Dynamic Kinetic Resolution of Ethyl 1,2,3,4-Tetrahydro-β-carboline-1-carboxylate: Use of Different Hydrolases for Stereocomplementary Processes

Article abstract of DOI:10.1002/ejoc.201700571

Both enantiomers of 1,2,3,4-tetrahydro-β-carboline-1-carboxylic acid have been prepared by dynamic kinetic resolution of the corresponding ethyl ester (±)-1. CAL-B-catalysed hydrolysis of (±)-1·HCl in NH₄OAc buffer (pH 8.0, 30 °C) provided amino acid (R)-2·HCl with 98 % ee and 90 % yield in 20 min. The hydrolysis with Alcalase in borate buffer (pH 8.0, 30 °C) showed S selectivity and the product (S)-2·HCl was obtained with 60 % ee and 66 % yield in 45 h. The absolute configuration of (S)-2 was determined by TDDFT electronic circular dichroism and optical rotation calculations.

Full text of DOI:10.1002/ejoc.201700571

A Journal of
Accepted Article
Title: Dynamic kinetic resolution of ethyl 1,2,3,4-tetrahydro-β-
carboline-1-carboxylate. Use of different hydrolases for
stereocomplementary processes.
Authors: Rita Megyesi, Attila Mándi, Tibor Kurtán, Eniko Forro, and
Ferenc Fülöp
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201700571
Link to VoR: http://dx.doi.org/10.1002/ejoc.201700571
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European Journal of Organic Chemistry
10.1002/ejoc.201700571
FULL PAPER
Dynamic kinetic resolution of ethyl 1,2,3,4-tetrahydro-β-carboline-
-carboxylate. Use of different hydrolases for
stereocomplementary processes.
1
[
a]
[b]
[b]
[a]
[a,c]
Rita Megyesi, Attila Mándi, Tibor Kurtán, Enikő Forró and Ferenc Fülöp*
Abstract: Both enantiomers of 1,2,3,4-tetrahydro-β-carboline-1-
carboxylic acid were prepared through dynamic kinetic resolution of
the corresponding ethyl ester [(±)-1]. CAL-B catalysed hydrolysis of
both the promastigote and amastigote forms of Leishmania
infantum.[9] Hagmann et al. prepared a tetrahydro-β-carboline
derivative, which was
a
highly potent sstr3 antagonist
(
±)-1·HCl in NH
4
OAc buffer (pH = 8.0, 30 °C) in 20 min provided
(somatostatin receptor 3, potential important receptor in treatment
of the type 2 diabetes) and induced little or no QTc prolongation
in CV dog models.[10] Based on the above results, an intensive
structure-activity relationship study was carried out at the C3
position of tetrahydro-β-carbolines to find the balance of sstr3
antagonist activity and hERG off-target liability.[ Boc-protected
amino acid [(R)-2·HCl] with 98% ee and 90% yield. The hydrolysis
with Alcalase in borate buffer (pH = 8.0, 30 °C) in 45 h showed S-
selectivity and the product (S)-2·HCl was obtained with 60% ee and
6
6% yield. The absolute configuration of (S)-2 was determined by
11]
TDDFT-ECD and –OR calculations.
1,2,3,4-tetrahydro-β-carboline-1-carboxylic acid [(±)-2] was used
as a component of the UGI-4CR reaction for the preparation of
diketopiperazine-fused tetrahydro-β-carbolines.[12] Molero and co-
workers prepared several peptide hybrids containing an
indoloquinolizidine core. On the basis of binding assay, a number
of them were found to show high affinity at dopamine
Introduction
Drugs, such as vincristine or vinblastine containing tetrahydro-β-
carboline core, have been used as chemotherapeutical agents for
a long time.[ New manzamine alkaloids (acanthomanzamine C-
E) containing β-carboline moiety, were isolated from the marine
sponge Acanthostrongylophora ingens.[3] Glucose-derived β-
carbolines (tangutorids A-F) were found in the fruit juice of
receptors.[13]
4´-Hydroxy-3´,5´-dimethoxyphenyl-tetrahydro-β-
1,2]
carboline-3-carboxylic acid RGB (Arg-Gly-Asp) peptide
conjugates possess free-radical scavenging and anti-thrombotic
activities.[14]
(3S)-1-Methyl-1,2,3,4-tetrahydro-β-carboline-3-
carboxylic acid was used as a scaffold for the synthesis of
Nitraria tangutorum and tangutorid
A
exhibited strong α-
[15]
promising anti-thrombotic dipeptide analogues.
