Efficient kinetic resolution of amino acids catalyzed by lipase AS 'Amano' via cleavage of an amide bond

Article abstract of DOI:10.1016/j.tetasy.2012.08.017

Herein the efficient kinetic resolution of non-natural alpha-amino acids catalyzed by lipase AS 'Amano' via cleaving the amide bond is reported. The starting materials were the corresponding amino acid amides and the amino acids were generated with ees of up to 99% with E values of >600. These results indicated that the lipase AS 'Amano' could be a powerful amide hydrolase for the kinetic resolution of amino acid starting from the corresponding amino acid amides.

Full text of DOI:10.1016/j.tetasy.2012.08.017

Tetrahedron: Asymmetry 23 (2012) 1338–1342
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Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Efficient kinetic resolution of amino acids catalyzed by lipase AS ‘Amano’
via cleavage of an amide bond
Bo Wang a,b, , Yanfeng Liu a, , Dela Zhang a, Yuhong Feng a, Jiacheng Li a
⇑
⇑
a Key Laboratory of Tropical Biological Resources of Ministry of Education, Haikou 570228, Hainan University, PR China
b KTH Royal Institute of Technology, Division of Biochemistry, School of Biotechnology, AlbaNova University Center, SE-106 91 Stockholm, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 July 2012
Accepted 31 August 2012
Herein the efficient kinetic resolution of non-natural alpha-amino acids catalyzed by lipase AS ‘Amano’
via cleaving the amide bond is reported. The starting materials were the corresponding amino acid
amides and the amino acids were generated with ees of up to 99% with E values of >600. These results
indicated that the lipase AS ‘Amano’ could be a powerful amide hydrolase for the kinetic resolution of
amino acid starting from the corresponding amino acid amides.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
has been demonstrated to influence the enzymatic cleavage of
unnatural amide substrates.16 The majority of KR reactions for pure
Non-natural amino acids are compounds of growing interest for
their incorporation in non-ribosomal peptides (NRPs), a class of
microbial natural products, which include several valuable small
molecule therapeutics (antibiotics and immunosuppressants) for
the preparation of new cyclic and linear peptidic drugs, for the syn-
thesis of peptide segments with predictable folding properties and
compounds not subject to protease action.1–8 There have been a
plethora of procedures developed for the preparation of enantio-
pure amino acids.4–7,9,10 Among them, enzymatic kinetic resolution
(KR) is a classical method and has been used as a practical way for
both small-scale preparations and industrial purposes. Amino acid
acylase and penicillin G acylase have been known for a long time to
be efficient biocatalysts for the KR of amino acids starting from the
corresponding amino acid amides.11–14 Lipases however, generally
do not cleave amide bonds. Activity toward amide bonds is highly
untypical for lipases due to the higher activation energy required
for the formation of the enzyme-bound tetrahedral transition state
amino acid isomers catalyzed by lipases start with the correspond-
ing amino acid esters.17–23 Lipases with the ability to hydrolyze the
amide bond of a substrate in high enantioselectivity with a broad
substrate scope are less common and have seldom been used for
the KR of amino acids using amino acid amides as the starting
material.
Herein we report a new powerful lipase AS ‘Amano’ for the effi-
cient KR of amino acids via cleaving the C(O)–N bond of amino acid
amides, which is commercially available, easily obtainable, and has
a broad substrate scope. Through this method, the corresponding
amino acids can be obtained with up to 99% ee and with an E value
of up to >600.
We found that lipase AS ‘Amano’ could hydrolyze 2-acetamido-
2-phenylacetic acid (2-APA) via cleavage of the amide bond of
2-APA, to yield the corresponding amino acid, 2-amino-2-phenyla-
cetic acid in 47% yield and 94% ee in HEPES solution (pH, 7.0) after
being left overnight (Scheme 1). This result that the lipase can
efficiently break the bond of the amides encouraged us to further
explore its function as an amide hydrolase for the preparation of
amino acids.
of amides in comparison to the more polar ester substrates.15
A
structural explanation for the ester versus amide specificity may
be the efficiency of the protonation of the leaving group, which
NHAc
NHAc
NH2
OH
OH
OH
Lipase AS "Amano"
+
Buffer solution
O
O
O
Scheme 1. Lipase catalyzed KR of (RS)-2-acetamido-2-phenylacetic acid.
