Synthesis of a psilocin hapten and a protein-hapten conjugate

Article abstract of DOI:10.1211/002235702320402116

Derivatives of psilocin with ω-functionalized alkyl spacers in position 1 of the indole ring were synthesized as haptens for use in a radioimmunoassay. Whereas the psilocin analogues with a 3-aminopropyl and a 4-aminobutyl moiety at the indole nitrogen decomposed during synthesis, the analogous 3-carboxypropyl psilocin derivative proved to be stable. This compound was coupled to bovine serum albumin (BSA) using the N-hydroxysuccinimide ester-mediated conjugation. The protein-hapten conjugate was characterized by matrix-assisted laser desorption ionization mass spectrometry. The mass spectrometry data indicated an average incorporation ratio of 4-5 molecules of psilocin hapten per molecule of BSA.

Full text of DOI:10.1211/002235702320402116

Journal of
JPP 2002, 54: 1265–1270
# 2002 The Authors
Received March 19, 2002
Accepted June 7, 2002
ISSN 0022-3573
Synthesis of a psilocin hapten and a protein–hapten
conjugate
Christian Albers, Matthias Lehr, Justus Beike, Helga Kohler
and Bernd Brinkmann
Abstract
Derivatives of psilocin with -functionalized alkyl spacers in position 1 of the indole ring were
synthesized as haptens for use in a radioimmunoassay. Whereas the psilocin analogues with a 3-
aminopropyl and a 4-aminobutyl moiety at the indole nitrogen decomposed during synthesis, the
analogous 3-carboxypropyl psilocin derivative proved to be stable. This compound was coupled to
bovine serum albumin (BSA) using the N-hydroxysuccinimide ester-mediated conjugation. The
protein–hapten conjugate was characterized by matrix-assisted laser desorption ionization mass
spectrometry. The mass spectrometry data indicated an average incorporation ratio of 4–5 molecules
of psilocin hapten per molecule of BSA.
Introduction
The indole derivatives psilocybin and psilocin (Figure 1) were ®rst identi®ed as
Psilocybe
constituents of hallucinogenic mushrooms of the genus
(Hofmann et al 1958 ;
Hofmann & Troxler 1959). Later, these indoles were also found in a variety of other
species of fungi (Stijve & Kuyper 1985 ; Gartz 1986 ; Semerdzieva et al 1986). Although
psilocybin is the major component in comparison with psilocin in mushrooms, its
phosphoric ester group is cleaved rapidly and extensively after ingestion of the
mushrooms (Horita & Weber 1961, 1962 ; Kalberer et al 1962). These ®ndings indicate
that psilocybin acts as a prodrug and that its metabolite psilocin represents the
pharmacologically active agent (Hasler et al 1997).
Institute of Pharmaceutical and
Medicinal Chemistry, University
¨
of Munster, Hittorfstrasse 58-62,
The abuse of psilocybin-containing hallucinogenic fungi has become an increasing
problem in recent years (MusshoΠet al 2000). Several methods have been described for
the detection of psilocybin and psilocin in fungi, for example thin layer chromatography
(Beug & Bigwood 1981), high-performance liquid chromatography (HPLC) with UV
(Beug & Bigwood 1981 ; Sottolano & Lurie 1983 ; MusshoŒ et al 2000), ¯ uorescence
(Christiansen et al 1981) or electrochemical detection (Wurst et al 1992), gas
chromatography-mass spectrometry (GC-MS) (Wurst et al 1992 ; Keller et al 1999) and
capillary zone electrophoresis (Pedersen-Bjergaard et al 1997). Current methods for the
determination of psilocin in body ¯ uids are REMEDi HS as a drug screening method
(Sticht & Kaferstein 2000), HPLC with electrochemical detection (Kysilka 1990 ; Hasler
et al 1997 ; Lindenblatt et al 1998), GC-MS (Sticht & Kaferstein 2000 ; Grieshaber et al
2001) and liquid chromatography electrospray ionization mass spectrometry (Bogusz
2000). Whereas the determination of psilocin in urine can be performed without a
sample clean up (Kysilka 1990), its analysis in plasma requires an extraction procedure
followed by derivatization of the extracts. In forensic toxicology analyses, an
immunoassay is often used (Carter et al 2000 ; De Letter et al 2001), because it oŒers the
opportunity to detect a drug of abuse in plasma or urine sensitively without pre-
treatment of the samples. However, such a method has not yet been published for
psilocin.
