Efficient chemoenzymatic synthesis of enantiomerically pure β-heterocyclic amino acid derivatives

Article abstract of DOI:10.1016/S0957-4166(00)00463-8

Enantiomerically pure (S)-pyrazolylalanine and its derivatives are nonproteinogenic amino acids with antidiabetic activity. A short and effective enantioselective synthesis of these compounds is described, using a kinetic resolution by an acylase from Aspergillus sp.

Full text of DOI:10.1016/S0957-4166(00)00463-8

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 11 (2000) 4719–4724
Efficient chemoenzymatic synthesis of enantiomerically pure
b-heterocyclic amino acid derivatives
Val e´ rie Rolland-Fulcrand,* Nicolas Haroune, Marie-Louise Roumestant and Jean Martinez
Laboratoire d’Aminoacides, Peptides et Prot e´ ines, Universit e´ Montpellier I et II, Place E. Bataillon,
34095 Montpellier Cedex 5, France
Received 17 October 2000; accepted 17 November 2000
Abstract
Enantiomerically pure (S)-pyrazolylalanine and its derivatives are nonproteinogenic amino acids with
antidiabetic activity. A short and effective enantioselective synthesis of these compounds is described,
using a kinetic resolution by an acylase from Aspergillus sp. © 2001 Elsevier Science Ltd. All rights
reserved.
1
. Introduction
(
S)-b-Pyrazolylalanine is a naturally occurring amino acid which was first isolated by Shinano
1
and Kayo from the pressed juice and the seeds of watermelon, Citrullus vulgaris, and has
subsequently been found in other cucurbit plants. Cucurbit seeds contain significant amounts of
2
3
g-
L
-glutamyl-b-pyrazolylalanine together with free b-pyrazolylalanine. Several racemic synthe-
4
,5
6
7
ses have been described, a resolution procedure and two enantiospecific syntheses have been
reported, the sign of the specific rotation for the (S)-enantiomer being different. Monteiro et al.
have reported an efficient synthesis of a series of b-heterocyclic amino acids by Michael addition
of heterocyclic nucleophiles to commercially available N-di-tert-butyloxycarbonyl dehydro-
alanine methylester. After total deprotection they obtained racemic mixtures of these amino
5
acids.
We present herein a short and efficient strategy to obtain enantiomerically pure b-heterocyclic
amino acids in nearly quantitative yields.
*
Corresponding author. E-mail: rolland@crit.univ-montp2.fr
0
957-4166/00/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(00)00463-8

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V. Rolland-Fulcrand et al. / Tetrahedron: Asymmetry 11 (2000) 4719–4724
2
. Results
Needing appreciable quantities of the two enantiomerically pure isomers for biological activity
studies, we describe a short and practical synthesis of either enantiomer of b-pyrazolylalanine
a, b-triazolylalanine 3b and azaindolylalanine 3c which can be performed on a gram scale
Scheme 1).
3
(
−
4
Scheme 1. i: K CO , CH CN/0.5% H O. ii: HCl 6N. iii: Aspergillus sp. acylase, phosphate buffer pH 7.2/5×10
M
2
3
3
2
CoCl2
Reaction of an excess of pyrazole a, 1,2,4-triazole b or 7-azaindole c on commercially
available methyl-2-acetamidoacrylate in CH CN at 60°C in the presence of K CO led directly
3
2
3
to the potassium salt of N-acetyl-b-heterocyclic alanines 1a, 1b and 1c in 95%, 90% and 78%
isolated yields, respectively. The presence of nearly 0.5% water in acetonitrile was sufficient to
perform also the saponification under basic conditions. Acidic deprotection (6N HCl) gave
racemic b-heterocyclic alanines 2a, 2b and 2c in quantitative yields. These compounds were
necessary to determine the conditions for a complete separation of the two enantiomers on
chiral column chromatography.
Resolution of racemic N-acetyl-b-pyrazolylalanine methyl ester with a-chymotrypsin in
phosphate buffer was unsuccessful, suggesting that b-pyrazolylalanine bears a close resemblance
8
to histidine. Resolution of N-acetyl-b-pyrazolylalanine with acylase from Aspergillus sp. gave
outstanding results yielding the two enantiomerically pure b-pyrazolylalanine enantiomers 3a
and 5a with excellent e.e. (99%+1%); the enantiomeric purity was checked on chiral Crown Pack
+
CR .
(
S)-b-Triazolyl alanine 3b is known as an important metabolite in plants of the fungicide
Myclobutanil. A biosynthetic pathway from O-acetyl- -serine and 1,2,4 triazole catalyzed by a
cysteine synthase present in higher plants has been reported. N-Acetyl-b-triazolylalanine was
obtained in 95% yield using the same experimental conditions and the resolution with Asper-
L
9