⇑
Corresponding authors. Tel./fax: +86 898 6618 6568 (Y.L.); tel./fax: +46 702 89
3099 (B.W.).
E-mail addresses: bo.wang@biotech.kth.se (B. Wang), chiralchem@hainu.edu.cn
(Y. Liu).
0957-4166/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetasy.2012.08.017

B. Wang et al. / Tetrahedron: Asymmetry 23 (2012) 1338–1342
1339
After obtaining the optimized conditions for the KR reaction
catalyzed by Amano lipase AS, an array of racemic -phenylalanine
2. Results and discussion
a
amides, 2-acetamido-3-phenylpropanoic acid, and its derivatives
(Table 2) were subjected to KR reactions promoted by lipase AS
‘Amano’ under the optimized conditions described above. The five
substrates employed were successfully and efficiently resolved,
yielding the corresponding a-amino acids. It was found that when
the hydrogen atom on the opposite position of the aromatic ring
Initial studies were performed to find a suitable buffer solution
for the KR reactions catalyzed by lipase AS ‘Amano’. Three different
buffer solutions at concentrations of 20 and 50 mM (Table 1) were
thus tested employing amino acid amide 2-APA as the model sub-
strate, the concentration of 2-APA was 20 mM. The KR results are
summarized in Table 1; when KR reactions were carried out in
phosphorus buffer solution (PBS) at the concentration of 50 mM,
an excellent ee value of 97% could be achieved with a conversion
of 37% with an E value of 119 after being stirred for 4 h (Table 1,
entry 2) at 35 °C. When reactions were performed in the other
two buffer solutions or in pure water, lipase AS ‘Amano’ displayed
generally lower enantioselectivities, leading to E values of less than
80, although a higher conversion could be achieved (Table 1, entry
5). Among the buffer solutions tested, PBS at a concentration of
50 mM was demonstrated to be the best and thus was chosen for
the KR reactions in further studies.
was substituted by a halogen atom, no obvious enhancement in
the ee values of the corresponding product a-amino acids was ob-
served. If the hydrogen at the same position was replaced with a
methyl or nitro group, which are considered to cause more steric
hindrance than a halogen, the amino acid generated was obtained
in excellent ee values of up to 98% (Table 2, entries 4 and 5), and a
slight increase in enantioselectivity. It seemed that the increased
steric hindrance (within the acceptance of Amano lipase AS) of
the motif of the substrate contributed to the enantioselectivity of
the lipase. It was also proposed that the active site of the lipase
from lipase AS ‘Amano’ functioned very similarly to some second-
ary-alcohol lipases and esterases, for instance lipase B from Can-
dida antarctica (CALB) and the esterase from Escherichia coli
BioH,26–30 which contain a big pocket and a smaller one. When
the biotransformation takes place, both the big and smaller groups
of the substrates fit well within the accordingly big and small pock-
ets of the lipase’s active site. When a larger group within the accep-
tance of the active site was introduced to the substrate, it could
thus fit more tightly within the pockets of the enzyme’s active site,
resulting in a higher enantiomeric excess of the corresponding
amino acid.
Table 1
Reaction solution screening for KR of 2-APA catalyzed by lipase AS ‘Amano’a
Entry
Solution
Concentration (mM)
Conversion (%)
eep
Ec
37
78
119
65
75
31
65
b
1
2
3
4
5
6
7
H2O
PBS
—
20
50
20
50
20
50
11
32
37
34
39
29
36
94
96
97
95
95
91
90
HEPES
Tris–HCl
a
Reaction conditions: substrate concentration, 20 mM, lipase AS ‘Amano’, 135 U
(10 mg), temperature, 35 °C, reaction time, 4 h.
b
Conversions and product ee values were determined by HPLC analysis equipped
with a chiral AD-H column run at room temperature, flow rate, 1 mL/min, mobile
phase, iso-propanol/n-hexane, 1:10.