[
4]
glucosidase inhibitory activity. An apoptosis inducer effect of
evodiamine (from Evodia rutaecarpa) via activation of JNK and
PERK in the human ovarium cancer cells was described.[5] (3S)-
Dynamic kinetic resolution (DKR) is a very effective way to
prepare a single enantiomer of a chiral compound, because
100% theoretical yield is attainable in these reactions. A relatively
1
,2,3,4-Tetrahydro-β-carboline-3-carboxylic acid isolated from
Cichorium endivia L. induced apoptosis in human colorectal
tumor cell line (HCT-8) in
dose-dependent manner.[6]
large number of articles and reviews have been published about
the application of enzymatic DKR.[16-22] Several DKR studies are
a
based on the use of metal catalysts, such as ruthenium[23,24] or
palladium[25] complexes, while others use non-metal racemising
Anticancer activity of phenyl-1,4-β-carboline hybrids was also
reported[
7]
and other N2-acyl-1,6-substituted-tetrahydro-β-
[26]
agents like zeolites. When the asymmetric centre has an acidic
carbolines showed selective inhibitory activity against breast
cancer reactive protein (ABCG2).[8] 1-Phenyl-2,3,4,9-tetrahydro-β-
carboline derivatives were shown to be potent inhibitors against
proton, in situ base-catalysed racemisation might occur.[17] For
instance, spontaneous racemisation without addition of external
auxiliaries (acid and base or metal) have been described for DKR
of dimethyl (1,3-dihydro-2H-isoindol-1-yl)phosphonate,[
27]
8-
amino-5,6,7,8-tetrahydroisoquinole,[28] 1,3-dihydro-2H-isoindole-
ester[29]
-carboxylic acid methyl and (±)-cis-N-
(alkoxycarbonyl)cyclopentane-1,2-diamines. Enantiomers of
[
a]
R. Megyesi, E. Forró, F. Fülöp,
University of Szeged
Institute of Pharmaceutical Chemistry
Eötvös u. 6, 6720 Szeged, Hungary
E-mail: fulop@pharm.u-szeged.hu
A. Mándi, T. Kurtán
1
[
30]
1
,2,3,4-tetrahydroisoquinoline-1-carboxylic acid[31] and its 6-
[32]
6-methoxy[32] and 6,7-dimethoxy[33] analogues have
hydroxy,
[
b]
c]
been prepared through lipase-catalysed DKR hydrolysis of the
corresponding ethyl esters. At first view, present DKR and DKR
University of Debrecen
Department of Organic Chemistry
P. O. Box 400, 4002 Debrecen
F. Fülöp,
MTA-SZTE Stereochemistry Research Group
Hungarian Academy of Sciences
Eötvös u. 6, 6720 Szeged, Hungary
of
hydrochloric
salt
of
ethyl
6-hydroxy-1,2,3,4-
[
[32]
tetrahydroisoquinoline-1-carboxylate
apparently look similar,
but the structures of starting esters are very different: the ring
with active carboxylate at position 1 in tetrahydroisoquinoline
skeleton is condensed with a benzylic ring, in β-carboline
skeleton with
considerably the racemisation.
a heteroaromatic ring, which can influence
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
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European Journal of Organic Chemistry
10.1002/ejoc.201700571
FULL PAPER
Our aim was to develop efficient dynamic kinetic enzymatic
strategies for the preparation of both enantiomers of 1,2,3,4-
Synthesis of racemic ethyl 1,2,3,4-tetrahydro-β-carboline-1-
carboxylate hydrochloride [(±)-1·HCl] and 1,2,3,4-tetrahydro-
β-carboline-1-carboxylic acid hydrochloride [(±)-2·HCl]
tetrahydro-β-carboline-1-carboxylic
hydrolysis of ethyl 1,2,3,4-tetrahydro-β-carboline-1-carboxylate
Scheme 1). The planned work promised a real challenge since in
acid,
through
directed
(
(±)-1·HCl was synthesized through Pictet-Spengler reaction of
tryptamine hydrochloride (3) and ethyl glyoxylate,[ while (±)-
2·HCl was prepared through cyclization of tryptamine
hydrochloride and glyoxylic acid[35] according to slightly modified
literature procedures (Scheme 2).
34]
the β-carboline skeleton the ring with active carboxylate at
position 1 is fused with a heteroaromatic ring as against the
benzene ring in the earlier investigated tetrahydroisoquinolines,
which may influence considerably the racemisation.
