Alcaligenes faecalis penicillin G acylase-catalyzed enantioselective acylation of dl-phenylalanine and derivatives in aqueous medium

Article abstract of DOI:10.1016/j.tetlet.2011.08.056

A new strategy based on enantioselective acylation properties of relatively unknown penicillin G acylase from Alcaligenes faecalis has been developed for the production of pharmacologically interesting enantiomerically pure d-phenylalanine. In order to get high reaction rate and enantioselectivity, two key factors (pH and temperature) and eight different acyl donors were optimized, and the optimal acylation reaction was carried out at pH 10, 35 °C, using phenylacetamide as the acyl donor. This enantioselective acylating method is also illustrated by the effective production of five different p-substituted phenylalanine derivatives in enantiopure.

Full text of DOI:10.1016/j.tetlet.2011.08.056

Tetrahedron Letters 52 (2011) 5398–5402
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Alcaligenes faecalis penicillin G acylase-catalyzed enantioselective acylation
of DL-phenylalanine and derivatives in aqueous medium
⇑
⇑
Xiangyu Gong, Erzheng Su , Pixiang Wang, Dongzhi Wei
State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology,130 Meilong Road, Shanghai 200237, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A new strategy based on enantioselective acylation properties of relatively unknown penicillin G acylase
Received 8 April 2011
Revised 31 July 2011
Accepted 9 August 2011
Available online 16 August 2011
from Alcaligenes faecalis has been developed for the production of pharmacologically interesting enanti-
omerically pure D-phenylalanine. In order to get high reaction rate and enantioselectivity, two key factors
(pH and temperature) and eight different acyl donors were optimized, and the optimal acylation reaction
was carried out at pH 10, 35 °C, using phenylacetamide as the acyl donor. This enantioselective acylating
method is also illustrated by the effective production of five different p-substituted phenylalanine deriv-
atives in enantiopure.
Keywords:
Penicillin G acylase
Acylation
Ó 2011 Elsevier Ltd. All rights reserved.
DL-Phenylalanine
Aqueous medium
Enantiopure
D
-phenylalanine and its p-substituted derivatives
N-phenylacetyl-DL-amino acids in aqueous medium.14 Neverthe-
(Fig. 1) are important chiral building blocks for the preparation
less, additional chemical phenylacetyl-protection and deprotection
of some pharmaceuticals.
D
-phenylalanine is an intermediate in
steps are required if an D-amino acid is the desired product, which
make PGA mediated hydrolysis method tedious and inefficient
(Scheme 1). Cole showed that PGA can also be applied in the syn-
the synthesis of antiviral Fosamprenavir1 and Saquinavir,2 antineo-
plastic Clientide,3 antidiabetic Nateglinide4 and anticoagulant Mel-
agatran.5
D
-Tyrosine [4-hydroxy-
of the Tractocile, a potent abortion prevention agent.6 4-Methyl-
-phenylalanine is used in the synthesis of enkephalinol analogs.7
In addition, 4-fluoro- -phenylalanine, 4-chloro- -phenylalanine
and 4-nitro- -phenylalanine are precursors of antiemetic Aprepit-
D-phenylalanine] is a constituent
thetic direction and thus provided a facile route of
D-amino acids
production based on enzymatic acylation of
a-amino functionality
D
using phenylacetic acid analogs as acylating agents.15 PGA cata-
lyzed acylation reaction is always competed by hydrolytic reaction
in aqueous medium. The hydrolysis of acylating agents and pro-
D
D
D
ant, antineoplastic Abarelix and analgesic Zolmitriptan, respec-
duced N-phenylacetyl-
acylation to completion and thereby leads to relatively low enan-
tiomeric excess value of the remaining substrates -amino
L-amino acids brings the difficulty to drive
tively.8–10
In this regard, the development of methods to access enantio-
(D
pure
D
-phenylalanine and its p-substituted derivatives is of great
acids).16 Many strategies have been proposed to favor acylation
over hydrolysis. Using organic solvents as the reaction medium is
the most often and thoroughly studied method.17–20 However,
massive amount of enzyme is required to perform the acylation
and the reaction time tends to be long because of the low activity
of PGA in organic solvents. Besides, amino acids must be first
interest. Methods combined with biocatalytic technologies have
been described for this purpose. For instance, Isabel group and Tak-
ashi group had, respectively, reported synthesizing methods in bio-
catalytic ways, which were from the corresponding DL-amino acid
amides with L-amidase and from DL-amino acid esters using prote-
ase.11,12 However, additional chemical hydrolysis steps and neces-
sity of preparing DL-amino acid amides or esters before enzymatic
hydrolysis limit these processes in industrial application.
O
O
O
OH
OH
OH
OH
Penicillin G acylase (PGA) has traditionally been used as an
industrial catalyst for the production of semi-synthetic antibiot-
ics.13 Moreover, PGA is also used in the synthesis of enantiopure
NH2
NH2
NH2
HO
O
O
O
L-amino acids through enantioselective enzymatic hydrolysis of
OH
OH
NH2
NH2
NH2
⇑
Cl
O2Na
Corresponding authors. Tel.: +86 21 64251803; fax: +86 21 64250068 (E.S.);
tel.: +86 21 64252078; fax: +86 21 64250068 (D.W.).
F
E-mail addresses: ezhsu@ecust.edu.cn (E. Su), dzhwei@ecust.edu.cn (D. Wei).
Figure 1. D-Phenylalanine and some p-substituted derivatives.
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.08.056

