Use of novel haptens in the production of antibodies for the detection of tryptamines

Article abstract of DOI:10.1039/c8ra02528b

Tryptamines are a group of hallucinogenic drugs whose detection in body fluids could be simplified by immunochemical assay kits. Antibodies for these assays are obtained by the immunization of laboratory animals with conjugates of a hapten similar to the target analyte and a suitable protein. Therefore we synthesized novel haptens derived from tryptamine-based drugs, with N,N-dimethyltryptamine (DMT), 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) and N,N-diisopropyltryptamine (DiPT) selected as the target analytes. Their structures were modified with a short linker ended with a carboxylic group. The haptens were conjugated with bovine serum albumin (BSA) and rabbits were immunized with the conjugates. The obtained polyclonal antibodies showed good reactivity and the LOD of the constructed ELISAs was in the range 0.006-0.254 ng mL^-1. Thus, they are suitable for the development of immunochemical assay kits.

Full text of DOI:10.1039/c8ra02528b

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Use of novel haptens in the production of
antibodies for the detection of tryptamines†
Cite this: RSC Adv., 2018, 8, 16243
ad
ac
bd
c
Michal Maryska,
Lucie Fojtıkova, Radek Jurok, Barbora Holubova,
´ ´ ´
ˇ
a
ad
Oldrich Lapcık
and Martin Kuchar
*
ˇ
ˇ´
ˇ
Tryptamines are a group of hallucinogenic drugs whose detection in body fluids could be simplified by
immunochemical assay kits. Antibodies for these assays are obtained by the immunization of laboratory
animals with conjugates of a hapten similar to the target analyte and a suitable protein. Therefore we
synthesized novel haptens derived from tryptamine-based drugs, with N,N-dimethyltryptamine (DMT), 5-
methoxy-N,N-dimethyltryptamine (5-MeO-DMT) and N,N-diisopropyltryptamine (DiPT) selected as the
target analytes. Their structures were modified with a short linker ended with a carboxylic group. The
haptens were conjugated with bovine serum albumin (BSA) and rabbits were immunized with the
conjugates. The obtained polyclonal antibodies showed good reactivity and the LOD of the constructed
Received 22nd March 2018
Accepted 24th April 2018
DOI: 10.1039/c8ra02528b
ꢀ1
ELISAs was in the range 0.006–0.254 ng mL . Thus, they are suitable for the development of
rsc.li/rsc-advances
immunochemical assay kits.
There are currently only two methods for the detection of
tryptamines. The rst involves the use of specic colour tests,
1
. Introduction
Tryptamines, a group of hallucinogenic drugs derived from which are based on the reaction of specic reagents forming
tryptamine, include both natural and synthetic compounds. coloured products with indole-derived compounds. But, with so
The best known tryptamine is dimethyltryptamine (DMT), the many different NPS now on the drug scene, colour tests are not
4
active compound of ayahuasca, the ritual beverage, which has reliable because of their low specicity. The more commonly
traditionally been used in several South American indigenous used second method involves the analysis of tryptamines by
1
cultures. Other traditional natural tryptamines are psilocybin liquid chromatography coupled with mass spectrometry (LC-
5,6
and psilocin, active compounds of the Psilocybe mushrooms MS). This method is precise, capable of determining multiple
also known as ‘magic mushrooms’. Over the past few decades, targets in one run, offers low detection limits and can be used
lots of synthetic derivatives of tryptamine have been prepared. for various matrices, especially body uids. However, it is also
Their rise in popularity has been attributed to Alexander Shul- demanding with respect to cost, sample preparation and anal-
gin, whose book TiHKAL (Tryptamines I have known and loved) ysis time. Furthermore, LC-MS-based techniques cannot be
2
described the synthesis and effects of many of them. These used in the eld.
synthetic tryptamines belong to the group of novel psychoactive
Because of the above-mentioned disadvantages, attention
substances (NPS), which are produced to give ‘legal highs’ and has focused on immunochemical methods, such as ELISA
3
bypass legal restrictions. Although there is widespread use of (enzyme-linked immunosorbent assay) and LFIA (lateral ow
NPS on the drug scene, options for their detection, including for immunosorbent assay). Antibodies for immunochemical
the detection of tryptamines, remain rather limited.
detection are usually produced by the immunization of labo-
ratory animals. Because tryptamines are haptens, i.e. molecules
too small to be immunogenic on their own, they must be
conjugated to a carrier protein prior to immunization. To do
a
Department of Chemistry of Natural Compounds, University of Chemistry and this, molecules of the target analytes are modied with short
Technology, Technick a´ 5, 166 28 Praha 6 – Dejvice, Czech Republic. E-mail: martin.
