Structure Elucidation and Spectroscopic Analysis of Chromophores Produced by Oxidative Psilocin Dimerization

Article abstract of DOI:10.1002/chem.202101382

Psilocin (1) is the dephosphorylated and psychotropic metabolite of the mushroom natural product psilocybin. Oxidation of the phenolic hydroxy group at the C-4 position of 1 results in formation of oligomeric indoloquinoid chromophores responsible for the

Full text of DOI:10.1002/chem.202101382

Accepted Article
Accepted Article
Title: Structure Elucidation and Spectroscopic Analysis of
Chromophores Produced by Oxidative Psilocin Dimerization
Authors: Claudius Lenz, Sebastian Dörner, 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.202101382
Link to VoR: https://doi.org/10.1002/chem.202101382
01/2020

10.1002/chem.202101382
Chemistry - A European Journal
RESEARCH ARTICLE
Structure Elucidation and Spectroscopic Analysis of
Chromophores Produced by Oxidative Psilocin Dimerization
Claudius Lenz,[a] Sebastian Dörner,[a] Alexander Sherwood,[b] Dirk Hoffmeister*[a]
[a]
C. Lenz, S. Dörner, Prof. Dr. D. Hoffmeister
Department Pharmaceutical Microbiology at the Hans-Knöll-Institute
Friedrich-Schiller-Universität
Beutenbergstrasse 11a, 07745 Jena (Germany)
E-mail: dirk.hoffmeister@leibniz-hki.de
[b]
A. Sherwood, Ph.D.
The Usona Institute
2800 Woods Hollow Road, Madison, WI 53711 (USA)
Supporting information for this article is given via a link at the end of the document.
Abstract: Psilocin (1) is the dephosphorylated and psychotropic
metabolite of the mushroom natural product psilocybin (2). Oxidation
of the phenolic hydroxy group at the C-4 position of 1 results in
formation of oligomeric indoloquinoid chromophores responsible for
the iconic blueing of bruised 2-producing mushrooms. Based on
previous NMR experiments, the hypothesis included that the 5,5’-
coupled quinone dimer of 1 was the primary product responsible for
the blue color. To test this hypothesis, we synthesized ring-methylated
1 derivatives to provide stable analogs of 1 dimers able to be
completely characterized. The chemically oxidized derivatives were
spectroscopically analyzed and compared to computationally-derived
absorbance spectra. Experimental evidence did not support the
original hypothesis. Rather, the blue color was shown to stem from
the quinoid 7,7’-coupled dimer of 1.
(5,5’-, 7,7’-, or 5,7’-coupled) that possibly exist within the product
mixture in the initial phase of 1 oxidation.
Despite numerous efforts to identify the main blue chromophore
in 1 oligomerization,[4,5] the structure(s) that confer the hue on
bruised Psilocybe mushrooms had been elusive, as was the
mechanism by which the colored matter is formed. From previous
mass spectrometry and NMR-based studies, the 5,5’-coupled
product was initially hypothesized to contribute the blue color,
given that coupling at the C-5 position was the major reaction in
the investigated enzymatic oxidation of 1.[3] Furthermore,
literature on analogous chromophores plausibly suggested a
rather reddish color for the 7,7'-coupled quinoid dimers.[6]
However, the mixture was intractable and too reactive, and rapid
oligomerization occurred. Therefore, a single defined dimer could
not be isolated and only indirect conclusions could be drawn.
Furthermore, the color and corresponding saturation of bruised
mushrooms changes both temporally and even from specimen to
specimen. This implies the presence of multiple chromophores,
which complicates a detailed analysis of individual components of
the hue.