¨
D-48149 Munster, Germany
Christian Albers, Matthias Lehr
Institute of Legal Medicine,
University of Munster, von-
Esmarch-Strasse 86, D-48149
Munster, Germany
¨
¨
¨
Justus Beike, Helga Kohler,
Bernd Brinkmann
Correspondence: M. Lehr,
Institute of Pharmaceutical and
Medicinal Chemistry, University
¨
of Munster, Hittorfstrasse 58-62,
¨
D-48149 Munster, Germany. E-
mail: lehrm!uni-muenster.de
Acknowledgement: Mass
spectrometric analysis was
performed by Jens Grote and
Simone Konig in the Integrated
Functional Genomics group of
the IZKF of the University of
¨
Here, we describe the synthesis of a psilocin hapten and its coupling to bovine serum
albumin (BSA). This BSA±hapten conjugate can be used for the development of a
radioimmunoassay for psilocin.
¨
Munster.
1265

1266
Christian Albers et al
N N
, -dimethyltryptamine (398 mg, 1 53 mmol)in dry DMF
(14 mL) was then added dropwise. After stirring the mix-
.
°
ture for 30 min at 0 C, 4-bromobutyronitrile (228 mg,
1 53 mmol) dissolved in dry DMF (10 mL) was added in
.
the same way. The mixture was stirred for an additional
30 min at the same temperature and overnight at room
temperature. The mixture was diluted with 5% aqueous
NaHCO3 solution and extracted with diethyl ether. The
organic phase was dried over MgSO4 and the solvent
evaporated. Silica gel chromatography with dichloro-
Figure 1 Structures of psilocybin and psilocin.
}
methane ethyl acetate triethyl amine (8:1:0.2, v v v)
gave as a waxy solid (237 mg, 49% ). H NMR (CDCl3):
}
} }
1
4
.
.
.
.
.
.
.
2 14 (s, 6H), 2 19±2 28 (m, 4H), 2 53±2 61 (m, 2H), 2 99±
3 07 (m, 2H), 4 22 (t, J 7 Hz, 2H), 5 19 (s, 2H), 6 56 (d,
Materials and Methods
.
¯
.
.
¯
¯
. ¯ .
8 Hz, 1H), 6 80 (s, 1H), 6,91 (d, J 8 Hz, 1H), 7 10 (t,
. .
8 Hz, 1H), 7 32±7 43 (m, 3H), 7 49±7 53 (m, 2H);
+
J
J
General
.
.
N
N«
-Ethyl- -(3-dimethylaminopropyl)carbodiimide
}
.
MS (EI) m z: 361 0 (M ).
(EDAC)polymer was purchased from Fluka (Taufkirchen,
Germany). BSA was a gift from Dade Behring (Marburg,
Germany). Silica gel chromatography was performed using
silica gel 60 (70±230 mesh) from Merck (Darmstadt, Ger-
many); 1H NMR spectra were obtained on a Varian Gem-
ini 200 NMR spectrometer. Chemical shifts are reported in
units (ppm) relative to tetramethylsilane. The electron
beam ionization (EI) mass spectra were obtained on a
Thermo Finnigan GCQ (San Jose, USA). The high res-
olution mass spectrum applying electrospray ionization
(HR-ESI-MS) was recorded on a Micromass Quattro-LC
(Manchester, UK).
4-[4-Benzyloxy-3-(2-dimethylaminoethyl)indol-1-
yl]butan-1-amine (5)
4
.
A solution of (133 mg, 0 37 mmol) in methanol (10 mL)
was added to Raney-Nickel (158 mg) in a hydrogenation
¯ ask. The mixture was stirred under a balloon ®lled with
H2 for 38 h at room temperature. The catalyst was removed
by ®ltration through a glass ®lter. The solvent was eva-
5
porated to give as a yellow waxy solid (116 mg, 86% ).
H NMR (CDCl3 ): 1 38±1 52 (m, 2H), 1 77±1 91 (m, 2H),
1
.
.
.
.
.
.
.
.
.
.
2 15 (s, 6H), 2 60±2 73 (m, 4H), 3 02±3 10 (m, 2H), 4 05 (t,
¯
1H), 6 88±6 94 (m, 1H), 7 03±7 12 (m, 1H), 7 33±7 53 (m,
. . ¯ .
