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D. Hoffmeister

Chemoenzymatic Synthesis of 5-Methylpsilocybin: A Tryptamine with Potential Psychedelic Activity

February 21, 2022 / Mushrooms / Psilocybin, Papers, Pharmacology & Chemistry / Leave a Comment / By / , , , ,

Abstract

A novel analogue of psilocybin was produced by hybrid chemoenzymatic synthesis in sufficient quantity to enable bioassay. Utilizing purified 4-hydroxytryptamine kinase from Psilocybe cubensis, chemically synthesized 5-methylpsilocin (2) was enzymatically phosphorylated to provide 5-methylpsilocybin (1). The zwitterionic product was isolated from the enzymatic step with high purity utilizing a solvent-antisolvent precipitation approach. Subsequently, 1 was tested for psychedelic-like activity using the mouse head-twitch response assay, which indicated activity that was more potent than the psychedelic dimethyltryptamine, but less potent than that of psilocybin.

Fricke, J., Sherwood, A. M., Halberstadt, A. L., Kargbo, R. B., & Hoffmeister, D. (2021). Chemoenzymatic Synthesis of 5-Methylpsilocybin: A Tryptamine with Potential Psychedelic Activity. Journal of natural products, 84(4), 1403–1408. https://doi.org/10.1021/acs.jnatprod.1c00087

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Injury-Triggered Blueing Reactions of Psilocybe “Magic” Mushrooms

February 6, 2022 / Mushrooms / Psilocybin, Papers, Pharmacology & Chemistry / Leave a Comment / By / , , , , , , ,

Abstract

Upon injury, psychotropic psilocybin-producing mushrooms instantly develop an intense blue color, the chemical basis and mode of formation of which has remained elusive. We report two enzymes from Psilocybe cubensis that carry out a two-step cascade to prepare psilocybin for oxidative oligomerization that leads to blue products. The phosphatase PsiP removes the 4-O-phosphate group to yield psilocin, while PsiL oxidizes its 4-hydroxy group. The PsiL reaction was monitored by in situ 13 C NMR spectroscopy, which indicated that oxidative coupling of psilocyl residues occurs primarily via C-5. MS and IR spectroscopy indicated the formation of a heterogeneous mixture of preferentially psilocyl 3- to 13-mers and suggest multiple oligomerization routes, depending on oxidative power and substrate concentration. The results also imply that phosphate ester of psilocybin serves a reversible protective function.

Lenz, C., Wick, J., Braga, D., García-Altares, M., Lackner, G., Hertweck, C., Gressler, M., & Hoffmeister, D. (2020). Injury-Triggered Blueing Reactions of Psilocybe “Magic” Mushrooms. Angewandte Chemie (International ed. in English), 59(4), 1450–1454. https://doi.org/10.1002/anie.201910175

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Iterative l-Tryptophan Methylation in Psilocybe Evolved by Subdomain Duplication

August 11, 2018 / Biology & Ethnobotany, Mushrooms / Psilocybin, Neuroscience, Papers, Pharmacology & Chemistry, Publications / By / , , , ,

Abstract

Psilocybe mushrooms are best known for their l-tryptophan-derived psychotropic alkaloid psilocybin. Dimethylation of norbaeocystin, the precursor of psilocybin, by the enzyme PsiM is a critical step during the biosynthesis of psilocybin. However, the “magic” mushroom Psilocybe serbica also mono- and dimethylates l-tryptophan, which is incompatible with the specificity of PsiM. Here, a second methyltransferase, TrpM, was identified and functionally characterized. Mono- and dimethylation activity on l-tryptophan was reconstituted in vitro, whereas tryptamine was rejected as a substrate. Therefore, we describe a second l-tryptophan-dependent pathway in Psilocybe that is not part of the biosynthesis of psilocybin. TrpM is unrelated to PsiM but originates from a retained ancient duplication event of a portion of the egtDB gene that encodes an ergothioneine biosynthesis enzyme. During mushroom evolution, this duplicated gene was widely lost but re-evolved sporadically and independently in various genera. We propose a new secondary metabolism evolvability mechanism, in which weakly selected genes are retained through preservation in a widely distributed, conserved pathway.

