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. 2020 Jul 2;26(37):8281-8285.
doi: 10.1002/chem.202000134. Epub 2020 Jun 8.

Scalable Hybrid Synthetic/Biocatalytic Route to Psilocybin

Janis Fricke  1 , Robert Kargbo  2 , Lars Regestein  3 , Claudius Lenz  1 , Gundela Peschel  3 , Miriam A Rosenbaum  3 , Alexander Sherwood  2 , Dirk Hoffmeister  1
Affiliations

Scalable Hybrid Synthetic/Biocatalytic Route to Psilocybin

Janis Fricke et al. Chemistry. .

Abstract

Psilocybin, the principal indole alkaloid of Psilocybe mushrooms, is currently undergoing clinical trials as a medication against treatment-resistant depression and major depressive disorder. The psilocybin supply for pharmaceutical purposes is met by synthetic chemistry. We replaced the problematic phosphorylation step during synthesis with the mushroom kinase PsiK. This enzyme was biochemically characterized and used to produce one gram of psilocybin from psilocin within 20 minutes. We also describe a pilot-scale protocol for recombinant PsiK that yielded 150 mg enzyme in active and soluble form. Our work consolidates the simplicity of tryptamine chemistry with the specificity and selectivity of enzymatic catalysis and helps provide access to an important drug at potentially reasonable cost.

Keywords: bioorganic chemistry; biosynthesis; enzymes; fermentation; kinase; phosphorylation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Scheme 1
Scheme 1
Dual repair and biosynthetic function of P. cubensis PsiK (red). The enzyme catalyzes 4‐hydroxtryptamine phosphorylation to norbaeocystin during 1 biosynthesis or repairs 1 by re‐phosphorylation of 2. In the mushrooms, the methyltransferase PsiM (grey) completes 1 biosynthesis after the phosphorylation step.
Figure 1
Figure 1
UHPLC‐MS analysis of in vitro activity assays with recombinantly produced PsiK and its variant PsiKD224A. Top trace: overlaid chromatograms of authentic 1 and 2 standards. N: negative control with heat‐treated enzyme.
Figure 2
Figure 2
Size‐exclusion chromatography with native and heat‐treated 4‐hydroxytryptamine/psilocin kinase PsiK. Both heat‐inactivated and active PsiK eluted at 15.9 mL (corresponding to ca. 43 kDa). The calculated mass of monomeric N‐terminally His‐tagged PsiK is 44.0 kDa. Calibration trace: a) 669 kDa (thyroglobulin), b) 440 kDa (ferritin), c) 158 kDa (aldolase), d) 75 kDa (conalbumin), e) 43 kDa (ovalbumin).
Figure 3
Figure 3
A) Progress of the gram‐scale synthetic/biocatalytic production of 1 from 2, as verified by UHPLC and HR‐ESIMS analyses of an aliquot. Top trace: overlaid individual chromatograms of authentic 1 and 2. B) 3D plot of UHPLC diode array and total ion chromatogram (TIC) data of the singly chromatographed 1 production assay. TIC trace a) ESI (+); trace b) ESI (−) mode.
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