Table 2
c
KR results of various phenylalanines catalyzed by lipase AS ‘Amano’a
E
values were calculated from eep and ees following the formula:
E = ln[(1 ꢀ c)(1 + eep)]/ln[(1 ꢀ c)(1 ꢀ eep)], c = ees/(ees + eep).
Entry Product
Conversionb (%) ee (%)
E
In order to determine the optimum pH value of lipase AS ‘Ama-
no’ in PBS for the KR of 2-APA, various pH values within the range
of 4.0–9.0 at intervals of 1.0 were then evaluated, and other condi-
tions such as enzyme loading, PBS, and substrate concentration
were identical. The profile of the PBS pH value versus the product
ee value is shown in Figure 1. It was indicated that when the model
KR reaction was carried out at pH 6.5, the corresponding amino
acid could be generated in 97% ee with an E value of up to >140
(Fig. 1). Based on the above results, we found that lipase AS ‘Amano’
had the optimum pH value of 6.5, which was in agreement with
that previously described by Murty et al. and Pera and co-work-
ers.24,25 Under such conditions, the lipase can display its highest
enantioselectivity and activity toward its substrate 2-APA.
O
1
41
37
97
96
141
OH
O
NH2
OH
OH
2
89
92
NH2
Cl
O
3
38
31
43
96
98
99
NH2
Br
O
OH
4
154
225
NH2
H3C
O
OH
5
NH2
O2N
a
Reactions were performed on a 5 mL scale, substrate concentration, 20 mM,
lipase AS ‘Amano’, 135 U (10 mg), reaction temperature, 35 °C, buffer solution
concentration, 50 mM, pH 6.5, and reaction time, 4 h.
b
Unless otherwise stated, conversions and ee values were determined by HPLC
analysis equipped with a chiral AD-H column, flow rate, 1 mL/min, mobile phase,
iso-propanol/n-hexane, 1:10; and HPLC analysis were run at 35 °C.
Figure 1. Profile of E value versus pH value in the KR of 2-APA catalyzed by lipase
AS ‘Amano’ (blue dotted line represents the trend line).

1340
B. Wang et al. / Tetrahedron: Asymmetry 23 (2012) 1338–1342
To better and further demonstrate the proposed assumption,
Lipase AS
Marker
"Amano"
we employed another group of amino acids, with no adjacent
methylene to the carbon atom on the existing amine group. Of
the substrates listed in Table 3, there was an aliphatic amino acid
bearing a small (less sterically hindered) group so as to compare
with ones carrying aromatic rings. When an aliphatic amino acid
was used as the substrate, the corresponding product gave an ee
value of 91% (Table 3, entry 4); whereas, if the amino acid amide
bearing one or more aromatic rings, the ee value of the amino acid
generated increased to 99% (Table 3, entry 3). However, if the sub-
strate carries a very large (highly steric hindered) group, for in-
stance, 2-acetamido-3,3-dimethylbutanoic acid (Table 3, entry 5),
no product (2-amino-3,3-dimethylbutanoic acid) was detected. It
was proposed that the more hindered group, tert-butyl group is
too big for the active pocket of the lipase to accommodate, leading
to no activity of lipase AS ‘Amano’ toward the 2-acetamido-3,3-
dimethylbutanoic acid being measured.
110 kDa
65 kDa
45 kDa
35 kDa
33 kDa
Table 3
Results of Amano lipase AS catalyzed KR of amino acid amidesa
Entry Product
Conversionb
(%)
ee
(%)
E
Figure 2. Protein electrophoresis for investigating the purity of the commercial
lipase AS ‘Amano’, from which we can see that the commercial lipase AS ‘Amano’
powder contains only one enzyme, and indicates that no possible contamination
was found.