Blei, F., Fricke, J., Wick, J., Slot, J. C., & Hoffmeister, D. (2018). Iterative l‐Tryptophan Methylation in Psilocybe Evolved by Subdomain Duplication. ChemBioChem, 19(20), 2160-2166., 10.1002/cbic.201800336.
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Production Options for Psilocybin: Making of the Magic

July 16, 2018 / Biology & Ethnobotany, Mushrooms / Psilocybin, Papers, Pharmacology & Chemistry, Publications / By / , , , ,

Abstract

The fungal genus Psilocybe and other genera comprise numerous mushroom species that biosynthesize psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine). It represents the prodrug to its dephosphorylated psychotropic analogue, psilocin. The colloquial term “magic mushrooms” for these fungi alludes to their hallucinogenic effects and to their use as recreational drugs. However, clinical trials have recognized psilocybin as a valuable candidate to be developed into a medication against depression and anxiety. We here highlight its recently elucidated biosynthesis, the concurrently developed concept of enzymatic in vitro and heterologous in vivo production, along with previous synthetic routes. The prospect of psilocybin as a promising therapeutic may entail an increased demand, which can be met by biotechnological production. Therefore, we also briefly touch on psilocybin’s therapeutic relevance and pharmacology.

Fricke, J., Lenz, C., Wick, J., Blei, F., & Hoffmeister, D. (2018). Production Options for Psilocybin: Making of the Magic. Chemistry–A European Journal., 10.1002/chem.201802758

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Biocatalytic production of psilocybin and derivatives in tryptophan synthase-enhanced reactions

May 11, 2018 / Mushrooms / Psilocybin, Neuroscience, Papers, Pharmacology & Chemistry, Publications / By / , , ,

Abstract

Psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine) is the main alkaloid of the fungal genus Psilocybe, the so-called “magic mushrooms.” The pharmaceutical interest in this psychotropic natural product as a future medication to treat depression and anxiety is strongly re-emerging. Here, we present an enhanced enzymatic route of psilocybin production by adding TrpB, the tryptophan synthase of the mushroom Psilocybe cubensis, to the reaction. We capitalized on its substrate flexibility and show psilocybin formation from 4-hydroxyindole and l-serine, which are less cost-intensive substrates, compared to the previous method. Furthermore, we show enzymatic production of 7-phosphoryloxytryptamine (isonorbaeocystin), a non-natural congener of the Psilocybe alkaloid norbaeocystin (4-phosphoryloxytryptamine), and of serotonin (5-hydroxytryptamine) by means of the same in vitro approach.

Blei, F., Baldeweg, F., Fricke, J., & Hoffmeister, D. (2018). Biocatalytic Production of Psilocybin and Derivatives in Tryptophan Synthase‐Enhanced Reactions. Chemistry–A European Journal, 24(40), 10028-10031., 10.1002/chem.201801047.
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Identification of ω-N-Methyl-4-hydroxytryptamine (Norpsilocin) as a Psilocybe Natural Product

September 20, 2017 / Mushrooms / Psilocybin, Papers, Pharmacology & Chemistry, Publications / By / , ,

Abstract

We report the identification of ω-N-methyl-4-hydroxytryptamine (norpsilocin, 1) from the carpophores of the hallucinogenic mushroom Psilocybe cubensis. The structure was elucidated by 1D and 2D NMR spectroscopy and high-resolution mass spectrometry. Norpsilocin has not previously been reported as a natural product. It likely represents the actual psychotropic agent liberated from its 4-phosphate ester derivative, the known natural product baeocystin. We further present a simple and artifact-free extraction method that prevents dephosphorylation and therefore helps reflect the naturally occurring metabolic profile of Psilocybe mushrooms in subsequent analyses.
Lenz, C., Wick, J., & Hoffmeister, D. (2017). Identification of ω-N-Methyl-4-hydroxytryptamine (Norpsilocin) as a Psilocybe Natural Product. Journal of Natural Products, 80(10), 2835-2838. 10.1021/acs.jnatprod.7b00407
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Enzymatic synthesis of psilocybin

August 25, 2017 / Mushrooms / Psilocybin, Papers, Pharmacology & Chemistry / By / , ,

Abstract

Psilocybin is the psychotropic tryptamine-derived natural product of Psilocybe carpophores, the so-called “magic mushrooms”. Although its structure has been known for 60 years, the enzymatic basis of its biosynthesis has remained obscure. We characterized four psilocybin biosynthesis enzymes, namely i) PsiD, which represents a new class of fungal l-tryptophan decarboxylases, ii) PsiK, which catalyzes the phosphotransfer step, iii) the methyltransferase PsiM, catalyzing iterative N-methyl transfer as the terminal biosynthetic step, and iv) PsiH, a monooxygenase. In a combined PsiD/PsiK/PsiM reaction, psilocybin was synthesized enzymatically in a step-economic route from 4-hydroxy-l-tryptophan. Given the renewed pharmaceutical interest in psilocybin, our results may lay the foundation for its biotechnological production.
Fricke, J., Blei, F., & Hoffmeister, D. (2017). Enzymatic synthesis of psilocybin. Angewandte Chemie International Edition. 10.1002/anie.201705489
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