Scalable Hybrid Synthetic/Biocatalytic Route to Psilocybin

Article abstract of DOI:10.1002/chem.202000134

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.

Full text of DOI:10.1002/chem.202000134

A Journal of
Accepted Article
Title: Scalable Hybrid Synthetic/Biocatalytic Route to Psilocybin
Authors: Janis Fricke, Robert Kargbo, Lars Regestein, Claudius Lenz,
Gundela Peschel, Miriam Rosenbaum, Alexander Sherwood,
and Dirk Hoffmeister
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.202000134
Link to VoR: http://dx.doi.org/10.1002/chem.202000134
Supported by

10.1002/chem.202000134
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Scalable Hybrid Synthetic/Biocatalytic Route to Psilocybin
Janis Fricke, [a] Robert Kargbo,[b] Lars Regestein,[c] Claudius Lenz,[a] Gundela Peschel,[c] Miriam A.
Rosenbaum,[c] Alexander Sherwood,[b] Dirk Hoffmeister*[a]
Abstract: Psilocybin, the principal indole alkaloid of Psilocybe
kinase gene psiK, opened up new avenues for the production of
1. Heterologously produced enzymes were used to
mushrooms, is currently undergoing clinical trials as a medication
against treatment-resistant depression and major depressive disorder. biocatalytically synthesize 1 in vitro.[7] In vivo production in
The psilocybin supply for pharmaceutical purposes is met by synthetic
chemistry. We replaced the problematic phosphorylation step during
synthesis by 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.
microbial hosts has also proven successful.[8] In addition, the in
vivo approach is attractive due to its scalability to pilot-scale
bioreactors and the availability of cofactors. However, in view of
registration as a pharmaceutical, the use of a cell-based
production system entails intensive procedures to remove cellular
components and debris that may be potentially allergenic or toxic.
Furthermore, unlike the in vitro or synthetic approaches, the
targeted compound needs to be purified from complex matrices,
which may involve a cell lysate or broth.
Herein, we describe the first combined chemical/biocatalytic
synthesis of 1. This cell-free in vitro procedure combines the facile
synthetic access to
2 with side product-free quantitative
Introduction
conversion through PsiK-mediated biocatalysis in a subsequent
step, thus eliminating heavy metal-dependent hydrogenolysis and
low yielding phosphorylation chemistry to produce gram-scale
amounts of 1. To facilitate future development of the
biotechnological processes, we present a thorough biochemical
characterization of P. cubensis PsiK and
heterologously produce and purify three-digit milligram amounts
of PsiK in soluble and active form.
The indole alkaloid psilocybin (1, Scheme 1) is the principal
natural product of hallucinogenic Psilocybe mushrooms.[1]
Pharmacologically, it represents the stable prodrug to the actual
active, yet unstable 4-dephosphorylated analog psilocin (2) which
targets mainly the human 5-HT2A-receptor as a partial agonist.[2]
Modern scientific therapeutics re-discovered 1 as a valuable drug
candidate.[3] Phase 2 clinical trials have shown favorable results
in the treatment of several indications, and 1 has received
Breakthrough Therapy designation by the United States Food and
Drug Administration (US FDA) in support of both treatment-
resistant depression and major depressive disorder. Other
indications include cancer-related existential distress and
substance dependencies.[4]
a protocol to
O
P
O
P
OH
O
OH
O
HO
HO
PsiM
CH3
N
NH2
CH3
N
H
N
H
2 SAH 2 SAM
psilocybin (1)
norbaeocystin
ADP
ATP
Given its status as a candidate drug, the demand for medical-
grade 1 is increasing. Traditionally, this compound is accessible
via chemical synthesis.[1,5-6] Identification of the 1 biosynthetic
genes and enzymes in Psilocybe mushrooms, including the
ADP
PsiK (repair)
ATP
PsiK
(biosynthesis)
Pi
OH
OH
CH3
N
NH2
CH3
N
N
[a]
J. Fricke, C. Lenz, Prof. Dr. D. Hoffmeister
Department Pharmaceutical Microbiology at the Hans-Knöll-Institute
Friedrich-Schiller-Universität
H
H
psilocin (2)
4-hydroxytryptamine
Beutenbergstrasse 11a, 07745 Jena (Germany)
E-mail: dirk.hoffmeister@leibniz-hki.de
R. Kargbo, Ph.D., A. Sherwood, Ph.D.
Usona Institute
2780 Woods Hollow Road, Madison, WI 53711
Dr. L. Regestein, Dr. G. Peschel, Prof. Dr. M. A. Rosenbaum
Bio Pilot Plant
[b]
[c]
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
phosphorylation step.