NH2
OH
1
44
48
96
95
117
115
O
lipase AS ‘Amano’ represents a powerful and highly enantioselec-
tive amide hydrolase.
NH2
OH
F3C
2
3. Conclusion
O
In conclusion, we have introduced a new powerful lipase func-
NH2
tion as an amide hydrolase for the efficient KR of a variety of
a-
OH
amino acids starting from the corresponding amino acid amides.
Most of the generated amino acids were obtained in high ee and
E values (up to 99% ee and E of >600). The differences in enantiose-
lectivities with respect to the substrates were also discussed, indi-
3
4
47
39
0
99
91
—
>600
41
O
NH2
cating
a
similar mechanism to most of the sec-alcohol
OH
enantiopreferred lipases. Meanwhile, the optimal pH value of li-
pase AS ‘Amano’ was also investigated. It was found that lipase
AS ‘Amano’ could be employed for the preparation of chiral amino
acids and their derivates in high enantiomeric excess, which is
thought to have potential in industrial application. Further studies
on the applications of lipase AS ‘Amano’ for the preparation of
chemical intermediates are currently under investigation.
O
NH2
OH
5
—
O
a
Reactions were performed on a 5 mL scale, substrate concentration, 20 mM,
4. Experimental section
4.1. Materials
lipase AS ‘Amano’, 135 U (10 mg), reaction temperature, 35 °C, buffer solution
concentration, 50 mM, pH 6.5, and reaction time, 4 h.
b
Unless otherwise stated, conversions and ee values were determined by HPLC
analysis equipped with a chiral AD-H column, flow rate, 1 mL/min, mobile phase,
iso-propanol/n-hexane, 1:10; and HPLC analysis was run at 35 °C.
All of the amino acids listed in Tables 2 and 3 were purchased
from Sigma Aldrich (China Mainland) and used without further
purification. The amino acid amides were prepared from the corre-
sponding amino acids and acetic anhydrides following the litera-
ture.31–33 Lipase AS ‘Amano’ was donated by Amino Enzyme Inc.
(Shanghai, China Mainland) as a white powder.
The possible contamination of lipase AS ‘Amano’ with an ami-
dase34 was evaluated by performing protein electrophoresis to
check if amidase or other amide hydrolases, which can cleave the
C–N bond of the amide, exist in the powder of commercial lipase
AS ‘Amano’; our results showed that only one enzyme with a
molecular weight of about 33 kDa was detected (Fig. 2). This indi-
cated that no other enzyme existed except for lipase AS ‘Amano’.
The lipase itself can effectively hydrolyze amino acid amides via
cleavage of the amide bond.
All of the amino acid amides listed in Tables 2 and 3 (except en-
try 5) were efficiently and successfully resolved by lipase AS ‘Ama-
no’, some of which were obtained in high conversions, excellent ee
values of up to 99% and with E values of up to >600, indicating that
4.2. General
1H and 13C nuclear magnetic resonance spectra (1H NMR) were
recorded on a Bruker AV400 instrument operating at the frequency
of 400 MHz for 1H NMR. All chemical shifts (d) are quoted in parts
per million (ppm) and reported relative to an internal tetramethyl-
silane (TMS, d 0.00) standard. The following abbreviations were
used to define the multiplicities: br, broad; s, singlet; d, doublet;

B. Wang et al. / Tetrahedron: Asymmetry 23 (2012) 1338–1342
1341
t, triplet; q, quartet; p, pentet; and m, multiplet. Yields and ee val-
ues were determined by HPLC analysis. The E values were calcu-
4.4.4. (S)-2-Amino-3-(p-tolyl)propanoic acid (Table 2, entry 4)
Obtained as a white solid, ee = 98%, mp: 275–279 °C, 1H NMR
(D2O, 400.13 MHz): 2.36 (s, 3H), 3.13–3.19 (q, J = 6.8 Hz, 1H),
3.32–3.35 (q, J = 7.2 Hz, 1H), 4.01–4.07 (q, J = 6.0 Hz, 1H), 4.73 (s,
1H), 7.40–7.53 (m, 4H), 12.5 (s, 1H). 13C NMR (D2O, 100.62 MHz):
lated
from
eep
and
ees
following
the
formula:
E = ln[(1 ꢀ c)(1 + eep)]/ln[(1ꢀc)(1ꢀeep)], c = ees/(ees + eep). HPLC
was performed with an Agilent 1100 instrument equipped with a
Chiralpak AD-H column (150 ꢁ 4.6 mm, Daicel Chemical Indus-
tries, LTD) at the wavelength of 254 nm run at room temperature,
flow rate, 1 mL/min, mobile phase, iso-propanol/n-hexane, 1:10.