V. Rolland-Fulcrand et al. / Tetrahedron: Asymmetry 11 (2000) 4719–4724
4721
gillus sp. acylase gave the two b-triazolylalanine enantiomers 3b and 5b, respectively in 45% and
4
2% yield after recrystallization (e.e.=99%+1%).
The azaindolylalanine is an isostere of the fluorescent probe, 7-azatryptophan, which was
1
0
prepared by a chemoenzymatic pathway. The N-acetyl-azaindolylalanine 1c was synthesized in
8% yield and the (S)-azaindolylalanine 3c was obtained in 38% final yield after recrystallization
7
using the same experimental conditions as for the 3a synthesis (e.e.=99%+1%).
This very short synthesis (only two steps) is an efficient route to a series of enantiomerically
pure unnatural amino acids. In order to use this strategy in combinatorial chemistry, we have
11
performed this synthesis of racemic compounds on solid support.
3
. Experimental
Thin-layer chromatography (TLC) was performed on Merck precoated silica gel 60F254
plates and spots were visualized by ultraviolet light or/and iodine vapour. The spots were
revealed also by 0.2% ninhydrin solution. HPLC was performed on Waters HPLC system with
+
a 486 UV detector apparatus on a Crown Pack CR column. Melting points were obtained on
a B u¨ chi 510 apparatus and were not corrected. Optical rotations were recorded with a
Perkin–Elmer 141 polarimeter at the sodium D line. Mass spectra were measured by Electro-
spray on Micromass apparatus (Manchester, UK), Platform model ESI (eluent H O/CH CN
2
3
1
0
.1% TFA (1/1) (vol/vol)). H NMR Spectra were recorded on a Brucker spectrometer AC 250.
3
.1. Potassium salt of (RS)-N-acetylpyrazolylalanine 1a
Pyrazole (2.1 g, 30 mmol) was dissolved in 35 ml acetonitrile containing 0.1% H O and K CO
2
2
3
(
3.4 g, 24.6 mmol) was added. The mixture was stirred for 10 min. Then methyl-2-acet-
amidoacrylate (2 g, 13 mmol) was added and the mixture was stirred for 48 h at 60°C. The
disappearance of the methyl-2-acetamidoacrylate was followed by TLC in ethyl acetate (R =
f
0
.85). After filtration, the solvent was evaporated under reduced pressure. The excess of pyrazole
was eliminated by precipitation in a mixture of MeOH/Et O. The potassium salt of (RS)-N-
2
acetylpyrazolylalanine 1a was directly obtained in 90% yield.
3
.2. Acylase from aspergillus genus (TCI) purification
−
1
The commercially available acylase from Aspergillus sp. from TCI (10 000 U g ) was eluted
−
4
on a Sephadex G50 Gel-filtration column with distilled water and a solution of CoCl (5×10
2
M). The elution was followed on a Waters refractometer and checked by Bradford test. The
enzyme was lyophilized and stored at −18°C. Only 30% in weight of protein was obtained from
the crude commercial powder. Preliminary enzyme purification was necessary to facilitate
(
S)-amino acid and (R)-N-acetyl amino acid separation.
3
.3. Enzymatic resolution by acetyl group hydrolysis: (S)-i-pyrazolylalanine 3a
−
4
The substrate 2a (2.5 mmol) was dissolved in phosphate buffer with 5×10 M CoCl (15 ml,
2
0
.1 M; pH 7.2). Then the purified acylase (20 mg) dissolved in buffer (0.5 ml) was added. The
reaction was stirred at room temperature. When the pH decreased to 6.5, aliquots of 6N NaOH