Results and Discussion
Scheme 1. Directed DKR hydrolysis of (±)-1
Scheme 2. Synthesis of (±)-1·HCl and (±)-2·HCl
obtained with ee >99% and a yield of 91% in 3 h.[32] In view of
CAL-B catalysed R-selective hydrolysis of (±)-1·HCl
these results, the CAL-B catalysed hydrolysis of ethyl 1,2,3,4-
tetrahydro-β-carboline-1-carboxylate hydrochloride [(±)-1·HCl]
was performed. First, the reaction was carried out in aqueous
In a very recent publication,[32] DKR of ethyl 6-methoxy-1,2,3,4-
tetrahydroisoquinoline-1-carboxylate has been described by
using CAL-B (Candida antarctica lipase B) as catalyst in
aqueous NH OAc buffer (pH = 8.5, 25 °C). The pronounced
4
instability of the racemic substrate could be overcome by the
use of the hydrochloric salt of the ester in DKR when the desired
4
NH OAc buffer (0.1 M), at pH = 8.0 at 30 °C (Table 1, entry 1).
The CAL-B catalysed hydrolysis of (±)-1 led to the (R)-2 with an
excellent ee of 97% and only 5 min. were required to reach a
conversion of 97% (entry 1).
6-methoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid was
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European Journal of Organic Chemistry
10.1002/ejoc.201700571
FULL PAPER
Table 1. CAL-B catalysed hydrolysis of (±)-1·HCl.a)
Entry
1
Buffer
pHb)
Temperature (°C)
30
Time (min)
5
ee
21
6
s
(%)c)
ee
97
97
89
87
90
90
89
90
94
p
(%)c)
Conv. (%)c)
97
99
95
99
94
99
96
99
>99
NH
NH
NH
4
OAc
4
OAc
4
OAc
8.0
15
5
15
5
15
5
15
5
rac.
rac.
rac.
rac.
14
2
3
4
9.0
9.0
30
4
borate
borate
8.0
9.0
30
30
66
5
-
a)
±)-1·HCl (0.0175 mmol, 4.9 mg) in 0.1 M buffer solution, 30 mg mL-1 CAL-B. b) pH was adjusted with NH OH or NaOH aqueous solution. c) According to
4
(
HPLC (Experimental section).
Table 2. Preparative-scale resolutions of (±)-1·HCl.a)
(%)b)
Yield (%)
90
25
α
D
Entry
1
Enzyme
CAL-B c)
Time
Product
ee
P
20 min
(R)-2·HCl
98
-22 (H
2
O, c 0.2)
-12 (EtOH, c 0.2)
2
Alcalase d)
45 h
(S)-2·HCl
60
66
+6.5 (EtOH, c 0.32)
a(±)-1·HCl (0.36 mmol, 100 mg) in 15 mL buffer solution, pH = 8.0, 30 mg mL enzyme, 30 °C. b)According to HPLC. c)in 0.1 M NH
-1
OAc buffer, pH was adjusted
4
d)
with NH
4
OH aqueous solution. in 0.1 M borate buffer, pH was adjusted with NaOH aqueous solution. In all of the analysed samples (HPLC), took at intervals
from the preparative-scale enzymatic mixtures the remaining substrate 1 was found to be in near racemic form.
Since the pH of the buffer solution can influence both enzyme
activity and enantioselectivity,[33] the pH was changed from 8.0 to
Alcalase-catalysed S-selective hydrolysis of (±)-1·HCl
9
.0. The ee of (R)-2 slightly decreased (89% after 5 min.), while
the reaction rate did not change significantly (entry 2 vs. 1).
Next, 0.1 M borate buffer was used instead of NH OAc buffer (pH
8.0 and 9.0). While a similar decrease in both ee and
Application of Alcalase in DKR through ester hydrolysis is a
known method.[36,37] Recently, Alcalase-catalysed DKR of N-tert-
butyloxycarbonylphenylalanine with benzylamine in a continuous-
flow system has also been reported.[38]
4
=
P
conversion was observed at pH = 8.0 (entry 4 vs. 1), the
decrease in the above parameters at pH = 9.0 was less
significant (entry 5 vs. 2).