1
biosynthesis after the
Leibniz Institute for Natural Product Research and Infection Biology
- Hans-Knöll-Institute
Beutenbergstrasse 11a, 07745 Jena (Germany)
Supporting information for this article is given via a link at the end of
the document
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10.1002/chem.202000134
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Results and Discussion
nearly inactive (< 5%) with Ca2+, Cu2+, Ni2+, Mn2+, or Zn2+. PsiK
followed a sigmoid kinetic response to increasing 2 and 4-
hydroxytryptamine concentrations, with K0.5 values of 72 µ
67 µ
, and a kcat of 16.1 s-1 and 4.5 s-1 for these substrates (Figure
S2). This translates into catalytic efficiencies (kcat/K0.5) of 2.24
105 and 6.67 104 M-1 s-1, respectively, with 2 and 4-
hydroxytryptamine as acceptor substrates. The K0.5 value for ATP
was 89 µ . The above values identified PsiK’s repair function as
more than three times as efficient as the biosynthetic one. Size
exclusion chromatography showed that active PsiK is monomeric
(Figure 2). This situation is reminiscent to glucokinase, which has
M and
First, we produced PsiK in a small scale as a hexahistidine-tagged
fusion using E. coli KRX
×
pJF23[6a] and investigated its steady
M
×
state kinetic properties. Phosphate-buffered in vitro assays were
analyzed for formation of 1 by UHPLC-MS. Previous work
confirmed a dual biosynthetic and protective role for PsiK as it
accepts both 4-hydroxytryptamine and 2 as substrates (Scheme
1). The former represents the phosphate acceptor during
biosynthesis. The repair reaction, which is the relevant reaction
for our study, includes phosphorylation of 2 back to 1, thus
removing instable and oligomerization-prone 2 from the cell.
Oligomeric 2 causes the blueing reaction of injured “magic”
mushrooms and binds to proteins which would be fatal to the
intact fungal mycelium.[9] To protect cell integrity, we expected
that the velocity of the repair direction should at least equal that
of the biosynthetic direction.
×
M
a single binding site for its phosphate acceptor substrate (D-
glucose), yet shows a sigmoid kinetic, which is a characteristic of
positive cooperativity.[11]
With a future large-scale process development in mind, a stirred-
tank fermentation and purification regime was established. E. coli
KRX
× pJF23 was performed batch-wise in a 75 L reactor, filled
with 30 L yeast-peptone-glucose medium. After harvest, cell
disruption and FPLC-based Ni2+ affinity chromatography, 150.2
mg pure PsiK was obtained (Figure S3).
In its native form, P. cubensis PsiK is a 40.4 kDa enzyme of 362
aa. Sequence alignment with other characterized or putative
small molecule kinases (Figure S1) identified a canonical ATP
binding domain that comprises a conserved motif 57KHA59. In the
structure of the Arabidopsis thaliana (mouse-ear cress) 5-
methylthioribose kinase (Protein Data Bank, accession # 2PYW),
the equivalent lysine residue in the motif 63KQA66 ionically
interacted with the α-phosphoryl group of ADP. In the Arabidopsis
enzyme, the conserved aspartate residue, D255 was shown to
coordinate bivalent Mg2+ cations in the active site.[10] The
equivalent residue in PsiK is D224.
a
d
c
e
b
calibration
PsiK
1
PsiK (heat-treated)
2
5
10
V/mL
15
20
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).
PsiKD224A
PsiK
N
1
2
3
t/min
Subsequently, we tested PsiK’s storability and stability at -20°C in
phosphate buffer, which was amended with 10% glycerol, for 30
d and we found the PsiK activity was fully retained (Figure S4).
For the use as substrate in a PsiK-based production assay, 2 was
synthesized via a recently published protocol,[6] essentially
following Speeter-Anthony chemistry[12] Commercial 4-
acetoxyindole underwent Friedel-Crafts acylation and amidation
to yield 3-(2-(dimethylamino)-2-oxoacetyl)-1H-indol-4-yl acetate,
which was reduced by lithium aluminum hydride to afford 2 in 73%
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.
