Journal of Molecular Liquids 392 (2023) 123479

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Journal of Molecular Liquids

journal homepage: www.elsevier.com/locate/molliq

Investigation of the structure, stability, and relative solubility of psilocybin

in water and pure organic solvents: A molecular simulation study
Lucas Paul a , Cyril T. Namba-Nzanguim b,c , Aidani Telesphory d , Jehoshaphat Oppong Mensah e ,
Denis Mteremko f , Rene Costa g , Saidi Mohamedi Katundu h , Lucas P. Kwiyukwa i ,
Naserian Daniel Kambaine h,j , Julius Juvenary i , Sixberth Mlowe a , Geradius Deogratias i ,
Daniel M. Shadrack k , Andrew S. Paluch a,l,∗
a
Department of Chemistry, Dar es Salaam University College of Education, P.O. Box 2329, Dar es Salaam, Tanzania
b
Department of Chemistry, University of Buea, P.O. Box 63, Buea CM-00237, Buea, Cameroon
c
Center for Drug Discovery, Faculty of Science, University of Buea, P. O. Box 63, Buea, Cameroon
d
Department of Science and Laboratory Technology, Dar es Salaam Institute of Technology, P.O. Box 2958, Dar es salaam, Tanzania
e
Department of Chemistry, Kwame Nkrumah University of Science and Technology, PMB, Kumasi, Ghana
f
Department of Biological and Food Sciences, The Open University of Tanzania, Tanzania
g
Department of Physical and Environmental Sciences, The Open University of Tanzania, Tanzania
h
Department of Chemistry, College of Natural and Mathematical Sciences, The University of Dodoma, P.O. Box 338, Dodoma, Tanzania
i
Chemistry Department, College of Natural and Applied Sciences, University of Dar es Salaam, P.O. Box 35061, Dar es Salaam, Tanzania
j
Iphytos Company Tanzania LTD, P. O. Box 15014, Arusha, Tanzania
k
Chemistry Department, Faculty of Natural and Applied Sciences, St John’s University of Tanzania, Dodoma, Tanzania
l
Department of Chemical, Paper and Biomedical Engineering, Miami University, Oxford, OH 45056, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Psilocybin is an indole-based secondary metabolite found naturally in mushrooms which possesses several
Psilocybin pharmacological effects. Recently, a large number of experimental investigations have been conducted to
Structure characterize the pharmacology of psilocybin and its derivatives, and to develop synthetic pathways to
Stability
manufacture psilocybin. Nonetheless, current research on the physical characterization of psilocybin is limited in
Solubility
part due to legal restrictions. In the present study, we investigate two unique tautomers of psilocybin as depicted
Solvation
in the 2D chemical structure of psilocybin presented in the recent literature. Using a combination of electronic
structure calculations and molecular simulation, we are able to identify and characterize the thermodynamically
preferred tautomer. We additionally computed the solvation free energy and investigated the solvation structure
of psilocybin in water and 35 organic solvents. We find that hydrogen bonding between psilocybin and the
solvent dominates the solvation process. Considering the thermodynamically preferred tautomer, we find that
the solubility in water is greater than all of the studied organic solvents.

1. Introduction
Psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine, Fig. 1), is an
indole-based secondary metabolite found in “magic mushrooms.” Psilo-
cybin was first isolated by Albert Hofmann in 1957 and then syn-
thesized in 1958 [1]. It is produced by basidiomycetes species and
extracted from several genera of fungi like the mushroom, Psilocybe
mexicana.
Psilocybin has shown a wide range of pharmacological applications
including neuroprotective effects [2], mental disorders [3], and has
been found to greatly affect the mind, reduce anxiety and have hallu-
cinogenic activity [4]. Recently, psilocybin has been approved by the
US Food and Drug Administration (FDA) for clinical studies for the
treatment of anxiety, depression and certain addictions [4]. Previous
clinical trials have suggested that psilocybin is expected to have a great
impact on the treatments of psychiatric disorders.
Despite its known beneficial effects, psilocybin has clinical limita-
tions related to (1) current research on the physical characterization
of psilocybin are limited, and (2) neurotoxic activity [2]. Additionally,
psilocybin is a prodrug metabolized in vivo to psilocin by dephosphory-

* Corresponding author.
E-mail address: PaluchAS@MiamiOH.edu (A.S. Paluch).

https://doi.org/10.1016/j.molliq.2023.123479
Received 19 July 2023; Received in revised form 24 October 2023; Accepted 30 October 2023
Available online 7 November 2023
0167-7322/© 2023 Elsevier B.V. All rights reserved.
L. Paul, C.T. Namba-Nzanguim, A. Telesphory et al.
Fig. 1. The chemical structure of psilocybin. The letter in parentheses is used
to indicate form “A” or “B”, labels introduced in this work to differentiate the
two tautomers. Form A is the common structure depicted in the literature, and
form B is the zwitterionic form reported as the product in ref. [7].
lation. To address this problem, it requires that psilocybin is dissolved
in an appropriate solvent and/or requires the use of nano-particles to
reduce the neurotoxic activity and enhance specific delivery. In addi-
tion to recent clinical investigations, recent successful efforts have been
made to develop synthetic routes to manufacture psilocybin [5–10].
Despite this, work to physically characterize psilocybin is limited,
in part due to legal restrictions. As a simple example, two different 2D
chemical structures of psilocybin have been used recently in the lit-
erature on the development of synthetic routes for synthesis. See for
example, ref. [9] versus [7]. In ref. [7], the 2D structure differs from
the rest of literature, wherein the authors report the product as zwit-
terionic psilocybin. Moreover, a knowledge and understanding of the
solubility of psilocybin in a range of solvents is lacking. Psilocybin is
known to be soluble in water, slightly soluble in methanol and acetone,
and insoluble in chloroform and hexane [3,11]. While knowledge of the
solubility is necessary to overcome clinical limitations in addition to
knowledge of aqueous solubility as it relates to bioavailability, pharma-
ceuticals in general need to satisfy various solubility criteria before they
may be manufactured at an industrial scale at a reasonable expense, and
then formulated for delivery [12–14]. With regards to production, the
synthesis is carried out in solution. Once synthesized, psilocybin must
be separated and purified. Alternatively, it may be necessary to selec-
tively extract psilocybin from its natural environment. Subsequently,
psilocybin needs to be formulated for delivery. If a tablet is to be made
to be administered orally, solvents will be necessary for the crystal-
lization process. Each of these processes can have different solubility
requirements, complicating design. In a typical manufacturing process,
solvents account for 80–90% of the utilized material [12,14]. Given the
large and complex chemistry of psilocybin, finding suitable solvents for
each stage of the manufacturing process using experimentation alone
would be challenging.
In the present study, we use a combination of electronic structure
calculations and atomistic molecular simulation to determine and char-
acterize the thermodynamically favored structure of psilocybin. We
additionally perform solvation free energy calculations for psilocybin
in water and 35 organic solvents to determine the relative solubility
and subsequently to probe the underlying solvation mechanism. The
use of molecular simulation is advantageous in that it is not only able
to quantitatively predict macroscopically observed properties of inter-
est, but can offer insight into the underlying molecular-level driving
forces [15–19].
2. Methodology
In this work, we seek to understand the structure, stability, and rel-
ative solubility of psilocybin (form A and B, see Fig. 1) in water and the
35 organic solvents which ref. [12] lists as the most commonly used in
the chemical industry (see Tables 1 and 2). This is accomplished with
a combination of free energy calculations and detailed structural anal-
ysis. In all cases, we will have a single solute (psilocybin form A or
B) molecule infinitely dilute in solution. The premise of the solvation
free energy calculations is depicted in Fig. 2. The solvation free energy,
Δ𝐺1solv , corresponds to the change in Gibbs free energy upon taking the
solute from a non-interacting ideal gas phase to solution at the same
2
Journal of Molecular Liquids 392 (2023) 123479
Fig. 2. A simple illustration representative of the solvation free energy calcula-
tions performed in this work. The solvation free energy of the solute (compo-
solv solv
nent 1) is computed in arbitrary solvent I and II, Δ𝐺1,I and Δ𝐺1,II , respectively,
which is related to the transfer free energy of the solute from solvent I to
tran
II, Δ𝐺1,I→II tran
. In turn, Δ𝐺1,I→II is directly related to equilibrium concentration
(moles/volume or mass/volume) of the solute in solvent II and I, 𝑐1,II and 𝑐1,I ,
respectively, where 𝑅 is the molar gas constant and 𝑇 is the absolute tempera-
ture.
Fig. 3. A simple illustration representative of the free energy calculations per-
formed to compare psilocybin form A and B. First, calculations are performed
to compute the Gibbs free energy of form A and B in the ideal gas phase, from
which we compute the Gibbs free energy of reaction for the interconversion of
rxn,ig
form A to B in an ideal gas, Δ𝐺A→B . This is combined with the solvation free
energy of form A and B in a common solvent, Δ𝐺Asolv and Δ𝐺Bsolv , respectively,
to obtain the Gibbs free energy of reaction for the interconversion of form A
rxn rxn
to B in solution, Δ𝐺A→B . In turn, Δ𝐺A→B is directly related to the equilibrium
concentration (moles/volume or mass/volume) of form B and A, 𝑐B and 𝑐A , re-
spectively, where 𝑅 is the molar gas constant and 𝑇 is the absolute temperature.
molar concentration. In this way, Δ𝐺1solv is a direct measure of the
solute-solvent intermolecular interactions. A negative value of Δ𝐺1solv
indicates that the solute prefers to be in solution (compared to the non-
interacting ideal gas state); the more negative the value of Δ𝐺1solv the
greater the affinity of the solute for the solution. When Δ𝐺1solv is com-
puted for the same solute in multiple solvents, the difference in Δ𝐺1solv
between two solvents may be related to the corresponding transfer free
energy, which in turns can be related to the macroscopically observable
relative concentration (mass or molar) of the solute at equilibrium (or
relative solubility) [20,21].
In Fig. 2 we depict the case of a common solute (psilocybin form A
or B) in multiple solvents. When comparing psilocybin forms A and B,
both forms will have a different ideal gas reference state. The premise
of quantitatively comparing forms A and B are depicted in Fig. 3. Elec-
tronic structure calculations are performed to compute the Gibbs free
energy of forms A and B in the ideal gas phase (vacuum). From this,
we can compute the Gibbs free energy of reaction for the interconver-
L. Paul, C.T. Namba-Nzanguim, A. Telesphory et al. Journal of Molecular Liquids 392 (2023) 123479