The Alcalase-catalysed hydrolysis of (±)-1·HCl was first
attempted under the same conditions that were applied in the
CAL-B catalysed preparative-scale resolution of (±)-1·HCl that is
The DKR of (±)-1·HCl performed at 4 °C, delivered the product in
using NH OAc buffer at pH = 8.0 and 30 °C (Table 2, entry 1). As
4
a similar fast reaction with excellent ee
P
(entry 3 vs. 2).
expected, Alcalase showed a complementary stereoselectivity in
comparison with CAL-B but the hydrolysis yielded (S)-2 with a
relatively low ee of 53% in 24 h (Table 3, entry 1). In order to
increase the ee of (S)-2, the quantity of enzyme used in the
reaction was increased from 30 to 60 mg mL . Unfortunately, the
ee dramatically decreased (ee = 42%, conv. = 31%, in 3 h; ee =
12%, conv. > 99%, in 24 h).
On the basis of the above results, the preparative-scale DKR
reaction of (±)-1·HCl was carried out in aqueous NH OAc buffer
at pH = 8.0 at 30 °C. Because of the very poor solubility of the
resulting (R)-2 in both aqueous and organic solvent, its
hydrochloric salt [(R)-2·HCl] was isolated (ee = 98% and yield =
4
-1
90%). Results are summarized in Table 2, entry 1.
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European Journal of Organic Chemistry
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FULL PAPER
Table 3. Alcalase-catalysed hydrolysis of (±)-1·HCl.
a)
Entry
1
Buffer
pHb)
Temperature (°C)
30
Time (h)
1
ee
s
(%)c)
ee
p
(%)c)
Conv.
c)
(%)
NH
NH
NH
NH
4
OAc
4
OAc
4
OAc
4
OAc
8.0
rac.
33
42
53
55
47
66
46
48
40
65
65
60
48
40
99
97
>99
31
90
57
99
32
99
46
>99
2
1
3
4
2
3
4
5
9.0
8.0
9.0
8.0
9.0
30
4
rac.
-
24
rac.
rac.
rac.
rac.
rac.
rac.
rac.
-
72
4
48
72
6
24
4
24
borate
borate
30
30
6
a)
±)-1·HCl (0.0175 mmol, 4.9 mg) in 0.1 M buffer solution, 30 mg mL-1 Alcalase. b) pH was adjusted with NH OH or NaOH aqueous solution. c)According to HPLC
4
(
(Experimental section).
When the pH was changed from 8.0 to 9.0, a much faster
reaction was observed (97% conversion reached after 1 h) with
only a minor improvement of ee (entry 2 vs. 1).
The reaction performed at 4 °C afforded a promising ee of 66%
at a conversion of 30%, which, however, drooped to 46% at a
conversion of 90% (entry 3). The same reaction carried out at
pH = 9.0 did not result in a better ee (entry 4 vs. 3).
(%)b)
(%)b)
Entry
1
Solvent
Time (h)
3
ee
s
ee
p
Conv.
(%)
b)
t-BuOH c)
rac.
rac.
4
-
-
-
-
18
98
17
48
2
3
2
4
2
t-BuOMe d)
4
Changing the NH OAc buffer to borate buffer (pH = 8.0) and
4
64
working under the above conditions, (S)-2 could be isolated with
an increased ee of 65% (entry 5 vs. 1). A decrease in ee of (S)-2
to 48% was observed upon increasing the pH from 8.0 to 9.0
a)(±)-1·HCl (0.0175 mmol, 4.9 mg), 30 mg mL Alcalase. b)According to HPLC
-1
c)
(
Experimental section). Mixture of 950 μL t-BuOH and 50 μL NH
4
OAc buffer,
40 °C. d)1 mL t-BuOMe with added Et
N (10 μL) and H
O (2 equiv.), 30 °C.
(
entry 6 vs. 5).
3
2
Preparation of a large number of enantiomeric amino acids,
through Alcalase-catalysed DKR of the corresponding amino
esters, performing the hydrolysis in 95% t-BuOH/5% aqueous
buffer (pH = 8.5), has been described in the literature.[36] On the
basis of the these observations, the DKR of (±)-1·HCl was
Considering the results of the above preliminary experiments,
the Alcalase-catalysed preparative-scale DKR was performed in
0.1 M aqueous borate buffer (pH = 8.0, 30 °C). The hydrolysis
was stopped at the 99% conversion after 45 h. The results are
summarized in Table 2, entry 2. To the best of our knowledge,
Alcalase catalysed such DKR transformation when the racemic
substrate is used as hydrochloric salt has not been described
yet.
