Psoralen Compounds

Psoralen, or derivatives of 9-methoxy-7H-furo[3,2-g]chromen-7-one tricyclic ring structures, are used as photoreactive groups in crosslinkers, biotinylation compounds, and nucleic acid probes. Psoralens have been used for many years as photochemotherapy agents for treatment of psoriasis and vitiligo (Smith and Barker, 2006). Psoralens react when exposed to UV light 

Psoralens can react by two different routes upon photoactivation (Parsons, 1980 Pathak, 1984). The first route is through the well-known photoreaction mechanism that principally involves intercalation within double-stranded DNA or RNA with the formation of adducts with adjacent thymine bases. The furan-side and pyrone-side rings in psoralen both can form cycloaddition products with the 5,6-double bond of thymine to create a crosslink between two DNA strands (Reaction 57) or to a lesser extent, within double-strand regions of RNA. 

Furan-side and pyrone-side adducts to the 5,6-double bond of thymine residues 

The following sections briefly describe three cycloaddition reactions that can be used to form bioconjugates. These reactions represent highly specific reactant pairs that have a chemoselec-tive nature, meaning they mainly react with each other and not other functional groups, such as those found on biomolecules. For a complete discussion of chemoselective ligation reactions, see Chapter 17. 

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Tab. 4.5 Singlet excitation energies of a small subset of psoralen compounds. Experimental data in aqueous solution given in parentheses [99]. Experimental absorption coefficients are denoted w=weak (<8000), i = intermediate (8000-15000), and s = strong (>15000).

Addition of coumarins to plant tissues can also dramatically effect PAL levels. Pre-soaking pea pods in a solution of the photodynamic coumarin 4.5, 8-trimethylpsoralen, followed by 4 minutes irradiation with 366 nm u.v. light, doubles the PAL activity 3 hours later and increases it twelve-fold 20 hours after. A contrasting effect of added coumarin on PAL activity has been noted in tobacco suspension cultures, where scopoletin addition inhibits PAL synthesis. The growth regulator lAA has the same effect, and, at 1 mmol 1" concentrations, reduces PAL activity by 53 %. Although the effect of scopoletin and lAA is probably simple end-product inhibition, that of the photodynamic coumarin must be at a different level, perhaps on plant DNA psoralen compounds, for example, are known to form stable complexes with the DNA base thymine. 

Some new photobiological and pharmacological aspects of psoralen compounds are reported in this chapter. 

It has been found that the compound 8-geranoxy psoralen is present in citrus oils, particu-lariy lemon and lime oils. This compound can be isolated from the oil by a process which involves primarily absorption on an adsorbent material followed by elution with a suitable solvent. 

Figure 11.15 The photoreactive compound psoralen-PEC>3-biotin can intercalate into double stranded DNA or RNA segments and covalently link to thymine bases via a photoreaction process.

Psoralens, or furocoumarins, are a class of heterocyclic aromatic compounds used in photochemotherapy treatment of a variety of skin diseases such as psoriasis, vitiligo, mycosis fungoides, polymorphous light eruption, and more [68-71]. The compounds are present in numerous plants throughout the world. In photoche-... 

In the oxygen-independent Type III reactions the excited/sensi-tized psoralen donates its excitation energy directly to, or reacts with, the target compound. This occurs if the substrate and the target compound (e.g., DNA) are already in close proximity or intercalated. The reactions will proceed very rapidly via the excited singlet state, and are, typically, cyclization reactions or electron-transfer between the sensitizer and the target. In addition, the psoralen can be ionized, either directly or via the excited state, and react with the target compound in the form of a radical cation. Furocoumarins are also employed in treatment of cutaneous T-cell lymphoma and some infections connected with AIDS, by so-called photopheresis processes [71, 74-76]. In this case, peripheral blood is exposed to, e.g., photoactivated (sensitized) 8-methoxypsoralen (8-MOP) in an extracorporeal flow system. This... 

Several modifications of the basic parent compounds have also been suggested, primarily by methyl or methoxy substituents (cf. Fig. 4.13). Two of the most active and widely used psoralens are 8-methoxypsoralen (8-MOP) and trimethylpsoralen (TMP). With the exception of 3-CP, the substituents reduce the ionization energies by 5-10 kcal mol-1 compared with the unsubstituted parent compounds (Tab. 4.4). In bulk water the effects are somewhat smaller. The largest effects are observed for TMP and AMT. The IP of flavin are very similar to those of the unsubstituted psoralen. 

