Enantioselective acylation of chiral amines catalysed by aminoacylase I

Article abstract of DOI:10.1016/S0957-4166(01)00563-8

Aminoacylase (E.C. 3.5.1.14) from Aspergillus melleus mediated the acylation of the primary amino group in a range of primary arylalkylamines and amino alcohols in anhydrous organic medium. The commonly used vinyl and isopropenyl esters proved to be unsuitable acyl donors because rapid uncatalysed aminolysis occurred in the presence of these additives. The unwanted aminolysis reaction could be suppressed by performing the enzymatic reaction in tert-butyl methyl ether medium with methyl 2-methoxyacetate as the acyl donor. We found that chiral amines were acylated with poor to moderate enantioselectivity, in contrast with the quantitative enantiodiscrimination that is commonly observed with the corresponding alcohols.

Full text of DOI:10.1016/S0957-4166(01)00563-8

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 12 (2001) 3267–3271
Enantioselective acylation of chiral amines catalysed by
aminoacylase I
Maxim I. Youshko,† Fred van Rantwijk and Roger A. Sheldon*
Laboratory of Biocatalysis and Organic Chemistry, Delft University of Technology, Julianalaan 136,
2628 BL Delft, The Netherlands
Received 19 November 2001; accepted 13 December 2001
Abstract—Aminoacylase (E.C. 3.5.1.14) from Aspergillus melleus mediated the acylation of the primary amino group in a range
of primary arylalkylamines and amino alcohols in anhydrous organic medium. The commonly used vinyl and isopropenyl esters
proved to be unsuitable acyl donors because rapid uncatalysed aminolysis occurred in the presence of these additives. The
unwanted aminolysis reaction could be suppressed by performing the enzymatic reaction in tert-butyl methyl ether medium with
methyl 2-methoxyacetate as the acyl donor. We found that chiral amines were acylated with poor to moderate enantioselectivity,
in contrast with the quantitative enantiodiscrimination that is commonly observed with the corresponding alcohols. © 2002
Published by Elsevier Science Ltd.
1. Introduction
herein a study on the enantioselective acylation of a
variety of arylalkylamines using a range of acyl donors.
Aminoacylase I (N-acyl-
L
-amino acid amidohydrolase,
E.C. 3.5.1.14) from Aspergillus sp. is a readily available
and inexpensive enzyme with a relaxed substrate toler-
ance.1 It is used in the industrial production of enan-
2. Results and discussion
tiopure
The related aminoacylase from porcine kidney also
mediates the reverse reaction, the acylation of -amino
L
-amino acids from their N-acyl derivatives.2,3
We initially attempted to acylate representative chiral
amines, such as 1-aminoindane 1 (see Fig. 1), with vinyl
acetate or butyrate, which has successfully been used in
the resolution of chiral alcohols.8,9 It became clear that
the uncatalysed aminolysis of this activated ester was
undesirably fast, in particular at synthetically relevant
concentrations;10 isopropenyl esters and acetoxyacetone
could not be used for the same reason. Hence, a range
of prospective acyl donors was subjected to enzymatic
as well as uncatalysed aminolysis by 1; the reaction
rates are compiled in Table 1. In general, the cat/uncat
ratio increased with decreasing activity of the donor,
but a very low acyl transfer rate, such as observed with
methyl butanoate, was evidently undesirable. Methyl
L
acids,4,5 in organic medium. Herrado´n et al. subse-
quently showed that aminoacylase from Aspergillus
melleus, in anhydrous organic medium, efficiently
catalyses the—irreversible—acyl transfer from vinyl
acetate to alcohols and amines.6,7 Further investigations
revealed that secondary arylalkanols are acylated with
high, often near-absolute, enantioselectivity8,9 and a
productivity surpassing that of the corresponding
lipase-catalysed reactions.9
The use of aminoacylase as an amine acylation catalyst
has received scant attention,4–6 despite the fact that
enantiopure amines are of considerable interest both as
chiral building blocks and auxiliaries. We present
* Corresponding author. Tel.: +31 15 278 2683; fax: +31 15 278 1415;
e-mail: secretariat-ock@tnw.tudelft.nl
† Present address: Belozersky Institute of Physico-Chemical Biology,
Lomonosov Moscow State University, 119899 Moscow, Russia.
Figure 1. Acylation of 1-aminoindane (1).
0957-4166/01/$ - see front matter © 2002 Published by Elsevier Science Ltd.
PII: S0957-4166(01)00563-8