X. Gong et al. / Tetrahedron Letters 52 (2011) 5398–5402
5399
NH
COOH
Cl
NaOH
+
O
COOH
NH2
O
PGA
COOH
NH
NH
OH
+
+
NH2
O
COOH
COOH
O
COOH
O
COOH
OH
6 M HCl
reflux
NH
+
NH2
O
O
Scheme 1. Production of enantiomerically pure
D
-phenylalanine by PGA hydrolysis.
transformed into esters to render them soluble in organic solvents.
So the use of aqueous medium is preferred option from the stand-
point of green chemistry. We had successfully developed an ap-
Table 1
Effect of pH and temperature on the A. faecalis PGA catalyzed acylation of DL-
phenylalanine
proach for the production of
D-b-phenylalanine in aqueous
Entry
pH
Temperature (°C)
ta (min)
eep (%)
ees (%)
cc (%)
b
b
medium based on enantioselective acylation property of PGA from
Escherichia coli, which proved that high enantiomeric excess value
of substrate and improved synthesis/hydrolysis ratio (s/h ratio)
could be achieved through optimization of acylation conditions
involving temperature and pH.21 Besides, we also found alkaline
medium like pH 10 was more suitable for acylation reaction cata-
1
2
3
4
5
6
7
8
9
8
9
30
30
30
30
20
25
30
35
40
280
240
200
300
240
240
200
220
300
>99
>99
>99
>99
>99
>99
>99
>99
>99
40
74
86
77
83
84
86
92
91
29
43
46
43
45
46
46
48
48
10
11
10
10
10
10
10
lyzed by PGA since ionization of a-amino functionality of b-phen-
ylalanine was reduced which made it a more efficient nucleophile
than water towards acyl-enzyme intermediate. And thus synthetic
efficiency was improved. So a PGA which is stable and active in alk-
ality is favorable. In respect of this, our attention was drawn to-
wards Alcaligenes faecalis PGA.
a
b
c
Reaction time at maximum DL-phenylalanine conversion.
Determined by chiral HPLC.
Deduced from the ee of the substrate (ees) and the product (eep): c = ees/
(ees + eep).
A. faecalis PGA is structurally similar to the well studied PGA
from E. coli.22 Compared with E. coli PGA, however, A. faecalis
PGA bears a broader pH optimum of 8–10 and maintains nearly
80% of its maximum catalytic activity at pH 11.23 Besides, A. faecalis
PGA displays higher thermostability (due to the presence of a
disulfide), higher catalytic activity and s/h ratio.24 All of these show
that the A. faecalis PGA may be an ideal biocatalyst for amino acids
acylation reaction. Dorel and co-workers have investigated the A.
faecalis PGA catalyzed enantioselective acylation of amines in
aqueous medium.23 To our knowledge, nevertheless, enantioselec-
tive acylation of amino acids catalyzed by A. faecalis PGA in aque-
ous medium has not been reported. Herein, we report a simple
methodology for obtaining enantiomerically pure D-phenylalanine
and derivatives, which is based on the principle of enantioselective
acylation of racemic amino acids with A. faecalis PGA immobilized
on oxirane acrylic beads (diam. 150–200 lm) in aqueous medium
(Scheme 2). In this work, the main influence parameters were opti-
mized and the acylating agents were screened.
studies were focused on the effects of pH and temperature on
the acylation of DL-phenylalanine, and the results are summarized
in Table 1. In all entries eep were above 99% and not affected by pH
or temperature, while ees was relatively lower. This is mainly be-
cause of the hydrolysis of phenylacetamide and produced N-phen-
ylacetyl-L
-phenylalanine.16 The maximum ees (92%) and substrate
conversion (48%) were attained at pH 10 and 35 °C. Relatively
higher pH and temperature could lead to higher conversion of sub-
strate and ees except for entries 4 and 9 because the stability of
immobilized PGA can be affected by harsh conditions (pH 11 and
40 °C). The results could be explained to a large extent on the basis
of diffusion limitation mechanism. As we known, in immobilized
preparations, diffusion limitation in the carrier tends to increase
hydrolysis due to depletion of the reactants and accumulation of
the products in the carrier beads.25 In this work, A. faecalis PGA
was immobilized on porous oxirane acrylic beads (diam. 150–
The reaction conditions were optimized using DL-phenylalanine
(1a) as substrate and phenylacetamide (2a) as acyl donor. Our
200
lm). Most of A. faecalis PGA molecules were immobilized in-
side the porous structure of the carrier beads. Reactants (phenyl-
COOH
NH
NH2
COOH
OH
PGA
+
+
+
NH2
O
COOH
O
NH2
O
Scheme 2. Production of enantiomerically pure
D-phenylalanine by PGA acylation.

