Application of aminoacylase I to the enantioselective resolution of α-amino acid esters and amides

Article abstract of DOI:10.1016/j.tetasy.2004.05.018

Aminoacylase I from Aspergillus melleus, a readily available and inexpensive enzyme mainly used in the industrial production of enantiopure L-amino acids from their N-acetyl derivatives, is shown to hydrolyze the esters and amides of natural and non-natural amino acids with high enantioselectivity (for the ester hydrolysis, E is up to 76, in case of amides E >300). The reaction rates of amide and ester hydrolysis are comparable, and in some cases these conversions proceeded even faster than 'traditional' aminoacylase- catalyzed hydrolysis of N-acetyl derivatives thus providing new possibilities for the resolution of the corresponding racemates. This novel approach provides an alternative route for the biocatalytic production of optically active amino acids and their derivatives.

Full text of DOI:10.1016/j.tetasy.2004.05.018

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 1933–1936
Application of aminoacylase I to the enantioselective
resolution of a-amino acid esters and amides
Maxim I. Youshko,a,* Luuk M. van Langen, Roger A. Sheldon and Vytas K. Svedas
b
c
a
ꢀ
a
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia
b
CLEA Technologies BV, Julianalaan 136, 2628 BL Delft, The Netherlands
Laboratory of Biocatalysis and Organic Chemistry, Delft University of Technology, Julianalaan 136, 2628 BL Delft, The Netherlands
c
Received 13 April 2004; accepted 17 May 2004
Abstract—Aminoacylase I from Aspergillus melleus, a readily available and inexpensive enzyme mainly used in the industrial
production of enantiopure -amino acids from their N-acetyl derivatives, is shown to hydrolyze the esters and amides of natural and
L
non-natural amino acids with high enantioselectivity (for the ester hydrolysis, E is up to 76, in case of amides E >300). The reaction
rates of amide and ester hydrolysis are comparable, and in some cases these conversions proceeded even faster than ‘traditional’
aminoacylase-catalyzed hydrolysis of N-acetyl derivatives thus providing new possibilities for the resolution of the corresponding
racemates. This novel approach provides an alternative route for the biocatalytic production of optically active amino acids and
their derivatives.
ꢀ
2004 Elsevier Ltd. All rights reserved.
1
. Introduction
The fact that the activity of aminoacylase is not re-
stricted to N-acyl transfer reactions gives new possibil-
ities for the application of these enzymes in
enantioselective conversions.
Aminoacylase I (AC, N-acyl-L-amino acid amidohy-
drolase, E.C. 3.5.1.14), is a readily available and inex-
1
pensive enzyme with relaxed substrate specificity that is
used in the industrial production of enantiopure
L
-
Herein we report on the highly effective and enantiose-
lective hydrolysis of esters and amides of different amino
acids catalyzed by aminoacylase I from Aspergillus
melleus. This approach gives a new alternative route for
the biocatalytic production of optically active amino
acids and their derivatives, which nowadays are broadly
used as physiologically active compounds, chiral auxil-
iaries or synthons and as intermediates in pharmaceu-
tical, food and agrochemistry.
2;3
amino acids from their N-acyl derivatives. In organic
and water-organic medium, aminoacylase has been
shown to mediate the reverse reaction––the acylation of
-amino acids ––and also perform enantioselective
irreversible acyl transfer from an activated acyl donor to
4;5
L
6–8
9;10
alcohols and amines.
To date, nearly all scientific
and industrial applications of the aminoacylase I family
of enzymes have exploited the ability of these catalysts
to cleave or synthesize the N-acyl amino moiety.
Recently, the ability of acylase I to perform enantio-
selective conversions of carboxylic acid derivatives has
been demonstrated, for example, butanolysis and
2. Results and discussion
11;12
enantioselective hydrolysis of carboxylic acid esters.
Another example of esterase activity of aminoacylases
In spite of the fact that esterase activity of aminoacylase
I towards carboxylic and amino acids derivatives has
13
has been reported, where acylase I was used to
hydrolyze N-acetyl- -phenylalanine alkyl esters to
give -phenylalanine.
11
D
,
L
recently been reported, no systematic investigations
describing its scope and potential applicability have
been performed. Therefore, we have chosen a number of
alkyl- and aryl-substituted amino acid esters and amides
(see Fig. 1) as potential substrates for aminoacylase
I-catalyzed enantioselective hydrolysis.
