& NaturalProducts |ReviewsShowcase| Production Options for Psilocybin:Making of the Magic Janis Fricke, ClaudiusLenz, JonasWick,Felix Blei, and Dirk Hoffmeister*[a] Chem. Eur.J.2019, 25,897 –903 biotechnological productioninvitro and in aheterologous Abstract: The fungal genus Psilocybe and other genera system in vivo. We also review synthetic routes to 1 and in- comprise numerousmushroom species that biosynthesize clude aview back on the history of 1 and ahead to its future psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine). It as avaluable therapeutic. represents the prodrug to its dephosphorylated psycho- tropic analogue,psilocin. The colloquial term “magic mushrooms” for these fungi alludes to their hallucinogenic Spotlights on Psilocybin’sHistory effects and to their use as recreational drugs.However, clinical trials have recognized psilocybin as avaluable can- “The first thing which they ate at the gathering was small, didate to be developed into amedication against depres- dark mushrooms(…). These are inebriating and induce vi- sion and anxiety. We here highlight its recently elucidated sions to be seen and even provoke sensuousness.” biosynthesis, the concurrently developed concept of enzy- (Bernardino de Sahagffln, 1499–1590) matic in vitro and heterologousinvivo production, along with previous synthetic routes. The prospect of psilocybin In his eminent Historiageneral de las cosas de la Nueva as apromising therapeutic may entail an increased Espana (General History of the Things of New Spain), the Fran- demand, which can be met by biotechnological produc- ciscan friar Bernardino de Sahagfflnprovided an extraordinary tion. Therefore, we also briefly touch on psilocybin’s thera- ethnographic account on the indigenous people of today’s peutic relevance and pharmacology. Mexico,their culture, and their ceremonies. His report also documentsthe phenomenal pharmacological effects of psy- chotropic and hallucinogenic tryptamine-like alkaloids:psilocin (4-hydroxy-N,N-dimethyltryptamine, 2,Figure 1) is the actual Introduction psychotropic principle and unstabledephosphorylated follow- up product of 1,which is more stable and the biosynthetically For decades, pharmaceutical chemistry has recognized micro- made metabolite. Minor mushroom alkaloids include norbaeo- bial natural products as avaluable source for new drugs and cystin (3), baeocystin (4), norpsilocin (5), and the quaternary drug leads.[1] Research has primarily been driven by the quest ammonium salt of 1,aeruginascin (6)asnon-, mono-, and tri- for new anti-infectives and anticancer compounds. Fungal me- methylated congeners.[4] tabolites have also served as immunosuppressants and lipid- Botanist Richard E. Schultes (1915–2001) investigated the lowering agents.[2] Depression and cancer-related anxiety are lost and misinterpretedidentityofthe “plant”that causedthe surely not among the conditions that would readily be associ- effectsdescribed in the ancientreports from CentralAmerica[5] ated with afungal product as apromising treatment option. and the associated mushroomsofthe genus Panaeolus (which However,psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine, includes speciesthat produce 1). Ethnomycologist R. Gordon 1,Figure 1), currently entering phase III clinical trials, is exactly Wasson (1898–1986) and his wife Valentina (1901–1958) col- lected specimens and had the opportunity to participate in a mushroomritual of native Mexicans.The fungi were identified by mycologist Roger Heim (1900–1979) as members of the genera Stropharia, Conocybe,and Psilocybe.[6] He also provided chemistAlbert Hofmann(1906–2008)atSandoz Laboratories with Psilocybe mexicana fruiting bodies, who succeeded in iso- lating 1 and 2 and in elucidating their structures.[4a–c] Subse- quently,close to 200 species, in the above genera and in Gym- Figure 1. Chemical structures of psilocybin and related alkaloids from Psilo- nopilus, Pluteus,and Inocybe species, have been confirmed as cybe speciesand other “magic mushrooms”. producersof1.The content of 1 dependsonthe extraction and workup method, as it may decompose to 2.[4e] Reported that.[3] It is the major metabolite of the hallucinogenic so- values are typically about 0.85%ofthe dry mass for Psilocybe called magic mushrooms and undoubtedly represents one of serbica,approximately 1.0%for P. semilanceata,and around the most prominentnaturalproducts. We present the recently 1.5–1.8 %inP. azurescens.