3268
M. I. Youshko et al. / Tetrahedron: Asymmetry 12 (2001) 3267–3271
Table 1. Effect of the acyl donor on the aminoacylase
I-mediated acylation of 1-aminoindane 1a
Table 2. Acylation of chiral amines 1 by methyl 2-
methoxyacetate mediated by aminoacylase I. Effect of the
reaction medium on the enantiomeric ratioa
Acyl donor
Initial rate
Ratio
cat/uncat
(mM min−1
)
Amine
Solvent (E)
1,2-Dimethoxyethane
Methyl butanoate
Allyl acetate
0.04
0.2
0.3
0.6
1.7
1.8
5.5
\300
\300
\300
120
140
10
Hexane
TBME
Ethyl 2-(methylamino)acetate
Ethyl 2-methoxyacetate
Methyl 2-methoxyacetate
2,2,2-Trifluoroethyl butanoate
Acetoxyacetone
1
2
3
9.7
2.7
7.8
9.3
3.0
8.4
10.2
3.4
8.0
B1
a Reaction conditions: amine (50 mM), acyl donor (60 mM),
aminoacylase I (50 U), solvent (5 mL), rt.
a Reaction conditions: 1 (50 mM), acyl donor (60 mM), aminoacylase
I (50 U), hexane (5 mL), rt.
solved the reagents of interest with only a small
decrease in reaction rate.
2-methoxyacetate11 provided the best compromise and
was used in all subsequent experiments; ethyl 2-
methoxyacetate and 2-(methylamino)acetate could also
be useful acetyl donors.
A variety of arylalkylamines (1–8, see Fig. 3) was
acylated in the presence of aminoacylase I under these
conditions. Compounds 1, 3, 6 and 8, which had only
an amine functionality, were resolved with moderate
enantioselectivity (E=7–9, see Table 3). In contrast, the
same enzyme mediated the acylation of 1-phenyl-
alkanols with near-absolute enantioselectivity.9 1-(1-
Naphthyl)ethylamine reacted much more slowly than
the other compounds, which is presumably due to steric
hindrance from the bulky naphthyl substituent.
We next investigated the effects of changing the reac-
tion medium on the acylation of 1. It has been shown
that hydrophobic solvents, such as hexane or toluene,
are the preferred reaction media for aminoacylase I-
catalysed transesterification,8,9 but the limited solubility
of many reagents of interest in such hydrophobic sol-
vents seriously limits their practical utility. We per-
formed the acylation of 1 by methyl 2-methoxyacetate
in a variety of solvents and found that the initial rate
correlates linearly with log P over a wide range (see Fig.
2). In contrast, the nature of the solvent had a negligi-
ble effect on the enantiodiscrimination of 1 as well as
1-amino-2-indanol 2 and 1-phenylethylamine 3 (see
Table 2). tert-Butyl methyl ether (TBME) was selected
as a compromise solvent because it adequately dis-
The series also included three amino alcohols 2, 4 and
7 to assess the effects of a primary hydroxyl group on
the chemo- and enantioselectivity of the biocatalyst. We
found that the selectivity for amino acylation was abso-
lute but the enantioselectivity was very poor. The enan-
tiomers of phenylglycine methyl ester 5 were also
poorly discriminated.
Figure 2. Acylation of 1-aminoindane by methyl 2-methoxy-
acetate in the presence of aminoacylase I; relationship of
initial rate and log P. Abbreviations: ACN: acetonitrile,
DME: 1,2-dimethoxyethane, t-BuOH: tert-butyl alcohol, i-
PrOMe: isopropyl methyl ether, TBME: tert-butyl methyl
ether.
Figure 3. Structures of amines 1–8 showing the preferentially
converted enantiomer.

