Minireviews ChemPlusChem doi.org/10.1002/cplu.202000581 1 2 3 Taking Different Roads: l-Tryptophan as the Origin of 4 5 Psilocybe Natural Products 6 [a] [b] [b] [a] 7 Claudius Lenz, Alexander Sherwood, Robert Kargbo, and Dirk Hoffmeister* 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 ChemPlusChem 2021, 86, 28–35 28 © 2020 The Authors. ChemPlusChem published by Wiley-VCH GmbH Wiley VCH Mittwoch, 30.12.2020 2101 / 181786 [S. 28/35] 1 Minireviews ChemPlusChem doi.org/10.1002/cplu.202000581 1 Psychotropic fungi of the genus Psilocybe, colloquially referred highlighted. Psilocybin and its congeners, the heterogeneous 2 to as „magic mushrooms”, are best known for their l- blue-colored psilocyl oligomers, alongside β-carbolines and 3 tryptophan-derived major natural product, psilocybin. Yet, N,N-dimethyl-l-tryptophan, are presented as well as current 4 recent research has revealed a more diverse secondary knowledge on their biosynthesis is provided. The multidiscipli- 5 metabolism that originates from this amino acid. In this nary character of natural product research is demonstrated, and 6 minireview, the focus is laid on l-tryptophan and the various pharmacological, medicinal, ecological, biochemical, and evolu- 7 Psilocybe natural products and their metabolic routes are tionary aspects are included. 8 9 1. Introduction In this minireview, we focus on the biosynthetic routes of L- 10 tryptophan-derived natural products in Psilocybe mushrooms. 11 l-Tryptophan (Figure 1) is an intriguing biomolecule in its own After looking at their biosynthesis we move on to brief forays 12 right. Among the 20 canonical proteinogenic amino acids, it is into research areas alongside the chemistry and into the 13 the only bicyclic structure, and has as the highest number of C- evolutionary aspects that encompass these compounds. 14 atoms.[1] Its de novo biosynthesis from d-erythrose-4-phosphate Usually, these fungi (e.g., Psilocybe cyanescens, Figure 2) are 15 and phosphoenol pyruvate requires 13 steps and 12 enzymes, synonymous with their psychedelic effects due their capacity to 16 more than for any other proteinogenic amino acid, and with an produce psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine, 17 average of 1.1%, its abundance in proteins is usually the lowest Scheme 1).[4] This metabolite is the direct precursor of the 18 of all amino acids.[1] The biogenesis of neurotransmitters and psychotropic psilocin (Figure 1) that earned these fungi a cult 19 hormones such as serotonin (5-hydroxytryptamine, 5-HT, Fig- status and is the reason why they have been dubbed „magic 20 ure 1) or melatonin begins with l-tryptophan.[2] From the mushrooms”. However, recent research has identified a more 21 perspective of natural product chemistry, a virtually limitless diverse set of indole compounds that were not previously 22 arsenal of bioactive plant and microbial metabolites originates recognized. These variable yet relatively simple compounds 23 from this particular amino acid, among them ergotamine, illustrate how natural product biosynthesis has evolved to 24 quinine, and ajmaline.[3] create structural diversity by transforming one generic precur- 25 sor l-tryptophan into bioactive molecules. 26 27 28 29 2. l-Tryptophan-derived natural products in 30 Psilocybe species 31 32 2.1. Monomeric psilocybin and its congeners 33 34 2.1.1. The route from l-tryptophan to psilocybin 35 36 A hallmark feature of Psilocybe mushrooms and other genera is 37 the biosynthesis of their signature natural product psilocybin.[4] 38 Psilocin, the dephosphorylated metabolite formed immediately 39 upon ingestion, subsequently acts as a ligand and partial 40 agonist of the 5-HT receptor in humans, thereby profoundly 41 2A impacting human perception and consciousness.[5] For millen- 42 nia, humans have made use of this phenomenal mushroom 43 l pharmacology, which is deeply rooted in Central American 44 Figure 1. Structures of -tryptophan, Psilocybe indole alkaloids, and other bioactive indoleethylamines. cultures for spiritual and divinatory purposes. The historical, 45 pharmacological, and medicinal aspects have been reviewed.[5,6] 46 [a] C. Lenz, Prof. Dr. D. Hoffmeister The isolation and structure elucidation of psilocybin was 47 Department Pharmaceutical Microbiology at the Hans-Knöll-Institute first described in the late 1950‘s by Albert Hofmann and 48 Friedrich-Schiller-Universität colleagues at Sandoz Laboratories.[4] They used [β-14C]-l- 49 Beutenbergstrasse 11a, 07745 Jena (Germany) E-mail: [email protected] tryptophan for investigative experiments that proved psilocy- 50 [b] Dr. A. Sherwood, Dr. R. Kargbo bin’s origin from this building block.[7] Less than a decade later, 51 The Usona Institute Agurell and Nilsson utilized the same concept, used various 52 2800 Woods Hollow Road, radiolabeled precursors and presented a sequence of biosyn- 53 Madison, 53711, WI (USA) © 2020 The Authors. ChemPlusChem published by Wiley-VCH GmbH. This is thetic events which metabolize l-tryptophan to psilocybin. 