linkers containing a suitable functional group. Using bufote-
nine and serotonin as haptens, Skerritt et al. reported the
kuchar@vscht.cz; Fax: +420-220-444-422; Tel: +420-220-444-432
b
Department of Organic Chemistry, University of Chemistry and Technology, Technicka´
development of an ELISA kit for the detection of DMT and 5-
5
, 166 28 Praha 6 – Dejvice, Czech Republic
7
c
MeO-DMT in Phalaris plants. However, this assay is limited
Department of Biochemistry and Microbiology, University of Chemistry and
a´ 3, 166 28 Praha 6 – Dejvice, Czech Republic
only to these natural tryptamines and is not selective between
Technology, Technick
d
Forensic Laboratory of Biologically Active Substances, University of Chemistry and them. Yamaguchi et al. prepared monoclonal antibodies
8
Technology, Technick a´ 3, 166 28 Praha 6 – Dejvice, Czech Republic
against psilocin for identication of magic mushrooms. These
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c8ra02528b
antibodies showed cross reactivity with DMT, but their limit of
1
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detection was relatively high. Thus far, no other immuno- bound to the protein. The average values were calculated from
chemical assay has been reported that targets either natural or the peak with highest intensity.
synthetic tryptamines.
The development of LFIA kits for the detection of synthetic
and other tryptamines is needed to screen potential users. To
provide such a kit, we rst synthesized novel haptens carrying
a short linker with a carboxyl group for the production of
antibodies selective to different tryptamines. Rabbits were
immunized with conjugates of these haptens with bovine serum
albumin (BSA). The antibodies obtained from the immuniza-
tion showed expedient sensitivity and selectivity for individual
tryptamines, and, thus, appear to provide a viable basis for LFIA
development.
2
.2 Immunisation and antiserum preparation
Four different immunisation conjugates (conjugate I–IV) were
used. Antisera were produced in rabbits and obtained according
to the procedure described previously. Stock solutions of
antisera were prepared by dissolving 1 mg of lyophilisate in 1 ml
of PBS and were stored at ꢀ20 C.
11
ꢁ
2.3 Indirect competitive ELISA
ELISA microtiter plates were coated with the coating conjugates
(
appropriately diluted in the carbonate–bicarbonate buffer; 100
ꢁ
ml per well) and incubated at 4 C overnight. The following day,
the plates were washed with the PBS-Tw (4 times, 350 ml per
well). The suitable standard (the concentration range of 0–500
2. Materials and methods
0
ꢀ1
Ethyl 2-bromoacetate was obtained from Merck and N,N -dicy-
clohexylcarbodiimide (DCC) was obtained from Fluka. Diiso-
butylaluminium hydride (DIBAL-H) solution in hexane, oxalyl
chloride and dry DMF were purchased from Acros. Other
reagents were purchased from Sigma-Aldrich. Dry THF,
dichloromethane and diethyl ether were dried with molecular
sieves. All reactions were carried out under argon atmosphere.
Thin layer chromatography (TLC) was performed on aluminium
backed sheets coated with 60F 254 silica gel from Merck.
Column chromatography was performed on silica (0.045–0.200
mm) from Merck. Reverse phase chromatography was carried
out using a CombiFlash Rf 200 apparatus (Teledyne ISCO) with
prepacked Redisep Rf Gold C18 columns (packed with C18-
reverse phase silica gel). NMR spectra were recorded on a Var-
ng mL in the PBS; 50 ml per well) was added into microtiter
plates followed by the solution of appropriate antiserum
(diluted in the PBS-0.1% BSA (w/v); 50 ml per well) and incubated
at room temperature for 1 hour. Microtiter plates were washed
again (4 times, 350 ml per well) and GAR-Po (diluted 1 : 10 000 in
the PBS-Tw; 100 ml per well) was added and incubated at room
temperature for 1 h. Aer the washing step, the TMB substrate
solution was added (100 ml per well) and incubated at room
temperature for 10 min. The enzyme reaction was stopped by
ꢀ
1
addition of 2 mol L H SO (50 ml per well) and the absorbance
2
4
was measured at 450 nm.
2.4 Calibration standard curve
1
13
Sigmoid calibration standard curves were obtained by plotting
the mean values of absorbance against the logarithm of stan-
dard concentrations through a four-parameter logistic equation
ian Gemini 300 (300 MHz for H; 75 MHz for C) or Agilent 400-
1
13
MR DD2 (400 MHz for H; 100 MHz for C) spectrometers. High
resolution mass spectra were measured on a LTQ Orbitrap XL
11
as described previously. The limit of detection (LOD) was
dened as the concentration of an analyte corresponding to the
maximum assay signal minus 3ꢂ standard deviation (SD) in
accordance with the calibration curve (the blank was calculated
from 3 parallel determinations with the absence of an analyte).
The IC50 corresponded to the concentration of analyte giving
(
Thermo Fischer Scientic) spectrometer using ESI ionization
technique. Mass spectra of hapten–protein conjugates were
measured on a Bruker Autoex Speed MALDI-TOF/TOF spec-
trometer. Microplate reader uQuant BIO-TEK was from Inc.