The main effects of substitution compared with the parent compounds is reduction of the various excitation energies by 0.1 eV and (usually) increased transition probabilities. We note, however, that there is a significant difference between the energies needed for excitation of the free psoralens in aqueous solu-... 

Other coumarin-related compounds include furans, found in moldy sweet potatoes, and the furan coumarin complexes (furanocoumarins) found in parsnip leaves (psoralens). They are photoreactive compounds (primary photosensitizers) that exacerbate sunburn to psoralen exposed skin. 

A well-investigated group of potent phototoxic compounds are the/wra-nocoumarins (psoralens) (Fig. 11.16), compounds that are also used as fish poisons. Furocoumarins absorb ultraviolet at around 330 nm and are converted to triplet stages these, in turn, produce singlet oxygen that damages amino acids (Fig. 11.17). 

Because the resistance of polyxenes to the toxic effects of linear furanocoumarins apparently does not extend to the angular furanocoumarins (22), we have undertaken comparative metabolic fate studies with a representative of each of these furanocoumarin classes. Tritlated psoralen or isopsoralen (Figure 1) was administered as before to last stage polyxenes caterpillars, and the distribution, elimination, and biochemical fate of the compounds determined (28). 

The disposition patterns of psoralen and Isopsoralen in polyxenes under the parameters studied were not dramatically different. As Indicated in Table III, there were no appreciable differences in the rate of excretion of radioactivity by caterpillars treated with the two compounds. In body tissues, however, levels of total radioactivity in Isopsoralen-treated caterpillars were consistently about twice those observed in psoralen-treated insects (Table IV). Further, levels of unmetabolized parent compounds retained in body tissues (where toxic effects would be expressed) were on the order of 3 times as high in caterpillars treated with the angular furanocoumarin, isopsoralen (Table V). 

The MCI spectrum of psoralen (Fig. 9.25) indicates that resonance capture is occurring so that most of the ion current is carried hy the molecular ion at m/z 186. The associated GC trace indicates that ionisation is ca 10 times more efficient for the same amount of psoralen in comparison with El. In addition, since most of the ion current is channelled into the molecular ion, an analytical method which selectively monitored the molecular ion of psoralen would be ca 40 times more sensitive if NICI conditions were used rather than El. The technique only works for compounds which are electron capturing. The small ion at m/z 218 indicates that the psoralen has also formed an adduct with traces of oxygen present in the instrument, i.e. true chemical ionisation. 

Linear furanocoumarins (psoralens) inhibit P450s as mechanism-based inactivators (suicide inhibitors). Thus, species that produce psoralens may have evolved C4H enzymes with enhanced tolerance to these compounds. Recombinant C4H from the psoralen-producing species R. graveolens showed markedly slower inhibition kinetics with psoralens, and possibly biologically significant tolerance, compared to C4H from a species that does not produce the compounds (H. tuberosus) ... 

Recognition of the photosensitizing effect of the naturally occurring furanocoumarin psoralin (desmethoxy (3-6)) led to trials of its utility for the treatment of skin diseases such as psoriasis. The partial effectiveness of this compound led to the preparation of synthetic analogues. The two commercially available drugs, methoxsalen (3-8) and trioxsalen (4-6), are used in a procedure that goes by the acronym PUVA (psoralen and UVA irradiation) for the treatment of psoriasis and other skin diseases. 

Substitution at some positions of psoralene reduce the activity, which decreases as the chain becomes longer (the 3-methyl derivative is not very active).76 Some compounds, such as 5,9-dihydroxypsoralene,77 are used as radiosensitizing drugs, and trioxasalene (2,5,9-trimethylpsora-lene) is used as a radioprotective.300- 13... 

Phenolics. The phenolics of citrus represent a varied and widely studied class of compounds. They range from the lipid-soluble methoxylated coumarins and psoralens to the water-soluble glycosides of the flavanones and flavones. They include the intensely bitter naringin and the highly insoluble hesperidin. Different classes within the group have characteristic UV spectra which have been used to detect adulterations in juices and oils. 

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