54 an open access article under the terms of the Creative Commons Attribution These authors proposed a five-step biosynthesis via decarbox- 55 Non-Commercial NoDerivs License, which permits use and distribution in ylation to tryptamine as the initial step, followed by two 56 any medium, provided the original work is properly cited, the use is non- commercial and no modifications or adaptations are made. methyltransfers, then 4-hydroxylation, and 4-O-phosphorylation 57 ChemPlusChem 2021, 86, 28–35 www.chempluschem.org 29 © 2020 The Authors. ChemPlusChem published by Wiley-VCH GmbH Wiley VCH Mittwoch, 30.12.2020 2101 / 181786 [S. 29/35] 1 Minireviews ChemPlusChem doi.org/10.1002/cplu.202000581 efficiency. Therefore, the cell is protected from psilocin, as it is 1 readily oxidized and oligomerizes (see section 2.2).[11] 2 3 4 2.1.2. Psilocybin’s clinical promise 5 6 Archaeological evidence has suggested that psilocybin in mush- 7 rooms has likely been consumed by humans for thousands of 8 years.[15] Given this familiarity and its demonstrated suitable 9 pharmacological properties, such as wide therapeutic index, 10 oral bioavailability, and acceptable duration of action, psilocy- 11 bin as a prodrug to the actual active compound psilocin is 12 considered an ideal material to explore in controlled clinical 13 trials.[16] With more than ten completed clinical trials over the 14 past decade, the therapeutic potential of psychedelics has 15 drawn considerable attention, particularly in the field of 16 Figure 2. Psilocybe cyanescens, a psilocybin and β-carboline producer that occurs in Europe and North America. This species grows on lignin-rich psychiatry.[17] For example, participants treated with psilocybin 17 substrates, such as wood chips, used to mulch plant beds or park areas. The for alcohol use disorder (AUD)[18] or tobacco addiction[19] have 18 mushrooms typically grow in clusters and appear in late fall when the both demonstrated impressive improvements in cessation post- 19 temperature has dropped below ca. 10 °C (50 °F). The mature specimen shows the characteristic wavy and dark-edged cap. treatment. Psilocybin-assisted therapy has also demonstrated 20 efficacy in the treatment of depression and anxiety in cancer 21 patients[20] as well as in an open-label study on treatment 22 resistant depression (TRD).[21] In response to the promising early 23 as the terminal step.[8] Recent work identified the genes clinical data, psilocybin has been granted breakthrough desig- 24 encoding psilocybin biosynthesis enzymes in Psilocybe and nation by the United States Food and Drug Administration 25 other genera.[9] The metabolic pathway was shown by the (FDA) for both TRD and major depressive disorder (MDD) in 26 activity of four Psilocybe cubensis enzymes (PsiD, PsiH, PsiK, and 2018 and 2019, respectively. Both disorders represent condi- 27 PsiM), which provide l-tryptophan decarboxylase, indole-4- tions with an unmet need where patients have not improved 28 monooxygenase, kinase, and N-methyltransferase activity, using conventional antidepressant treatments. 29 respectively.[9a,10,11] Characterization of these enzymes confirmed Currently available treatment options for adversely affected 30 l-tryptophan as the first substrate in the pathway. This research mental health fall short on several metrics and the need for 31 led to a revised biosynthetic sequence in which methylation, new approaches is urgent and highly desirable. Psilocybin- 32 rather than phosphorylation, concludes the biosynthesis assisted treatment is attractive because it requires only a small 33 (Scheme 1). In the case of aeruginascin (Figure 1), the quater- dose, has been shown to be non-addictive, and has demon- 34 nary ammonium derivative of psilocybin found mainly in strated potential efficacy with possibly a once in a lifetime 35 Inocybe mushrooms,[12] a third N-methyltransfer takes place. treatment.[17] Collectively, psilocybin does not fit well into a 36 Collectively, these results identified norbaeocystin and traditional model of profit-driven pharmaceutical development. 37 baeocystin[13] as biosynthetic intermediates, and norpsilocin[14] Furthermore, because psilocybin is naturally-occurring and well- 38 (Figure 1) as a shunt product. Contrasting the previously described, patentability possibilities are minimal. For these 39 assumed role as psilocybin’s direct precursor, psilocin is not a reasons, early developmental efforts and gaps in funding have 40 pathway intermediate at all. Rather, the combined enzyme been met philanthropically by non-profit organizations such as 41 specificities cooperatively prevent psilocin formation.[9a,11] Most the Multidisciplinary Association for Psychedelic Studies (MAPS), 42 intriguingly, the kinase PsiK plays a dual biosynthetic and Usona Institute, and The Heffter Research Institute.[22] A unifying 43 protective role: during biosynthesis, it phosphorylates 4-hydrox- goal of these organizations has been to further the under- 44 ytryptamine to norbaeocystin.