21.7, 37.4, 57.1, 127.3, 131.1, 134.9, 135.8, 174.0. ½a D20
¼ ꢀ5:7 (c
ꢂ
1.0, hydrochloric acid).
4.4.5. (S)-2-Amino-3-(4-nitrophenyl)propanoic acid (Table 2,
entry 5)
4.3. General procedure for the preparation of amino acid
amides
Obtained as a white solid, ee = 99%, mp: 242–245 °C 1H NMR
(D2O, 400.13 MHz): 3.10–3.16 (q, J = 6.0 Hz, 1H), 3.33–3.38 (q,
J = 7.2 Hz, 1H), 3.99–4.05 (q, J = 6.8 Hz, 1H), 4.70 (s, 1H), 7.49–
7.54 (d, J = 8.0, 2H), 8.19–8.23 (d, J = 8.0, 2H), 12.7 (s, 1H). 13C
NMR (D2O, 100.62 MHz): 37.9, 56.9, 124.6, 129.7, 140.9, 141.8,
To a solution of amino acid (50 mmol) in a 50 mL solution
(25 mL DMF and 25 mL H2O), 52 mmol of acetic anhydride and tri-
ethylamine (60 mmol, 6.06 g) were added. The solution was stirred
at room temperature overnight. After reaction, the solution was
evacuated at reduced pressure to remove DMF. The residue was
added to 30 mL of H2O, stirred at rt for 40 min, after which the
pH value of the solution was changed to 7.0 with a solution of so-
dium carbonate and extracted with 20 mL of ethyl acetate three
times. The combined organic solution was washed with saturated
brine and then dried over anhydrous magnesium sulfate. The or-
ganic solution was evacuated to remove the organic solvent, and
the residue was chromatographed to afford pure amino acid
amides.
174.7. ½a 2D0
¼ þ8:1 (c 1.08, methanol).
ꢂ
4.4.6. (S)-2-Amino-2-phenylacetic acid (Table 3, entry 1)
Obtained as a white solid, ee = 96%, mp: 294–297 °C, 1H NMR
(D2O, 400.13 MHz): 4.79 (s, 1H), 4.82 (s, 2H), 7.46–7.52 (m, 5H),
11.2 (s, 1H). 13C NMR (D2O, 100.62 MHz): 58.5, 127.9, 129.4,
129.6, 134.1, 173.2. ½a D20
¼ þ135 (c 0.95, hydrochloric acid).
ꢂ
4.4.7. (S)-2-Amino-2-(3-(trifluoromethyl)phenyl)acetic acid
(Table 3, entry 2)
Obtained as a white solid, ee = 95%, 1H NMR (D2O, 400.13 MHz):
4.76 (s, 1H), 5.03 (s, 2H), 7.33 (s, 1H) 7.44–7.59 (m, 3H), 11.7 (s,
1H). 13C NMR (D2O, 100.62 MHz): 61.5, 124.0, 128.1, 129.6,
4.4. General procedure for the KR of amino acid amides
Kinetic resolution of (RS)-2-acetamido-2-phenylacetic acid (Ta-
ble 2, entry 1). A 25 ml round-bottomed flask was charged with
131.3, 132.0, 133.7, 136.9, 172.4. ½a D20
¼ þ11:4 (c 1.05, water).