4722
V. Rolland-Fulcrand et al. / Tetrahedron: Asymmetry 11 (2000) 4719–4724
were added until the optimum pH 7.2 for the best enzyme activity. The reaction could be
monitored by a pHstat.
The enantioselective hydrolysis was followed by HPLC chromatography on a chiral column
+
Crown Pack CR with a solution of H O/perchloric acid pH 1.6 at 0°C as eluent. The mixture
2
was stirred during 32 h without hydrolysis of the (R)-N-Ac-b-pyrazolylalanine 4a and the
reaction was quenched with 6N HCl until pH 3. The products were separated by elution on ion
exchange resin (Dowex 50WX8). The solvent was evaporated under reduced pressure and the
residue was dissolved in a solution (10 ml) of distilled water/methanol (4/6) (vol/vol). The ion
exchange resin (500 mg) was previously washed 2 times with distilled water and 2 times with
methanol. The solution was mechanically stirred with resin for 30 min. The binding of the free
(
S)-amino acid was followed by its disappearance from TLC with ethanol/NH OH (4/1) as
4
eluent (R =0.4) and ninhydrine. To follow the binding, chiral chromatography could also be
f
used. The resin-bound-amino acid (S) was washed with distilled water (15 ml) and filtrated (2
times). The (R)-N-Ac-b-pyrazolylalanine 4a and the acylase were in the filtrate. (S)-b-Pyrazolyl-
alanine 3a was eluted with a solution of 2 M NH OH (10 ml) and the resin was washed with
4
distilled water. Compound 3a was obtained in 45% yield after evaporation under reduced
pressure and recrystallization in a mixture of water/ethanol (1/9) (vol/vol). The purity of
(
S)-b-pyrazolylalanine 3a was checked by chiral chromatography (purity >99.5%). The condi-
tions of an efficient separation of the two enantiomers, obtained by complete hydrolysis of
RS)-N-Ac-b-pyrazolylalanine 2a with 6N HCl under reflux, had been performed previously.
7
(
7a
[
h] =−68 (c=9.7 H O) (lit. [h] =−72.0 (c=1.0 H O); mp=239–241°C (lit. mp 241–243°C).
D
2
D
2
1
H NMR (D O): l ppm 4.25 (t, 1H, J=5.05 Hz); 4.75 (d, 2H, CH , J=5.05 Hz); 6.5 (t, 1H,
2
2
+
J=2.1 Hz); 7.75 (dd, 2H, J=8.34 Hz and 2.1 Hz). ESI: [M+H] =156. Anal. calcd for
C H N O , 1/2 H O, C, 43.89; H, 6.1; N, 25.5; found: C, 43.88; H, 5.45; N, 25.1.
6
9
3
2
2
+
HPLC on chiral Crown Pack CR optimized conditions: wavelengh=200 nm; flow=0.3 ml
−
1
min ; pH=1.6 (HClO /milliQ H O); elution at 0°C.
4
2
Retention time
12 min
14 min
10 min
7.5 min
(
(
(
(
RS)-N-Ac-b-Pyrazolylalanine methyl ester
R)-N-Ac-b-Pyrazolylalanine 4a
S)-b-Pyrazolylalanine 3a
R)-b-Pyrazolylalanine 5a
3
.4. (R)-i-Pyrazolylalanine 5a
R)-N-Ac-b-Pyrazolylalanine 4a (1.25 mmol) was hydrolyzed in 6N HCl (10 ml) under reflux
(
overnight. Then the mixture was neutralized with 6N NaOH in MeOH (10 ml) and NaCl salts
precipitated. After filtration the solution was concentrated to give a white solid (95%).
(
R)-b-Pyrazolylalanine 5a (1.2 mmol) was bound on Dowex 50WX8 ion exchange resin (500
mg), washed with water and eluted with 2 M NH OH (10 ml). After evaporation the residue was
4
crystallized in a mixture of water/ethanol (1/9) (vol/vol) in 90% yield. The purity of (R)-b-pyra-
zolylalanine 5a was checked by chiral chromatography (purity >99.5%). [h] =+67 (c=10 H O);
D
2
+
mp=240–242°C; ESI: [M+H] =156.