4
carried out in a mixture of t-BuOH and 0.1 M NH OAc aqueous
buffer (95:5) (pH = 8.5) at 40 °C. The reaction reached a
conversion of 98% after 24 h, but amino acid (S)-2 could not be
detected in the reaction mixture (Table 4, entry 1).
3
t-BuOMe as reaction medium was also probed with Et N as
additive to promote the liberation of base (±)-1 from its
hydrochloride salt. Again, amino acid (S)-2 was not formed
Experiments with regard to the racemisation of the separated
(
R)- and (S)-amino acids, under the enzymatic reaction
(
entry 2).
conditions were performed (Supporting information). While
practically no racemisation of (R)-amino acid over 20 min (Fig
S1), a significant racemisation of (S)-amino acid over 45 h (Fig
S2) could be detected.
Table 4. Alcalase-catalysed hydrolysis of (±)-1·HCl in organic solvents.a)
Determination of the absolute configuration (S)-2
Although enantiomers of 2 were produced earlier by HPLC
separation using chiral stationary phase,[
39,40]
the absolute
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European Journal of Organic Chemistry
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FULL PAPER
configuration of 2 has not been correlated yet with chiroptical
parameters such as specific rotation and electronic circular
dichroism (ECD).
In order to elucidate the absolute configuration of the
enantiomers of 2, the ECD spectrum of CAL-B derived 2·HCl
was recorded in water and TDDFT-ECD calculations were
performed for the arbitrarily chosen (S) enantiomer of the
cation.[
41,42]
The preliminary Merck Molecular Force Field
low-energy
conformers. DFT reoptimization of these conformers at various
levels [B3LYP/6-31G(d) in vacuo, B97D/TZVP[43,44] PCM/H
and CAM-B3LYP/TZVP[45,46] PCM/H
O] yielded 2, 4 and 4 low-
energy conformers over 1% Boltzmann population, respectively
Table 5). The first two major computed conformers were found
(
MMFF) conformational search resulted in 9
2
O
2
(
to be identical in both the in vacuo and solvent-model
calculations with a total population of ca. 91-99%. However, the
gas-phase ECD calculations performed on the gas-phase
conformers at various levels gave moderate to good agreement
with the experimental spectrum, while the solvent model ECD
calculations for the solvent model optimized conformers gave
Figure 1. Experimental ECD spectrum of the anionic form of 2 in H
compared with the PBE0/TZVP PCM/H O ECD spectrum computed for the
CAM-B3LYP/TZVP PCM/H O conformers of the anionic form of (S)-2. Bars
represent the rotational strength values of conformer A.
2
O
2
2
moderate mirror-image agreement (see SI). These contradictory Conclusions
results were attributed to the different estimation of the
electronic transition moment of the charged chromophore by the
gas-phase and solvent model methods. For the unambiguous
configurational assignment, OR calculations were also
performed on both the gas-phase and the CAM-B3LYP/TZVP
PCM/MeCN optimized conformers at various levels.[47,48] All the
combinations of levels resulted in positive average optical
rotation values, which were opposite to the experimental
negative specific rotations observed in MeOH and water (Table
Directed dynamic kinetic enzymatic strategies were developed
for the preparation of both enantiomers of 1,2,3,4-tetrahydro-β-
carboline-1-carboxylic acid [(R)-2 and (S)-2], through the
hydrolysis of ethyl 1,2,3,4-tetrahydro-β-carboline-1-carboxylate
(±)-1. Due to the high instability of (±)-1, its hydrochloric salt [(±)-
1·HCl] was successfully used in the enzymatic reactions. CAL-B
catalysed DKR resolution of (±)-1·HCl carried out in 0.1 M
NH OAc aqueous buffer (pH = 8.0, 30 °C) gave amino acid (R)-
4
5
and 6). This result confirmed that the solvent model ECD
2·HCl with an excellent enantiomeric excess of 98% and high
yield of 90%. Alcalase-catalysed DKR resolution of (±)-1·HCl in
0.1 M borate buffer (pH = 8.0, 30 °C) afforded (S)-2·HCl with an
ee 60% and a yield of 66%. It is to highlight that the ring with
agreement (see SI) is the method of choice to determine the
absolute configuration. To further verify this result, experimental
ECD of the anionic form of 2 (obtained by addition of NaOH)
was measured, which was found to be identical with that of the
cationic form. Furthermore, TDDFT-ECD protocol was carried
out for the arbitrarily chosen (S) enantiomer of the anion.
active carboxylate at position
1
is condensed with
a
heteroaromatic pyrrole ring as against the benzene ring in the
earlier investigated tetrahydroisoquinolines, which influenced
considerably the racemisation. TDDFT-ECD and OR
calculations allowed correlating the specific rotation and ECD
spectrum with the absolute configuration of 2. To the best of our
knowledge, the absolute configurations for the present
enantiomers were determined for the first time in the literature.