Table 1
A summary of the dimensionless solvation free energy of psilocybin form A in water and the
35 organic solvents which ref. [12] lists as the most commonly used in the chemical industry.
The solvents are classified by chemical functionality following ref. [12]. The subscript in the
reported solvation-free energy corresponds to the uncertainty in the three decimal places.
The solvation-free energy is broken down into the Coulombic (Coul, 𝑚 = 10 to 14) and LJ
(𝑚 = 0 to 10) contribution, where the total solvation-free energy corresponds to the sum of
these two contributions.

Δ𝐺1solv ∕ (𝑅𝑇 ) form A

Solvent Class CAS Coul LJ Total

ethanol alcohol 64-17-5 −25.350043 −18.808050 −44.158066

butanol alcohol 71-36-3 −23.627206 −20.638061 −44.265215
2-ethylhexanol alcohol 104-76-7 −22.674078 −22.163087 −44.837117
isobutanol alcohol 78-83-1 −21.796093 −20.784064 −42.580157
isopropanol alcohol 67-63-0 −24.168087 −20.158056 −44.326104
methanol alcohol 67-56-1 −26.714045 −16.313037 −43.027058
propanol alcohol 67-63-0 −23.134052 −20.058056 −43.192076
propylene glycol alcohol 57-55-6 −29.416057 −18.134090 −47.550107
acetone ketone 67-64-1 −22.795024 −19.630041 −42.425048
methyl ethyl ketone ketone 78-93-3 −21.539049 −20.608045 −42.147067
methyl isobutyl ketone ketone 108-10-1 −19.690048 −21.377078 −41.067092
methyl isopropyl ketone ketone 563-80-4 −21.299033 −21.121051 −42.420061
mesityl oxide ketone 141-79-7 −24.213046 −20.920055 −45.133072
ethylene bromide halogenated 106-93-4 −9.838026 −23.690065 −33.528070
chloroform halogenated 67-66-3 −12.272027 −23.389045 −35.660052
1,2-dichloroethane halogenated 107-06-2 −11.056022 −22.434046 −33.489051
dichloromethane halogenated 75-09-2 −13.179015 −22.080039 −35.259042
tetrachloroethylene halogenated 127-18-4 −0.582004 −24.652054 −25.234054
carbon tetrachloride halogenated 56-23-5 −0.368002 −24.342052 −24.728052
dimethylformamide amide 68-12-2 −31.142040 −19.276065 −50.418076
1,4-dioxane ether 123-91-1 −19.411031 −20.985077 −40.396083
butyl ether ether 142-92-1 −9.215028 −22.568061 −31.783067
ethyl ether ether 60-29-7 −12.440047 −21.689038 −34.128060
diisopropyl ether ether 108-20-3 −11.200038 −21.978061 −33.178072
tetrahydrofuran ether 109-99-9 −16.351064 −22.582053 −38.932083
tert-butyl methyl ether ether 1634-04-4 −12.748030 −21.858050 −34.607059
dimethyl sulfoxide sulfur 67-68-5 −33.576059 −17.353073 −50.930094
pyridine amine 110-86-1 −20.101035 −21.413049 −41.514060
acetonitrile nitrile 75-05-8 −18.675026 −20.027039 −38.702047
ethyl acetate esters 141-78-6 −22.748037 −20.724054 −43.471065
cyclohexane aliphatic 110-82-7 −0.170002 −23.339072 −23.509072
hexane aliphatic 110-54-3 −0.130001 −22.749042 −22.879042
toluene aromatic 108-88-3 −10.362031 −22.687051 −33.050059
xylene aromatic 1330-20-7 −10.333031 −23.371060 −33.704068
water water 7732-18-5 −42.718037 0.729091 −41.989098

rxn,ig
sion of form A to B in an ideal gas, Δ𝐺A→B . This can be combined with Simulations were performed for psilocybin (form A and B, see Fig. 1)
the solvation free energy of form A and B in a common solvent, to ob- in water and the 35 organic solvents which ref. [12] lists as the most
tain the Gibbs free energy of reaction for the interconversion of form commonly used in the chemical industry (see Tables 1 and 2). Psilo-
rxn . The Gibbs free energy of reaction in turn
A to B in solution, Δ𝐺A→B cybin form A corresponds to the common structure depicted in the
is related to the macroscopically observable relative concentration (or literature, and form B is the zwitterionic form reported as the product in
population) of form B relative to form A [20]. ref. [7]. Force fields were selected following the work of Caleman et al.
[24,25]. We first attempted to use the all-atom version of the optimized
3. Computational details potential for liquid simulations (OPLS-AA) [26–30], where the LigPar-
Gen web server was used to generate force field parameters for psilo-
3.1. Molecular simulation cybin [31,32]. However, bonded parameters were missing to model the
phosphate group, which led to instabilities in the simulations. As a re-
sult, psilocybin and the organic solvents were modeled with the General
All interactions were modeled using a “class I” potential energy
( ) AMBER Force Field version 2 (GAFF2) as implemented in the AMBER 20
function where all non-bonded intermolecular interactions 𝑈nb were
simulation suite [33–35] with AM1-BCC partial charges [36,37]. Param-
accounted for using a combined Lennard-Jones (LJ) plus fixed point
eters were generated using antechamber and converted from AMBER to
charge model of the form [22,23]
GROMACS format using ParmEd. Single molecule 3D structures of psilo-
[( ) ( )6 ]
𝜎𝑖𝑗 12 𝜎𝑖𝑗 cybin and the 35 organic compounds were generated with Open Babel
( ) 1 𝑞𝑖 𝑞𝑗
𝑈nb 𝑟𝑖𝑗 = 4𝜀𝑖𝑗 − + (1) 2.3.2 [38,39] by performing a systematic conformation search to iden-
𝑟𝑖𝑗 𝑟𝑖𝑗 4𝜋𝜀0 𝑟𝑖𝑗
tify the lowest energy conformer followed by geometry optimization,
where 𝑟𝑖𝑗 is the separation distance between sites 𝑖 and 𝑗, 𝜀𝑖𝑗 is the well- all using the General Amber Force Field (GAFF) [34] with Gasteiger
depth of the LJ potential, 𝜎𝑖𝑗 is the distance at which the LJ potential is partial charges [40]. Water was modeled using the rigid TIP4P/2005
zero, and 𝑞𝑖 and 𝑞𝑗 are the partial charges of sites 𝑖 and 𝑗, respectively. model [41].