7 Hz, 2H), 5 18 (s, 2H), 6 55 (d, J 8 Hz, 1H), 6 81 (m,
.
5H); MS (EI) m z: 365 2 (M ).
J
.
.
.
.
.
Synthesis of the psilocin hapten
+
}
.
Dibenzyl ²3-[4-benzyloxy-3-(2-dimethylaminoethyl)indol-
-
1-yl]propyl ´amine (2)
.
NaH (25 mg, 0 62 mmol) was dissolved in dry dimethyl-
formamide (DMF; 8 mL) at 0 C and stirred for 30 min at
[4-Benzyloxy-3-(2-dimethylaminoethyl)indol-1-yl]butyric
acid (7)
°
N N
this temperature. The solution of 4-benzyloxy- , -di-
methyltryptamine (Yamada et al 1998 ; Nichols & Frescas
.
1999) (62 mg, 0 21 mmol) in dry DMF (14 mL) was added
dropwise and the mixture was stirred for further 30 min
4
.
To a solution of (237 mg, 0 65 mmol) in ethanol (10 mL),
a solution of 10% aqueous potassium hydroxide (12 mL)
was added with stirring. The mixture was heated under
m
re¯ ux for 10 h. The solution was acidi®ed with 2 hy-
°
at 0 C. The solution of dibenzyl(3-bromopropyl)amine
(Nagle et al 2000) (165 mg, 0 52 mmol) dissolved in dry
DMF (10 mL) was then added in the same way. The result-
ing mixture was stirred for 30 min in an ice bath, then for
drochloric acid and evaporated under reduced pressure.
The residue was triturated with dichloromethane to dis-
solve the product. The solution was separated and the
.
7
solvent was evaporated under vacuum giving as a yellow
waxy solid (97 mg, 36% ). H NMR (dimethylsulfoxide-
1
°
4 h at 60 C, and subsequently overnight at room tempera-
ture. The mixture was diluted with 5% aqueous NaHCO3
solution and extracted with diethyl ether. The organic
phase was dried over MgSO4 and evaporated. Silica gel
.
¯
.
¯
.
d6): 2 02 (quint, J 7 Hz, 2H), 2 29 (t, J 7 Hz, 2H), 2 64
(s, 6H), 3 21±3 32 (m, 4H), 4 21 (t, J 7 Hz, 2H), 5 29 (s,
.
.
.
¯
.
.
.
2H), 6 72±6 78 (m, 1H), 7 12±7 20 (m, 2H), 7 25 (s, 1H),
7 43±7 69 (m, 5H); MS (EI) m z: 380 2 (M ).
.
.
.
+
}
chromatography with dichloromethane methanol (1:1)
produced as a yellow waxy solid (83 mg, 74% ). H NMR
}
.
.
.
1
2
.
(CDCl3): 1 94 (quint, J 7 Hz, 2H), 2 13 (s, 6H), 2 44±
2 56 (m, 4H), 2 92±3 00 (m, 2H), 3 57 (s, 4H), 4 00 (t, J
¯
.
.
.
.
.
.
.
¯
4-[3-(2-Dimethylaminoethyl)-4-hydroxyindol-1-yl]butyric
acid (8)
.
7 Hz, 2H), 5 18 (s, 2H), 6 43 (s, 1H), 6 51 (d, J 8 Hz,
1H), 6 84 (d, J 8 Hz, 1H), 7 04 (t, J 8 Hz, 1H), 7 27±
.
.
¯
.
¯
.
¯
.
7
.
A solution of (75 mg, 0 18 mmol)in 95% ethanol (10 mL)
+
}
7 53 (m, 15H); MS (EI) m z: 531 2 (M ).
.
.
}
}
was added to 40 mg Pd C (10% , w w) in a hydrogenation
¯ ask. The mixture was stirred under a balloon ®lled with
H2 for 2 h at room temperature. The catalyst was removed
by ®ltration through a cotton pad under nitrogen. The
4-[4-Benzyloxy-3-(2-dimethylaminoethyl)indol-1-
yl]butyronitrile (4)
.
NaH (119 mg, 3 0 mmol) was added to dry DMF (8 mL) at solvent was evaporated under reduced pressure giving as
0 C and stirred for 30 min. The solution of 4-benzyloxy- a waxy solid (49 7 mg, 95% ). IR (KBr): 2900, 1720, 1460,
8
°
.