Winooski, USA and 96-well polystyrene microtiter plates Costar
9
018 were purchased from Corning Inc., USA. Peroxidase
50% inhibition of the asymptotic maximum. The linear working
labelled goat anti-rabbit antibody (GAR-Po) was obtained from
Nordic Immunological Laboratories, Netherlands. Analytical
standards psilocin and psilocybin were obtained from THC
Pharm GmbH The Health Concept, Germany. Analytical stan-
dards of DMT, 5-MeO-DMT, DiPT and 5-MeO-DiPT were
range corresponded to the analyte concentration causing the
20–80% inhibition of the maximum assay signal.
2.5 Specicity of antibodies
9
prepared according to literature in purity $98% (LC). Sero-
To verify the ability of the antibodies to react with similar
epitopes on different tryptamines, the cross-reactivity (CR) tests
were performed. The percent CR (CR (%)) was calculated from
IC50 obtained from the calibration curves: (IC50 of target drug)/
tonin was purchased from Sigma-Aldrich. Other tryptamines 5-
MeO-DALT, 4-HO-MET, acetylpsilocin, aMT and 5-MeO-DIPT
were generously donated by the Institute of Criminalistics
Prague.
(IC50 of tested compound) ꢂ 100.
3
. Results and discussion
2.1 Preparation of conjugates
10
Following the procedure we described previously,
the To design the structure of haptens, DMT, 5-MeO-DMT and DiPT
prepared haptens I–IV were conjugated to BSA using activated were selected as target analytes and their structures were
ester method. Obtained conjugates I–IV were analyzed by modied with a short alkyl chain ended with a carboxylic group.
MALDI-TOF to determine the number of hapten molecules Four different haptens were prepared, differing in a position of
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Fig. 1 Designed haptens I–IV.
the linker (Fig. 1). Hapten I (1a) with the linker on the amino Arylhydrazinium chlorides 7a,b were prepared in high yields
group was derived from DMT. Haptens II–IV (1b–d) with the from aniline derivatives 8a,b by diazotation and subsequent
13
linker in position 5 of indole ring were derived from 5-MeO- reduction of diazonium salts with SnCl
DMT (haptens II and III) and DiPT, respectively (hapten IV). As an acetal component of the Fischer reaction, we used N,N-
Synthesis of hapten I (1a) started from indole (2) and used dimethylamino-1,1-dimethoxybutane (9) and N,N-
a sequence of reactions developed by Speeter and Anthony to diisopropylamino-1,1-dimethoxybutane (10). Compound 9 was
2 2
$2H O.
9,12
obtain N-methyltryptamine (5).
Alkylation of 5 with ethyl-4- prepared in two steps from ethyl-4-chlorobutyrate (11) (Fig. 4).
bromobutyrate and subsequent hydrolysis of ester 6 yielded
hapten I (1a) (Fig. 2).
Because the same sequence of reactions could not be used in
the synthesis of acetal 10, we developed a different approach
We used an approach based on the Fischer reaction for the starting from succinic anhydride (13) (Fig. 5). Reaction of 13
synthesis of haptens II–IV (1b–d). Haptens were obtained with diisopropylamine and subsequent reduction of acid 14
directly from suitable arylhydrazines and amino acetals (Fig. 3). with LiAlH led to the amino alcohol 15. Swern conditions were
4
Fig. 2 Synthesis of hapten I.
Fig. 3 Direct synthesis of haptens II–IV using Fischer reaction.
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Fig. 4 Preparation of N,N-dimethylamino-1,1-dimethoxybutane (9).
Fig. 5 Synthesis of N,N-diisopropylamino-1,1-dimethoxybutane (10).
used for oxidation of 15 to aldehyde, which was immediately
acetalized to obtain 10 in moderate yield.
Antisera were collected by immunization of rabbits with all
of prepared conjugates. The indirect competitive format of
11
Haptens I–IV (1a–d) were conjugated with bovine serum ELISA was used for antiserum testing. First, checkerboard
albumin (BSA) using the methodology previously employed in titrations were performed and suitable immunoreagent
10,14
our group.
Conjugates I–IV were submitted to MALDI-TOF concentrations were determined when the maximum absor-
analysis and the number of hapten molecules was determined bance ranged from 1.2 to 1.9. Then the calibration curves with
as follows: 13 for conjugate I, 31 for conjugate II, 28 for conju- target analyte were constructed. The antibody with the highest
gate III and 37 for conjugate IV.
sensitivity to the appropriate analyte was selected (on the basis
of the lowest IC50 values) for each immunisation conjugate and
further tests were made. In this study, four different ELISAs
(ELISA I–IV) for the detection of tryptamine-based drugs were
successfully developed. Titration standard curves are shown in
Fig. 6 and analytical parameters of methods are summarized in
the table (Table 1).
The specicity of obtained antibodies was evaluated with 102
new psychoactive substances and non-hallucinogenic
compounds: 11 synthetic or natural tryptamines, 18 phenyl-
ethylamines, 13 piperazines, 18 synthetic cannabinoids, 27
cathinones, 16 non-hallucinogenic substance. Cross-reactivity
values for tryptamines are summarized in Table 2. Positive
cross-reactivity values were obtained only for synthetic trypt-
amines and psilocin. No cross-reactivity was observed with
other tested compounds. Our results show that synthesized
haptens and immunogens are functional and that they can be
used for the production of antibodies. These antibodies can be
applied in the development of immunoanalytical methods for
the tryptamine-based drug detection.