To verify its function, we engineered the cloned psiK cDNA to
replace D224 by alanine. PsiKD224A was catalytically inactive when
2 was offered as substrate and not even traces of 1 were detected
(Figure 1). This result suggests a similar cation-coordinating role
for D224 in the active site.
overall yield. When 2 mM (725 mg) of 2, 4 mM ATP, and 1.89 µM
PsiK were used, we observed a near-complete phosphorylation
reaction after 10 min, and quantitative conversion to 1 within 20
min (Figure 3A). Workup included preparative reversed-phase
Optimum turnover was found at 40°C and at pH 7.0. Furthermore,
PsiK activity depended on Mg2+. In contrast, with Co2+ as divalent
cation, the turnover dropped to 11.5% and the enzyme remained
chromatography and
a
subsequent solvent/antisolvent
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10.1002/chem.202000134
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precipitation in acidic (pH 4) H2O and acetone. After washing the Conclusions
precipitate, 893 mg (i.e., an isolated yield of 88.5%) of pure 1 were
obtained (Figure 3B). The identity of the biocatalytically produced
We expect our results will motivate bioprocess research to further
develop and scale up the presented protocol. Therefore, the
current work is directed towards further upscaling, and optimizing
the post-biocatalysis work-up. We conclude our research helps
provide 1 at a reasonable cost, particularly to support non-profit
endeavors that are geared towards re-introducing this invaluable
natural product to pharmaceutical and clinical use.
1
1 was confirmed by H and 13C NMR spectroscopy (Figure S5,
Table S1) which yielded data that was consistent with published
values.[13]
The structural elucidation, and synthesis of 1 and 2 were first
described by Albert Hofmann.[1] Various improved protocols have
been developed since,[5] however, the synthesis has still
remained problematic due in part to scalability in large scale
manufacturing, instability during the isolation of 2, in-process
Experimental Section
impurities during their synthesis, hydrolysis of
1 during
recrystallization and the lability of the O,O-dibenzyl ester
intermediate. Regardless of the protocol, the final deprotection
involves a heavy metal (Pd/C), which is not preferred from a
regulatory affairs perspective for medical grade compounds.
Furthermore, our approach avoids corrosive and pyrophoric n-
butyllithium. Our route to provide upper milligram to gram
amounts of 1 combines the simplicity of traditional tryptamine
chemistry with the power of biocatalysis to eliminate heavy metals
and the notoriously problematic phosphorylation step, while also
circumventing cell-based production. Demand for 1 is expected to
increase. To translate from gram- to kilogram-scale, further
optimization, such as reduced solvent use and an alternative to
the preparative reversed phase HPLC-based purification
procedure, may be required. Also, selective filtration-precipitation-
recrystallization may help make this process more amenable to
scale-up. With these changes and given the outstanding
selectivity of the enzymatic phosphorylation step, we expect
drastically reduced pricing for kilogram-scale 1 production.
General procedures: Molecular biology methods followed the instructions
of the manufacturers of kits and enzymes (NEB, Promega). Chemicals,
media ingredients and solvents were purchased from Carbolution
Chemicals, Roth, Sigma-Aldrich, VWR, and Deutero, except 2 which was
synthesized following a published procedure.[6] Authentic 1 was isolated
from P. cubensis carpophores.[14]
Site-directed mutagenesis: The gene encoding PsiKD224A was created
by PCR-amplifying psiK from plasmid pJF23.[7a] The psiK gene was
amplified in two fragments that overlapped in the portion to be mutated.
The 5’-end of fragment 1 and the 3’-end of fragment 2 overlapped with the
polylinker of plasmid pET28a, linearized by BamHI and HindIII restriction.
The fragments were assembled using the NEBuilder HiFi DNA Assembly
Kit to yield plasmid pJF70. Oligonucleotides to amplify fragment 1 (448 bp)
were oJF159 (5´-CATGGCGGCCCTGTGGAGTG-3´) and oJF161 (5´-
CTCGAGTGCGGCCGCAAG-3´). Fragment 2 (699 bp) was amplified
using primers oJF160 (5´-CCACAGGGCCGCCATGACAAG-3´) and
oJF162 (5´-CAGCAAATGGGTCGCGGATC-3´). For PCR, 200 µm each
dNTP, 1 ng template DNA, and 1 U Phusion DNA polymerase (NEB) in HF
buffer and MgCl2, supplied with the enzyme was used. Thermal cycling
was for both fragments: initial hold at 98°C for 1 min, 32 cycles of 98°C for
60 s, 67°C for 30 s, and 72°C for 60 s, and a terminal hold at 72°C for 5
min. The PCR products were purified by agarose gel electrophoresis and
subsequent elution with Promega’s Wizard SV Gel and PCR Clean-Up
System.
