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, were identified in extracts of perennial ryegrass (Lolium perenne) seed that was infected with the endophytic fungus Neotyphodium lolii. The lolicines 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 biosynthetic precursors of several groups of more complex indole-diterpenoids, suggesting that the lolicines might be biosynthetic precursors of the lolitrem group of indole-diterpenoid neurotoxins. During purification of 1b and 2b, a third compound, lolitrem N (4c), was isolated as its propionate (4d) and identified as 35-epilolitriol. A further late-eluting compound was isolated as its acetate from the same fraction as the lolicines. This compound was found to be identical with lolitriol 10-O-acetate (4b) prepared from lolitrem B (3a). This finding confirms lolitriol (4a) as a natural constituent of endophyte-infected perennial ryegrass, and is consistent with the proposal that lolitriol is a biosynthetic precursor of lolitrems A (9), B (3a), and E (10). Detailed examination of the (1)H NMR spectra of 4b revealed the presence of 31-epilolitrem N 10-O-acetate (4f) as a naturally occurring contaminant, indicating that 31-epilolitrem N (4e) is a metabolite of endophyte-infected ryegrass. Analysis of the NMR spectra of 3a led to a reassignment of the C-19-C-21 resonances of the lolitrems, and identified 31-epilolitrem F (3c) as a natural constituent of L. perenne.