1267
(1 8 mg 500 L); 0 5 L of the sample solution was spotted
Synthesis of psilocin derivatives
1
1260, 770, 730 cm ; H NMR (CH3OD): 2 05 (quint, J
1
-
}
7 Hz, 2H), 2 25 (t, J 7 Hz, 2H), 2 89 (s, 6H), 3 22±3 34 with 0 5 L of the matrix solution onto the MALDI target.
.
¯
.
.
.
(m, 2H), 3 44±3 51 (m, 2H), 4 10 (t, J 7 Hz, 2H), 6 41 (d,
¯
.
¯
.
.
.
.
.
.
.
. . .
7 Hz, 1H), 6 84±6 95 (m, 2H), 7 00 (s, 1H); MS (EI)
¯
J
+
}
m z: 290 2 (M ); HR-ESI-MS for C16H22N2O3 H : calcu-
lated 291 1709 ; found 291 1738.
+
.
-
.
.
Results and Discussion
8
The HPLC system for the analysis of consisted of a
Hewlett-Packard Series II liquid chromatograph equipped
with Hewlett-Packard Series 1050 variable wavelength
detector. Separation was achieved on a Merck LiChrospher
A speci®c antibody is needed for the development of an
immunoassay. With a molecular weight of 204, psilocin is
too small to initiate an immune response on its own.
Antibodies for psilocin can only be raised against a protein±
hapten conjugate, whereby the hapten should have high
similarity with psilocin. Characteristic structural features
of psilocin are the phenolic hydroxy group in position 4
and the (dimethylamino)ethyl side chain in position 3 of
the indole ring system. In the immunogen, these structures
should be accessible for antibody generation.
¬
60 RP select B column, 4 mm (internal diameter)
250 mm, particle size 5 m. The mobile phase consisted of
}
.
m
0 02 KH2PO4 acetonitrile (gradient from 95:5 to 75:25,
v v). The ¯ ow rate was 1 mL min 1 and the injected sample
-
}
volume was 20 L. The UV absorbance was monitored at
8
266 nm. The purity of was greater than 90% .
We therefore decided to prepare psilocin derivatives
with an alkyl spacer in position 1 ending with a primary
amine. The coupling of this hapten to the protein should be
Preparation of the immunogen
.
Dry chloroform (3 6 mL) was added to polymer-supported achieved via glutaraldehyde condensation.
EDAC (383 mg) under nitrogen. A solution of (10 4 mg,
8
.
N N
The synthesis started from 4-benzyloxy- , -dimethyl-
.
0 036 mmol) in dry DMF (0 9 mL) was then added, fol- tryptamine ( ). This was reacted with NaH and dibenzyl-
lowed by the addition of a solution of -hydroxysuc- (3-bromopropyl)amine in DMF to yield
.
1
N
2
(Figure 2).
.
.
.
cinimide (3 7 mg, 0 032 mmol) in dry DMF (0 9 mL). The Subsequently, the benzyl-protecting groups at the phenolic
reaction mixture was stirred for 40 h. The polymer was hydroxy moiety and the amine functionality of this com-
removed by ®ltration through a glass ®lter. The ®ltrate was pound should be removed in one step by hydrogenolysis at
}
concentrated under reduced pressure to remove the chloro- atmospheric pressure with Pd C as a catalyst. Surprisingly,
form and then added to a solution of BSA (148 mg, a mixture of several products was obtained during this re-
.
. m
.
0 002 mmol) in phosphate buŒer (0 1 , pH 7 0, 15 mL) action as shown by TLC and HPLC analysis. In the
under nitrogen. After stirring overnight, the mixture was 1H NMR spectrum of this mixture, signals for the propyl
puri®ed by gel chromatography using PD10 desalting spacer and the dimethylaminoethyl side chain were de-
columns from Amersham Pharmacia Biotech (Braun- tectable in the expected integration ratio. However, in
. m
schweig, Germany). Elution was performed with 0 1
comparison with these signals, theintegrals forthearomatic
.
phosphate buŒer (pH 7 0). The eluate was dialysed against protons were not high enough. This result indicated that
bidistilled water (3 days). The water was then removed by oxidation of the phenolic ring system had probably oc-
lyophilization to yield 122 mg of the hapten±BSA con- curred.