A
(1)
285.0993
1
2
0
*
100 300 500
Heterologous production of PsiK: PsiK and PsiKD224A were produced in
E. coli KRX (Promega), transformed with expression plasmids pJF23 or
pJF70. Small-scale production and purification was carried out as
described.[15] To produce seed cultures for pilot-scale fermentations
(below), E. coli KRX × pJF23 was grown overnight in 20 mL LB medium,
amended with kanamycin (50 µg mL-1), and shaken at 37°C and 180 rpm.
Cells were centrifuged (11,000 x g, 10 min, 4°C), washed with phosphate
buffered saline (PBS), and stored in 10% (v/v) glycerol at -80°C until use
as inoculum.
m/z
30
20
10
285.0995
*
100 300 500
1
2
3
m/z
t/min
1000
800
600
B
400
200
0
Pilot-scale PsiK production: The inoculum was transferred to a 2 L
shake flask, filled with 200 mL LB medium, for 12 h, at 37°C and 180 rpm.
This seed culture was inoculated with an OD600 of 0.05 and transferred to
the main culture with a final OD600 of 3.5. The main batch culture (30 L,
37°C) was carried out in a 75 L stirred tank reactor (C75, bbi, Berlin,
Germany), using YPD medium (20 g L-1 glycerol, 20 g L-1 tryptone, 10 g L-
yeast extract). The dissolved oxygen tension was controlled by the
stirring rate at a minimum value of 20%. The initial OD600 was 0.023. PsiK
production was induced after 10 h by adding 1 g L-1
L-rhamnose. Upon
b
a
600
3
500
2
400
/nm
1
300
1
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.
induction, the temperature was shifted to 16°C. For fermentation details,
see Figure S6. The fermentation continued for another 14 h before the
broth was centrifuged at 14,000 × g for 30 min at 4°C. Subsequently, the
biomass was suspended in 5 L lysis buffer (50 m
M sodium dihydrogen
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phosphate, 300 m
M
NaCl), amended with 20 m
M
imidazole, homogenized
evaporated under reduced pressure. For chromatographic purification of
1, the residue was dissolved in H2O:acetonitrile (ACN, 9:1, v/v) with 0.1%
trifluoroacetic acid (TFA), which yielded 893 mg (88.5%) of pure 1.
at 850 bar in a high-pressure Gaulin flow homogenizer which was rinsed
with another 1 L of lysis buffer, centrifuged at 14,000  g for 45 min at 4°C
and finally filtered (0.45 µm). PsiK was further purified by immobilized
metal affinity chromatography on
a FPLC system (ÄktaPilot, GE
Preparative liquid chromatography: 1 was purified on an Agilent 1260
series instrument, equipped with a Zorbax Eclipse XDB C18 column (250 ×
21.2 mm, 10 µm). Solvent A was 0.1% TFA in H2O, solvent B was ACN. A
linear gradient 5-100% B within 20 min at a flow of 20 mL min-1 was applied.
Further workup included solvent/antisolvent precipitation in 20 mL acidic
(pH 4) H2O and 30 mL acetone. The precipitate was washed with cold
acetone. To verify purity, 1H and 13C NMR spectra of purified 1, dissolved
in D2O, were recorded at 500 and 125 MHz, respectively, and 300 K on a
Bruker Avance III spectrometer. Chemical shifts were referenced to
residual non-deuterated solvent (δH 4.80 ppm).
Healthcare), equipped with a FineLINE Pilot 35 column filled with 200 mL
Ni-Sepharose FF. The flow rate was 20 mL min-1. The system was
equilibrated in lysis buffer before 6 L of supernatant were loaded. The
column was washed step-wise with a gradient of 30 m
volumes), 40 m (1 cv), and 50 m imidazole (2 cv), using the same buffer.
The final elution was conducted with 500 m imidazole, resulting in a
200 mL fraction. The eluted enzyme was rebuffered on a Amicon Ultra 15
centrifugal filter unit (Merck), equilibrated with phosphate buffer (50 m
M (2 column
M
M
M
M
,
pH 7.0), and stored at -80°C with 10% glycerol until used for production of
1. The protein concentration was determined using Bradford’s method.[16]
The total yield was 150.2 mg pure PsiK. Prior to use for assays, PsiK was
rebuffered by FPLC (below).