B3LYP/6-31G(d) in vacuo and CAM-B3LYP/TZVP PCM/H
reoptimization of the 4 preliminary MMFF conformers resulted in
and 3 low-energy conformers over 1% Boltzmann population.
2
O
3
ECD spectra computed at various levels for both sets of
conformers gave nice mirror-image agreement with the
experimental spectrum (Figure 1) allowing the unambiguous
elucidation of the absolute configuration of ()-2 as (R).
According to literature data for related -carboline derivatives,
ECD is mainly governed by the helicity of the saturated six-
membered ring.[49] In our case, however, the positive charge in
the ring of the cationic form had substantial contribution to the
electronic transition moment vector, which was estimated in
different ways in the gas-phase and by the solvent model. On
the other hand, the ring of the anionic form contained no charge
resulting in similar ECD spectra in both the gas-phase and the
solvent model.
Experimental Section
General information
Candida antarctica lipase
specification: ≥ 5000 Ug ) was a product of Sigma-Aldrich. Alcalase
B
(CAL-B, L4777, Lot: SLBL2553V,
-
1
(cross linked enzyme aggregation from subtilisin Carlsberg, WA22337,
-
1
Lot: 1340239, specification: = 12.5 Ug ) was from Fluka. For the
1
experiments, solvents of the highest analytical grade were used. H NMR
and 13C NMR spectra were measured on Bruker Avance DRX 400.
Elemental analysis was performed with a PerkinElmer CHNS-2400 Ser II
elemental analyzer. PerkinElmer 341 polarimeter was used for the
determination of optical rotation. Melting points were measured with a
Koffler apparatus. The pH of buffer solutions was determined with an
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European Journal of Organic Chemistry
10.1002/ejoc.201700571
FULL PAPER
Orion 210 A+ pH meter. ECD spectra were recorded on a JASCO J-810
spectropolarimeter. New Brunswick Scientific Innova 4000 incubator
shaker with 150 shaking speed (rpm) was used for the enzymatic
reactions.
18.26, 41.59, 54.09, 63.68, 107.55, 111.74, 118.26, 119.77, 122.34,
123.06, 126.05, 137.75, 166.26 ppm. Anal. calcd. for: C14
2 2
H17ClN O : C
59.89, H 6.10, N 9.98, found: C 59.90, H 6.08, N 9.97.
Synthesis of 1,2,3,4-tetrahydro-β-carboline-1-carboxylic acid [(±)-2]
Analytical methods
Tryptamine hydrochloride (1.0 g, 5.1 mmol) was suspended in 30 mL
The hydrolysis was followed by HPLC equipped with Chiralpak IA column
2
H O in a three-neck, round-bottomed flask. Glyoxylic acid monohydrate
(Daicel, 4.6 mm×250 mm). Conversions were determined after calibration
(0.47 g, 5.1 mmol, 1 equiv.) dissolved in 10 mL H O was added dropwise.
2
using an external standard. Samples were prepared with the following
procedure. After reaction, the solvent was evaporated off (20 µL) then
The mixture was to 15 °C, and then the pH was set to 4 with 50% NaOH.
The reaction was stirred at rt. for 24 h and the crystallised product [(±)-2]
was filtered (0.6 g, yield: 59%, m.p.: 230-232 °C, beige crystals). Due to
the low solubility of the amino acid [(±)-2], its hydrochloride salt was
1
00 µL ethanol, 10 µL of the standard solution of (4-phenyl-3-buten-2-
one (0.017 M, solvent: EtOH) and 200 µL eluent was added. The eluent
was n-hexane:isopropyl alcohol (60:40, + 0.1% Et NH), flow rate: 0.5 mL
min , detection at 280 nm at rt. Retention times (min): standard: 8.4, (±)-
: 13.6 and 16.7. Under these conditions the product amino acid was not
detectable. The ee values of amino acids were determined, after
derivatization with Boc O, in the presence of Et N (5 µL). In this case, the
solvent was also removed before derivatization. The eluent was n-
2
prepared. (±)-2 (0.1 g, 0.46 mmol) was dissolved in 15 mL H
2
O, then 4
-
1
mL 10% hydrochloric acid was added. Product (±)-2·HCl (brown crystals)
1
isolated after solvent evaporation was recrystallised from EtOH and Et
(1:25) (0.1 g, yield: 86%, m.p.: 184-186 °C, yellow crystals).