3
L. Paul, C.T. Namba-Nzanguim, A. Telesphory et al. Journal of Molecular Liquids 392 (2023) 123479

Table 2 All of the molecular dynamics simulations were performed using

A summary of the dimensionless solvation free energy of psilocybin form B in GROMACS 2020.2 [51–54]. For psilocybin and the organic solvents,
water and the 35 organic solvents which ref. [12] lists as the most commonly all bond lengths involving hydrogen were constrained using P-LINCS
used in the chemical industry. The solvents are classified by chemical function- [44,55,56]. Water was modelled as completely rigid using the SET-
ality following ref. [12]. The subscript in the reported solvation-free energy
TLE algorithm [57,58]. The Verlet neighbor list was used, and LJ in-
corresponds to the uncertainty in the three decimal places. The solvation free
teractions were cut-off at 1.4 nm with long-range analytic dispersion
energy is broken-down into the Coulombic (Coul, 𝑚 = 10 to 14) and LJ (𝑚 = 0 to
10) contribution, where the total solvation free energy corresponds to the sum
corrections applied to the energy and pressure [22,23,44,45]. Lorentz-
of these two contributions. Berthelot mixing rules were used for unlike LJ sites [22]. The elec-
trostatic terms were evaluated with the smooth particle-mesh-Ewald
Δ𝐺1solv ∕ (𝑅𝑇 ) form B
method (SPME) with tin-foil boundary conditions [44,45,59] with real
Solvent Class Coul LJ Total space interactions truncated at 1.4 nm. The SPME B-spline was order
ethanol alcohol −46.657133 −22.787044 −69.444140 4, the Fourier spacing was 0.12 nm, and the relative tolerance between
butanol alcohol −42.796187 −24.796059 −67.591196 long and short-range energies was 10−8 . The equations of motion were
2-ethylhexanol alcohol −38.394289 −26.578085 −64.972302
integrated with a timestep of 2 fs, the time constant for the thermostat
isobutanol alcohol −44.116061 −23.252063 −67.368124
isopropanol alcohol −44.147106 −23.915056 −68.062120 was 1 ps and the time constant for the barostat was 4 ps.
methanol alcohol −50.022126 −19.335035 −69.356131 All of the GROMACS force field files used in the present study along
propanol alcohol −43.908204 −24.120054 −68.028211 with sample GROMACS input files are provided in the Supporting In-
propylene glycol alcohol −54.710139 −22.722085 −77.432163 formation accompanying the electronic version of this manuscript.
acetone ketone −35.067053 −23.777040 −58.844067
methyl ethyl ketone ketone −32.279052 −24.702045 −56.980069
methyl isobutyl ketone ketone −29.405078 −25.694054 −55.098095 3.1.1. Free energy calculations
methyl isopropyl ketone ketone −29.996056 −25.367051 −55.363076 The solvation free energy, Δ𝐺1solv , for the solute infinitely dilutes in
mesityl oxide ketone −31.792069 −25.498054 −57.290088 water and the 35 organic solvents at 298.15 K and 1 bar was calcu-
ethylene bromide halogenated −20.256049 −29.021063 −49.277080 lated using a multi-stage free energy perturbation method [60–63] with
chloroform halogenated −26.138057 −27.886044 −54.025072 the multi-state Bennett’s acceptance ratio method (MBAR) [64–67] fol-
1,2-dichloroethane halogenated −22.768041 −26.832045 −49.600061
lowing our previous work [18,68–70]. A “soft-core” potential was used
dichloromethane halogenated −27.917031 −26.348038 −54.265049
tetrachloroethylene halogenated −0.989007 −29.626052 −30.615052 to couple/decouple the solute-solvent intermolecular LJ interactions.
carbon tetrachloride halogenated −0.701004 −29.104051 −29.805051 Stage (𝑚) dependent decoupling parameters, 𝜆LJ 𝑚 and 𝜆𝑚 , controlled
elec

dimethylformamide amide −42.772090 −24.510063 −67.281109 the LJ and electrostatic intermolecular interactions, respectively. The
1,4-dioxane ether −30.064055 −25.936076 −56.001094 decoupling parameters varied from 0 to 1. When 𝜆LJ 𝑚 = 𝜆𝑚 = 1, the so-
elec

butyl ether ether −14.288062 −26.896054 −41.184082 lute is fully coupled to the system. When 𝜆𝑚 = 𝜆𝑚 = 0, the solute is
LJ elec

ethyl ether ether −19.920032 −25.417036 −45.337050 decoupled from the system. The use of a soft-core potential is impor-
diisopropyl ether ether −17.226052 −26.031044 −43.257068
tant in that while it gives the correct limiting value of the LJ potential,
tetrahydrofuran ether −26.136037 −27.123051 −53.260062
tert-butyl methyl ether ether −20.115044 −25.934047 −46.049064
it additionally allows nearly decoupled molecules to overlap with fi-
nite energy (and hence finite probability), thereby increasing the phase
dimethyl sulfoxide sulfur −46.081137 −22.763069 −68.844153
space overlap between neighboring states. The soft-core potential had
pyridine amine −33.589054 −26.086050 −59.675074
the form [71–73]
acetonitrile nitrile −34.442033 −24.388037 −58.830049
( )
ethyl acetate esters −32.742155 −24.970052 −57.712164 𝑈LJ
sc
𝑟𝑖𝑗 ; 𝑚
cyclohexane aliphatic −0.399002 −27.948065 −28.348065
hexane aliphatic −0.340002 −26.782041 −27.122041 ⎧ ⎫
⎪ 𝜎𝑖𝑗12 𝜎𝑖𝑗6 ⎪
toluene aromatic −19.776046 −27.187048 −46.963066 = 4𝜆LJ
𝑚 𝜀𝑖𝑗 ⎨ [ ] − [ ( ) ] ⎬ (2)
( ) 2
xylene aromatic −19.026066 −27.741057 −46.767087 ⎪ 1 − 𝜆𝐿𝐽 𝛼 𝜎 6 + 𝑟6 1 − 𝜆𝐿𝐽
𝑚 𝛼𝐿𝐽 𝜎𝑖𝑗 + 𝑟𝑖𝑗 ⎪
6 6
⎩ 𝑚 𝐿𝐽 𝑖𝑗 𝑖𝑗 ⎭
water water −76.021347 −4.117084 −80.138357
where 𝛼LJ is a constant, which had a value of 1/2. The electrostatic term
in the intermolecular potential was decoupled linearly as
While either Monte Carlo or molecular dynamics could be used to ( ) 1 𝑞𝑖 𝑞𝑗
compute configurational properties, here we use molecular dynamics. 𝑈elec 𝑟𝑖𝑗 ; 𝑚 = 𝜆elec
𝑚 4𝜋𝜀 𝑟 (3)
0 𝑖𝑗
All of the simulations consisted of a single solute (psilocybin form A
or B) molecule infinitely dilute in solution. The number of solvent At each stage 𝑚, an independent MD simulation was performed. For
molecules was chosen to obtain a cubic box with an edge length of each stage 𝑚 the total simulation time was 22 ns with the first 2 ns dis-
approximately 4.6 nm at 298.15 K and 1 bar. Initial structures were carded from analysis as equilibration. The change in the Hamiltonian
generated using Packmol [42,43]. This was followed by 3,000 steepest with the current configuration between stage 𝑚 and the other stages is
descent minimization steps to remove any bad contacts that might have computed every 0.20 ps. This is saved for subsequent post-simulation
resulted from the packing. We next performed two steps of equilibra- analysis with MBAR [67] to determine Δ𝐺1solv . This analysis was per-
tion in an 𝑁𝑃 𝑇 ensemble at 298.15 K and 1 bar with the equations of formed using the Python implementation of MBAR (PyMBAR) and the
motion integrated using the Verlet leap-frog algorithm [22,23,44,45]. GROMACS analysis script distributed with it [74]. The analysis script
First, we performed a 2 ns equilibration using the Berendsen thermo- has implemented an autocorrelation analysis so that only uncorrelated
stat and barostat [44–46]. This was followed by 12 ns of equilibration samples are used to determine Δ𝐺1solv and the corresponding uncertainty
using the stochastic velocity rescaling thermostat [44,47–49] and the [75–77].
Parrinello-Rahman barostat [50]. The final structure from this series A total of 15 different stages were used for the free energy calcula-
of simulations was then used as the initial structure for our free en- tions where 𝑚 = 0 corresponds to a non-interacting (ideal gas) state and
ergy calculations, as will be described momentarily. The final 𝑁𝑃 𝑇 𝑚 = 14 is a fully interacting system. From 𝑚 = 1 to 10 the LJ interactions
equilibration for psilocybin form A and B in ethanol, propylene glycol, were increased from 𝜆LJ 𝑚 = 0.1 to 1.0 in 10 equal increments of 0.1. Elec-
acetone, chloroform, carbon tetrachloride, pyridine, toluene, cyclohex- trostatic interactions were increased in a square root fashion following
ane, and water was continued for an additional 100 ns which was used 𝜆elec
𝑚 ={0.50, 0.71, 0.87, 1.00} from 𝑚 = 11 to 14. The change in free en-
for subsequent structural analysis. ergy in going from 𝑚 = 0 to 10 (non-interacting to full LJ) we refer to