Introduction
Tryptamines, in particular when 4- or 5-hydroxylated, represent a
group of potent psychotropic compounds of natural or synthetic
origin. Most prominently, psilocin (1, 4-hydroxy-N,N-
dimethyltryptamine, Figure 1) causes profound changes in
perception and consciousness.[1] It is the pharmacologically active
dephosphorylated analog of psilocybin (2, Figure 1) and is formed,
e.g., upon ingestion of so-called ‘magic’ mushrooms of the genus
Psilocybe and other genera. The pharmaceutical relevance of 2
as a candidate drug against therapy-refractory depression and
anxiety has impressively been underlined by clinical studies.[2]
Fungal tryptamines occur as oligomers as well. In bruised or
injured Psilocybe mushrooms, 2 is dephosphorylated as well to 1,
whose indolic 4-OH group is subsequently oxidized. This short
cascade, catalyzed by the phosphatase PsiP and the laccase
Psilocin (1): R1=OH, R2=R3=H
R1
1
2
3
2
Psilocybin ( ): R =OPO3H2, R =R =H
4
R2
5
N
Bufotenin (3): R1=R3=H, R2=OH
5-Methylpsilocin (4): R1=OH, R2=CH3, R3=H
N
7
R3
H
1
2
3
5
7-Methylpsilocin ( ): R =OH, R =H, R =CH3
Figure 1. Chemical structures of natural tryptamines, and synthetic C-5 and C-
7 methylated psilocin derivatives.
PsiL, prepares 2 via 1 for instant oligomerization into
a
heterogeneous quinoid set of compounds that accounts for the
distinctive blue hue of Psilocybe fruiting bodies.[3] Given their
protein-precipitating and radicalic oxidation properties, these
oligomers may add a possible second layer of bioactivity - beyond
the receptor-binding nature of monomeric 1 - that perhaps
protects the fungus from feeding invertebrates. Coupling between
two monomers preferentially occurs on positions C-5 and C-7,
which implies three possibilities for primary dimeric regioisomers
We sought more profound insight into the oxidation chemistry of
1 by precise characterization and analysis of the individual
chromophores produced by its initial dimeric products. A pivotal
report by Laatsch describes research to reveal structure-color
correlations by analogously oxidizing specifically designed 4-
hydroxyindole model compounds which carried specific
methylations to interrupt product oligomerization at the dimeric
level.[4] Inspired by this report, and supporting the recently
1
This article is protected by copyright. All rights reserved.

10.1002/chem.202101382
Chemistry - A European Journal
RESEARCH ARTICLE
accomplished chemoenzymatic synthesis of 5-methylpsilocybin,[7]
we synthesized strategically 5- and 7-methylated 1 derivatives
that were oxidatively dimerized into stable derivatives that could
be isolated and characterized. To discern differences in oxidation
and reaction behavior of hydroxylated tryptamines, the 5-hydroxy
isomer of 1, bufotenin (3), was also chemically dimerized in a
parallel approach and investigated for spectroscopic features.
While the latter is yellow (λmax = 432 nm), 4a showed an intense
violet color. Its absorption spectrum is similar to the one obtained
from 1 oxidation, i.e., 1x, with a maximum at λ = 583 nm (Figure
2B).
Results
Chromophore analysis – Our previous study on 1 oxidation used
mass spectrometry, along with IR and in situ 13C NMR
spectroscopy, and identified a heterogeneous mixture of psilocyl
oligomers (up to 13-mers) as products. We also recognized C-5
as the primary coupling position and therefore proposed the 5,5’-
dimer being primarily responsible for the blue color.[3] During
chromatographic and mass spectrometric analysis of the blue
product mixture, an unstable compound (hereafter referred to as
1x) was observed as well, whose ion mass was m/z 405 [M+H]+,
which is compatible with a didehydrodimer of 1. However, the
exact
coupling
position(s)
remained
unknown.
The
simultaneously recorded UV/Vis spectrum of 1x suggested a
potential contribution to the blue coloration, which prompted us to
re-investigate the structure of the chromophores. The longest
absorption maximum appeared at λ = 600 nm (Figure 2A). This
wavelength corresponds to orange light absorption and,
consequently, to
a blue hue. Consistently, computational
rendering of the full corresponding visible transmission spectrum
returned azure RGB-colors (Figure 2A, Figure S1) for this species.