Fig. 6 Typical ELISA standard curves (A) ELISA I (standard DMT); (:)
ELISA II (standard 5-MeO-DMT); (▬) ELISA III (standard 5-MeO-DMT);
(-) ELISA IV (standard DiPT).
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Table 1 Analytical parameters of assembled ELISAs
Linear working range
a
b
ꢀ1
ꢀ1
c
b
ꢀ1
IC50 ꢃ SD (ng ml
)
(ng ml
)
LOD ꢃ SD (ng ml
)
ELISA I
0.52 ꢃ 0.17
27.3 ꢃ 5.20
0.29 ꢃ 0.07
3.36 ꢃ 0.28
0.11–4.24
4.08–241.96
0.03–4.93
0.27–45.13
0.017 ꢃ 0.005
0.254 ꢃ 0.059
0.006 ꢃ 0.002
0.034 ꢃ 0.005
ELISA II
ELISA III
ELISA IV
a
b
c
IC50, 50% intercept. SD, standard deviation. LOD, limit of detection.
a
Table 2 Cross-reactivity data for developed ELISA
mixture was reuxed for 7 hours. Then the mixture was cooled
with an ice bath and decomposed according to Fieser workup.
Precipitated salts were ltered of, washed several times with
Cross-reactivity (%)
Compound
DMT
ELISA I
ELISA II
ELISA III
ELISA IV THF and the ltrate was evaporated. The residue was dissolved
in dichloromethane (200 ml), washed with water (3 ꢂ 200 ml)
100.00
0.57
1.90
2.00
2.16
0.14
0.06
3.60
53.48
100.00
0.95
0.09
5.87
0.07
2.81
225.71
0.07
<0.001
<0.001
38.89
100.00
2.46
0.21
20.08
0.04
23.04
149.63
0.08
<0.001
0.43
0.09
0.04
0.19
100.00
0.05
<0.001
<0.001
0.10
<0.001
<0.001
88.43
4
and brine (150 ml), and the organic layer was dried with MgSO .
5
5
-MeO-DMT
-MeO-DALT
Purication of the crude product by column chromatography
CH Cl : MeOH, 5 : 1) afforded N-methyltryptamine (5) as an
(
DiPT
-HO-MET
2
2
ꢁ
16
ꢁ
4
off-white solid (717 mg, 42%). Mp ¼ 62–64 C (lit. 81–83 C);
1
aMT
O-Acetylpsilocin
Psilocin
Psilocybin
Serotonin
3
H NMR (300 MHz, CDCl ) d: 2.44 (s, 3H), 2.88–3.02 (m, 4H),
7
7
.04 (d, 1H, J ¼ 2.3 Hz), 7.09–7.15 (m, 1H), 7.17–7.23 (m, 1H),
.35–7.39 (m, 1H), 7.62–7.66 (m, 1H), 8.06 (s br, 1H).
<0.001
<0.001
0.01
Ethyl 4-[N-[2-(1H-indol-3-yl)ethyl]-N-methyl]aminobutanoate (6).
5
-MeO-DiPT
To a mixture of N-methyltryptamine (5) (523 mg, 3.0 mmol) and
NaI (450 mg, 3.0 mmol) in isopropyl alcohol (15 ml) was added
DIPEA (620 mg, 835 mL, 4.8 mmol) and ethyl-4-bromobutyrate
a
DMT:
N,N-dimethyltryptamine,
5-MeO-DMT:
5-methoxy-N,N-
dimethyltryptamine, 5-MeO-DALT: N,N-diallyl-5-methoxytryptamine,
DiPT: N,N-diisopropyltryptamine, 4-HO-MET: 4-hydroxy-N-methyl-N- (878 mg, 645 mL, 4.5 mmol) and resulting solution was heated
ethyltryptamine, aMT: a-methyltryptamine, 5-MeO-DiPT: 5-methoxy-
N,N-diisopropyltryptamine.
ꢁ
to 60 C overnight. Solvent was removed on rotavap. The residue
was dissolved in dichloromethane (75 ml) and washed with
saturated NaHCO
ml). Organic phase was dried with MgSO
Purication by column chromatography (CH Cl : MeOH,
3
solution (75 ml), water (75 ml) and brine (75
4
and evaporated.
4
. Experimental
2
2
1
0 : 1) afforded the titled compound 5 as a light-brown viscous
4
.1 Synthesis of haptens
.1.1 Hapten I
-(1H-indol-3-yl)-2-oxoacetyl chloride (3). Compound 3 was
1
oil (570 mg, 65%). H NMR (300 MHz, CDCl ) d: 1.25 (t, 3H, J ¼
3
4
2
7
.3 Hz), 1.86 (qui, 2H, J ¼ 7.2 Hz), 2.32–2.41 (m, 5H), 2.52 (t, 2H,
J ¼ 7.2 Hz), 2.72–2.81 (m, 2H), 2.92–3.01 (m, 2H), 4.13 (q, 2H, J ¼
9
prepared according to literature from indole (2) (2.00 g, 17.1
mmol) as a yellow solid (3.31 g, 93%).
