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The natural product hypericin was tested in recent years as a biological photosensitizer with a potential for viral and cellular photodamage. We thus studied extensively its spectroscopy and membrane partitioning. Absorption, fluorescence excitation and emission spectra of the sodium salt (HyNa) were measured in 36 protic and aprotic, polar and apolar, solvents. Electronic transition bands as well as vibrational progressions were identified. Aggregation in some nonpolar solvents and protonation in organic acids were demonstrated. Modeling solvatochromism was done by Lippert equation, by the ET(30) parameter and by the Taft multiparameter approach. In all cases, separation into protic and aprotic solvents gave much better fits to the models. 13C chemical shift data could also be correlated with solvent polarity. They correlated best with Lippert's delta f polarity measure, but tended to fall into two distinct solvent groups--each along different lines--corresponding to protic and aprotic media, respectively. This interesting phenomenon suggests that in the case of the charged and slightly water soluble HyNa, two mechanisms of solvation are involved, each resulting in its own line equation. In aprotic media, dipole-dipole interaction is the predominant solvation mechanism. In protic solvents, the most effective means of solvation is likely to be hydrogen bonding. When intercalated into the liposomal phospholipid bilayer, HyNa is oriented at an angle to the interface, thus experiencing a gradient of solvent polarities: a highly polar environment (similar to methanol) for C-2/5, suggesting that they lie not far from the interface; a moderately polar environment (similar to that of n-propanol) for C-6a/14a, which are somewhat deeper within the bilayer; and a more lipophilic environment (akin to n-hexanol) for C-10/11. The fluorescence excitation peak in liposomes also correlates with an aprotic medium of relatively high polarity, as might be excepted from a molecule in a shallow position in the bilayer.

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Low temperature base catalyzed autoxidation (BCA) of the A-ring of 21-acetoxypregn-5-ene-3,20-dione 20-ethylene ketal (7) resulted in the saponification of the ester with the concomitant formation of 2,21-dihydroxypregna-1,4-diene-3,20-dione 20-ethylene ketal (8). Continued BCA at ambient temperature, converts the latter to 1,21-dihydroxy-2-oxaprogesterone 20-ethylene ketal (9), which is reduced by NaBH4 to the 2-oxasteroid, 21-hydroxy-2-oxaprogesterone 20-ethylene ketal (10). Treatment of enol 8, lactol 9, and lactone 10 with aqueous acid generates the corresponding deprotected analogs 2,21-dihydroxypregna-1,4-diene-3,20-dione (enol 11), 1,21-dihydroxy-2-oxaprogesterone (lactol 12), and 2-oxacortexone (2-oxadesoxycorticosterone, 21-hydroxy-2-oxaprogesterone, lactone 13). In bovine spermatozoa, neither 2-oxasteroid ketal 10 nor its deprotected analog 13 stimulated Ca2+ uptake. In high concentration (0.5 mM), the inhibition of Ca2+ uptake is only 37% for 13, as compared to 83% found with the parent steroid, cortexone (desoxycorticosterone, 21-hydroxyprogesterone, 5). The difference in molecular structure between 13 and 5 indicates the importance of the oxygen atom in ring A in achieving the protective effect of the steroid. Ketalization of the C-20 carbonyl is not important for protection. Thus it seems that by replacing C-2 by an oxygen atom we can reduce the biological damage caused by relatively high concentrations of steroid treatment. These results are highly significant when treatment of patients with high doses of steroids is considered.

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--2-(Dimethylamino) fluorene (1a) and 5-benzoyloxy-2,3,7,8,12,13,17,18-octaethylporphyrin (4) react with superoxide anion radical (generated from KO2/18-crown-6 polyether) in aprotic media. Yet, when incorporated into the lipid bilayer of dimyristoyl phosphatidylcholine liposomes, these two substrates are inert to superoxide, generated enzymatically (xanthine oxidase/acetaldehyde) or radiolytically (60Co or 137Cs source/formate solution). On the other hand, 7-acetoxy-4-methylcoumarin (6), which reacts with superoxide in aprotic media yielding the corresponding 4-methylumbelliferone (7), also gives the same product when incorporated within the liposomal bilayer and reacted with radiolytically or enzymatically generated superoxide. In the latter case, the reaction is inhibited by SOD. NMR studies indicate that in contradistinction to the highly lipophilic 1a and 4, which presumably lie well within the lipid bilayer, 7 lies in a highly polar region of the bilayer. These results suggest that superoxide anion does not penetrate deep into the liposomal bilayer; nevertheless, superoxide reactions can, indeed, be observed, provided the active site of the substrate lies at or near the lipid-water interface.

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The superoxide-mediated base catalyzed autoxidation of alpha-oxo enols is initiated by the deprotonation of the labile hydroxyl group. Thus, the reaction of O2-. (generated from KO2/crown ether in aprotic media) with 3-hydroxycoumarin (1), followed by a CH3I-workup, generates products 2-4 via a deprotonation-oxidation sequence complicated by a competing saponification of the lactone linkage. The related coumarin reductone (alpha-oxo enediol) 8 is rapidly oxidized by O2-., HO- and t-butoxide to the corresponding triketone, which in turn undergoes further oxidation and rearrangement ultimately yielding (upon methyl iodide workup) products 9-14. When the O2-. mediated oxidation is carried out under argon in completely degassed solutions, large amounts (greater than 20%) of monodeprotonation product (detected as 9) accumulate. These results are discussed in light of the differing mechanisms proposed by Sawyer and Afanas'ev for the interaction of O2-. with the reductone ascorbic acid.

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