Therefore, we hypothesized most of the blue tint during the initial
1 oxidation cascade is caused by 1x, as other chromatographic
peaks of the oxidation mixture with absorption at long
wavelengths showed a much lower intensity.[3]
We sought to clarify which of the three hypothetical 1-
didehydrodimers 1a (7,7'-coupled), 1b (5,5'-coupled) and 1c (5,7'-
coupled) possessed absorption properties matching those of 1x,
the putative 1 didehydrodimer that initially attracted our attention
during LC-DAD-MS analysis. The unstable dimers 1a-1c
appeared not readily accessible in pure form via synthesis. Thus,
we designed an analogy model and used comparative
calculations. The experimental design aimed at selectively
interrupting 1 oxidation on the dimeric level before oligomerization
Figure 2. Visual range diode array absorption spectra of quinoid dimers of 1
and of their 5- and 7-methylated congeners 4 and 5, and comparison with their
theoretical spectra calculated with time-dependent density functional theory
(dashed lines). The shading under the curves represents the actual substance
colors under the chromatographic conditions (acetonitrile/H2O + 0.1 % formic
acid) depicted as color gradients between two rendered RGB values, obtained
by two different computational methods (HDTV for I and Wα for II, see
Supporting Information). The right y-axis refers to this gradient. The
experimental spectrum in panel A represents the elusive putative dimer 1x,
which was later shown to be consistent with 1a (=7,7’-coupled unmethylated 1),
i.e., the main dimeric blue compound of 1 oxidation. Panel B: dimer 4a (=7,7’-
coupled, 5,5’-dimethylated 4); panel C: dimer 5a (5,5’-coupled, 7,7’-
dimethylated) and theoretical spectrum of 1b (5,5’-coupled unmethylated 1);
panel D: dimer 6 (5,7’-coupled, 7,5’-dimethylated 4 and 5).
proceeds, using 5- and 7-methylated congeners of
1 to
strategically block the reactive positions on the indole core. Due
to the methyl-blocked positions, the ring cannot further engage in
oligomerization. Consequently, the dimers become amenable to
experimental determination of spectroscopic features and
subsequent comparison with in silico predicted spectra,
calculated by time-dependent density functional theory (TD-DFT).
Next, we oxidized 5. From published observations with analogous
oxidations of methylated naphthols,[4,6] we hypothesized that a
symmetric 5,5’-coupled ortho-dimer would absorb at longer
wavelengths than the 7,7’-coupled para dimer. In fact, in the
We synthesized 5-methylpsilocin (4, Figures 1 and S2a-b) and 7-
methylpsilocin (5, Figures and S3a-b) from the respective
methylated indoles by Speeter-Anthony tryptamine synthesis
(Scheme 1).[8] Upon oxidation of 4 by FeIII in MeOH/H2O, we
obtained two stable products that were identified by NMR
spectroscopy. The symmetric quinoid para-dimer 4a (Figures 2B
and S4a-e) was found, along with the less abundant monomeric
para-quinone 4b (Figures 3 and S5a-e).
initially obtained green solution, LC-DAD-MS identified
a
didehydrodimer (λmax = 703 nm, m/z 433 [M+H]+) as the major
product. Its absorption spectrum (Figure 2C), derived from diode
array data, corresponded much less to the spectrum obtained
from didehydrodimer 1x, compared to the spectrum obtained from
7,7-coupled didehydrodimer 4a. Furthermore, it proved to be
unstable and its color faded rapidly, leading to a near-colorless
solution within minutes. After acetylation of the lyophilized
oxidation products, using acetic anhydride and pyridine in ethyl
2
This article is protected by copyright. All rights reserved.

10.1002/chem.202101382
Chemistry - A European Journal
RESEARCH ARTICLE
acetate, derivative 5b (Scheme 2, Figures S6a-d) was isolated as
the major product (Figure S7). From the characterization of
derivative product 5b, we concluded the ortho-coupled quinoid 5a
to represent the initially formed dimeric green chromophore,
which is easily reduced to its hydroquinoid form to enable
acetylation.
experimental spectra were recorded in a mainly aqueous acidified
solvent mixture.
Figure 3. A) Hypothetical dimeric quinoids obtained upon 5- and/or 7-coupling
of 1 (1a-1c), as well as their respective methylated congeners used in this study.
B) Identified primary oxidation products formed upon 3 oxidation by FeIII in
MeOH/H2O.
Scheme 1. Hydroxytryptamines, intermediates, and analogs, synthesized by
Speeter-Anthony chemistry from 5-methyl-1H-indol-4-ol 7, 7-methyl-1H-indol-4-
ol 8, and 1H-indol-5-ol 9, respectively, as initial educts.
