Besides 1 and its congeners, other amino‐acid derived natural products have not been reported yet from Psilocybe mushrooms. Therefore, their secondary metabolomes appear surprisingly little understood, despite 60 years of intensive research. We addressed this knowledge gap and describe here an in‐depth re‐analysis of natural‐product profiles of five Psilocybe species. In all of them, we identified β‐carbolines as their products, i.e., a metabolic profile reminiscent of the active principles of ayahuasca.

Another entheogen that has traditionally been consumed in spiritual and healing ceremonies is a psychotropic brew, known by its vernacular name ayahuasca, a Quechua term literally meaning “vine of the souls”. Unlike Psilocybe mushrooms, it is not the product of a single biological species. Rather, ayahuasca consists of leaves of N , N ‐dimethyltryptamine (DMT, 3 , Scheme 1 ) producers, e.g., Psychotria viridis (Rubiaceae, coffee family). 9 Compound 3 is inactive when taken up orally, but becomes neuroactive in the presence of MAO A inhibitors that prevent 3 degradation in the human gut (Scheme 1 ). Such inhibitors are present in ayahuasca as well, because its second ingredient is the bark of the jungle vine Banisteriopsis caapi (Malpighiaceae), which produces β‐carbolines, which are strong reversible MAO inhibitors. 8 Ayahuasca's synergism, caused by two separate species, has empirically been discovered in pre‐Columbian times by South American natives. 9 It compensates the fact that synchronous production of a bioactive compound and the inhibitor of its own degradation as enhancer in one single species is unprecedented for psychotropic natural products.

Since ancient times, vision‐inducing, consciousness‐altering natural products, so‐called entheogens, have been used for spiritual purposes. The producing plants or fungi have accompanied humankind and impacted the genesis of culture and religion. 1 Indisputably, mushrooms producing psilocybin ( 1 , Scheme 1 ) rank among the most prominent entheogens and were considered the “flesh of the gods” (teonanacatl) by the Aztecs. 1 Numerous species within the fungal genus Psilocybe and other genera biosynthesize 1 which represents the phosphorylated prodrug to the psychotropic agent psilocin ( 2 ), 2 first described by Albert Hofmann and co‐workers sixty years ago. 3 Subsequently, N‐methylated l‐tryptophan as well as indoleethylamines, i.e., the intermediates of 1 baeocystin, norbaeocystin, and norpsilocin were discovered. 4 Compound 2 interferes with serotonergic neurotransmission because it acts as a partial agonist primarily on the 5‐hydroxytryptamine (5‐HT) 2A ‐receptor. 5 The perceptual and somatic effects include synesthesia, visual hallucinations, dilated pupils, and others. 6 The effects last for several hours before they subside when 2 is eliminated both renally through O‐glucuronylation and by formation of 4‐hydroxyindol‐3‐yl‐acetaldehyde (Scheme 1 ). The latter process is catalyzed by the monoamine oxidase isozyme A (MAO A), 7 a mitochondrial flavin‐dependent enzyme that oxidatively deaminates serotonin and other biogenic and neuroactive amines. Consequently, MAO inhibitors generally increase the pharmacological effects of such bioactive amines.

Results and Discussion

In the course of metabolic profiling of carpophores of Psilocybe mexicana, we routinely extracted with methanol, using a published protocol,4c and analyzed the crude extracts by LC‐HR‐ESI‐MS. As expected, 1, its immediate biosynthetic precursors baeocystin and norbaeocystin, and low amounts of its dephosphorylated follow‐up compound 2 were detected. However, we also identified two very minor mass spectrometric signals that showed retention times and masses dissimilar to those of authentic standards of 1 and its precursors (Figure 1 A). These signals appeared at t R =4.53 min (m/z=183.0916 [M+H]+) and at t R =4.89 min (m/z=213.1022 [M+H]+). We hypothesized that β‐carbolines may account for these signals as the observed masses are in good agreement with that of harmane (4, Figure 1) and harmine (5).10 Upon exposure to UV light, β‐carbolines fluoresce.11 Therefore, we repeated the analysis, this time using an acidic aqueous mushroom extract and an HPLC instrument interfaced to a fluorescence detector, excitation was at λ=340 nm, emission was recorded at λ=410 nm. The signals were detected again, and authentic 4 and 5 standards showed identical retention times and masses (Figure 1 B).

Figure 1 Open in figure viewer PowerPoint A) Chromatography of methanolic P. mexicana extracts. Top trace: overlaid extracted ion chromatogram (mass tolerance=0.1 ppm) for the masses of norbaeocystin (m/z=257.0680 [M+H]+, t R =1.33 min), baeocystin (m/z=271.0836 [M+H]+, t R =1.43 min), psilocybin (1, m/z=285.0992 [M+H]+, t R =1.53 min), and psilocin (2, m/z=205.1333 [M+H]+, t R =3.01 min). Below, extracted ion chromatograms for the masses of harmane (4, m/z=183.0916 [M+H]+) and harmine (5, m/z=213.1022 [M+H]+). Bottom: UV/Vis chromatogram (recorded at λ=300 nm, portion from 4.25–5.25 min expanded) and mass spectra. B) HPLC analysis with fluorescence detection. Upper trace: overlaid chromatograms of authentic 4 and 5, lower trace: acidic aqueous P. mexicana mushroom extract. C) HPLC analysis with fluorescence detection. Upper trace: overlaid chromatograms of authentic 4–7, traces a–d: carpophores of P. cyanescens, P. cubensis FSU12410, P. cubensis FSU12407, and P. semilanceata, respectively. Trace e: P. mexicana sclerotia, traces f and g: P. mexicana and P. cubensis mycelium. D) Chemical structures of β‐carbolines identified as Psilocybe natural products during this study, and of known Psilocybe indole alkaloids baeocystin, norbaeocystin, and norpsilocin.

