MPP=0.05+/-0.01, TMP=0.13+/-0.003 and 8-MOP=10.79+/-1.85 micromol/L. Regardless of the limitations of the in vitro model, our results suggested that the lower IC(50) values of TMP and MPP might be of clinical importance.     

Photochemical and Photobiological Studies of a Furonaphthopyranone as a Benzo-spaced Psoralen Analog in Cell-free and Cellular DNA

Abstract— Photobiological activities of the benzo-spaced psoralen analog furonaphthopyranone 3 have been investigated in cell-free and cellular DNA. The molecular geometry parameters of 3 suggest that it should not form interstrand crosslinks with DNA. With cell-free DNA no evidence for crosslinking but also not for monoadduct formation was obtained; rather, the unnatural furocoumarin 3 induces oxidative DNA modifications under near-UVA irradiation. The enzymatic assay of the photosensitized damage in cell-free PM2 DNA revealed the significant formation of lesions sensitive to formamidopyrimidine DNA glyco-sylase (Fpg protein). In the photooxidation of calf thymus DNA by the furonaphthopyranone 3, 0.29±0.02% 8-oxo-7,8-dihydroguanine (8-oxoGua) was observed. With 2'-deoxyguanosine (dGuo), the guanidine-releasing photooxidation products oxazolone and oxoimidazolidine were formed predominately, while 8-oxodGuo and 4-HO-8-oxodGuo were obtained in minor amounts. The lack of a significant D₂O effect in the photooxidation of DNA and dGuo reveals that singlet oxygen (type II process) plays a minor role; control experiments with tert -butanol and mannitol confirm the absence of hydroxyl radicals as oxidizing species. The furonaphthopyranone 3 (E_red= -1.93±0.03V) should act in its singlet-excited state as electron acceptor for the photooxidation of dGuo (δG_ET ca – kcal/mol), which corroborates photoinduced electron transfer (type I) as a major DNA-oxidizing mechanism. A comet assay in Chinese hamster ovary (CHO) AS52 cells demonstrated that the psoralen analog 3 damages cellular DNA upon near-UVA irradiation; however, no photosensitized mutagenicity was observed in CHO AS52 cell cultures     

Synthesis and biological evaluation of glycosylated psoralen derivatives

A novel route for the efficient synthesis of a target psoralen moiety, 4,4′-dimethylxanthotoxol, has been developed, which need only four steps using cheap pyrogallol as a starting material. Subsequently, a range of new glycosylated psoralen derivatives were synthesized in good yields with simple procedures and mild reaction conditions. The experiment of biological activity shows that some of the glycosylated psoralen derivatives have antiproliferative activities against human cancer cell lines. A strong photo-induced antiproliferative effects were found under UVA. All of the glycosylated psoralen derivatives exhibited antioxidant activities against the oxidation of DNA induced by Cu²⁺/glutathione (GSH). Further experiment also demonstrates that the introduction of sugar moieties in some glycosylated psoralen derivatives can improve their antioxidant activities significantly.     

PHOTOBIOLOGICAL PROPERTIES OF A NOVEL, NATURALLY OCCURRING FUROISOCOUMARIN, CORIANDRIN

The photobiological properties of a novel, naturally occurring furoisocoumarin isolated from coriander and named coriandrin are described. Photosensitized lethal and mutagenic effects in bacteria indicate that it is more active than psoralen. It is a weak frameshift mutagen in the dark. Mammalian cells in tissue culture are photosensitized more actively with coriandrin than with psoralen even though preliminary evidence from interrupted radiation experiments and DNA analysis suggest that coriandrin does not form DNA interstrand crosslinks. Sister chromatid exchanges were induced with a unit dose of 1.1 x 10(-2) with coriandrin; the value for psoralen is 3 x 10(-3). Coriandrin appears to be metabolized more rapidly than furocoumarins by liver mixed function oxidases. Skin photosensitizing activity is very weak compared with psoralen, a surprising observation considering its potency in biological test systems.     