L
*
Corresponding author. Tel.: +7-095-939-4484; fax: +7-095-939-2355;
e-mail: youshko@belozersky.msu.ru
0
957-4166/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.05.018

1934
M. I. Youshko et al. / Tetrahedron: Asymmetry 15 (2004) 1933–1936
Table 2. Aminoacylase I-catalyzed enantioselective hydrolysis of
R1
O
CH3 NH2
HN
amino acid amides
NH2
H3C
R
H3C
Compound Time Preferred
(
Conversion Ee
configuration (%) (%)
s
E
h)
O
3
5
5
7
1
1
1.0
0.5
1.0
1.5
1.0
2.5
L
L
L
L
L
L
49.2
50.2
49.3
50.6
50.1
48.5
96.3 >300
97.8 320
95.7 >300
1
2
3
4
. R=OCH3, R1=H
. R=OC2H5, R1=H
. R=NH2, R1=H
5
a
>99
>99
92.0
>300
>300
240
. R=OH,
R1=Ac
0
3
R1
Reaction conditions: 10 mM of starting racemic compound, pH 7.5,
R1
R
HN
HN
2
5 ꢁC, 50 U of aminoacylase I.
For immobilized aminoacylase I.
R
a
O
O
HO
In contrast with the ester substrates, all the tested amino
acid amides showed excellent enantioselectivity (E
>240). From Table 2 it can be seen that both alkyl- and
aryl-substituted amino acid amides were hydrolyzed
with extremely high enantioselectivity. In some cases
(compounds 3, 7 and 10) it was very hard to detect even
6
7
8
. R=OCH3, R1=H
. R=NH2, R1=H
. R= OH, R1=Ac
9. R=OCH3, R1=H
10. R=NH2, R1=H
11. R=OH, R1=Ac
R1
O
HN
traces of
illustrated in Figure 2 for the example of aminoacylase-
D
-enantiomeric hydrolysis (E >300). This is
R
OEt
catalyzed hydrolysis of compound 10. It is clear that
NH2
O
HO
over the course of the reaction only the
amide was converted to the corresponding
acid, whereas the hydrolysis of the -form was negligi-
L
-form of the
L
-amino
1
1
3. R=NH2, R1=H
4. R=OH, R1=Ac
1
2
D
ble. Similar results were obtained with substrates 3, 7
and 13.
Figure 1. Structures of converted amino acid derivatives.
Hydrolytic reactions were performed in aqueous solu-
tions at pH 7.5 for the amides (which is close to the
maximum catalytic activity of aminoacylase I) and
pH 6.5 in the case of esters (to suppress the nonenzy-
matic ‘blank’ hydrolysis). The results summarized in
Tables 1 and 2 show that in both cases the aminoacylase
was able to conduct enantioselective hydrolysis of the
racemic substrates.
5
4
3
2
1
0
Table 1. Aminoacylase I-catalyzed enantioselective hydrolysis of
amino acid esters
Compound
Time Preferred
(h) configuration (%)
Conversion Ee
s
E
2
0
40
60
80
100
120
(%)
Time, min
1
2
3.2
4.0
6.0
5.0
5.0
5.5
L
L
L
L
L
L
53
52
49
56
48
51
92
97
90
88
50
82
33
76
90
15
5
a
Figure 2. Reaction curves for aminoacylase I-catalyzed hydrolysis of
compound 8. s -HPGA, d -HPGA, -HPG, r -HPG. Reac-
tion conditions: pH 7.5, 25 ꢁC, 50 U of aminoacylase I.
2
D
L
ꢀ
D
L
9
6
1
2
21
Reaction conditions: 10 mM of starting racemic compound, pH 6.5,
We also performed the enantioselective hydrolysis of
compounds 2 and 5 using a commercially available
immobilized preparation of aminoacylase I. As can be
seen from Tables 1 and 2 that the reactions were cata-
lyzed by the immobilized enzyme proceeded as well as
with the native enzyme (in both cases, enantioselectivity
was slightly higher compared to the native enzyme-cat-
alyzed hydrolysis).
2
5 ꢁC, 50 U of aminoacylase I.
a
For immobilized aminoacylase I.