We analyzed acidic aqueous extracts of other Psilocybe species by HPLC and fluorescence detection (Figure 1 C) to investigate if β‐carbolines were present in those fungi as well. Compound 4 and, in lower quantities, 5 were found (t R =2.98 and 3.16 min) in carpophores of P. cyanescens, P. semilanceata, and of two P. cubensis isolates, as well as in P. mexicana (both sclerotia and mycelium), and in P. cubensis mycelium. In addition to the above‐mentioned β‐carbolines, we detected norharmane (6, t R =2.85 min, Figure 1) and perlolyrine (7, t R =3.49 min), and identified them by their masses (m/z=169.0763 and 265.0974 [M+H]+) and by comparison with synthetic standards. The latter compound is known as a plant alkaloid from Codonopsis pilosula (Campanulaceae, bellflower family).12 Overall, the β‐carboline pattern was quantitatively and qualitatively inhomogeneous among species, yet indicated that their occurrence is i) more widespread within the genus Psilocybe and ii) independent of the developmental stage. For final evidence that Psilocybe fungi contain β‐carbolines, we purified the two major compounds from P. cubensis carpophores. Subsequent 1D and 2D NMR spectroscopy resulted in spectra (Figures S1–S10, Table S1, Supporting Information) that were identical to reported data for 4 and 5.13

Biosynthetically, β‐carbolines derive from tryptamine and have been isolated from plants, bacteria, and various fungi including basidiomycetes.10, 14 To confirm that the compounds are intrinsic Psilocybe products, we carried out stable‐isotope labeling with 13C 11 ‐l‐tryptophan and P. mexicana mycelium in liquid axenic culture under controlled laboratory conditions, along with an unlabeled control, and detected 4, 6, and 7 again. In the stable‐isotope‐treated cultures, the masses of the carbolines expectedly increased by ten mass units (Figure 2). This is compatible with the incorporation of ten 13C atoms, i.e., a 13C 10 ‐tryptamine moiety. Thus, we had excluded a carboline source other than Psilocybe’s intrinsic cellular metabolism.

Figure 2 Open in figure viewer PowerPoint LC‐MS analysis of P. mexicana mycelial extracts after 13C stable‐isotope labeling. The generic labeling pattern is shown by red carbon atoms. UHPLC chromatograms were recorded at λ=300 nm. Top trace: overlaid chromatograms of standards 4–10. Center trace: culture grown with unlabeled l‐tryptophan (control). Bottom trace: culture grown in the presence of 13C 11 ‐l‐tryptophan. Below, HR‐ESI‐MS spectra are shown. Blue: spectra for t R =4.26–4.28 min with coeluting 6 and the isomer of 8 (panel A: unlabeled, panel B: 13C‐labeled situation). Green: spectra for t R =4.50–4.52 min showing 4 and 9/10 coeluting, panel C: unlabeled, panel D: 13C‐labeled. Red: spectra for t R =4.94 min showing 7, panel E: unlabeled, panel F: 13C‐labeled. Upper right: UV/Vis spectra of 4 and collective spectra of the β‐carbolines, detected at t R =4.50 min.

We detected two further compounds in minor quantities. The first one whose mass was identical to that of harmol (8, m/z=199.0869 [M+H]+) was eluted at t R =4.26 min. However, authentic 8 showed an shorter retention time (t R =3.99 min, Figure 2), which points to an isomer of 8 as Psilocybe metabolite. P. mexicana mycelium also contained a compound at t R =4.89 min (m/z=213.1025 [M+H]+). Even though this molecular mass is identical to that of 5, the retention time was not, as this unidentified compound virtually co‐eluted with 4 at t R =4.53 min.

This mass is consistent with that of cordysinins C and D (9 and 10), i.e., enantiomeric β‐carbolines described from the caterpillar fungus Ophiocordyceps sinensis.15 Comparison with a synthesized mixture of 9 and 10 confirmed that one of those compounds, or both, is a P. mexicana metabolite as well.

P. cubensis FSU12410 mycelia and carpophores were used to quantify the concentration of 4, i.e., the major β‐carboline in the fungal biomass (Figure 1 C, Table S2, Supporting Information). Although mycelia showed a concentration of 21 μg g−1 dried biomass, we found a 100‐fold lower concentration in the carpophores (0.2 μg g−1). Sclerotia of P. mexicana contained 1.4 μg g−1 4 and 1.6 μg g−1 5. Next, we used MALDI imaging to investigate the spatial distribution of 4 in fungal mycelium. An actively growing P. cubensis culture was screened for a compound with m/z 183.1(±0.7) Da, which corresponds to 4 (Figure 3). The signals of maximum intensity localized to the hyphal tips while more mature areas showed low abundance.

Figure 3 Open in figure viewer PowerPoint MALDI‐MS imaging of P. cubensis mycelium. The image was taken to detect m/z 183.1(±0.7) Da, i.e., the mass of 4 [M+H]+, and a portion was overlaid on a photograph of the mycelium. Peripheral areas of the mycelium showed highest abundance (red). The image was digitally optimized for brightness which sets the maximum intensity to 60 % of the initial image.