4
L. Paul, C.T. Namba-Nzanguim, A. Telesphory et al.
as the LJ contribution, Δ𝐺1solv,LJ , and the change in free energy in go-
ing from 𝑚 = 10 (full LJ with no electrostatic) to 14 (full LJ and full
electrostatic) we refer to as the Coulombic contribution, Δ𝐺1solv,Coul . The
total solvation-free energy is the sum of these two contributions. Put dif-
ferently, the LJ contribution corresponds to the change in free energy
in going from a state with no solute-solvent intermolecular interactions
(𝑚 = 0) to a state with solute-solvent LJ intermolecular interactions (but
no electrostatic interactions, 𝑚 = 10). Within the state 𝑚 = 0, the solute
may be found anywhere in the system with equal probability. It fol-
lows that an interesting feature of the LJ contribution is that it captures
the change in free energy resulting from the solute cavity formation
in solution. The Coulombic contribution corresponds to the change in
free energy in going from a state with solute-solvent LJ intermolec-
ular interactions but no electrostatic interactions (𝑚 = 10) to a state
with full solute-solvent LJ and electrostatic intermolecular interactions.
The Coulombic contribution will therefore capture the effect of solute-
solvent intermolecular hydrogen bonding.
The simulation parameters for the free energy calculations were the
same as the last step of equilibration except the equations of motion
were integrated with the GROMACS “stochastic dynamics” integrator,
corresponding to stochastic or velocity Langevin dynamics integrated
with the leap-frog algorithm [44,45,78]. The time constant for the
stochastic thermostat was 1.0 ps. This change is necessary as a local
thermostat is required to correctly control the temperature of the de-
coupled and weakly coupled solute molecule.
Sample GROMACS input files are provided in the Supporting Infor-
mation accompanying the electronic version of this manuscript.
3.2. Electronic structure calculations
Electronic structure calculations were performed to determine the
Gibbs free energy of forms A and B in the ideal gas phase. The ini-
tial 3D molecular structures of forms A and B were generated using
the Avogadro package [79]. The geometries were brought to the min-
imum conformational energy using the Universal Force Field (UFF)
[80] available within the software. In each case, a geometry optimiza-
tion and frequency calculation was subsequently performed with the
B3LYP/cc-PVTZ level of theory/basis set using Gaussian 16 [81,82];
the equilibrium geometries were confirmed by the absence of imagi-
nary frequencies. From the frequency calculation the ideal gas-phase
Gibbs free energy was computed as the “Sum of electronic and thermal
free energies” [83].
4. Results and discussion
4.1. Solvation free energy calculations
The computed values of Δ𝐺1solv along with the LJ and Coulombic
contribution for psilocybin form A in water and the 35 organic solvents
are summarized in Table 1. Based on the structure of psilocybin, we ex-
pect hydrogen bonding to be important in the solvation process. This is
reflected in our results where for the aliphatic alkanes (cyclohexane and
hexane) and the non-hydrogen containing halogens (tetrachloroethy-
lene and carbon tetrachloride) the Coulombic contribution to Δ𝐺1solv is
close to 0, and as a result, the value of the total Δ𝐺1solv is greater (or less
negative) than all of the others. Moreover, consider the comparison of
chloroform and carbon tetrachloride. The two molecules differ in that
chloroform contains a single hydrogen which is a weak hydrogen bond
donor [84,85]. When the hydrogen is replaced with chlorine (to give
carbon tetrachloride), the Coulombic contribution and the total Δ𝐺1solv
increase by approximately 12𝑅𝑇 and 11𝑅𝑇 , respectively.
Comparing the alcohols and ketones, we find that the Coulombic
contribution and the total Δ𝐺1solv values are comparable. Whereas al-
cohols are capable of both donating and accepting a hydrogen bond,
ketones are acceptors only. The most negative values of Δ𝐺1solv are
in dimethylformamide (DMF) and dimethyl sulfoxide (DMSO), both of
5
Journal of Molecular Liquids 392 (2023) 123479
which are strong hydrogen bond acceptors. This could possibly suggest
that psilocybin form A is primarily acting as a hydrogen bond donor,
or that there is a competition between interactions wherein psilocybin
form A acts as a hydrogen bond donor and acceptor leading to no net
difference in the ketones and alcohols. We will clarify this point mo-
mentarily with detailed structural analysis.
The most negative value of the Coulombic contribution to Δ𝐺1solv is
for water. On the other hand, the LJ contribution to Δ𝐺1solv in water
is the only case where Δ𝐺1solv is positive, indicative of the difficulty
disrupting the hydrogen bonding network of water to accommodate
the solute. A positive value of the LJ contribution to Δ𝐺1solv indicates
that psilocybin form A would prefer to be in a non-interacting ideal
gas state than in water with full LJ interactions (but no intermolecular
electrostatic interactions). This again underscores the important role of
hydrogen bonding in the solvation of psilocybin.
The computed values of Δ𝐺1solv along with the LJ and Coulombic
contribution for psilocybin form B in water and the 35 organic solvents
are summarized in Table 2. We again find that hydrogen bonding plays
an important role. For the aliphatic alkanes and the non-hydrogen con-
taining halogens, the Coulombic contribution to Δ𝐺1solv is again close to
0. However, we find that the effect of hydrogen bonding is noticeably
more important with psilocybin form B as compared to form A. Going
from chloroform to carbon tetrachloride, with form B the Coulombic
contribution and the total Δ𝐺1solv increase by approximately 25𝑅𝑇 and
24𝑅𝑇 , respectively, which is more than double that observed for form
A.
Comparing the alcohols and ketones for form B, the Coulombic con-
tribution and the total Δ𝐺1solv for the alcohols is now more negative
than the ketones; this could suggest that psilocybin form B is a stronger
hydrogen bond acceptor than donor, leading to more favorable interac-
tions in the alcohols as compared to the ketones. Moreover, the most
negative value of the Coulombic contribution and the total Δ𝐺1solv both
now correspond to water. Interestingly, for water, the LJ contribution
to Δ𝐺1solv is now also negative.
Fig. 4 is a parity plot of the Coulombic and LJ contribution, and the
total Δ𝐺1solv for psilocybin form B versus form A. In all cases, the contri-
butions and total Δ𝐺1solv for form B are less than form A. This indicates
a greater stabilization of form B in the solution as compared to form
A. Considering the Coulombic contribution, we find that the values for
forms B and A are well correlated with an 𝑅2 of 0.93. Moreover, the
line of best fit has a slope of 1.62. The change in the Coulombic con-
tribution is systematic; the more negative the Coulombic contribution
for form A, the greater the decrease for form B. This corroborates the
finding that hydrogen bonding is important in the solvation of psilo-
cybin, with the effect greatest for form B. The Coulombic contribution
decreases on average by 12.3𝑅𝑇 , with a standard deviation of 7.4𝑅𝑇 .
The largest decrease is for water (33.3𝑅𝑇 ), and the smallest decrease is
for hexane (0.2𝑅𝑇 ). Considering the LJ contribution, we find that the
values for forms B and A are very well correlated with an 𝑅2 of 0.98.
For this case, the slope of the line of best fit is 1. The LJ contribution
decreases on average by 4.3𝑅𝑇 with a standard deviation of 0.6𝑅𝑇 .
4.2. Free energy of form A and B
Using electronic structure calculations, the Gibbs free energy of form
B relative to form A in the ideal gas phase is –8.53𝑅𝑇 , which corre-
sponds to the Gibbs free energy of reaction for the interconversion of
rxn,ig
forms A to B, Δ𝐺A→B . The negative value indicates that form B is the
preferred form. Form B corresponds to the zwitterionic form of psilocy-
rxn,ig
bin reported in ref. [7]. The magnitude is significant, where Δ𝐺A→B is
related to the macroscopically observable relative molar (or mass) con-
ig ig
centration of form B (𝑐B ) relative to form A (𝑐A ) in the ideal gas-phase
( rxn,ig )
ig
𝑐B Δ𝐺A→B
= exp − = 5.08 × 103 (4)
ig
𝑐A 𝑅𝑇
L. Paul, C.T. Namba-Nzanguim, A. Telesphory et al. Journal of Molecular Liquids 392 (2023) 123479