5400
X. Gong et al. / Tetrahedron Letters 52 (2011) 5398–5402
Table 2
Effect of different acyl donors on the A. faecalis PGA catalyzed acylation of DL-phenylalanine
R2
R2
R2
COOH
NH
R3
COOH
OH
PGA
+
+
+
NH2
D-1a
eesb(%)
O
COOH
O
NH2
O
R1
R1
R1
1a
Acyl donor
2a-2h
ta (min)
eepb(%)
>99
cc (%)
(s/h)0
d
Entry
1
equiv
2.5
NH2
220
92
72
57
48
2.3
O
O
2a
OCH3
2
3
2.5
2.5
140
>99
>99
42
36
1.9
1.5
2b
NH2
1800
O
HO
HO
2c
OCH3
4
5
2.5
2.5
380
340
>99
>99
49
30
33
23
0.58
O
2d
OH
NH2
No hydrolysis
O
O
2e
OH
OCH3
6
7
8
2.5
2.5
2.5
320
340
300
>99
>99
>99
19
60
37
16
38
26
0.082
2f
NH2
NH2
No hydrolysis
O
2g
NH2
OCH3
0.13
O
2h
a
b
c
Reaction time at maximum DL-phenylalanine conversion.
Determined by chiral HPLC.
Deduced from the ee of the substrate (ees) and the product (eep): c = ees/(ees + eep).
d
Determined as a ratio of initial rates for accumulation of both reaction products.
acetamide and
structure, in which the reaction was taken place and the product
(N-phenylacetyl- -phenylalanine) was generated, thus concentra-
tion gradients of both reactants and product were produced and
consequently equilibrium was reversed to hydrolysis. Moreover,
L
-phenylalanine) must diffuse into the porous
and higher ees and conversion of substrate were achieved. Inter-
estingly, this explanation offered support for choosing A. faecalis
PGA, which characterized with the broader pH stability range
and higher thermostability, as the biocatalyst for the acylation
reaction.
L
hydrolysis of N-phenylacetyl-
amide generated phenylacetic acid which induced a pH gradient
in the carrier. Since the pH affected the reaction equilibrium as
L
-phenylalanine and phenylacet-
Acyl donor is another important factor which influences the
acylation reaction rate and enantioselectivity. In this work, 4 pairs
of phenylacetic derivatives (2a and 2b, 2c and 2d, 2e and 2f, 2g and
2h) were used as acyl donors in order to provide general guidelines
for the choice of proper acyl donors in enantioselective acylation in
aqueous media. Each pair included an amide and an ester which
bore the same phenylacetic structure and generated the same syn-
thetic and hydrolytic products in the reaction. For instance, the
synthetic and hydrolytic products of acyl donors phenylacetamide
well as the ionization of
a-amino functionality of phenylalanine,
the pH gradient amplified the general effects of the concentration
gradient. Higher pH and temperature increased the solubility of
phenylacetic acid and N-phenylacetyl-L-phenylalanine in aqueous
medium, and thus diminished their accumulation in the carrier
beads of the immobilized PGA. In that way, s/h ratio was improved

