ISOLATION OF LOLICINE-A, LOLICINE-B, LOLITRIOL, AND LOLITREM-N FROM LOLIUM-PERENNE INFECTED WITH NEOTYPHODIUM-LOLII AND EVIDENCE FOR THE NATURAL OCCURRENCE OF 31-EPILOLITREM-N AND 31-EPILOLITREM-F

Lolicines A (1a) and B (2a), late-eluting lolitrem-like compounds, wer e identified in extracts of perennial ryegrass (Lolium perenne) seed t hat was infected with the endophytic fungus Neotyphodium lolii. The lo licines were isolated as their 11-O-propionates (1b and 2b) and their structures determined by mass spectrometry and one-and two-dimensional NMR spectroscopy. The structures of lolicines A and B were similar to those of paspaline (5a) and paspaline B (5b), compounds known to be b iosynthetic precursors of several groups of more complex indole-diterp enoids, suggesting that the lolicines might be biosynthetic precursors of the lolitrem group of indole-diterpenoid neurotoxins. During purif ication of 1b and 2b, a third compound, lolitrem N (4c), was isolated as its propionate (4d) and identified as 35-epilolitriol. A further la te-eluting compound was isolated as its acetate from the same fraction as the lolicines. This compound was found to be identical with lolitr iol 10-O-acetate (4b) prepared from lolitrem B (3a). This finding conf irms lolitriol (4a) as a natural constituent of endophyte-infected per ennial ryegrass, and is consistent with the proposal that lolitriol is a biosynthetic precursor of lolitrems A (9), B (3a), and E (10). Deta iled examination of the H-1 NMR spectra of 4b revealed the presence of 31-epilolitrem N 10-O-acetate (4f) as a naturally occurring contamina nt, indicating that 31-epilolitrem N (4e) is a metabolite of endophyte -infected ryegrass. Analysis of the NMR spectra of 3a led to a reassig nment of the C-19-C-21 resonances of the lolitrems, and identified 31- epilolitrem F (3c) as a natural constituent of L. perenne.