SINGLET OXYGEN GENERATION BY FUROCOUMARINS: EFFECT OF DNA AND LIPOSOMES

The quantum yield of singlet oxygen generation by aqueous furocoumarins was measured at 365 nm using the photosensitized inactivation of subtilisin Carlsberg as the probe with the following results: psoralen (0.18), 5-methoxypsoralen (0.013), and 8-methoxypsoralen (0.035). Singlet oxygen formation was significant for dark complexes of 8-MOP with calf thymus DNA and the covalent DNA photoadducts. Incorporation of 8-MOP in sonicated egg phosphatidylcholine liposomes did not inhibit photosensitization of subtilisin Carlsberg and also led to lipid peroxidation, with positive tests for the involvement of singlet oxygen. Peroxidation of the liposomes was inhibited by the presence of α-tocopherol and promoted by the presence of cholesterol in the membranes.     

PHOTOBIOLOGICAL ACTIVITY IN YEAST OF DERIVATIVES OF PSORALEN SUBSTITUTED AT THE 3,4 AND/OR THE 4'',5'' REACTION SITE

Abstract— Derivatives of psoralen substituted at the position 3 and/or 5' with various substituents (CN, CO-R or COO-R) have been newly synthesized. Their photobiological reactivity was determined by measuring the effects on survival and the induction of cytoplasmic 'petite' mutations (rho-) in the haploid yeast Saccharomyces cereuisiae after treatment with 365-nm irradiation in the presence of the different compounds. The effects were compared to those of known mono-functional compounds such as angelicin, 3-carbethoxypsoralen and 5,7-dimethoxycoumarin and bi-functional compounds such as psoralen and 8-methoxypsoralen for which the photoaffinity to DNA is well known. The derivative carrying a CN group in position 3 of the psoralen molecule exhibited effects comparable to those of the bi-functional agent 8-methoxypsoralen. Three derivatives substituted at position 3 showed a biological activity comparable to those of the mono-functional agent 3-carbethoxypsoralen. Two compounds substituted at position 5' by COOH or COOCH₃-groups were much less photobiologically active than the corresponding derivatives substituted in position 3. This suggested that the furanic site of the psoralen molecule may be photobiologically more active than the 3,4 site. Four newly synthesized angular derivatives, i.e. furo (2,3 f, g) coumarins, were found to be two to three times less active than angelicin.
At the dose rate of 365-nm irradiation used the measure of survival and rho^- induction per viable cell allowed the classification of some of the compounds as mono- and some as bi-functional agents in analogy to known mono- and bi-functional compounds.     

REPAIR OF 8-METHOXYPSORALEN INDUCED DNA INTERSTRAND CROSS-LINKS IN Tetrahymena thermophila

Abstract— The protozoan Tetrahymena thermophila was treated with 8-methoxypsoralen in combination with long wavelength ultraviolet irradiation and the DNA-repair response was studied. Following the treatment a lag-period in cell proliferation was observed, the duration of which was proportional to the amount of psoralen used, and both swelling and deformation of the cells were observed. The treatment with 3 μg 8-methoxypsoralen/mℓ and a light dose of 8 kJ m^-2 resulted in a 10-fold decrease in DNA and RNA synthesis activity, while the protein synthesis was only moderately affected. Using the same conditions the lag-period was 30 h, and during this time the psoralen induced DNA interstrand cross-links were removed. Alkaline elution experiments showed that the repair process involves DNA single strand scissions, whereas no double strand DNA scissions were detected.     

Psoralen Derivatives with Enhanced Potency

Psoralen is a furocoumarin natural product that intercalates within DNA and forms covalent adducts when activated by ultraviolet radiation. It is well known that this property contributes to psoralen’s clinical efficacy in several disease contexts, which include vitiligo, psoriasis, graft-versus-host disease and cutaneous T-cell lymphoma. Given the therapeutic relevance of psoralen and its derivatives, we attempted to synthesize psoralens with even greater potency. In this study, we report a library of 73 novel psoralens, the largest collection of its kind. When screened for the ability to reduce cell proliferation, we identified two derivatives even more cytotoxic than 4′-aminomethyl-4,5′,8-trimethylpsoralen (AMT), one of the most potent psoralens identified to date. Using MALDI-TOF MS, we studied the DNA adduct formation for a subset of novel psoralens and found that in most cases enhanced DNA binding correlated well with cytotoxicity. Generally, our most potent derivatives contain positively charged substituents, which we believe increase DNA affinity and enhance psoralen intercalation. Thus, we provide a rational approach to guide efforts toward further optimizing psoralens to fully capitalize on this drug class’ therapeutic potential. Finally, the structure–activity insights we have gained shed light on several opportunities to study currently underappreciated aspects of psoralen’s mechanism.     