2
O
2
3
1H NMR (400 MHz, MeOD-d4): δ = 3.00-3.18 (m, 2H, CH
2
), 3.56-3.77 (m,
-
1
hexane:isopropyl alcohol (85:15, + 0.1% TFA), flow rate: 0.5 mL min ,
detection at 230 nm at rt. Retention times (min) for Boc-protected
derivatives: (±)-1: 17.7, 24.5, (S)-2: 20.9 and (R)-2: 47.6.
2
7
7
1
2
H, CH ); 5.49 (s, 1H, CH); 7.04-7.10 (dt, 1H, J = 0.80 Hz, 7.32 Hz Ar);
.14-7.20 (dt, 1H, J = 0.88, 7.56 Hz, Ar); 7.40-7.45 (d, 1H, J = 8.12 Hz);
.48-7.52 (d, 1H, J = 7.84 Hz) ppm. 13C NMR (400 MHz, DMSO-d6): δ =
8.64, 41.18, 53.85, 107.63, 112.75, 118.95, 119.93, 122.95, 124.58,
Small-scale enzymatic resolution
2 2
126.33, 137.43, 168.26 ppm. Anal. calcd. for: C12H13ClN O : C 57.04, H
5.19, N 11.09, found: C 57.02, H 5.23, N 9.96.
Small-scale reactions were carried out in incubator shaker, in aqueous
buffer. Racemic substrate (±)-1·HCl (4.9 mg, 0.0175 mmol) was
CAL-B catalysed preparative-scale DKR reaction of (±)-1·HCl
dissolved in the buffer solution [0.1 M NH
acid with NaOH), pH = 8.0 or 9.0, 1 mL], and the pH was re-adjusted with
NH OH (25% aqueous solution) or NaOH (2 M aqueous solution) at the
start of the reaction. After the addition of lipase (30 mg CAL-B or 30 mg,
0 mg Alcalase), the reaction mixture was shaken at 4 °C or 30 °C in
4
OAc or borate buffer (boric
(
±)-1·HCl (100 mg, 0.36 mmol) was dissolved in 15 mL 0.1 M NH
4
OAc,
4
then pH = 8.0 was set with NH OH (25% aqueous solution). 450 mg
CAL-B was added and the reaction mixture was shaken in an incubator
shaker at 30 °C for 20 min. The reaction was stopped at 98% conversion.
4
6
incubator shaker with 150 rpm shaking speed.
The enzyme was filtered and washed with NH
4
OAc buffer (pH = 8.0,
3×20 mL). The solvent was evaporated then the residue was dissolved in
In organic solvents, substrate (±)-1·HCl (4.9 mg, 0.0175 mmol) was
2
20 mL H O and pH= 4 was set with 5% hydrochloric acid. The solvent
4
suspended in 950 µL t-BuOH, then 50 µL NH OAc was added (0.1 M, pH
was evaporated, the residue was dissolved in 25 mL EtOH and then
=
8.5 with 25% aqueous solution of NH OH). The mixture was shaken
4
filtered. Amino acid (R)-2·HCl isolated after solvent evaporation was
with 30 mg Alcalase at 40 °C (150 rpm shaking speed). Alternatively, the
reaction was performed in 1 mL t-BuOMe, in the presence of TEA (10 µL)
2
recrystallised from EtOH and Et O (1:2) to give light yellow crystals [81
25
25
mg, yield: 90%, ee = 98%, α
= 0.2), ECD (H O, λ [nm] (Δε), c 0.100 mM): 294sh (+0.12), 267 (+0.56),
25 (–4.40), 209 (+0.56), 198 (–0.79), m.p.: 181-183 °C]. The 1H NMR
400 MHz, MeOD-d4) spectroscopic data for (R)-2·HCl were similar to
D
= - 12 (EtOH, c = 0.2), α
D
2
= - 22 (H O, c
2
and 2 equiv H O (6.3 µL) with 30 mg Alcalase at 30 °C.
2
2
(
Synthesis of 1,2,3,4-tetrahydro-β-carboline-1-carboxylic acid ethyl
ester [(±)-1]
those for (±)-2·HCl.