M. I. Youshko et al. / Tetrahedron: Asymmetry 12 (2001) 3267–3271
3269
Table 3. Enantioselective acylation of arylalkylamines in the presence of aminoacylase Ia
Compound
Initial rate (mM h−1
)
Preferred enantiomer
Conversion (%)
E.e.amine (% S)
Eb
1
2c
3
4
5
6
7
8
47
4
34
37
18
3
R
1S,2R
R
53
37
57
46
52
27
54
47
72
26
78
12
56
28
24
56
9.3
3.0
8.4
1.3
5.4
9.1
1.8
7.3
R
S
Rd
R
47
35
Nd
a Reaction conditions: amine (15 mM), methyl methoxyacetate (20 mM), aminoacylase I (50 U), MTBE (5 mL), rt.
b Determined via numerical integration of e.e.S versus j (see Section 4).
c Reaction in 1,2-dimethoxyethane.
d Tentatively assigned on the basis of the HPLC retention time (see Section 4).
The difference in enantiodiscrimination of secondary
arylalkanols and the corresponding amines is particu-
larly striking and difficult to explain. Presumably, alco-
hols and amines bind differently in the active site.
Recent work has indicated that aminoacylase I also has
aminopeptidase activity12 and, hence, it seems plausible
that amines could also bind in the subsite that, in the
normal reaction pattern, receives the N-terminal group.
tylamine, isopropenyl acetate, ethyl methoxyacetate,
methyl methoxyacetate, N-acetyl- -methionine, racemic
L
1-phenylethylamine, (R)-(+)-1-phenylethylamine, (S)-
(−)-1-phenylethylamine, were purchased from Acros,
Belgium. Acetoxyacetone, (R)-(−)-1-aminoindane, S-
(+)-1-aminoindane,
(1S,2R)-(−)-1-amino-2-indanol,
(1R,2S)-(+)-1-amino-2-indanol, methyl butyrate, 2,2,2-
trifluoroethyl butyrate, vinyl acetate, vinyl butyrate
were purchased from Fluka. Racemic 1-(1-naph-
thylethylamine was from Lancaster, England. (R)- and
(S)-Phenylglycine methyl esters were kindly donated by
DSM Fine Chemicals, Netherlands. The organic sol-
vents and buffer compounds were commercial products
of analytical or HPLC grade.
The reaction temperature can profoundly affect the
steric course of enzymatic reactions13 and a temperature
decrease often,14–16 but not universally, results in
improved stereoselection. Thus, we investigated the
effects of the reaction temperature on rate and enan-
tioselectivity of the acylation of 1–3. As we expected,
the acylation rate decreased steeply as the temperature
was lowered. The enantiomeric ratio improved only by
a factor of 2, or less, for a 36°C temperature decrease,
which is in the same range as we previously observed
with lipase-mediated resolutions15,16 but is too low to be
of practical significance.
4.2. Analysis and equipment
The progress of the reactions was monitored by
reversed phase HPLC chromatography using
a
Chrompack 4.6×150 mm 5 m Nucleosil C-18 column,
with detection on a Waters 486 tunable absorbance
detector with Waters Millenium32 software. The hydro-
lysis of N-acetyl-L-methionine was monitored using
3. Conclusions
acetonitrile–aqueous 50 mM phosphate buffer pH 3.0
(15:85, v/v) as the eluent at a flow rate of 0.5 mL min−1
and detection at 210 nm. The mobile phase for moni-
toring the acylation of the amines and aminoalcohols
was prepared by adjusting a 0.68 g L−1 solution of
KH2PO4 in acetonitrile–water (30:70, v/v) containing
0.68 g L−1 sodium dodecylsulphate to pH 3.0 with
phosphoric acid. The flow rate was 0.5 mL min−1;
detection at 210 nm. The retention times of the prod-
ucts were compared with those of chemically prepared
samples.
Aminoacylase I from A. melleus mediates the acylation
of primary arylalkylamines with low to moderate enan-
tioselectivity. The amino group is preferentially
acylated in the presence of a primary alcohol function.
4. Experimental
4.1. Materials
Aminoacylase I from A. melleus, 1.3 U mg−1, was
purchased from Fluka. The activity of aminoacylase
was measured using the standard hydrolytic assay of
The enantiomeric purity of the unreacted amines 1–7
was analyzed by chiral HPLC using a 150×4 mm
Crownpak CR(+) column, with detection on a Waters
486 tunable absorbance detector at 215 nm with Waters
Millenium32 software (Table 5). The eluants used and
retention times found are compiled in Table 4. The
enantiomeric purity of the acylation product of 8 was
analyzed using 4.6×250 mm Chiralcel OD column, elu-
ant hexane–iso-propyl alcohol (95:5, v/v) at a flow rate
of 0.8 mL min−1.
N-acetyl
mmol of
L
-methionine.9 One unit (U) will liberate 1
L
-methionine per min.
Allyl acetate, 1-aminoindane, (R)-(−)-2-amino-2-
phenylethanol, (S)-(+)-2-amino-2-phenylethanol, (S)-
(−)-2-amino-3-phenyl-1-propanol, (R)-(+)-2-amino-3-
phenyl-1-propanol, 2-amino-4-phenylbutane, 2-hep-