Among the ester substrates, the highest enantioselec-
tivity (E ¼ 76) was observed for the short alkyl-substi-
tuted compound 2, while for the aryl-substituted amino
acid esters the enantioselectivity varied from poor
(
1
E ¼ 5, compound 6) to moderate (E ¼ 21, compound
2). It is worth noting that the enantioselectivity in-
As noted above, the aminoacylase I-catalyzed enantio-
selective hydrolysis of amino acid esters and amides can
be an alternative route for the biocatalytic production of
optically active amino acids and their derivatives. As a
creased significantly (more than two times) on switching
from the methyl to the ethyl ester of amino butyric acid
1
and 2.

M. I. Youshko et al. / Tetrahedron: Asymmetry 15 (2004) 1933–1936
1935
result, it was interesting to compare these new reactions
phenylalanine ethyl ester,
D
-())- and
L
-(+)-phenylgly-
with ‘traditional’ aminoacylase I-catalyzed cleavage of
N-acetyl amino acid derivatives. For this purpose, we
synthesized several N-acetylated amino acid derivatives
and compared the reaction rates and enantioselectivities
of their enzymatic hydrolysis with the corresponding
parameters for the hydrolysis of the carboxylic deriva-
tives (amides) of the same amino acids. As can be seen
from Table 3, the highest enantioselectivity was ob-
served for the aminoacylase I-catalyzed hydrolysis of
amino acid amides in all cases. Furthermore, in some
cases the conversion of these substrates proceeded faster
than the hydrolysis of the corresponding N-acetylated
compounds (compare compounds 7 and 8 and 10 and
cine amide, -())- and L-(+)-p-hydroxy-phenylglycine
D
methyl ester hydrochloride and
were purchased from Fluka.
D,L-tyrosine ethyl ester
D
-())- and
esters, -())- and
())- and -(+)-homophenylalanine amide were synthe-
sized in the Laboratory of Biocatalysis and Organic
Chemistry, TUDelft, Netherlands, -())- and -(+)-
phenylglycine, -())- and -(+)-phenylglycine methyl
ester and -())- and -(+)-p-hydroxy-phenylglycine and
-())- and -(+)-p-hydroxy-phenylglycine amide were
L
-(+)-Aminobutyric acid ethyl and methyl
D
L
-(+)-aminobutyric acid amide and
D-
L
D
L
D
L
D
L
D
L
purchased from DSM Fine Chemicals, The Nether-
lands. The organic solvents and buffer compounds were
commercial products of analytical or HPLC grade.
1
1), thus demonstrating the potential of the method for
the production of enantiomerically pure amino acids
and their derivatives.
4.2. Analysis and equipment
Table 3. Comparison of the enantioselectivity and the reaction rates
for the aminoacylase I-catalyzed hydrolysis of N-acetylated amino
acids and the corresponding amino acid amides
The progress of the reactions was monitored by reversed
phase HPLC chromatography using a Chrompack
4.6 · 50 mm 5 · Nucleosil C-18 column, with detection
on a Waters 486 tunable absorbance detector with
Compound
Initial rate,
mM min
Preferred
configuration
E
ꢁ
1
32
Waters Millenium software. The hydrolysis of N-acet-
yl- -methionine was monitored by using an acetonitrile–
aqueous 50 mM phosphate buffer pH 3.0 (15:85, v/v) as
3
4
7
8
0.33
0.41
0.35
0.04
0.21
0.03
0.09
0.62
L
L
L
L
L
L
L
L
>300
>300
>300
96
L
ꢁ
1
an eluent at flow rate 0.5 mL min and detection at
210 nm. The mobile phase for monitoring the hydrolytic
reactions of corresponding amides, esters and their
10
11
13
14
>300
110
240
N-acetylated derivatives was prepared by adjusting a
ꢁ1
70
0
.68 g L
solution of KH PO in acetonitrile–water
2 4
ꢁ
1
Reaction conditions: 10 mM of starting racemic substrate, pH 7.5,
5 ꢁC, 50 U of aminoacylase I.
(30:70, v/v) containing 0.68 g L sodium dodecylsulfate
to pH 3.0 with phosphoric acid. The flow rate was
0
2
ꢁ
1
.5 mL min ; detection at 210 nm. The retention times
of the products corresponded well to those of the
chemically prepared samples.