2
Anionic form: ECD (H O, λ [nm] (Δε), c 0.075 mM): 294sh (+0.23), 270
Tryptamine hydrochloride (3.0 g, 15.3 mmol) was dissolved in 60 mL
EtOH. The solution was cooled to 0 °C and 6 mL 50% toluene solution of
ethyl glyoxylate (2 equiv., 30.6 mmol) was added. The reaction mixture
was stirred at 0 °C (1 h) and then at room temperature (24 h). After the
reaction the solvent was evaporated off, the residue was dissolved in
(
+0.32), 229 (–2.53), 213 (+0.56), 207sh (–0.21).
Alcalase-catalysed preparative-scale DKR reaction of (±)-1·HCl
±)-1·HCl (100 mg, 0.36 mmol) was dissolved in 15 mL 0.1 M borate
water (90 mL) and the pH was set to 9 with saturated NaHCO
solution was extracted with EtOAc (3×100 mL) and the organic layer was
dried on anhydrous Na SO . Evaporation of the solvent yielded unstable
3
. The
(
buffer, then the pH = 8.0 was set with NaOH (25% aqueous solution).
After the addition 450 mg Alcalase the reaction mixture was shaken in an
incubator shaker at 30 °C for 45 h. The reaction was stopped at 99%
conversion. The enzyme was filtered and washed with borate buffer (pH
2
4
product (±)-1 (dark brown oil). Therefore, (±)-1 was dissolved in 20 mL
EtOH and then 10 mL ethanolic hydrochloric acid was added. The
residue after solvent evaporation gave product (±)-1·HCl. It was
crystallised from ethanol and diethyl ether (1:10) and recrystallised from
ethanol (3.6 g, yield: 84%, m.p.: 216-219 °C, beige crystals).
=
8.0, 3×20 mL). The solvent was evaporated, the residue was dissolved
in 20 mL H O and then the pH was set to 4 with 5% hydrochloric acid.
2
The solvent was evaporated, the residue was dissolved in 25 mL EtOH
and then filtered. After the evaporation of the solvent the residue was
crystallised from EtOH and Et O (1:2). The product [(S)-2·HCl] was
2
1H NMR (400 MHz, D
(
O) spectroscopic data for (±)-1·HCl: δ = 1.37-1.46
t, 3H, J = 7.16 Hz, CH ); 3.15-3.23 (m, 2H, CH ), 3.74-3.84 (m, 2H,
); 4.43-4.56 (m, 2H, CH ) 5.70 (s, 1H, CH); 7.26-7.33 (t, 1H, J = 7.40
2
2
5
3
2
isolated as yellow crystals [59 mg, yield: 66%, ee = 60%, α
D
= + 6.5
CH
2
2
(EtOH, c = 0.32), m.p.: 184-187 °C]. The 1H NMR (400 MHz, MeOD-d4)
Hz, Ar); 7.37-7.44 (t, 1H, J = 7.40 Hz, Ar); 7.60-7.64 (d, 1H, J = 8.24 Hz);
.69-7.75 (d, 1H, J = 7.92 Hz) ppm. 13C NMR (400 MHz, D
O): δ = 13.43,
spectroscopic data for (S)-2·HCl were similar to those for (±)-2·HCl.
7
2
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European Journal of Organic Chemistry
10.1002/ejoc.201700571
FULL PAPER
Computational section
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European Journal of Organic Chemistry
10.1002/ejoc.201700571
FULL PAPER
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European Journal of Organic Chemistry
10.1002/ejoc.201700571
FULL PAPER
Layout 2:
FULL PAPER
Enzymatic hydrolysis*
CAL-B
Alcalase
Rita Megyesi, Attila Mándi, Tibor Kurtán,
Enikő Forró, Ferenc Fülöp*
Page No. – Page No.
Dynamic kinetic resolution of ethyl
1
,2,3,4-tetrahydro-β-carboline-1-
Directed dynamic kinetic enzymatic strategies were developed for the preparation
of both enantiomers of 1,2,3,4-tetrahydro-β-carboline-1-carboxylic acid, through the
hydrolysis of ethyl 1,2,3,4-tetrahydro-β-carboline-1-carboxylate. CAL-B catalysed
DKR resolution gave R-enantiomer with an excellent enantiomeric excess of 98%
and high yield of 90%. Alcalase-catalysed DKR resolution afforded (S)-enantiomer
with an ee 60% and a yield of 66%.
carboxylate. Use of different
hydrolases for stereocomplementary
processes
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