ꢂ
(RS)-2-acetamido-2-phenylacetic acid at
a
concentration of
4.4.8. (S)-2-([1,10-Biphenyl]-4-yl)-2-aminoacetic acid (Table 3,
entry 3)
50 mM and 5 mL of phosphorus buffer solution at pH 6.5. Next,
4.2 mg of free enzyme powder of lipase AS ‘Amano’ was added to
the mixture. When this was completed, the resulting mixture
was stirred at 35 °C for 24 h. Yields and ee values of the products
were determined by HPLC analysis. When the reaction was com-
pleted, the solution was filtered through a pad of cotton. The solu-
tion was changed to pH 7.0, extracted with ethyl acetate three
times, and the combined organic solution was dried over anhy-
drous magnesium sulfate and then evacuated to remove the organ-
ic solvent. The resulting residues were purified by column
chromatography on silica gel with petroleum ether/ethyl acetate
(1:10–1:5) to afford the pure amino acids.
Obtained as a white solid, ee = 99%, mp: 267–273 °C, 1H NMR
(D2O, 400.13 MHz): 4.80 (s, 1H), 5.09 (s, 2H), 7.30–7.55 (m, 9H),
9.3 (s, 1H). 13C NMR (D2O, 100.62 MHz): 58.5, 127.1, 127.4,
127.9, 128.3, 129.1, 129.4, 129.6, 134.1, 171.2. ½a D20
¼ þ6:9 (c
ꢂ
0.95, dichloromethane).
4.4.9. (S)-2-Aminopentanoic acid (Table 3, entry 4)
Obtained as a white solid, ee = 91%, mp: >300 °C, 1H NMR (D2O,
400.13 MHz): 0.91–0.95 (t, J = 8.0 Hz, 3H), 1.31–1.34 (m, 2H), 1.75–
1.78 (q, J = 6.8 Hz, 2H), 3.53–3.56 (t, J = 6.0, 1H), 4.96 (s, 2H), 8.4 (s,
1H). 13C NMR (D2O, 100.62 MHz): 12.9, 19.1, 32.4, 53.8, 173.9.
½
a 2D0
ꢂ
¼ þ4:6 (c 1.0, water).
4.4.1. (S)-2-Amino-3-phenylpropanoic acid (Table 2, entry 1)
Obtained as a white solid, ee = 97%, mp: 253–257 °C, 1H NMR
(D2O, 400.13 MHz): 3.12–3.18 (q, J = 6.0 Hz, 1H), 3.29–3.34 (q,
J = 6.0 Hz, 1H), 4.00–4.03 (q, J = 6.0 Hz, 1H), 4.79 (s, 1H), 7.35–
7.47 (m, 5H), 12.1 (s, 1H). 13C NMR (D2O, 100.62 MHz): 36.4,
Acknowledgments
Financial support from the Twelfth Five-year Plan for National
Science and Technology (Grant number 2011BAE06B04-07), the
Hainan University Youth Fund (Grant number, QNJJ1004), and
the Important Plan of Science and Technology in Haikou city (Grant
number, 2011-0104 and 2009043) is gratefully acknowledged.
56.1, 127.7, 129.1, 129.4, 135.1, 173.9. ½a D20
¼ ꢀ25:9 (c 1.0, water).
ꢂ
4.4.2. (S)-2-Amino-3-(4-chlorophenyl)propanoic acid (Table 2,
entry 2)
Obtained as a white solid, ee = 96%, mp: 259–264 °C, 1H NMR
(D2O, 400.13 MHz): 3.11–3.16 (q, J = 7.2 Hz, 1H), 3.31–3.35 (q,
J = 6.0 Hz, 1H), 4.01–4.03 (q, J = 6.0 Hz, 1H), 4.83 (s, 1H), 7.39–
7.53 (m, 4H), 12.6 (s, 1H). 13C NMR (D2O, 100.62 MHz): 36.4,
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