Table 3
A summary of the dimensionless solvation free energy of psilocybin
form B relative to A, ΔΔ𝐺1solv ∕ (𝑅𝑇 ), and the dimensionless Gibbs free
energy of reaction for the interconversion of form A to B in solution,
rxn
Δ𝐺A→B ∕ (𝑅𝑇 ). The subscript in the reported values corresponds to the
uncertainty in the two decimal places.

A→B
Solvent Class ΔΔ𝐺1solv ∕ (𝑅𝑇 ) rxn
Δ𝐺A→B ∕ (𝑅𝑇 )
ethanol alcohol −25.2915 −33.8215
butanol alcohol −23.3329 −31.8629
2-ethylhexanol alcohol −20.1432 −28.6732
isobutanol alcohol −24.7920 −33.3220
isopropanol alcohol −23.7416 −32.2716
methanol alcohol −26.3314 −34.8614
propanol alcohol −24.8422 −33.3722
propylene glycol alcohol −29.8819 −38.4219
acetone ketone −16.4208 −24.9508
methyl ethyl ketone ketone −14.8310 −23.3710
methyl isobutyl ketone ketone −14.0313 −22.5613
methyl isopropyl ketone ketone −12.9410 −21.4810
mesityl oxide ketone −12.1611 −20.6911
ethylene bromide halogenated −15.7511 −24.2811
chloroform halogenated −18.3709 −26.9009
1,2-dichloroethane halogenated −16.1108 −24.6408
dichloromethane halogenated −19.0106 −27.5406
tetrachloroethylene halogenated −5.3807 −13.9107
carbon tetrachloride halogenated −5.0807 −13.6107
dimethylformamide amide −16.8613 −25.4013
1,4-dioxane ether −15.6113 −24.1413
butyl ether ether −9.4011 −17.9311
ethyl ether ether −11.2108 −19.7408
diisopropyl ether ether −10.0810 −18.6110
tetrahydrofuran ether −14.3310 −22.8610
tert-butyl methyl ether ether −11.440 9 −19.980 9
dimethyl sulfoxide sulfur −17.9118 −26.451 8
Fig. 4. A parity plot of the Coulombic (Coul, 𝑚 = 10 to 14) and LJ (𝑚 = 0 to 10)
contribution, and the total solvation free energy for psilocybin form B versus pyridine amine −18.1610 −26.6910
form A. (See Tables 1 and 2.) The solid line corresponds to 𝑦 = 𝑥 and is drawn acetonitrile nitrile −20.1307 −28.6607
as a reference, and 𝑚 and 𝑅2 correspond to the slope and squared correlation ethyl acetate esters −14.2418 −22.7718
coefficient, respectively, of the line of best fit to the data.
cyclohexane aliphatic −4.8410 −13.3710
hexane aliphatic −4.2406 −12.7806
rxn,ig toluene aromatic −13.9109 −22.4509
The value of Δ𝐺A→B can be combined with Δ𝐺1solv for psilocybin
xylene aromatic −13.0611 −21.6011
form A and B to determine the Gibbs free energy of reaction for the
rxn (see Fig. 3). A sum- water water −38.1537 −46.6837
interconversion of form A to B in solution, Δ𝐺A→B
mary of the computed values of Δ𝐺A→B rxn , along with Δ𝐺solv of form B
1
relative to form A, ΔΔ𝐺1solv , is provided in Table 3. In all cases, ΔΔ𝐺1solv precipitation [86]. This was a point made in ref. [7] wherein a synthetic
is negative as already discussed, corresponding to a greater stabilization process to was proposed to produce the zwitterionic form of psilocybin
of form B relative to A in solution. Since Δ𝐺A→B rxn = Δ𝐺rxn,ig + ΔΔ𝐺solv ,
A→B 1 (form B). The trend is consistent with the literature that psilocybin is
rxn is more negative than Δ𝐺 rxn,ig
we have that Δ𝐺A→B A→B
. This indicates that soluble in water, slightly soluble in methanol and acetone, and insol-
form B is thermodynamically preferred over form A, where in all cases uble in chloroform and hexane [3,11]. Interestingly, for form A (see
the preference is significant. Table 1), water is not the solvent in which psilocybin has the great-
Having identified form B as the thermodynamically preferred form est solubility. For form A, the solubility is greatest is the polar aprotic
of psilocybin, we revisit Table 2 for the Δ𝐺1solv of form B in the stud- solvents dimethyl sulfoxide and dimethylformamide.
ied solvents. As shown in Fig. 2, the relative value of Δ𝐺1solv is related
to the macroscopically observable relative molar (or mass) concentra- 4.3. Structural analysis
tion in solution at equilibrium. The value of Δ𝐺1solv is most negative
in water, indicating the psilocybin has the greatest solubility in wa- Psilocybin form A and B (see Fig. 1) are capable of forming inter-
ter. The next closest solvent is propylene glycol, for which the Δ𝐺1solv and intra-molecular hydrogen bonds, and are able to act as both a
differs by 2.706𝑅𝑇 , corresponding to a molar (or mass) concentration hydrogen bond donor and acceptor. The free energy calculations sug-
approximately 15 times greater in water than in propylene glycol. The gested that form B is a stronger hydrogen bond acceptor than donor,
difference is even greater in the common solvent ethanol, the next clos- leading to more favorable interactions in alcohols as compared to ke-
est solvent, where the relative concentration is over 44-thousand times tones. On the other hand, for form A, there was no major difference
greater in water than in ethanol. Based on these findings, the use of between solvation in the ketones and alcohols, suggesting that either
anti-solvent crystallization may prove a promising technique to pu- form A is a stronger hydrogen bond donor or that there is a competi-
rify psilocybin from aqueous solutions. Anti-solvent crystallization is tion between interactions wherein psilocybin form A acts as a hydrogen
a technique to recover the solute with a relatively high solubility from bond donor and acceptor leading to no net difference. To clarify these
solution, which avoids temperature control as in cooling crystallization. points, structural analysis was performed for psilocybin form A and B
Instead, a concentrated solution is mixed with another miscible solvent in ethanol, propylene glycol, acetone, chloroform, carbon tetrachloride,
in which the solubility is much lower, leading to supersaturation and pyridine, toluene, cyclohexane, and water to better understand the sol-