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M. I. Youshko et al. / Tetrahedron: Asymmetry 12 (2001) 3267–3271
Table 4. Temperature effects on rate and enantioselectivitya
Amine
Initial rate (mM h−1
)
E
4°C
20°C
40°C
4°C
20°C
40°C
1
16
1.5
12
47
4.3
34
120
4.5
100
13
5.0
10.0
9.6
3.4
8.2
9.1
2.5
7.8
2b
3
a Reaction conditions: amine (15 mM), methyl methoxyacetate (20
mM), aminoacylase I (50 U), MTBE (5 mL), rt.
b Reaction in 1,2-dimethoxyethane.
4.3. Acylation of amines
Acylations were carried out in 10 mL closed reaction
vessels at the appropriate temperature under permanent
stirring. The reactants were dissolved and the reaction
was initiated by adding 50 U aminoacylase I. Further
details are given in the table footnotes. Non-enzymatic
(blank) reactions were performed under the same condi-
tions but without enzyme. Samples were withdrawn in
the course of the reaction, centrifuged, and subjected to
HPLC analysis as described above.
Figure 4. Eq. (1) fitted through the datapoints of the
acylation of 3 at 20°C to calculate E. Abbreviations: j:
conversion of 3, e.e.S: enantiomeric excess of 3.
integrating Eq. (1) with minimization of the sum of
squares of deviations as criterium (Fig. 4).
de.e.S (1+e.e.S)(1−e.e.S)
=
(1)
dj
E+1
ꢀ ꢁ
4.4. Calculation of the enantiomeric ratio
(1−j)
−e.e.S
E−1
It is common practice to calculate E from a single
datapoint by applying Sih’s equations.17 We have
sought to increase the accuracy and reliability by calcu-
lating E via numerical integration of Eq. (1) with
subsequent regression analyses.18 Thus, E was deter-
mined by fitting the experimental point sets of e.e.S
versus j (where e.e.S is the enantiomeric excess, and j is
the conversion of the amine, see Fig. 2) by numerically
Acknowledgements
Financial support by Degussa-Hu¨ls AG and INTAS
(grant number YSF 2001/1-121 to MIY) is gratefully
acknowledged.
Table 5. Chiral HPLC of 1–7
Amine
Column
Mobile phase
Flow rate (mL min−1
)
Retention time (min)
R
S
1
2
3
4
5
6
7
Crownpak
CR(+)
Crownpak
CR(+)
Crownpak
CR(+)
Crownpak
CR(+)
Crownpak
CR(+)
Crownpak
CR(+)
Crownpak
CR(+)
Aqueous HClO4 pH 1, 20°C
Aqueous HClO4 pH 1, 20°C
Aqueous HClO4 pH 1, 20°C
Aqueous HClO4 pH 1, 20°C
Aqueous HClO4 pH 2, 20°C
MeOH–aqueous HClO4 pH 2 (5:95, v/v), 40°C
aqueous HClO4 pH 1, 20°C
0.6
0.6
0.6
0.6
0.6
0.6
0.6
60
15a
40
40
18b
29
23
15
18
37
135c
27
120c
30
a (1R,2S).
b (1S,2R).
c Configuration tentatively assigned on the basis of the retention time.

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3271
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