3. Conclusions
Aminoacylase I from A. melleus was shown to catalyze
the hydrolysis of the esters and amides of natural and
non-natural amino acids with high enantioselectivity.
The reaction rates of these conversions are comparable,
and in some cases much higher than those for the ‘tra-
ditional’ aminoacylase-catalyzed hydrolysis of N-acetyl
derivatives of the corresponding compounds.
Reaction curves and enantiomeric composition of the
reaction mixtures starting from the racemic compounds
were analyzed by chiral HPLC using a 150 · 4 mm
Crownpak CR (+)-column, with detection on a Waters
486 tunable absorbance detector at 210 nm with Waters
32
Millenium software. The enantiomeric ratio (E) of the
enantioselective conversions was calculated using sets of
experimental points of ee
s
or ee
p
versus n [where ee
s
(or
ee ) is the enantiomeric excess, and n is the conversion of
p
10
the initial substrate], as described earlier. The eluents
used and retention times found are compiled in Table 4.
4. Experimental
4
.1. Materials
4
.3. Chemical acylation of racemic amino acids
ꢁ
1
Acylase I from A. melleus, 1.3 U mg , and immobilized
ꢁ
1
(
on Eupergit C) acylase I from Aspergillus, 63 U g
moist material) were from Fluka. The activity of ami-
N-Acetylated compounds 6, 9 and 12 were prepared by
acylation of the corresponding amino acids with acetic
(
noacylase was measured using the standard hydrolytic
14
anhydride in an alkaline aqueous solution. Thus,
15 mmol of acetic anhydride was added dropwise to the
aqueous solution, containing 10 mmol of the corre-
sponding compound at pH 11 at 4 ꢁC. The reaction
mixture was stirred for about 30 min and then the pH
value adjusted to 1.5. The precipitated product was fil-
tered, washed with acidic ice-cold water and dried. The
yields were in the range of 80–85% (w/w).
9
assay of N-acetyl
L
-methionine. One unit (U) would
-methionine per min. -())- and
-methionine, -())-
-(+)-homophenylalanine,
-(+)-tyrosine and -tyrosine ethyl ester
hydrochloride were purchased from Acros, Belgium.
))- and -(+)-leucine amide, -())- and -(+)-homo-
liberate 1 lmol of
L
D
L-
(
+)-2-Aminobutyric acid, N-acetyl-
and -(+)-leucine, -())- and
-())- and
L
D
L
D
L
D
L
L
D
-
(
L
D
L

1936
M. I. Youshko et al. / Tetrahedron: Asymmetry 15 (2004) 1933–1936
Table 4. Chiral HPLC for compounds 1–14
ꢁ1
Compound
Column
Mobile phase
Flow rate (mL min
)
Retention time (min)
D
L
1
2
3
Crownpak CR(+)
Crownpak CR(+)
Crownpak CR(+)
Crownpak CR(+)
Crownpak CR(+)
Crownpak CR(+)
Crownpak CR(+)
Crownpak CR(+)
Crownpak CR(+)
Crownpak CR(+)
Crownpak CR(+)
Crownpak CR(+)
Crownpak CR(+)
Aqueous HClO
Aqueous HClO
Aqueous HClO
Aqueous HClO
Aqueous HClO
Aqueous HClO
Aqueous HClO
Aqueous HClO
Aqueous HClO
Aqueous HClO
Aqueous HClO
Aqueous HClO
4
4
4
4
4
4
4
4
4
4
4
4
pH 1.3, 20 ꢁC
pH 1.3, 20 ꢁC
pH 1.3, 20 ꢁC
pH 1.0, 20 ꢁC
pH 1.3, 20 ꢁC
pH 2.0, 20 ꢁC
pH 1.0, 20 ꢁC
pH 1.5, 20 ꢁC
pH 2.0, 20 ꢁC
pH 1.0, 20 ꢁC
pH 1.5, 20 ꢁC
pH 2.0, 20 ꢁC
0.6
0.6
0.6
0.6
0.6
0.6
0.6
0.6
0.6
0.6
0.6
0.6
0.6
5.0
12.9
25.3
3.7
9.5
2.5
2.1
5.6
a
4
3.2
9.7
5
6
7
14.3
5.6
24.9
26.1
33.7
19.7
21.8
28.4
35.7
37.0
a
8
9.1
8.4
9
1
1
1
1
0
1
2
3
3.8
5.8
a
18.5
19.5
MeOH–aqueous HClO
10:90, v/v), 20 ꢁC
MeOH–aqueous HClO
10:90, v/v), 20 ꢁC
4
pH 1.5
(
a
14
Crownpak CR(+)
4
pH 2.0
0.6
41.2
87.5
(
a
1
In these cases, the reactions were followed by monitoring the free
D
- and
L
-amino acids, accumulated over the course of enzymatic hydrolysis.