6
L. Paul, C.T. Namba-Nzanguim, A. Telesphory et al.
Fig. 5. Structural images for psilocybin form A and B with the atomic labels
used during structural analysis [87,88].
vation mechanism. Structural images for psilocybin and the solvents are
provided in Figs. 5 and 6, respectively, with the atomic labels used dur-
ing analysis and discussion here.
4.3.1. Connection matrix analysis (CMat)
First, to gain an overview the inter- (solute-solvent) and intra-
molecular (solute-solute) hydrogen bonding, we performed a connec-
tion matrix (CMat) analysis [87,88]. In Fig. 7 we show results for the
CMat analysis for the solvents capable for participating in hydrogen
bonding (ethanol, propylene glycol, acetone, pyridine, and water) and
as a counter example, carbon tetrachloride. Generally, the connection
matrix is made up of two parts; the left part shows the interaction con-
nection between the promising proton donor and acceptor while the
right side is the heat map which displays a measure of the interaction
strength. The interaction strength is quantified using the first peak of
the local density (i.e., the un-normalized radial distribution function),
where the greater the intensity (or density) and the shorter the dis-
tance, the stronger the interaction. A matrix box filled with a black
cross suggests there are no interactions within a cutoff distance of 350
pm. Within the analysis 1–2, 1–3, and 1–4 intra-molecular interactions
are also excluded, as are solvent-solvent intermolecular interactions.
Within Fig. 7 we place potential hydrogen bond accepting sites on the
rows, and potential hydrogen bond donating sites on the columns.
The CMat for psilocybin in carbon tetrachloride is found in Fig. 7
A and B for form A and B, respectively. As a solvent carbon tetrachlo-
ride is not capable of participating in hydrogen bonding. Both form A
and B display the existence of only intramolecular hydrogen bonding.
For form A, the phosphate H atoms (H1 and H2) show intramolecu-
7
Journal of Molecular Liquids 392 (2023) 123479
lar interactions with the (unprotonated) tertiary amine N (N1). On the
other hand, psilocybin form B exhibits stronger intramolecular interac-
tion between the equivalent O atoms (O3 and O4) with the H atom in
the ammonium (protonated tertiary amine, H2), in addition to a weaker
intramolecular interaction between O1 and H2. As discussed earlier, us-
ing electronic structure calculations we found that form B had a lower
free energy as compared to form A in the ideal gas state. From the anal-
ysis here in carbon tetrachloride, form B may be favored over A due to
the presence of stronger intramolecular interactions.
The CMat for psilocybin in acetone is found in Fig. 7 C and D for
form A and B, respectively. Acetone is a polar aprotic solvent that is
only capable of accepting hydrogen bonds. Psilocybin form A has two
phosphate hydrogen atoms (H1 and H2) which strongly, and pyrrole
H attached to the ring N (H3) which weakly interacts (intermolecular)
with the acetone O (O1). Form A also exhibits the same intramolecu-
lar interactions as found in carbon tetrachloride, only weaker. On the
other hand, for form B we observe the same strong intramolecular inter-
actions as in carbon tetrachloride. Additionally, weaker intermolecular
interactions exist between the acetone O atom (O1) and the phosphate
H atom (H1), and both H atoms bonded to an N atom (H2 and H3) with
different intensities.
The CMat for psilocybin in pyridine is found in Fig. 7 E and F for
form A and B, respectively. Similar to acetone, pyridine is a polar apro-
tic solvent. As a result, we find that for both forms the interactions are
analogous to the case of acetone. This is in support of the computed
values of Δ𝐺1solv discussed earlier (see Tables 1 and 2), wherein the nu-
merical values for acetone and pyridine did not differ significantly.
Next, the CMat for psilocybin in ethanol is found in Fig. 7 G and
H for form A and B, respectively. Ethanol is a polar protic solvent ca-
pable of both donating and accepting hydrogen bonds. As a result, we
observe both inter- and intra-molecular hydrogen bonding, where psilo-
cybin both donates and accepts hydrogen bonds. In form A, we observe
the same intermolecular interactions wherein psilocybin acts the hydro-
gen bond donor in addition to the same intramolecular interactions as
observed in acetone. However, we additionally observe hydrogen bond-
ing between the ethanol H atom (H1) and the O atoms in the phospate
group of psilocybin (O1, O2, O3, and O4), with the strongest interac-
tion with O4. Likewise for form B, we observe the same intermolecular
interactions wherein psilocybin acts the hydrogen bond donor in ad-
dition to the same intramolecular interactions, as observed in acetone.
However, we also observe hydrogen bonding between the ethanol H
atom (H1) and the O atoms in the phospate group of psilocybin (O1,
O2, O3, and O4). We also find that with the presence of these addi-
tional intermolecular hydrogen bonds, the intensity of the intramolec-
ular interactions decreases. Additionally, for form A the strength of the
interactions wherein psilocybin was a donor (H1,2–O1) and acceptor
(O4–H1,2) were comparable. On the other hand, for form B psilocybin
was a stronger acceptor (O3,4–H1) than donor (H1–O1), with an in-
tensity greater than that exhibited by form A. This corroborates with
the result of the free energy calculations, suggesting that form B is a
stronger hydrogen bond acceptor than donor, resulting in a lower sol-
vation free energy in the alcohols as compared to the ketones. This also
provides clarity in that form A participates in hydrogen bonding as both
an acceptor and donor with comparable strength. The competition be-
tween acting as a hydrogen bond donor and acceptor must therefore be
the reason of the only minor difference in the Columbic contribution to
the solvation free energy observed in the ketones and alcohols.
The CMat for psilocybin in water is found in Fig. 7 I and J for form A
and B, respectively. Also similar to ethanol, water is a polar protic sol-
vent, although we expect that water is a stronger hydrogen bond donor
and acceptor. We find that for both forms the interactions analogous
to the case of ethanol. As compared to ethanol, we do find that the
intensities (i.e., local densities) of the intermolecular hydrogen bonds
are higher, confirming stronger hydrogen bonds between psilocybin
and water as compared to ethanol. This is confirmed by the computed
values of Δ𝐺1solv discussed earlier. We found that the Coulombic con-
L. Paul, C.T. Namba-Nzanguim, A. Telesphory et al.
Fig. 6. Structural images for the select solvents with the atomic labels used during structural analysis [87,88].
tribution was much more negative in water as compared to ethanol.
Moreover, the LJ contribution in water was larger (less favorable) in
part as a consequence of the necessity to break strong water-water in-
termolecular hydrogen bonds in order to create a cavity for the solute
in solution. Moreover, the Coulombic contribution for form B in water
was more negative (i.e., more favorable) than A confirming that form B
is a stronger hydrogen bond acceptor. Additionally, while for form A in
ethanol the strength of the interactions wherein psilocybin was a donor
(H1,2–O1) and acceptor (O4–H1,2) were comparable, in water the case
where psilocybin is an acceptor is stronger. For form B, the same trend
is observed in water as in ethanol wherein psilocybin appears to be a
stronger hydrogen bond acceptor. If we consider the hydrogen bond
donating and accepting scale used by the solubility parameter based
method MOSCED (Modified Separation of Cohesive Energy Density),
the hydrogen bond donating and accepting strength of ethanol are com-
parable (12.58 and 13.29, respectively). On the other hand, water is
a much stronger hydrogen bond donor (52.78) than acceptor (15.86)
[89–91]. The result of water being a much stronger hydrogen bond
donor is likely why the intermolecular interaction wherein form A is
acting as the acceptor in water is stronger than the case where form A
is acting as a donor.
Finally, the CMat for psilocybin in propylene glycol is found in Fig. 7
K and L for form A and B, respectively. Similar to ethanol, propylene
glycol is a polar protic solvent. However, propylene glycol contains two
hydroxyl groups that is able to form both inter- and intra-molecular hy-
drogen bonds [91–96]. We find that for both forms the interactions are
analogous to the case of ethanol. We note that while the CMat shows a
lower intensity for the intermolecular hydrogen bonds between psilocy-
bin and propylene glycol (or local density maximum), this is the result
of psilocybin interacting with two hydroxyl groups.
4.4. Additional structural analysis
Additional structural analysis was performed in support of CMat,
with the results presented in the supporting information (SI) accompa-
nying the electronic version of this manuscript. First, results from the
radial distribution (RDF) are presented in Figures S1 and S2 of the SI,
for form A and B, respectively, and in Table S1. As compared to CMat,
the RDF corresponds to the local relative to bulk density as a func-
tion of distance. The results from the RDF analysis are in agreement
8
Journal of Molecular Liquids 392 (2023) 123479
with the CMat analysis. We note that the RDF intensity followed the
trend ethanol > propyl glycerol > pyridine > acetone > water > carbon
tetrachloride. We note that while in the CMat analysis we observed a
relatively large local density of water around psilocybin, the intensity of
the RDF is noticeably relatively smaller. Quantiatively this results from
the larger bulk density of water compared to the other solvents, and
is indicative that in water psilocybin is competing with strong water-
water intermolecular interactions. As noted earlier, this is reflected in
the results of the solvation free energy calculations.
To confirm the observation of hydrogen bonding between psilocybin
and the solvents, in Figure S3 of the SI we show the results of a com-
bined distribution function (CDF) analysis. For the CDF, we plot the
results of an angle distribution function (ADF) analysis versus a RDF
analysis, to show the angle versus distance for the interactions of inter-
est, with the scale indicative of the frequency of occurrence. A hydrogen
bond is characterized by a short interaction distance (taken here to be
less than 450 pm), and specific interaction angle (taken here to be be-
tween 130◦ and 180◦ or between 0◦ and 50◦ , depending on how the
angle is measured) [87,88]. The results of the CDF analysis confirm the
presence of hydrogen bonding.
Last, in Figure S4 of the SI we show the results of a spatial distribu-
tion function (SDF) analysis. Results are presented for psilocybin forms
A and B in ethanol and carbon tetrachloride. The solvents were selected
following the RDF analysis as the solvents with the largest and smallest
RDF intensities, respectively. The SDF if used to visualize the 3D sol-
vation of psilocybin by the solvent, where the iso-surfaces correspond
to local densities of a given value. The SDF analysis was visualized us-
ing the Visual Molecular Dynamics (VMD) software. For the case of
ethanol we observe clustering of the solvent around the polar regions
of psilocybin. On the other hand, for carbon tetrachloride, we observe
a propensity toward the non-polar regions of psilocybin.
4.5. Electronic structure calculations
4.5.1. Stability and vibrational spectra
The stability or possible existence of the two forms of psilocybin (A
and B) can be further characterized from the analysis of the optimized
geometries and vibrational frequencies computed using electronic struc-
ture calculations. The electronic structure calculation (i.e., DFT) results
showed that form B is relatively more stable than form A with a –21.15
L. Paul, C.T. Namba-Nzanguim, A. Telesphory et al. Journal of Molecular Liquids 392 (2023) 123479