[7] characterized biosynthesis enzymes of 1 and the concept of its Structurally,thesealkaloids are fairly simple and achiral, and they are closely relatedtothe neurotransmitterserotonin [a] J. Fricke, C. Lenz, J. Wick, F. Blei, Prof. Dr.D.Hoffmeister (5-hydroxytryptamine). Compound 1 has amolecular mass of Department Pharmaceutical Microbiology m/z=284.1 (C12H17N2O4P), yet it uniquelycombines two struc- Hans-Knçll-Institute Friedrich-Schiller-Universität tural elements that are, each in their own way,unusual. First, a Beutenbergstrasse 11a, 07745 Jena (Germany) 4-hydroxyindole moiety is avery rare and distinctive structural E-mail:[email protected] feature amongnaturalproducts. Second, 1 (and 3, 4,and 6 The ORCID identification number(s) for the author(s) of this articlecan be found under:https://doi.org/10.1002/chem.201802758. alike) features aphosphate ester,which is also comparatively Selected by the EditorialOffice for our Showcaseofoutstanding Review- rare in natural products,thoughisfrequently found in energy type articles (www.chemeurj.org/showcase). metabolism and cellular signaling. Chem. Eur.J.2019, 25,897 –903 898 Pharmacology of Psilocybin How Mushrooms Make Psilocybin Acloser look at the pharmacology of 1 clarifies the reason The historically earliestand still popular—albeit now illicit— behind the “magic” (i.e.,the hallucinogenic effects) of the way for humans to have access to 1 is to ingest the producing above fungi (Figure 2): after ingestion, cleavage of the phos- organism,that is, to use its biosynthetic capacity,but how do mushrooms produce 1? Its origin from l-tryptophan (8)was established early on.[9] Today’s knowledge on its biosynthesis is rooted in seminal work by pharmaceutical chemists Stig Agurell (1932–2018) and J. Lars Nilsson (1938–2014). They synthesizedvarious 14C- and 3H-radiolabeled presumed precursors, fed them to fungal my- celium,and traced incorporation into 1.Intheir work, these authors propose afive-stepbiosynthesis beginning with 8 that successively undergoes decarboxylation to tryptamine (9), re- peated methylation to N,N-dimethyltryptamine (10), 4-hydrox- ylation to yield 2,and 4-O-phosphorylation leading to the for- mation of 1 (Scheme 1).[10] On the basis of their results, these authors also hypothesize that alternative pathways to 1 may exist. Half acentury later,genomics greatly helped advance our understanding of the biosynthetic events. The genome se- quences of various species producing 1,including P. cubensis,a European isolate of the wider P. cyanescens species complex (P. serbica), aNorth Americanstrain of P. cyanescens sensu stric- to, Panaeolus cyanescens,and Gymnopilus dilepis have been re- ported.[11] These speciesshare aset of genes in an approxi- mately 11–22-kb portion in their genomes that encodes four biosynthesis enzymes along with transporters (Figure 3). Heter- ologous productionofthe putative decarboxylase PsiD, the kinase PsiK, and the methyltransferase PsiM in Escherichia coli and in vitro activity assays were performed. Aspergillus niger was used for in vivo characterization of PsiH, aputative P450 Figure 2. Mature fruitingbodies of a) P. cyanescens and b) P. semilanceata as monooxygenase. The activities found confirmed the hypothesis producersof1.c)Cultivated mushrooms of an unpigmented P. cubensis strain. d) Psilocybe sclerotia containing 1—Sclerotia are resilient fungalstruc- that these enzymes catalyze the formation of 1.Concurrently, tures that withstandunfavorable environmental conditions. the substrate specificity profiles revealed unexpected results regarding the order of biosynthetic eventsand the emerging optionofthe biotechnological productionof1 phate ester occurs, which convertsprodrug 1 into 2.[7c] The PsiD catalyzes the decarboxylation of 8 into 9 as the initial latter compound interferes with serotonergic neurotransmis- step but is not strictly specific,asitalso decarboxylates 4-hy- sion, as it agonistically acts on 5-hydroxytryptamine (5-HT)2A droxy-l-tryptophan (12), whichisadvantageous for the in vitro and 5-HT2C receptors (equilibrium dissociation constant, Ki = production of 1.Curiously,PsiD is entirely unrelated to known 6nm for the 5-HT2A receptor) and, to alesser extent,on5-HT1A fungal and plant aromatic l-aminoacid decarboxylases. Rather, receptors of the human nervoussystem.[7c,8] The threshold it belongs to the family of pyridoxal 5’-phosphate (PLP)-inde- dose for 1 is about 5mg, and uptake of 12–20 mg per os (oral pendentphosphatidylserine decarboxylases.[11a] [8] delivery) causes an altered state of consciousness. Alethal PsiH is aP450 monooxygenase that selectivelyhydroxylates dose can only be estimated theoretically.[7c] Somaticeffects in- the 4-position of 9 to produce 4-hydroxytryptamine (13)asa clude dilated pupils, alteredheart rate and bloodpressure, second biosynthetic step (Scheme1).[11a] among others. The perceptual effects become manifested in PsiK catalyzes the subsequent phospho-transfer step onto an alteredsense of time (it appears to be virtually standing 13 to yield 3.Opposite to the previousview, 2 is not the pre- still), synaesthesia,