Indole diterpenoid secondary metabolites produced by the Epichloë festucae var. lolii-Lolium perenne symbiosis

The indole diterpenoid intermediates of lolitrems in a crude extract of Lolium perenne (L. perenne) seed infected with Epichloë festucae var. lolii (E. festucae var. lolii) and their biological activity against porina larvae were investigated. Liquid-liquid partitioning of the crude extract, together with a series of bench columns (normal and reversed phase) and semi-preparative High Performance Liquid Chromatography (HPLC) yielded the novel compounds A – D (64 – 67). Mass spectrometry (MS) and Nuclear Magnetic Resonance (NMR) spectroscopy were used to determine the structures of these alkaloids. The indole moiety of known indole diterpenoids was modified to a six-membered ring in the novel compounds A – D (64 – 67), with the right side of these molecules similar to that of lolitrem B (3) and terpendole C (49). Compounds A – D (64 – 67) differ in the stereochemistry and substituent at C-2. The structural features of these compounds are very unusual. These contain a spiro centre that fuses the modified indole region to the right side, indicating that they may be artefacts rather than naturally occurring, which in turn, could explain why they are not detectable in the Liquid Chromatography Mass Spectrometry (LC-MS) analysis of fresh extract. However, further work would be required to confirm this. The known alkaloids, terpendole D (50), 13-desoxypaxilline (37) and paspaline (42) were also isolated, together with a potential emindole analogue. This is the first report of terpendole D (50) from this ryegrass-endophyte association. The presence of an emindole analogue suggested that further intermediates are yet to be isolated from this association as indicated in the LC MS analysis of the crude extract. However, due to the scarcity of the emindole analogue, complete structural confirmation was not possible. The biological activity of paxilline (36), lolitrem B (3), 13-desoxypaxilline (37), paspaline (42), terpendole D (50) and compounds A (64) and B (65) on porina larvae was determined using a semi-synthetic diet feeding bioassay. These compounds had not been previously tested on porina. Paxilline (36) had a dose-dependent effect on both food consumption and larval weight. Diet consumption and weight gain of porina larvae were significantly reduced by paxilline at concentrations of 2.5, 5 and 10 μg/g but not at concentrations of 1.0 μg/g, compared to dimethyl sulfoxide (DMSO) solvent control diet. Lolitrem B (3) had a similar effect on consumption and weight gain to that of paxilline (36) at concentrations of 5 and 10 μg/g but there was no indication that 13-desoxypaxilline (37), terpendole D (50), paspaline (42), compound A (64) or compound B (65) reduced feeding by porina larvae.