The structure of 4a (i.e., the 7,7’-coupled, 5,5’-methylated dimeric
compound) had been proven by NMR (Figure 3A, S4a-e). Using
the TD-DFT approach, a theoretical absorbance spectrum of its
(E)-isomer was calculated. The obtained first excitation energy
deviated by -0.12 eV (+34 nm) from the experimental value, which
is within the common error range of such calculations.[9]
Furthermore, the shoulder around λ = 490 nm in the spectrum of
4a (Figure 2B), contributing to its violet appearance, was
reproduced by the calculations. Furthermore, an analogous
theoretical absorption spectrum for putative structure 1x was
obtained and compared to the experimental result (Figure 2A).
The theoretical shift in the longest local maximum for 5,5’-
bismethylation of the para-dimer (i.e., 4a compared to 1a) was -
14 nm and therefore close to the experimentally observed value
of -17 nm. The assigned structures for the 5,5’-coupled dimer 5a
and the putative asymmetrical dimer 6 were also consistent with
the calculated theoretical absorbance spectra (Figure 2C-2D).
After combined oxidation of equimolar amounts of 4 and 5, we
detected another didehydrodimer (m/z 433) that was not found
following the separate oxidation of either substrate. The new
didehydrodimer demonstrated a yellowish-greenish color of low
saturation and produced broad absorption bands with maximum
absorption at λmax = 385 and 673 nm (Figure 2D). We tentatively
propose the 5,7’-coupled dimer 6 (Figures 3A and S8) as its
structure.
With the experimental absorption spectra obtained from
compounds 1x, 1a, 4a, 5a, and 1b (Figure 2), we revised our initial
hypothesis and preliminarily ascribed the quinoid 7,7’-dimeric
structure 1a (Figure 3B) to the principal didehydrodimer
responsible for the blue chromophore formed upon 1 oxidation.
This assignment contrasted our previous hypothesis that the 5,5’-
coupled dimer 1b was primarily responsible for the blue
chromophore. However, despite the similar relative profile in
absorbance spectra for 1a compared to 4a, the observed overall
shift of -17 nm in the longest local maximum from λ = 600 nm for
putative 1a (Figure 2A) to 583 nm for methylated derivative 4a
(Figure 2B) was larger than expected. For example, in analogous
para-naphthylidenequinones, the respective 2,2’-bismethylation
accounted for only -3 nm shift.[6b] We therefore corroborated our
structural assignments by the quantum chemical calculation of the
excitation energies at the B3LYP/def2-TZVP level of theory.
Considering the combined experimentally derived data and the
quantum chemical calculations, we concluded that the
prototypical blue chromophore of 1 oxidation products, and of
bruised Psilocybe mushrooms alike, is represented by the quinoid
7,7’-coupled dimer 1a (Figure 3A). Hence, our experimental data
does not support our previous hypothesis of the symmetric 5,5’-
coupled 1b as main component of the blue color. Still, both 1b
and the asymmetric 5,7’-dimer 1c may contribute to occasionally
occurring green tints and to general desaturation or darkening of
the primary blue color.
Quantum chemical calculation - This particular approach was
chosen based on results of a benchmark study that compared
computed absorption spectra of indigoid dyes with experimentally
generated data,[9] but by utilizing a more modern basis set in our
study. We modeled the respective diprotonated dimers in
aqueous medium (implicit solvent modeling), since the
Characterization of bufotenin oxidation products
– To
compare oxidation properties and propensity toward dimer
formation with naturally occurring C-4- versus C-5-
hydroxytryptamines, we investigated the oxidation products of
3
This article is protected by copyright. All rights reserved.

10.1002/chem.202101382
Chemistry - A European Journal
RESEARCH ARTICLE
bufotenin (3, Figures 1 and S9a-b), a regioisomer of 1. We used
size exclusion chromatography[10] to separate the mixture
obtained by 3 oxidation in aqueous MeOH using FeIII to provide
four main fractions (A-D, in order of elution, Figure S10). Putative
ortho-quinones (3c and 3e, Scheme 2) were further derivatized
with o-phenylenediamine, and the products were characterized.
the monomeric o-quinone 3e (λmax = 540 nm, m/z 219 [M+H]+,
Scheme 2) by direct treatment with o-phenylenediamine and
subsequent isolation of phenazine 3f (Scheme 2, Figures S14a-
d). Comparing oxidation products of 1 and 3, we infer that the
main coupling sites are switched from C-5 and C-7 in 4-HTs to C-
3 and C-4 in 5-HTs. In part, this can be viewed as a direct outcome
of respective stabilization patterns of the initially formed
hydroxytryptamine radicals. The obtained major oligomeric
quinoids of oxidized 4-HTs generally show lower first excitation
energies than those of 5-HTs, yielding blue to green colors in the
former versus yellow to orange colors in the latter.