7
1
.3 Hz), 7.03 (d, 1H, J ¼ 2.3 Hz), 7.08–7.14 (m, 1H), 7.15–7.22 (m,
13
H), 7.33–7.38 (m, 1H), 7.58–7.63 (m, 1H), 8.09 (s br, 1H);
C
2-(1H-indol-3-yl)-N-methyl-2-oxoacetamide (4). Glyoxylyl chlo-
NMR (75 MHz, CDCl
3
) d: 14.20, 22.29, 22.89, 32.07, 41.92, 56.56,
ride 3 (3.21 g, 15.5 mmol) was added in portions to an ice cold
aqueous solution of methylamine (20 ml, 40 wt%) and resulting
suspension was stirred at 0 C for 2 hours. The precipitate was
5
1
8.05, 60.32, 111.11, 114.02, 118.69, 119.15, 121.56, 121.87,
27.38, 136.20, 173.55; HRMS (ESI): m/z [M + H] calculated for
+
ꢁ
C H N O : 289.19105, found 289.19125.
1
7
24 2 2

ltered and washed with water and diethyl ether. Recrystalli-
4-[N-[2-(1H-indol-3-yl)ethyl]-N-methyl]aminobutanoic acid hydro-
zation of the crude product from THF/Et O gave the titled
2
chloride (1a, hapten I). To a mixture of ester 5 (433 mg, 1.5 mmol)
in 40% aqueous ethanol (20 ml) was added NaOH (66 mg, 1.65
mmol) and the solution was stirred at r.t. overnight. Then the
solution was acidied with 1 M aqueous HCl to pH ¼ 1, evap-
orated and the residue was puried by reverse-phase ash
compound 4 as a pale yellow solid (2.53 g, 81%). Mp ¼ 214–
ꢁ ꢁ
15
1
215 C (lit. 218–219 C); H NMR (300 MHz, DMSO-d ) d: 2.75
6
(
8
d, 3H, J ¼ 4.7 Hz), 7.22–7.30 (m, 2H), 7.50–7.57 (m, 1H), 8.19–
1
3
.26 (m, 1H), 8.68 (m br, 1H), 8.77 (s, 1H), 12.21 (s br, 1H);
) d: 25.57, 112.19, 112.56, 121.33,
22.53, 123.42, 126.25, 136.27, 138.54, 164.09, 182.08; HRMS
C
NMR (75 MHz, DMSO-d
1
6
chromatography (MeOH : H
2
O, gradient 5–100% MeOH) to
obtain the titled compound 1a (hapten I) as a white foam
+
(ESI): m/z [M + Na] calculated for C H N O : 225.06345,
1
1
1
10 2 2
(
246 mg, 63%). H NMR (300 MHz, DMSO-d ) d: 1.83–1,96 (m,
6
found 225.06329.
2
3
H), 2.35 (t, 2H, J ¼ 7.3 Hz), 2.80 (s, 3H), 3.06–3.20 (m, 4H), 3.21–
.31 (m, 2H), 6.97–7.04 (m, 1H), 7.06–7.13 (m, 1H), 7.25 (d, 1H, J
N-methyltryptamine (5). The solution of amide 4 (2.00 g, 9.9
mmol) in dry THF (180 ml) was added dropwise to an ice-cold
solution of LiAlH
under argon atmosphere. Aer complete addition, the reaction
¼
2.3 Hz), 7.37 (d, 1H, J ¼ 8.2 Hz), 7.63 (d, 1H, J ¼ 7.6 Hz), 11.00
4
(3.79 g, 100 mmol) in dry THF (180 ml)
13
(
6
s br, 1H); C NMR (75 MHz, DMSO-d ) d: 19.0, 19.8, 30.8, 39.3,
54.1, 55.2, 109.2, 111.5, 118.3, 118.5, 121.2, 123.2, 126.7, 136.2,
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+
ꢁ
1
2
73.6; HRMS (ESI): m/z [M + H] calculated for C15
61.15975, found 261.15979.