V. Rolland-Fulcrand et al. / Tetrahedron: Asymmetry 11 (2000) 4719–4724
4723
3
.5. (S)-i-Triazolylalanine 3b synthesis
R)-N-Acetyl-b-triazolylalanine 4b precipitated and (S)-b-triazolylalanine 3b was soluble at
(
−
1
pH 4 and 0°C in a 0.2N HCl solution. The concentration of products was around 50 g l .
The separation of both products was carried out by simple filtration. The (S)-b-triazolylala-
nine solution was concentrated under reduced pressure. The (R)-N-acetyl-b-triazolylalanine 4b
precipitated again and this procedure was repeated until one spot appeared by TLC or one
1
peak in chiral chromatography. [h] =−25 (c=10 H O); mp=233–234°C. H NMR (D O): l
D
2
2
ppm 4.25 (t, 1H, J=4.30 Hz); 4.8 (d, 2H, CH , J=4.25 Hz); 8.12 (s, 1H); 8.45 (s, 1H). ESI:
2
+
[
M+H] =157. Anal. calcd for C H N O , 1.5 H O, C, 32.8; H, 6.0; N, 30.6; found: C, 33.06;
5
8
4
2
2
H, 5.6; N, 31.0.
+
The purity of (S)-b-triazolylalanine was checked by chiral HPLC on Crown Pack CR
column optimized conditions: wavelengh=200 nm; flow=0.2 ml min ; pH=1.7 (HClO /
−
1
4
milliQ H O); elution at 0°C.
2
Retention time
(R)-N-Ac-b-Triazolylalanine 1b
(S)-b-Triazolylalanine 3b
(R)-b-Triazolylalanine 5b
7.0 min
5.0 min
6.0 min
3
.6. (S)-7-Azaindolylalanine 3c
The Michael addition was slower than with pyrazole a or triazole b and during the addition
the aminoester intermediate appears clearly on TLC [R =0.35 in ethyl acetate/diethyl ether
f
(
1/1)]. The reaction was stopped after 48 h at 60°C with the disappearance of the methyl-2-
acetamidoacrylate followed by TLC in ethyl acetate (R =0.85). The solvent was evaporated
f
under reduced pressure and the residue was chromatographed on silica gel in ethyl acetate/
diethyl ether (1/1) as eluent. The (RS)-N-acetylazaindolylalanine methyl ester (15%) and (RS)-
1
N-Ac-7-azaindolylalanine 1c (78%) were characterized by H NMR. The enzymatic hydrolysis
of 1c by Aspergillus sp. acylase was quantitative and the (S)-amino acid was easily obtained in
+
3
7% yield. [h] =−10 [c=10 H O/HCl (3N)]; ESI: [M+H] =206. Anal. calcd for C H N O ,
D
2
10 11
1
3
2
C, 58.5; H, 5.4; N, 20.5; found: C, 57.9; H, 5.2; N, 21.0; mp=278–280°C. H NMR
DMSO-d ) l ppm: 3.75 (dd, 1H, J=6.5 Hz and 3.25 Hz); 4.4 (dd, 1H, J=13.8 Hz and 6.5
(
6
Hz); 4.85 (dd, 1H, J=13.8 Hz and 3.25 Hz); 6.5 (d, 1H, J=3.4 Hz); 7.15 (dd, 1H, J=7.9 Hz
and 4.7 Hz); 7.6 (d, 1H, J=3.25 Hz); 8 (d, 1H, J=7.9 Hz); 8.3 (d, 1H, J=3.25); the purity of
+
(
S)-7-azaindolylalanine was checked by chiral HPLC on Crown Pack CR column optimized
−
1
conditions: wavelengh=200 nm; flow=0.5 ml min ; pH=2 (HClO /milliQ H O); elution at
4
2
0
°C.
Retention time
15 min
9.9 min
(R)-N-Ac-b-Azaindolylalanine 4c
(S)-b-Azaindolylalanine 3c
(R)-b-Azaindolylalanine 5c
12 min

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V. Rolland-Fulcrand et al. / Tetrahedron: Asymmetry 11 (2000) 4719–4724
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1
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.