Synthesis and In Vitro Biological Evaluation of Psoralen‐Linked Fullerenes

Photodynamic therapy (PDT) is a widely used medicinal treatment for the cancer therapy that utilizes the combination of a photosensitizer (PS) and light irradiation. In this study, we synthesized two novel C₆₀ fullerene derivatives, compounds 1 and 2 , with a psoralen moiety that can covalently bind to DNA molecules via cross‐linking to pyrimidine under photoirradiation. Along with several fullerene derivatives, the biological properties of several novel compounds have been evaluated. Compounds 1 and 2 , which have been shown to induce the production of hydroxyl radicals using several ROS detecting reagents, induced DNA strand breaks with relatively weak activities in the in vitro detection system using a supercoiled plasmid. However, the psoralen‐bound fullerene with carboxyl groups ( 2 ) only showed genotoxicity in the genotoxicity assay system of the umu test. Compound 2 was also seen to have cytotoxic activities in several cancer cell lines at higher doses compared to water‐soluble fullerenes.     

PHOTOADDITION OF 8-METHOXYPSORALEN TO E. coli DNA POLYMERASE I. ROLE OF PSORALEN PHOTO-ADDUCTS IN THE PHOTOSENSITIZED ALTERATIONS OF POL I ENZYMATIC ACTIVITIES

Abstract— We have previously demonstrated that 8-methoxypsoralen (8-MOP)‡ plus UVA is able to inactivate the three enzymatic activities of E. coli DNA polymerase I and that oxygen is required for these reactions (M. Granger et al. , (1982) Photochem. Photobiol. , 36 , 175–180). We now show that UV-A irradiation produces a covalent incorporation of the psoralen derivative into the enzyme either in the presence or in the absence of oxygen. The excited psoralen binds directly to the protein in an oxygen-independent reaction; no complex was detected in the absence of irradiation. Fluorescence measurements reveal that at least two photoadducts are formed.
The 8-MOP-photomodified enzyme is still fully active but further irradiation leads to an inhibition of the 5'→ 3' polymerase activity whereas the 5'→ 3' exonuclease activity is not affected. A major part of the inhibition reaction is shown to be oxygen-dependent but singlet oxygen quenchers have no effect on the kinetics. This oxygen-dependent reaction is attributed to a photosensitization, due to covalently bound 8-MOP, of neighbouring amino acids through an intermediate reactive oxygen species which is not singlet oxygen. The oxygen-independent reaction is attributed to a direct photosensitization through, for example, a radical mechanism.     

PHOTOTOXIC EFFECTS OF FOUR PSORALENS ON L1210 CELLS. THE CORRELATION WITH DNA INTERSTRAND CROSS-LINKING

L1210 mouse leukaemia cells were treated with psoralen [S-methoxy-(XMOP), 4,5′,8-trimethyI-(TMP), 4′-hydroxymethyl-4,5′,8-trimethyl-(HMT) or 4′-amino- methyl-4,5′,8-trimethylpsoralen (AMT)] in combination with long wavelength ultraviolct irradiation (Λ～ 365 nm). In order to investigate the relative photobiological activities of the psoralens, cell viability and DNA-synthesis activity as well as psoralen-DNA photoaddition and DNA interstrand cross-linking were measured after the treatment. In all assays the activity ranking order was found to he: TMP > HMT > AM7 > 8MOP. Furthermore, a direct correlation between phototoxicity, psoralen induced DNA interstrand cross-links and inhibition of DNA synthesis was indicated. Finally, psoralen uptake by the cells appears to be an important determinant for phototoxicity, whereas their DNA photoreactivity does not.