.1.2 Haptens II–IV
H
20
N
2
O
2
:
mixture was stirred at 0 C for 1 hour, then the layers were
separated and the aqueous one extracted with dichloromethane
(2 ꢂ 100 ml). Combined organic layers were washed with brine
4
Ethyl 2-[4-[(tert-butoxycarbonyl)amino]phenoxy]acetate (16). (300 ml) and dried with MgSO . Aer solvent removal, the
Compound 16 was prepared according to literature in two residue was dissolved in methanol (35 ml) acidied with few
steps from 4-aminophenol (17) (10.09 g, 100.0 mmol) as a white drops of concentrated sulfuric acid. The solution was stirred for
solid (11.29 g, 88% (2 steps)). Mp ¼ 53–55 C; H NMR (300 18 hours, diluted with dichloromethane (125 ml), washed with
) d: 1.27 (t, 3H, J ¼ 7.2 Hz), 1.48 (s, 9H), 4.26 (q, 2H, J 10% aqueous NaHCO
MHz, CDCl (100 ml), water (100 ml) and brine (100
7.2 Hz), 4.56 (s, 2H) 6.55 (s br, 1H), 6.78–6.85 (m, 2H), 7.26 (d, ml) and dried with MgSO4. Distillation under reduced pressure
4
1
7
ꢁ
1
3
3
¼
1
3
2
6
H, J ¼ 8.8 Hz); C NMR (75 MHz, CDCl ) d: 14.1, 28.3, 61.3, gave the titled acetal 12 as a colorless liquid (9.76 g, 78%). Bp ¼
3
ꢁ
21
ꢁ
1
5.9, 80.2, 115.1, 120.3, 132.4, 153.0, 153.6, 169.0; HRMS (ESI): 53–55 C (5 torr) (lit. 84–86 C (25 torr)); H NMR (300 MHz,
+
m/z [M + Na] calculated for C H NO : 318.13119, found CDCl ) d: 1.70–1.90 (m, 4H), 3.32 (s, 6H), 3.56 (t, 2H, J ¼ 6.3 Hz),
1
5
21
5
3
318.13149.
4.39 (t, 1H, J ¼ 5.4 Hz).
2
-(4-aminophenoxy)acetic acid (8b). Suspension of 16 (5.91 g,
0.0 mmol) in 1 M hydrochloric acid (50 ml) was stirred at 60 C was prepared according to literature from acetal 11 (4.86 g,
4-(N,N-dimethylamino)-1,1-dimethoxybutane (9). Compound 9
ꢁ
13
2
overnight. Aer cooling to r.t., the solution was extracted with 31.8 mmol) as a colorless liquid (4.32 g, 84%) aer distillation
ꢁ
13
ꢁ
dichloromethane (50 ml) and phases separated. Aqueous phase under reduced pressure. Bp ¼ 59–60 C (6 torr) (lit. 40 C (1
was treated with solid Na CO to adjust pH to 4–5 (according to torr)).
2
3
pH paper). The resulting precipitate was ltered off and dried,
4-(N,N-diisopropylamino)-4-oxobutanoic acid (14). To a solu-
yielding the titled compound 8b as an off-white solid (3.03 g, tion of succinic anhydride (13) (10.07 g, 100.0 mmol) in
ꢁ
18
ꢁ
1
9
1%). Mp ¼ 200–215 C (lit. 200–210 C); H NMR (300 MHz, dichloromethane (300 ml) was added diisopropyl amine
DMSO-d ) d: 4.46 (s, 2H), 6.45–6.52 (m, 2H), 6.58–6.65 (m, 2H). (21.8 ml, 300.0 mmol) and resulting solution was stirred for 20
General procedure A: preparation of arylhydrazinium chlorides. hours. Then the mixture was concentrated and 1 M hydro-
6
19
A procedure from the literature was modied as follows: chloric acid (250 ml) was added to the residue. Aqueous layer
suspension of aniline acid 8a,b (1 eq.) in concentrated hydro- was extracted with dichloromethane (3 ꢂ 150 ml) and
ꢀ1
ꢁ
chloric acid (3 ml mmol ) was cooled to ꢀ5 C and a solution combined organic layers were dried with MgSO4. Solvent
ꢀ
1
of sodium nitrite (1.05 eq.) in water (0.5 ml mmol ) was added removal offered the titled compound 14 as a light brown viscous
1
dropwise. Aer complete addition the mixture was stirred for 1 oil (16.50 g, 82%), which solidied upon standing in a fridge. H
ꢁ
hour at ꢀ5 C and then it was added dropwise to a solution of NMR (300 MHz, CDCl
3
) d: 1.16 (d, 6H, J ¼ 6.7 Hz), 1.29 (d, 6H, J
SnCl
2
$2H
2
O (3 eq.) in concentrated hydrochloric acid (1 ml ¼ 7.0 Hz), 2.56–2.64 (m, 4H), 3.46 (m br, 1H), 3.95 (sept, 1H, J ¼
13
ꢀ
1
ꢁ
mmol ) cooled to ꢀ20 C. Aer complete addition, the mixture 6.7 Hz), 11.02 (s br, 1H); C NMR (75 MHz, CDCl
3
) d: 20.3, 20.6,
ꢁ
was stirred for additional 2.5 hours at ꢀ20 C and then the 29.5, 29.8, 45.9, 48.4, 170.7, 176.7.