jugate. The conjugate was analysed by matrix-assisted laser
Since the hydrogenolysis of the benzyl-protecting groups
2
desorption ionization mass spectrometry (MALDI-MS) of the primary amine moiety of lasts much longer than
employing a TofSpec 2E Spectrometer from Micromass the deprotection of the phenolic group, we decided to
(Manchester, UK). The matrix was sinapinic acid. The generate the free -aminoalkyl side chain before cleaving
.
protein sample was dissolved in 0 1% tri¯ uoroacetic acid the 4-benzyloxy moiety. This would oŒer the opportunity
}
Figure 2 Synthesis of compounds 2 and 3. i. Dibenzyl (3-bromopropyl)amine, NaH, DMF; ii. Pd C, H2, MeOH, 48 h.

1268
Christian Albers et al
}
Figure 3 Synthesis of compounds 4, 5, 6, 7 and 8. i. 4-Bromobutyronitrile, NaH, DMF; ii. Raney-Nickel, H2, MeOH, 38 h; iii. Pd C, H2,
MeOH, 2 h; iv. aqueous KOH, ethanol.
to reduce the reaction time in the last step, probably under the conditions applied. Finally, deprotection of the
avoiding greater decomposition of the target compound. phenolic hydroxy group was carried out by hydrogenolysis
}
In order to synthesize a 4-benzyloxy- , -dimethyltryp- using Pd C as a catalyst. However, the reduction of the
tamine with a 1-(4-aminobutyl) side chain at the indole reaction time in this last step (2 h instead of 48 h) did not
N N
6
nitrogen, a 3-cyanopropyl residue was introduced at the lead to a pure compound . Similar side products were
1
3
.
indole nitrogen ®rst by reaction of with 4-bromobu- obtained as during the synthesis of
tyronitrile in DMF in the presence of NaH as base (Figure Finally, we decided to prepare a psilocin derivative with
3). The nitrile moiety of was then converted to a primary a 1-alkyl spacer ending with a carboxy group. The car-
4
5
amine ( ) by catalytic hydrogenation with Raney-Nickel as boxylic acid moiety would oŒer the possibility to couple
a catalyst. The benzyl-protecting group was not cleaved the hapten to BSA via an activated ester. Thus, the nitrile

1269
Synthesis of psilocin derivatives
100
50
0
%
30000
35000
55000
m/z
60000
65000
70000
75000
50000
40000
45000
Figure 4 Matrix-assisted laser desorption ionization mass spectra of bovine serum albumin (BSA) and the BSA±hapten conjugate.
4
functionality of was hydrolysed to a carboxy group with
KOH (Figure 3). The obtained intermediate was then and the generated hapten is not distinct from that of the
Since the maximum of the UV absorbance of psilocin
7
8
converted to the target by cleavage of the benzylether protein, UV spectroscopic determination of the ratio of
with Pd C-hydrogen. Decomposition of this substance incorporation of the hapten into BSA was not possible.
}
during the reaction did not occur, as shown by HPLC Therefore, we used MALDI-MS for the characterization
analysis.
of the bioconjugate (Adamczyk et al 1994). Mass spectra
The coupling of a hapten via a carboxylic acid group to were successfully recorded for BSA and the BSA±hapten
the amino groups of a protein is usually performed directly conjugate (Figure 4). For BSA and the conjugate, mol-
N N«
-dicyclohexylcarbodiimide (Jenner & ecular weights of 66497 and 67682 Da were measured,
using EDAC or
Law 1996). This process, however, leads to undesirable side respectively. This indicates an average incorporation ratio
,
N
acylurea formation on the protein (Adamczyk et al 1995). of BSA.
reactions such as cross-linking of the protein and
-
of 4±5 molecules of psilocin hapten (MW 290) per molecule
These disadvantages can be avoided by the use of pure Inconclusion, apsilocin±BSA immunogen was prepared,
active esters. Adamczyk et al (1995) described a method to which can be used to develop a radioimmunoassay for
N
produce pure -hydroxysuccinimide and penta¯ uoro- psilocin in human plasma.
phenol esters from carboxylic acid haptens by the ap-
plication of polymer-supported EDAC. We used this
N
method to generate the -hydroxysuccinimide ester of the
psilocin derivative . After activation with solid-supported
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8
N
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-
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N
8
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