Fraction
A
was yellow and contained an intractable
heterogeneous mixture of trimers and higher order products.
Fraction B was also yellow and contained the major product of the
reaction. LC-DAD-MS analysis identified a colorless compound
as primary component. Its mass (m/z 407 [M+H]+) pointed to a
dehydrodimer. Furthermore, a yellow species (λmax = 434 nm, m/z
421 [M+H]+), possibly derived from the former by further oxidation
was present in trace quantities. After lyophilization of fraction B,
the main dehydrodimer was isolated by semi-preparative HPLC
and identified by HRMS, 1H and 13C NMR as brachybotryne, i.e.,
4,4’-bibufotenin (3a, Figures 3B and S11a-d).[11] Fraction C was
orange and contained a near-colorless didehydrodimer (3b, very
weak visual absorption band at about λ = 460 nm, m/z 405 [M+H]+,
Figure S12) that autoxidatively produced a more strongly vis-
absorbing compound (3c, λmax = 462 nm, m/z 421 [M+H]+,
Scheme 2).
Discussion
Color changes or formation following oxidation of small molecule
natural products are known from other mushroom species as well.
Gyrocyanine is
a
L-tyrosine-derived diarylcyclopentenone
pigment that was isolated from various boletes. Upon mycelial
injury and concomitant exposure to air, an intensely blue anion is
formed.[12] A similar chromophore change is observed with
pulvinic acids, for example variegatic acid, which is a yellow
lactone pigment that turns into a blue quinone methide anion
chromophore when oxidized.[13] Contrasting 1, these natural
products remain monomeric as the blue chromophore is formed,
although oxidative dimerization is known to turn variegatic acid
into badione A and norbadione A, which confer the brownish color
on the cap of the producing mushrooms.
Recently, we presented an enzymatic cascade both for 2
biosynthesis[14] and for conversion of 2 to 1 and subsequent
oligomerization along with evidence for a complex mixture of
chromophoric structures.[3] We hypothesized that in higher
oligomers of 1, more than one of the respective chromophoric
systems is present, eventually leading to blueish polymers.
Building on these previous results, we now identified the
chromophore of 1a, the symmetrical 7,7'-didehydrodimer of 1, to
contribute most of the blue color in the reaction. 5,5’-couplings
may contribute a green tint by forming ortho-biindolylidenedione
chromophores, while asymmetrical (e.g., 5,7’-coupled) or higher
order chromophores may cause color desaturation or browning.
Purification of the methylated quinoid model reaction products
revealed that the p-quinoid structures were more stable than the
o-quinoids. The latter were prone to decolorization by follow-up
reactions and reduction to their hydroquinoid form, presumably
driven by the close proximity of the electron withdrawing
carbonyls and a more positive redox potential, as found for
analogous biphenols.[15,16] As the 5-position of the indole scaffold
appears kinetically preferred during coupling,[3] we assume these
instabilities explain why the blue color predominates: the
kinetically favored, yet unstable o-coupled green chromophores
fade rapidly while the more chemically stable para-quinoids
persist and confer the blue color.
Scheme 2. Oxidation products of 5- or 7-methylated 1 (compounds 4 and 5)
and of 3, including derivatives used for structural characterization. Color
shadings represent actual substance colors as rendered by the described
method (HDTV, see Figure 2 and Supporting Information).
The oxidation of 3 closely resembles that of serotonin.[10] Given
the yellow hue of the oxidation products of 3, they may contribute
to the yellowish appearance of basidiocarps of the False Death
Cap Amanita citrina, known to biosynthesize 3.[17] Research on
the natural occurrence of 3 oxidation products is scarce, and their
potential ecological functions have remained unknown. However,
numerous reports exist on the bioactivity and ecological function
After treatment with o-phenylenediamine, the phenazine
compound 3d (Scheme 2, Figures S13a-e) was isolated,
suggestive of 3b and 3c to represent the respective initial
oxidation products. Fraction D was purple and contained mainly
4
This article is protected by copyright. All rights reserved.