suspension was ltered. Solids were washed with cold ethanol
4-(N,N-diisopropylamino)butan-1-ol (15). Suspension of LiAlH
4
and diethyl ether and dried, yielding arylhydrazinium chlorides (6.83 g, 180.0 mmol) in dry THF (300 ml) was cooled with ice
a,b. bath and then the solution of amide 14 (8.05 g, 40.0 mmol) in
-(carboxymethyl)phenylhydrazinium chloride (7a). Was prepared dry THF (150 ml) was added dropwise. Aer complete addition,
according to general procedure A from aniline 8a (2.27 g, 15.0 the reaction mixture was reuxed for 5 hours. Then it was
7
4
ꢁ
20
mmol) as a white solid (2.86 g, 94%). Mp ¼ 207–210 C (lit. 228– cooled with an ice bath and decomposed according to Fieser
ꢁ
1
2
29 C); H NMR (300 MHz, DMSO-d
8.5 Hz), 7.16 (d, 2H, J ¼ 8.5 Hz), 8.20 (s br, 1H), 10.27 (s br, 3H); and the ltrate was evaporated. The residue was dissolved in
3
6
) d: 3.48 (s, 2H), 6.92 (d, 2H, J workup. Solids were removed by ltration, washed with THF
¼
1
C NMR (75 MHz, DMSO-d
6
) d: 39.9, 114.6, 128.1, 129.9, 144.3, dichloromethane (250 ml), washed with water (3 ꢂ 200 ml) and
+
1
73.0; HRMS (ESI): m/z [M] calculated for C H N O : 167.08150, brine (150 ml) and dried with MgSO . Distillation under
8
11
2
2
4
found 167.08124.
reduced pressure gave the titled amino alcohol 15 as a colorless
ꢁ
1
4
-(carboxymethoxy)phenylhydrazinium chloride (7b). Was liquid (4.91 g, 71%). Bp¼ 65–68 C (0.43 torr); H NMR (300
prepared according to general procedure from aniline 8b MHz, CDCl
3
) d: 1.04 (d, 12H, J ¼ 6.7 Hz), 1.58–1.71 (m, 4H),
ꢁ
(
(
836 mg, 5.0 mmol) as a white solid (937 mg, 86%). Mp ¼ 135 C 2.43–2.52 (m, 2H), 3.10 (sept, 2H, J ¼ 6.7 Hz), 3.48–3.58 (m, 2H),
1
13
decomp.); H NMR (300 MHz, DMSO-d
6
) d: 4.60 (s, 2H), 6.82– 6.06 (s br, 1H); C NMR (75 MHz, CDCl
3
) d: 20.0, 27.6, 32.4,
1
3
6.89 (m, 2H), 6.95–7.02 (m, 2H), 10.63 (s br, 3H); C NMR (75 44.9, 47.5, 62.7.
6
MHz, DMSO-d ) d: 64.9; 115.0, 116.9, 139.5, 153.1, 170.3; HRMS
4-(N,N-diisopropylamino)-1,1-dimethoxybutane (10). Dimethyl
+
(
ESI): m/z [M ꢀ NH ] calculated for C H NO : 166.04987, found sulfoxide (2.56 ml, 36.0 mmol) was added dropwise to a solution
3
8
8
3
166.04977.
of oxalyl chloride (2.57 ml, 30.0 mmol) in dry dichloromethane
ꢁ
4
-Chloro-1,1-dimethoxybutane (12). DIBAL-H (94 ml, 1 M (60 ml) cooled to ꢀ55 C and the mixture was stirred for addi-
solution in hexane, 94.0 mmol) was added dropwise to a solu- tional 10 minutes. Then the solution of alcohol 15 (2.60 g, 15
tion of ethyl-4-chlorobutyrate (11) (12.30 g, 81.7 mmol) in dry mmol) in dry dichloromethane (10 ml) was added and the
ꢁ
ꢁ
dichloromethane (125 ml) cooled to ꢀ78 C. Aer addition, the mixture was stirred at ꢀ55 C for additional 15 minutes. Then
ꢁ
reaction mixture was stirred for half hour at ꢀ78 C and then trimethylamine (8.9 ml, 60.0 mmol) was added and the mixture
poured into ice-cold 10% hydrochloric acid (160 ml). Resulting was allowed to warm to r.t. (1 hour). Reaction mixture was then
16248 | RSC Adv., 2018, 8, 16243–16250
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poured into water (130 ml), phases were separated and the (207 mg, 61%). H NMR (300 MHz, CD
3
OD) d: 1.26 (d, 12H, J ¼
aqueous one extracted with dichloromethane (2 ꢂ 100 ml). 6.5 Hz), 2.83–3.04 (m, 4H), 3.48 (sept, 2H, J ¼ 6.5 Hz), 3.57 (s,
Combined organic phases were dried with MgSO and evapo- 2H), 6.93 (s, 1H), 7.11 (dd, 1H, J ¼ 8.5 Hz, J ¼ 1.5 Hz), 7.24 (d,
4
1
2
1
3
rated. The residue was dissolved in methanol (50 ml), acidied 1H, J ¼ 8.5 Hz), 7.38 (s, 1H); C NMR (75 MHz, CD OD) d: 18.1,
3
with concentrated sulfuric acid (2.5 ml) and the mixture was 24.9, 46.6, 49.2, 55.9, 110.2, 112.3, 119.1, 124.4, 124.5, 128.1,
stirred at r.t. for 18 hours. Then the mixture was diluted with 129.8, 136.7, 180.9.