H NMR (300 MHz, DMSO-d ) of 4: d (ppm) 0.95 (t,
Acknowledgements
6
3
COCH
H, CH CH ), 1.78 (m, 2H, CH CH ), 1.93 (s, 3H,
), 4.1 (d, 1H, CHCO
2 3 2 3
3
2
H), 8.43 (d, 1H, NH), 11.9
Financial support from INTAS (grant number 03-56-25)
is gratefully acknowledged.
(
s, 1H, CO H).
2
1
H NMR (300 MHz, DMSO-d
H, COCH ), 5.4 (d, 1H, CHCO
aromatic protons), 8.5 (d, 1H, NH), 12.6 (s, 1H,
6
) of 8: d (ppm) 1.92 (s,
3
3
2
H), 7.38 (m, 5H,
References and notes
1
CO
1
1
2
H). H NMR (300 MHz, DMSO-d
.89 (s, 3H, COCH ), 5.35 (d, 1H, CHCO
H, PhOH), 7.11–7.44 (dd, 4H, C H (OH)), 8.54 (d, 1H,
6
) of 11: d (ppm)
1
. Chenault, H. K.; Dahmer, J.; Whitesides, G. J. Am. Chem.
Soc. 1989, 111, 6354–6364.
2. Bommarius, A. S.; Drauz, K.; Klenk, H.; Wandrey, C.
3
2
H), 6.57 (s,
6
4
NH), 12.7 (s, 1H, CO
2
H).
Ann. N.Y. Acad. Sci. 1992, 672, 126–136.
3
. Sato, T.; Tosa, T. Bioprocess. Technol. 1993, 16, 3–4.
4. Kos ꢁa ry, J.; Sisak, C.; Szajani, B.; Boross, L. Biocatalysis
994, 11, 329–337.
1
H NMR (300 MHz, DMSO-d ) of 14: d (ppm) 1.89 (s,
6
1
3
PhCH
H, COCH ), 1.97 (m, 2H, CH CH ), 2.62 (m, 2H,
H), 7.26 (m, 5H, aromatic
H).
3
2
2
5
6
7
8
9
. Yokoigawa, K.; Sato, E.; Esaki, N.; Soda, K. Appl.
Microbiol. Biotechnol. 1994, 42, 287–289.
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367–370.
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A. Tetrahedron: Asymmetry 2000, 11, 1801–1808.
. Herrad oꢁ n, B.; Valverde, S. Synlett 1995, 6, 599–602.
0. Youshko, M. I.; Van Rantwijk, F.; Sheldon, R. A.
Tetrahedron: Asymmetry 2001, 12, 3267–3271.
1. Liljeblad, A.; Lindborg, J.; Kanerva, L. T. Tetrahedron:
Asymmetry 2000, 11, 3957–3966.
2. Liljeblad, A.; Aksela, R.; Kanerva, L. T. Tetrahedron:
Asymmetry 2001, 12, 2059–2066.
3. Kitamura, T.; Matsumoto, Y.; Yoshimura, N.; Kuraray,
C. O.; Ltd, EP 0 198 397 A2, 08.04.1986.
14. Chattaway, F. D. J. Chem. Soc. 1931, 2495–2496.
2
), 4.15 (q, 1H, CHCO
protons), 8.1 (d, 1H, NH), 12.38 (s, 1H, CO
2
2
4
.4. Enzymatic hydrolytic reactions
Enzymatic reactions were carried out in a 10 mL closed
reaction vessel at the appropriate temperature and pH
values under permanent stirring. The reactants were
dissolved in 50 mM Tris buffer with reactions initiated
by adding 50 U of aminoacylase I. Further details are
given in the notes to the tables. Nonenzymatic (blank)
reactions were performed under the same conditions but
without adding enzyme. Samples were withdrawn over
the course of the reaction, centrifuged and subjected to
HPLC analysis as described above.
1
1
1
1