Acknowledgements
Size-exclusion chromatography: FPLC (Äkta Pure 25, GE Healthcare)
fitted to a Superdex 200 increase 10/300 GL column with 24 mL bed
volume was used i) to determine if PsiK was active as a mono- or multimer,
and ii) to rebuffer PsiK. Prior to sample loading, PsiK was concentrated on
a centrifugal filter unit (10 kDa cut off, Amicon Ultra 15, Merck). Elution
was with phosphate buffer (10 mM sodium dihydrogen phosphate, 140 mM
NaCl, pH 7.5) at a flow of 0.5 mL min-1. For rebuffering and subsequent
use in assays, PsiK was eluted with assay buffer (below).
We thank Karin Burmeister, Karsten Willing, Andrea Perner and
Heike Heinecke (Hans-Knöll-Institute Jena) for technical
assistance, and for recording high-resolution mass and NMR
spectra, respectively. C.L. acknowledges a doctoral fellowship by
the International Leibniz Research School (ILRS) for Microbial
Interactions. This work was supported by the Deutsche
Forschungsgemeinschaft (DFG grant HO2515/7-1 to D.H.) and
by the Usona Institute (Madison, WI). D.H. is also supported by
the DFG Collaborative Research Center ChemBioSys 1127.
In vitro PsiK assays: Assays to detect activity or to record kinetics were
set up in sodium dihydrogen phosphate buffer (50 m
β-mercaptoethanol in a final volume of 50 µL. ATP and MgCl2 or chlorides
of other bivalent cations (2 m each) and either 4-hydroxytryptamine or 2
(1 m ) were added as substrate, PsiK was added at 1 µ . The reactions
M, pH 7.0) and 3 mM
M
Keywords: biosynthesis • enzymes • fermentation • kinase •
M
M
psilocybin
were incubated for 10 min at 40°C. Subsequently, the assays were
lyophilized, the residue was dissolved in 50 µL MeOH, centrifuged (11,000
 g, 10 min, 4°C), filtered, and chromatographically analyzed (below). To
determine optima, pH and temperature were varied between pH 6-8 and
20-50°C. To investigate the storability of PsiK, aliquots were stored with or
without additional glycerol (10%, v/v) at 4, -20 and -80°C and analyzed by
the above assay after 14 and 30 days.
[1]
[2]
a) A. Hofmann, R. Heim, A. Brack, H. Kobel, Experientia 1958, 14, 107–
109; b) A. Hofmann, R. Heim, A. Brack, H. Kobel, A. Frey, H. Ott, T.
Petrzilka, F. Troxler, Helv. Chim. Acta 1959, 42, 1557–1572.
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2438−2447.
[3]
[4]
[5]
F. X. Vollenweider, M. Kometer, Nat. Rev. Neurosci. 2010, 11, 642–651.
M. W. Johnson, R. R. Griffiths, Neurotherapeutics 2017, 14, 734–740.
a) D. E. Nichols, S. Frescas, Synthesis 1999, 6, 935–938; b) O. Shirota,
W. Hakamata, Y. Goda, J. Nat. Prod. 2003, 66, 885–887, c) S. Bartolucci,
M. Mari, G. Di Gregorio, G. Piersanti, Tetrahedron 2016, 72, 2233–2238;
d) H. Sakagami, K. Ogasawara, Heterocycles 1999, 51, 1131–1135; e)
C. Hu, H. Qin, Y. Cui, Y. Jia, Tetrahedron 2009, 65, 9075–9080; f) N.
Gathergood, P.J. Scammells, Org. Lett. 2003, 5, 921–923.
A. M. Sherwood, P. Meisenheimer, G. Tarpley, R. B. Kargbo, Synthesis,
2020, 52, 688-694,
Analytical liquid chromatography: UHPLC-ESIMS-analyses of in vitro
assays were performed on an Agilent Infinity 1290 chromatograph with a
Luna Omega Polar C18 column (100 × 2.1 mm, 1.6 µm particle size,
Phenomenex), and coupled to an Agilent 6130 Single Quadrupole mass
detector. The flow was 0.5 mL min-1. Diode array detection was between
λ=200-600 nm. Chromatograms were extracted at λ=280 nm. Solvent A
was 0.1% (v/v) formic acid in H2O, solvent B was acetonitrile (ACN). The
linear gradient was initially 1% B, increased to 10% within 3 min, and to
100% B in 1 min. LC-HRESIMS analysis in positive and negative mode
[6]
[7]
a) J. Fricke, F. Blei, D. Hoffmeister, Angew. Chem. Intl. Ed. 2017, 56,
12352–12355; Angew. Chem. 2017, 129, 12524–12527; b) H.T.