dichloromethane (150 ml), cooled with ice bath and 20% NaOH
solution was added (100 ml). Phases were separated, the
aqueous one was diluted with water (50 ml) and extracted with
5
. Conclusion
dichloromethane (2 ꢂ 75 ml). Combined organic phases were
We successfully used haptens with novel structures to produce
polyclonal antibodies against various tryptamines. The con-
structed ELISAs have low detection limits. Some of the anti-
bodies show good reactivity not only with the target analytes,
but also with psilocin and 5-MeO-DiPT. Although the antibodies
have not yet been characterized in complex matrices, they
appear to be suitable for the development of immunochemical
assay kits. In our next work, we will focus on the establishment
of an ELISA for the detection of tryptamines in human body
washed with brine (200 ml) and dried with MgSO . Distillation
4
under reduced pressure gave the titled amino acetal 10 as
ꢁ
1
colorless liquid (1.74 g, 54%). Bp ¼ 51–55 C (0.24 torr); H NMR
300 MHz, CDCl
) d: 0.98 (d, 12H, J ¼ 6.6 Hz), 1.36–1.51 (m, 2H),
.53–1.63 (m, 2H), 2.39 (t, 2H, J ¼ 7.3 Hz), 2.99 (sept, 2H, J ¼ 6.5
(
3
1
1
3
Hz), 3.31 (s, 6H), 4.37 (t, 1H, J ¼ 5.9 Hz); C NMR (75 MHz,
CDCl ) d: 20.7, 26.1, 30.3, 44.7, 48.2, 52.6, 104.7.
General procedure B: preparation of haptens II–IV. A procedure
from the literature was modied as follows: to a 4% sulfuric
acid, which was rst heated to 50 C and bubbled with argon,
3
13

uids. We believe that the outcome of our work could lead to
ꢁ
LFIA kits designed for the on-site testing of NPS users.
arylhydrazinium chloride 7a,b (1 eq.) and then amino acetal 9 or
1
0 (1.2 eq.) were added and the resulting mixture was heated to
ꢁ
Conflicts of interest
80
C for 3.5 hours. Aer cooling to r.t., the mixture was
neutralized with concentrated ammonia solution. Water was
removed under reduced pressure and the residue was treated
with ethanol (10 ml mmol ) and ltered to remove most of the
None.
ꢀ
1
inorganic salts. Filtrate was evaporated, 1 M hydrochloric acid Acknowledgements
ꢀ
1
(
10 ml mmol ) was added and resulting solution was evapo-
This work was supported by the Ministry of Interior of the Czech
Republic (projects VG20122015075 and VI20172020056 ) and co-
funded with nancial support from specic university research
rated again. Purication of crude product by reverse-phase ash
chromatography (water/methanol, gradient 5–100% of meth-
anol) gave haptens 1b–d (haptens II–IV).
(MSMT No. 20/2015, MSMT No. 20-SVV/2016 and MSMT No. 20-
2-[3-[2-(N,N-dimethylamino)ethyl]-1H-indol-5-yl]acetic acid hydro-
SVV/2017).
chloride (1b, hapten II). Was prepared according to general
procedure B from arylhydrazinium chloride 7a (608 mg, 3.0
mmol) and acetal 9 (580 mg, 3.6 mmol) as a colorless glassy
References
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solid (475 mg, 56%). H NMR (300 MHz, CD OD) d: 2.87 (s, 6H),
3
3
.11–3.21 (m, 2H), 3.30–3.39 (m, 2H), 3.66 (s, 2H), 7.07 (dd, 1H,
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1
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2
1
3
7
.51 (s, 1H); C NMR (75 MHz, D
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solid (238 mg, 53%). H NMR (300 MHz, D
2
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2
.90–3.00 (m, 2H), 3.08–3.18 (m, 2H), 4.26 (s, 2H), 6.73 (dd, 1H,
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1
¼ 8.8 Hz, J
2
¼ 2.1 Hz), 6.99 (d, 1H, J ¼ 2.1 Hz), 7.10 (s, 1H),
1
3
7
5
1
2
.21 (d, 1H, J ¼ 8.8 Hz); C NMR (75 MHz, D O) d: 20.1, 42.7,
2
7.4, 67.7, 100.9, 108.2, 112.3, 112.9, 124.8, 126.6, 131.7, 152.0,
+
77.2; HRMS (ESI): m/z [M] calculated for C H N O :
1
4
18 2 3
63.13902, found 263.13912.
-[3-[2-(N,N-diisopropylamino)ethyl]-1H-indol-5-yl]acetic acid
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1
16250 | RSC Adv., 2018, 8, 16243–16250
This journal is © The Royal Society of Chemistry 2018