Isolation, structure elucidation and testing of secondary metabolites from fungi of the genus Lactarius and Mycena

Abstract

This thesis is about the isolation and structure elucidation of so far uninvestigated secondary metabolites from fruiting bodies of the mushrooms Lactarius circellatus, Lactarius trivialis and Mycena zephirus. In the Spiteller group L. circellatus has already been investigated before for its content of sequiterpenoids and five unknown and several known sequiterpenoids have been identified and their structures have been elucidated. However, their stereo configuration, the proton and carbon resonances in their NMR spectra have not been fully assigned so far. Therefore, NMR spectra of the previously isolated sequiterpenes were recorded again. Moreover, fruiting bodies of L. circellatus were extracted again and investigated for the presence of new compounds, resulting in the isolation of the so far unknown sequiterpenoid 12-hydroxy-15-carboxyblennin A (19) together with hydroxyblennin A (34). A careful analysis of the coupling constants in the 1H NMR, the analysis of the NOE correlations in the NOESY spectra, and a detailed analysis of the correlations in the HMBC spectra in respect to their dihedral angle according to the Karplus relationship allowed to determine the relative configuration of all isolated sequiterpenoids, including the so far unpublished new sequiterpenoids 12-hydroxy-15-carboxyblennin A (19), 7,15-dihydroxyblennin A (20), 2,3-epoxylactarorufin A (21),14-carboxy-deoxylactarorufin A (22), and 13-hydroxy-14-carboxy-deoxylactarorufin A (23). To determine the absolute configuration of the isolated sequiterpenoids, CD spectra were recorded and compared with the corresponding calculated CD spectra. By these means the absolute stereochemistry of the sequiterpenoids 19 to 23 could be determined. However, the measured and calculated CD spectra of 22 and 23 were not matching perfectly. So far, there have been no reports about pigments from L. circellatus, despite the fact that it exhibits on the surface of its cap a grayish green color similarly to L. blennius. Consequently, the pigment was isolated and its structure elucidated to be blennione (35), the same pigment that is present in L. blennius. Blennione (35) was found to be much more abundant in L. circellatus than in L. blennius, the mushroom it was originally isolated from. Some biological tests were performed but blennione (35) displayed no activity. In addition, tiny amount of a pink pigment from L. blennius was found related to lilacinone (37), a pink pigment that was isolated and structurally elucidated before from L. lilacinus. Fresh fruiting bodies of Lactarius trivialis display an intensive violet color on the cap surface that is fading away upon ageing of the fruiting bodies. The responsibe pigments have not been investigated so far. Therefore, fruiting bodies of L. trivialis were subjected to a comprehensive investigation of their secondary metabolites with a focus on their pigments. Not entirely unexpectedly, the isolation of the pigments from the skin cap turned out to be difficult due to the low stability of the pigments, especially when methanol was used as a solvent. However, an extraction with acetone with subesquent column chromatography on Sephadex LH20 with acetone as a solvent followed by purification of the pigments with semi-preparative HPLC resulted in the isolation of five colored compounds, that were named trivialines A (47), B, C, D, and E. Due to their HR-ESI-MS data, the compounds five pigments are closely related to each other and show a high degree of unsaturation. Only in the case of trivialine A (47) and trivialine B the isolated amount was sufficient to record a full set of one and two dimensional NMR spectra. Consequently, the structure elucidation focussed on these two compounds, both exhibiting a molecular formula of C32H20N4O6. Due to the high degree of unsaturation, the low number of unexchangable protons, leading to a lack of corellations in the HMBC, the structure elucidation turned out to be very challenging. Nevertheless, for trivialine A (47) a tentative structure was proposed. Moreover, fruiting bodies of L. trivialis were investigated for the presence of new sesquiterpenoids. Thus, the new compound 14-hydroxylactarorufin B (53) was isolated and its structure elucidated. In addition, the lactarorufin B (27) and 14-hydroxyblennin A (52), both known from other mushrooms, were also identified to be present in L. trivialis. So far, 14-hydroxyblennin A (52) was reported only from Russula sanguinea, but the published NMR data fit perfectly to 15-hydroxyblennin A (34), while the NMR data from the corresponding sesquiterpene from L. trivialis fit very well to the structure of 14-hydroxyblennin A (52). Consequently, the structure in the above mentioned manuscript about R. sanguinea has to be revised to 15-hydroxyblennin A (34). Apart from mushrooms of the genus Lactarius, in this thesis also fruiting bodies of Mycena zephirus were investigated in respect to their secondary metabolite content and the structures of two compounds were elucidated to be N5-isopentylglutamine (54) and N5-2-methylbutylglutamine (55), respectively.