Enzymatic Route toward 6‐Methylated Baeocystin and Psilocybin

Abstract

Psilocybin and its direct precursor baeocystin are indole alkaloids of psychotropic Psilocybe mushrooms. The pharmaceutical interesti np silocybin as at reatment optiona gainst depression and anxiety is currently being investigated in advanced clinicalt rials. Here, we reportabiocatalytic route to synthesize 6-methylated psilocybin and baeocystin from 4-hydroxy-6methyl-l-tryptophan, which was decarboxylated and phosphorylated by the Psilocybec ubensis biosynthesis enzymes PsiD and PsiK. N-Methylation was catalyzed by PsiM. We further present an in silico structural model of PsiM that revealed aw ell-conserved SAM-binding core along with peripheraln onconserved elements that likely govern substrate preferences.The indole nucleus is ac ommon motif with bioactiven atural products that is, in numerous instances, modified duringb iosynthesis. Modifications include, for example, 7-chlorination in the case of rebeccamycin, prenyl transfer to various positions to make asterriquinones, or hydroxylation at C-5 and C-6 of serotonin and amanitins,r espectively.R eserpine, ibogaine, and harmaline are methoxy-substituted att hesec arbons. Dissimilar from the above positionsa nd substituents, psilocybin( 1, Scheme 1) features aunique 4-phosphoryloxy group.Compound 1 is the principal metabolite of Psilocybe carpophores ("magic mushrooms"), whereas baeocystin (2)a nd norbaeocystin (3), the immediate metabolic precursors, are present in minor quantities. [1] The dephosphorylated analogue of 1, psilocin (4), represents the pharmacologically active, that is, psychotropic compound as it is ap artial agonist at the human 5HT 2A receptor. [2] The pharmaceutical value of 1 in the treat-ment of end-of-life anxiety and therapy refractory depression has been documentedi nv ariousa dvanced clinicalt rials. [3] Syntheses of 1 congeners date back to the late 1950s. Following the discovery of 1 by Albert Hofmann and co-workers, various series of derivatives were synthesized,w hich also contained the 6-phosphoryloxy and 6-hydroxy isomerso f1 and 4,a long with a6 -methylated, side-chain-alkylated tryptamine. [4] Subsequent efforts were made to install bromine or af ormyl group at C-5-or C-7, vary the substituents at the w-N-atom of 4,o r add various substituentst ot he aromatic ring of N,N-dimethyltryptamine (5), to produce its 6-and 7-methyl analogues, among others. [5] Despite theser esultsa nd despite the synthesis of 2-methyl derivatives of 5 and N,N-diethyltryptamine, [6] none of the previouse fforts, using synthetic chemistry,h ad focused on introducing am ethyl substituent to the indole of 1 or 2.We report the in vitro synthesis of 6-methylpsilocybin (6, Scheme1)a nd, as main products,i ts immediate precursors 6methylnorbaeocystin (7)a nd 6-methylbaeocystin (8). We address the previous lack of ring-methylated 1 analogues and simultaneously provide am odel of the PsiM structure for insight into the origin of its methylating activity on 3 and 7.W ei dentified al oop structure as criticalf or methyl acceptors ubstrate selectivity.C oncu...

S‐Adenosyl‐l‐Methionine Salvage Impacts Psilocybin Formation in “Magic” Mushrooms

“…The iterative methylation of norbaeocystin to psilocybin (Figure A) by PsiM is thought to be a rate‐limiting critical step since N ‐methyltransferases are comparably slow enzymes with k cat values ranging between 0.12 h −1 to 0.58 s −1 . Previous studies demonstrated increased psilocybin yields, when Escherichia coli SAH nucleosidase and adenine deaminase were added to the PsiM reaction to irreversibly remove SAH from the chemical equilibrium . Hence, we hypothesized SAH could inhibit PsiM and ran assays in the presence of SAH, ranging between 1–16 m m .…”

Taking Different Roads: l‐Tryptophan as the Origin of Psilocybe Natural Products

“…Recent work identified the genes encoding psilocybin biosynthesis enzymes in Psilocybe and other genera [9] . The metabolic pathway was shown by the activity of four Psilocybe cubensis enzymes (PsiD, PsiH, PsiK, and PsiM), which provide l ‐tryptophan decarboxylase, indole‐4‐monooxygenase, kinase, and N ‐methyltransferase activity, respectively [9a,10,11] . Characterization of these enzymes confirmed l ‐tryptophan as the first substrate in the pathway.…”

A draft reference assembly of the Psilocybe cubensis genome

“…773,735 CCS reads were generated. Quast (Gurevich et al 2013) was used to assess the quality of the input fasta sequence file (N50 = 13.9Kb) and the output assembly fasta file (3.33Mb N50).…”

“…This compound has been classified as a “breakthrough therapy” for depression by the FDA ( Johnson and Griffiths 2017 ). The psilocybin pathway was identified by Fricke et al, but to date no public references exist in NCBI with N50s longer than 50kb ( Fricke et al 2017 ; Blei et al 2018 ; Fricke et al 2019a ; Fricke et al 2019b ; Blei et al 2020 ; Demmler et al 2020 ; Fricke et al 2020 ). A more contiguous genome assembly can assist in further resolution of the genetic diversity in the fungi’s secondary metabolite production.…”