10.1002/chem.202101382
Chemistry - A European Journal
RESEARCH ARTICLE
[3]
[4]
[5]
C. Lenz, J. Wick, D. Braga, M. García-Altares, G. Lackner, C. Hertweck,
M. Gressler, D. Hoffmeister, Angew. Chem. Int. Ed. 2020, 59, 1450–
1454; Angew. Chem. 2020, 132, 1466–1470.
of other mono- or dimeric indolic mushroom natural products.
Indigo, indirubin and isatin (an indoline) were observed in the
confrontation zone of two mushroom species.[18] Modified and
extended indole systems, as found in the iminoquinone
mycenarubin D of the mushroom Mycena haematopus[19] or in
the β-carboline infractopicrin of Cortinarius infractus,[20] were
recognized as active against soil bacteria and conferring a highly
bitter taste on the producer which deters feeding predators,
H. Laatsch, in Yearbook of the European College for the Study of
Consciousness 1997 (Eds.: H.-C. Leuner, M. Schlichting), Verlag für
Wissenschaft und Bildung, Berlin, 1998, pp. 241–256.
a) H. Blaschko, W. G. Levine, Biochem. Pharmacol. 1960, 3, 168–169;
b) H. Blaschko, W. G. Levine, Brit. J. Pharmacol., 1960, 15, 625–633; c)
W. G. Levine, Nature, 1967, 215, 1292–1293.
[6]
[7]
a) C. Göltner, H. Laatsch, Liebigs Ann. Chem. 1991, 1085–1089; b) A.
Kral, H. Laatsch, Z. Naturforsch. B 2014, 48, 1401–1407.
J. Fricke, A. M. Sherwood, A. L. Halberstadt, R. B. Kargbo, D.
Hoffmeister, J. Nat. Prod. 2021, 84, 1403–1408.
respectively. In contrast, the ecological advantage for
production in Psilocybe mushrooms remains obscure. Given that
binds to neurotransmitter receptors with high affinity,
2
1
monomeric 1, formed from 2, may protect the mushroom from
mycophagous insects by interfering with their behaviour.[21] An
alternative hypothesis includes that 1 is produced from 2 to fulfill
an ecological function as an oligomeric „defense-on-
demand“ mixture.[2b] Its polyphenolic properties may generate
reactive oxygen species and mediate binding to and precipitation
of proteins. Therefore, the chromophoric psilocyl oligomers may
exert an immediate deleterious effect on feeding insects and may
represent the true ecologically relevant molecules for which 2 is
accumulated as their chemically stable precursor.
[8]
[9]
M. E. Speeter, W. C. Anthony, J. Am. Chem. Soc. 1954, 76, 6208–6210.
E. A. Perpète, D. Jacquemin, J. Mol. Struct. THEOCHEM 2009, 914,
100–105.
[10] a) M. Z. Wrona, G. Dryhurst, J. Org. Chem. 1987, 52, 2817–2825; b) M.
Z. Wrona, G. Dryhurst, J. Pharm. Sci. 1988, 77, 911–917.
[11] M. A. dos S. Ribeiro, C. M. B. Gomes, A. S. N. Formagio, Z. V. Pereira,
U. Z. Melo, E. A. Basso, W. F. da Costa, D. C. Baldoqui, M. H. Sarragiotto,
Tetrahedron Lett. 2016, 57, 1331–1334.
[12] a) W. Steglich, A. Thilmann, H. Besl, A. Bresinsky, Z. Naturforsch. 1977,
32c, 46–48; b) M. Gill, W. Steglich, Prog. Chem. Org. Nat. Prod., 1987,
51, 1–317.
[13] W. Steglich, W. Furtner, A. Prox, Z. Naturforsch. 1968, 23b, 1044–1050.
[14] a) J. Fricke, F. Blei, D. Hoffmeister, Angew. Chem. Intl. Ed. 2017, 56,
12352-12355; Angew. Chem. 2017, 129, 12524–12527; b) F. Blei, F.