Reynolds, V. Vijayakumar, E. Gluck-Thaler, H. B. Korotkin, P. B.
Matheny, J. C. Slot, Evol. Lett. 2018, 2, 88–101.
with purified substances were performed on
a Thermo Accela
chromatograph coupled to an Exactive Orbitrap mass spectrometer, as
described.[14]
[8]
a) S. Hoefgen, J. Lin, J. Fricke, M. Stroe, D. J. Mattern, J. E. Kufs, P.
Hortschansky, A. A. Brakhage, D. Hoffmeister, V. Valiante, Metab. Eng.
2018, 48, 44–51; b) A. M. Adams, N. A. Kaplan, Z. Wei, J. D. Brinton, C.
S. Monnier, A. L. Enacopol, T. A. Ramelot, J. A. Jones, Metab. Eng. 2019,
56,111-119.
Gram-scale production of 1: Scaled-up production was carried out in a
1.8 L reaction in sodium dihydrogen phosphate buffer (50 m
Before adding enzyme and substrates, the reaction buffer was pre-
warmed to 40°C. Subsequently, 725 mg 2 (2 m ), 3.97 g ATP disodium
salt (4 m ), 1.46 g MgCl2 ꞏ 6 H2O (4 m ) and 422 mg β-mercaptoethanol
(3 m ) were added. The reaction was initiated by adding 149.7 mg of
M, pH 7.0).
M
M
M
[9]
C. Lenz, J. Wick, D. Braga, M. García-Altares, G. Lackner, C. Hertweck,
M. Gressler, D. Hoffmeister, Angew. Chem. Intl. Ed. 2020, 59, 1450-1454.
M
purified PsiK (1.89 µM). Samples (50 µL) were taken in triplicates in 10 min
intervals to monitor product formation by UHPLC-MS. After 90 min, the
solvent was evaporated under reduced pressure, and the residue was
dissolved in 500 mL MeOH, centrifuged (3,200  g, 15 min, 4°C) also to
remove precipitated protein and ADP, and filtered. The solvent was
[10] S.-Y. Ku, K. A. Cornell, P. L. Howell, BMC Struct. Biol. 2007, 7, 70.
[11] a) C. M. Porter, B. G. Miller, Bioorg. Chem. 2012, 43, 44–50, b) D.
Pollard-Knight, A. Cornish-Bowden, Mol. Cell. Biochem. 1982, 44, 71–
80.
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10.1002/chem.202000134
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FULL PAPER
[12] M. E. Speeter, W. C. Anthony, J. Am. Chem. Soc. 1954, 76, 6208–6210
[13] F. Blei, F. Baldeweg, J. Fricke, D. Hoffmeister, Chem. Eur. J. 2018, 24,
10028–10031.
[14] C. Lenz, J. Wick, D. Hoffmeister, J. Nat. Prod. 2017, 80, 2835–2838.
[15] J. Fricke, A. Sherwood, R. Kargbo, A. Orry, F. Blei, A. Naschberger, B.
Rupp, D. Hoffmeister, ChemBioChem 2019 (doi: 10.1002/
cbic.201900358).
[16] M. Bradford, Anal. Biochem. 1976, 72, 248-254.
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Entry for the Table of Contents
FULL PAPER
O
O
Beneficial bulk
psilocybin as a medication will entail an
increasing demand. We present a
–
Future use of
J. Fricke, R. Kargbo, L. Regestein, C.
Lenz, G. Peschel, M. A. Rosenbaum, A.
Sherwood, D. Hoffmeister*
N
H
gram-scale
synthetic/biocatalytic
Page No. – Page No.
approach in which the mushroom
kinase PsiK replaces tedious chemical
phosphorylation. A pilot-scale protocol
to make three-digit milligram amounts
of pure and active recombinant PsiK
available is also described.
THF
H
3C
CH3
N
H
PsiK
PsiK
H2 Pd/C
Cl
Scalable Hybrid
Synthetic/Biocatalytic Route to
Psilocybin
PsiK
O
C
Cl
LAH
C
O
O
P
HO
HO
O
CH3
N
CH3
N
H
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