Fig. 8. Comparison of the computed IR spectra of psilocybin form A and B.

Table 4
Vibrational modes of forms A and B calculated at the B3LYP/cc-PVTZ level of
theory in the gaseous phase. The symbols 𝜔 = wagging vibration, 𝛿 = bending
vibration, 𝜈 = stretching vibration and 𝜌 = rocking vibration (in plane).
A (cm−1 ) B (cm−1 ) Experimental (cm−1 ) Tentative assignment of vibrations
465.31 465.18 461.25 𝜔PO4
754.12 734.33 746.77 𝛿CCC(indole) + 𝜈NR2 + 𝜔CH(indole)
780.67 798.43 785.93 𝜌CH2
854.54 857.97 855.93 𝛿CCC(indole) +𝜈P−OH
938.44 891.25 920.69 𝜈P−OH
1050.94 1034.53 1042.33 𝜌OH
1102.01 1102.99 1101.24 𝜌CH(indole) + 𝜌CH2
1242.35 1221.98 1229.69 𝛿CCC + 𝜈C=N(indole)
1336.70 1287.23 1348.55 𝜈P=O+𝜌CH2
1446.72 1465.36 1442.19 𝜈C=C + 𝜈C=N(indole)
− 1823.24 − 𝜈N−H⋯O
3189.58 3184.56 3179.96 𝜈CH(benzene)

Each form belongs to the C1 symmetry point group consisting of

Fig. 7. The connection matrix (CMat) analysis of psilocybin form A and B in
carbon tetrachloride (CCl4 , A and B), acetone (C and D), pyridine (E and F),
ethanol (G and H), water (I and J), and propylene glycol (K and L). The first
column of figures corresponds to psilocybin form A, and the second column
corresponds to psilocybin form B.
kJ/mol free energy difference between them. The absence of imaginary
frequencies confirms that the obtained geometries during optimization
correspond to the minima on the potential energy surface. In order to
understand the differences in the two forms of psilocybin, their respec-
tive harmonic vibrational frequencies were analyzed, and the calculated
infrared (IR) spectra for both forms are presented in Fig. 8. The two
forms are very similar, only that B is obtained when hydrogen is ab-
stracted from the acidic moiety (phosphate group) to the basic moiety
(dimethylamino group) resulting in a zwitterionic product also known
as an inner salt.
36 atoms which accounts for 102 vibrational degrees of freedom. Con-
former A has been experimentally reported among the two conformers
and the IR spectral data are available for comparison [97]. Therefore,
in Table 4, we compare the calculated IR active vibrational modes for
form A with experimental IR frequencies and provide tentative spec-
tral assignments. Form A exhibits five bands with distinct intensities at:
170−540 cm−1 , 750−950 cm−1 , 1000−1660 cm−1 , 2900−3250 cm−1
and 3670−3830 cm−1 . It is worth noting that the calculated IR spectrum
for form A matches well with the experimentally reported spectra. For
form B, we observed bands with varying intensities at: 200-560 cm−1 ,
720−900 cm−1 , 960−1100 cm−1 , 1200−1680 cm−1 . Conformer B ex-
hibits a distinct peak at 1823.24 cm−1 originating from the abstraction
of a hydrogen atom from the phosphate group to the dimethylamino
group. Other bands observed include 3050−3200 cm−1 and 3650−3830
cm−1 . In the region below 2000 cm−1 form B exhibits higher intensity
than those of A and vice versa for vibrations above 3000 cm−1 .
The IR spectra of both forms revealed distinct stretching vibrations
𝜈, including the 𝜈N−H originating from the five-membered ring at 3677
cm−1 . Also, the 𝜈O−H for the two terminal OH groups in form A were ob-
served at 3817 cm−1 and 3826 cm−1 , while that of form B was detected
at 3830 cm−1 . The out of plane 𝜈O−H exhibits a higher intensity than the
one in-plane. The 𝜈C−H of the dimethylamine group were observed at
around 2900 cm−1 and 3042–3049 cm−1 for A and B, respectively. The
CH2 units exhibit 𝜈C−H at 3027(3042) cm−1 for A and 3089(3116) cm−1
for B. For form B, H is positioned between N and O, and one can observe
that form B exhibits a sharp peak with the highest intensity at 1823.24
cm−1 corresponding to N-H stretching with an average bond distance of