Baldeweg, J. Fricke, D. Hoffmeister, Chem. Eur. J. 2018, 24, 10028-
10031.
Conclusion
The dimerization of 1 is a key step of the spectacular blueing
reaction of Psilocybe magic mushrooms. Due to its dynamic
progress and subsequent oligo-/polymerization, the nature of the
initial blue compound(s) had remained elusive. Experimental and
computational evidence derived from 1 as well as with methylated
derivatives as surrogates proved the quinoid chromophore of the
7,7’-coupled dimer of 1 to confer the blue color on injured
mushrooms. Our work sheds light on an iconic phenomenon that
has intrigued natural product chemists, and amateurs alike, for
decades.
[15] B. Guan, P. Wan, J. Chem. Soc. Chem. Commun. 1993, 409–410.
[16] M. Jonsson, J. Lind, G. Merényi, J. Phys. Chem. A 2002, 106, 4758–
4762.
[17] J. A. Beutler, A. H. Der Marderosian, J. Nat. Prod. 1981, 44, 422–431.
[18] K. Krause, E.-M. Jung, J. Lindner, I. Hardiman, J. Poetschner, S.
Madhavan, C. Matthäus, M. Kai, R. C. Menezes, J. Popp, A. Svatoš, E.
Kothe, PLoS One 2020, 15, e0232145.
[19] J. S. Lohmann, S. Wagner, M. von Nussbaum, A. Pulte, W. Steglich, P.
Spiteller, Chem. Eur. J. 2018, 24, 8609–8614.
[20] a) W. Steglich, L. Kopanski, M. Wolf, M. Moser, G. Tegtmeyer,
Tetrahedron Lett. 1984, 25, 2341–2344; b) P. Spiteller, Chem. Eur. J.
2008, 14, 9100–9110.
[21] H. T. Reynolds, V. Vijayakumar, E. Gluck-Thaler, H. B. Korotkin, P. B.
Matheny, J. C. Slot, Evol. Lett. 2018, 2, 88–101.
Acknowledgements
We thank A. Perner, H. Heinecke, and V. Hänsch (Leibniz
Institute for Natural Product Research and Infection Biology,
Hans-Knöll-Institute Jena) for recording HRMS and NMR spectra,
and for support with chemical syntheses, respectively. We thank
O. Lenz (Otto Group, Dresden) for adapting the specrend source
code. C. L. acknowledges
a doctoral fellowship by the
International Leibniz Research School (ILRS) for Microbial
Interactions. This work was funded by the Deutsche
Forschungsgemeinschaft (DFG, German Research Foundation)
– Project-ID 239748522 – SFB 1127 and by the Usona Institute
(Madison, WI).
Keywords: bufotenin • chromophore • oxidation • psilocybin •
tryptamine
[1]
[2]
a) F. Tyls, T. Palenicek, J. Horacek, Europ. Neuropsychopharmacol.
2014, 24, 342–356; b) R. B. Kargbo, ACS Med. Chem. Lett. 2020, 11,
399–402.
a) D. E. Nichols, J. Antibiot. 2020, 73, 679–686; b) C. Lenz, A. Sherwood,
R. Kargbo, D. Hoffmeister, ChemPlusChem 2021, 86, 28–35; c) J. Fricke,
C. Lenz, J. Wick, F. Blei, D. Hoffmeister, Chem. Eur. J. 2019, 25, 897–
903.
5
This article is protected by copyright. All rights reserved.

10.1002/chem.202101382
Chemistry - A European Journal
RESEARCH ARTICLE
Entry for the Table of Contents
Self-made color frenzy: Psilocin oxidation causes the iconic blueing of Psilocybe “magic” mushrooms. To prevent oligomerization,
but allow for dimerization, 5- and 7-methylated psilocin monomers were synthesized. Chemical oxidation enabled spectroscopic
chromophore investigation of the dimeric gateway compounds and comparison to computationally-derived absorption spectra. The
7,7’-coupled quinoid psilocyl dimer was identified as major initial blue product, while the 5,5’-coupled dimer contributes a greenish
tint.
Twitter usernames: @UniJena, @LeibnizHKI
6
This article is protected by copyright. All rights reserved.