Singlet oxygen yields, optical properties, and phototoxicity of reduced derivatives of the photosensitizer cercosporin

The perylenequinone cercosporin (CR) is a singlet oxygen generating photosensitizer produced by Cercospora spp which plays a critical role in parasitism of plants by these fungi. Several lines of evidence suggest that the defense mechanism of Cercospora spp towards this toxin is the generation of a cell surface reducing environment that leads to transient reduction of CR. In order to demonstrate that reduced CR is less toxic, several derivatives of CR were synthesized. Hexaacetyl-dihydrocercosporin (HAC) was prepared by reductive acetylation of CR. Noranhydrocercosporin (NAC) resulted from dehydration of CR and tetramethyl-noranhydrodihydrocercosporin (TMNAC) was a product of reductive methylation of NAC. The perylenequinones, CR and NAC, absorb more light than their respective reduced derivatives, but are much less fluorescent: the relative fluorescence intensities of HAC, TMNAC, and dithionite-reduced CR were 80-160 times greater than that of CR and NAC. Also, CR and NAC were more efficient at generating singlet oxygen. As measured by time-resolved IR luminescence, the singlet oxygen quantum yields relative to CR (adjusted to 1.00) were 0.16, 0.19 and 0.73, respectively, for HAC, TMNAC, and NAC. Toxicity was measured by assaying for inhibition of growth of CR-sensitive fungi in constant light. The reduced derivatives were less toxic than their respective oxidized forms. None of the compounds showed significant growth inhibition in the dark with any of the fungi, or when assayed in the light with the CR-resistant fungus Cercospora kikuchii. A lipid peroxidation assay with methyl linolenate also showed that HAC was less active than CR. Thus, reduction of CR leads to greater fluorescence intensities, lower production of singlet oxygen and lower phototoxicity. These data support the hypothesis of transient cercosporin reduction as a mechanism of defense against cercosporin toxicity.