9
L. Paul, C.T. Namba-Nzanguim, A. Telesphory et al.
Fig. 9. The HOMO and LUMO charge density distribution, HOMO/LUMO ener-
gies and energy gap of psilocybin A and B. (Note that 1 eV = 96.485 kJ/mol
[100].)
Fig. 10. The electronic absorption spectra of psilocybin form A and B.
1.130 Å and a 1.422 Å O-H bond. Other characteristic vibrations and
assignments are summarized in Table 4.
The results of the electronic structure calculation reveal differences
in the geometrical parameters between the two forms. For instance, the
P-O bond lengths in the H2 PO4 group (form A) are 1.605, 1.467, 1.612
and 1.594 Å, while those in HPO4 (form B) are 1.652, 1.485, 1.616
and 1.529 Å. As can be observed hydrogen abstraction resulted in the
shortening of the P-O− bond and an increase in other P-O bonds.
4.5.2. Optical and electronic characteristics
Frontier molecular orbitals are widely used for the analysis of sta-
bility and chemical reactivity of molecules [98,99]. Fig. 9 shows the
calculated highest occupied molecular orbital (HOMO), lowest unoccu-
pied molecular orbital (LUMO) energies, energy gaps and HOMO and
LUMO charge density distribution. The electronic absorption spectra for
psilocybin A and B are presented in Fig. 10. Both HOMO and LUMO of
form B are up-shifted by 0.29 eV and 0.32 eV, respectively (Note that 1
eV = 96.485 kJ/mol [100]). The two forms depict comparable HOMO
and LUMO charge distributions. The simulated electronic spectra show
that both forms exhibit similar electronic transitions characterized by
two bands in the ultraviolet region of the electromagnetic spectrum.
The high energy bands were observed around 196–198 nm resulting
from 𝑛 → 𝜋 ∗ transitions and the lower energy bands were observed at
252 nm and 250 nm for A and B, respectively; the lower energy bands
are observed due to 𝜋 → 𝜋 ∗ transitions. At absorption maximum wave-
lengths, form B’s electronic spectra are slightly blue-shifted compared
to form A; consequently leading to an increased energy gap for B, as
seen in Fig. 10. The smaller energy gap for A indicates that form A can
10
Journal of Molecular Liquids 392 (2023) 123479
be relatively reactive when compared to form B. Based on this analysis,
it is recommended that further research should focus on characterizing
both forms of psilocybin. Investigating the chemical kinetic and ther-
modynamic properties of these two forms of psilocybin can shed light
on their respective chemical stability.
5. Summary and conclusion
Psilocybin is the active psychedelic compound known for hallucino-
genic properties present in “magic mushrooms.” Since its structural
elucidation and chemical synthesis in 1958, both synthetic and puri-
fied psilocybin have been subjected to clinical trials, with the findings
showing that the compound presents huge potential for the treatment of
various mental health disorders. However, as a result of legal measures
psilocybin use has been restricted to controlled medical and scientific
research in most of the world. Its clinical applications are hampered by
poor solubility in most organic solvents, and the ability to study and
investigate the physical properties of psilocybin are limited.
In this work we investigate two different tautomers of psilocybin (la-
beled here as form A and B). While form A corresponds to the tautomer
commonly depicted in the literature, form B corresponds to the zwitte-
rionic form reported recently in ref. [7]. We expect that the presence
of two different chemical structures in the literature results from the
limited ability to experimentally investigate the physical properties of
psilocybin. We use molecular simulation to investigate the interaction
and solvation of psilocybin in a wide range of solvents. We additionally
used electronic structure calculations (DFT) to reveal that form B is the
thermodynamically favored tautomer. The calculated vibrational char-
acteristics in this study accord well with experimentally observed data.
Both exhibit two absorption bands with the higher energy band at 196
nm and 198 nm while the lower energy bands were observed at 252
and 250 nm for A and B, respectively.
The propensity of psilocybin to solvate in solvents was deter-
mined by using molecular simulation solvation free energy calculations,
Δ𝐺1solv , which is related to the macroscopically observable relative
molar (or mass) concentration at equilibrium (or relative solubility)
[21]. The computed Δ𝐺1solv along with LJ and Coulombic contribu-
tion showed that form B had a higher stabilization in solvents than
form A. Hydrogen bonding has a strong influence in the solvation pro-
cess. We found that form B is a stronger hydrogen bond acceptor than
donor, leading to more favorable interactions in alcohols as compared
to ketones. On the other hand, for form A, there was no major dif-
ference between solvation in the ketones and alcohols. Form A acts
as a hydrogen bond donor and acceptor with comparable strength,
leading to a competition between interactions in solution. This solva-
tion mechanism was further confirmed using detailed structural analy-
sis.
Having identified form B as the thermodynamically preferred tau-
tomer, the results of the solvation free energy showed that the solubility
in water is greater than in any of the studied organic solvents. This could
present opportunities for anti-solvent crystallization to purify psilocybin
from an aqueous solution as suggested in ref. [7] wherein a synthetic
process to was proposed to produce the zwitterionic form of psilocybin
(form B). If one were interesting in improving the solubility relative to
water, future studies might consider the use of alternative solvents such
as ionic liquids or deep eutectic solvents. The present study suggests
that in order to increase the solubility relative to water, one would re-
quire a solvent that is a stronger hydrogen bond donor than water. The
authors acknowledge that the accuracy of DFT predictions can be influ-
enced by the choice of the level of theory. In this study, the reported
results are specifically based on a comparison with experimental data
for form A, revealing a standard deviation of 5.14 cm−1 . To improve
DFT predictions, it is recommended to evaluate different DFT function-
als; especially those specialized in modeling hydrogen bonding.
L. Paul, C.T. Namba-Nzanguim, A. Telesphory et al.
CRediT authorship contribution statement
Lucas Paul: Conceptualization, Formal analysis, Investigation,
Methodology, Project administration, Visualization, Writing – original
draft, Writing – review & editing. Cyril T. Namba-Nzanguim: For-
mal analysis, Investigation, Writing – original draft, Writing – review
& editing. Aidani Telesphory: Formal analysis, Investigation, Writing
– review & editing. Jehoshaphat Oppong Mensah: Formal analysis,
Investigation, Writing – review & editing. Denis Mteremko: Formal
analysis, Investigation, Writing – review & editing. Rene Costa: Formal
analysis, Investigation, Writing – review & editing. Saidi Mohamedi
Katundu: Formal analysis, Investigation, Writing – review & editing.
Lucas P. Kwiyukwa: Formal analysis, Investigation, Writing – review
& editing. Naserian Daniel Kambaine: Formal analysis, Investiga-
tion, Writing – review & editing. Julius Juvenary: Formal analysis,
Investigation, Writing – review & editing. Sixberth Mlowe: Formal
analysis, Investigation, Writing – review & editing. Geradius Deogra-
tias: Conceptualization, Formal analysis, Investigation, Methodology,
Visualization, Writing – original draft, Writing – review & editing.
Daniel M. Shadrack: Conceptualization, Formal analysis, Investiga-
tion, Methodology, Visualization, Writing – original draft, Writing –
review & editing. Andrew S. Paluch: Conceptualization, Formal anal-
ysis, Investigation, Methodology, Project administration, Resources,
Visualization, Writing – original draft, Writing – review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgements
All of the molecular dynamics simulations were performed at the
Ohio Supercomputer Center on the Pitzer Cluster [101]. Resources were
graciously provided to support a workshop on free energy calculations
by Andrew S. Paluch delivered at the Dar es Salaam University College
of Education (DUCE), Tanzania.
Appendix A. Supplementary material
Supplementary material related to this article can be found online
at https://doi.org/10.1016/j.molliq.2023.123479.
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