X. Gong et al. / Tetrahedron Letters 52 (2011) 5398–5402
5401
Table 3
A. faecalis PGA catalyzed acylation of phenylalanine derivatives
R
NH2
COOH
COOH
OH
PGA
+
+
+
NH
O
NH2
NH2
O
R
R
O
L-(3a-3f)
Product [
COOH
D-(1a-1f)
1a-1f
ta (min)
Entry
R
cb (%)
(s/h)0
L-(3a–f)]
Substrate [
Yieldd (%)
68
D
-(1a–f)]
[D
-(1a–f)] eef (%)
c
Yieldd (%)
eee (%)
eee (%)
1
2
3
4
5
6
H
220
240
220
220
180
340
48
47
48
49
49
49
2.3
1.6
1.8
2.9
2.6
2.7
90
92
91
85
88
95
>99
>99
99
99
99
92
88
91
94
94
94
98
92
95
>99
99
>99
OH
CH3
Cl
F
NO2
65
64
73
69
69
>99
a
b
c
d
e
f
Reaction time at maximum DL-phenylalanine conversion.
Deduced from the ee of the substrate (ees) and the product (eep): c = ees/(ees + eep).
Determined as a ratio of initial rates for accumulation of both reaction products.
Yields were calculated taking into account the percentage of conversion c.
Determined by chiral HPLC.
After recrystallization from water/ethanol.
(2a) and methyl phenylacetate (2b) were N-phenylacetyl-
L
-phen-
acylation of DL-phenylalanine in aqueous medium despite of their
remarkable s/h ratio.
Enantioselective acylation of various racemic p-substituted
phenylalanines was also investigated (Table 3). A. faecalis PGA
ylalanine and phenylacetic acid, respectively. Under the optimized
pH and temperature, phenylacetamide gave a conversion and ees of
48.7% and 91.6%, respectively (Table 2, entry 1), both of which were
the highest among all the acyl donors. Thus resulted the best acyl
donor for acylation in aqueous medium, followed by methyl phen-
ylacetate (Table 2, entry 2). Alessandra and co-workers reported
that the 4-hydroxyphenylacetic group was more preferentially ac-
cepted than phenylacetic group by PGA from E. coli when acylated
selectively acylated
their corresponding racemates in high ee (>99%), leaving the
remaining -p-substituted phenylalanines in moderate to high ee
L-isomer of p-substituted phenylalanines from
D
(88–94%) (Table 3, entries 1–5). Slight decrease in both conversion
and ees were observed when phenylalanine was substituted by
electron-donating groups at the p-position of benzene ring (Table
3, entries 1 and 2), which was due to the fact that N-phenylace-
L
-tyrosine ethyl ester in toluene.19 However, the result of this arti-
cle was otherwise. Both 4-hydroxyphenylacetamide (2c) and
methyl 4-hydroxyphenylacetate (2d) gave lower ees, conversion
and s/h ratio even with longer reaction time than phenylacetamide
and methyl phenylacetate (Table 2, entries 3 and 4). That maybe
because of the lack of a Ser residue at the bottom of acyl-binding
pocket rendered A. faecalis PGA less specific to 4-hydroxyphenyl-
acetic acid derivatives than E. coli PGA.24 The presence of a hydro-
tyl-4-methyl-
L
-phenylalanine and N-phenylacetyl-
L-tyrosine hy-
drated faster than N-phenylacetyl-
L
-phenylalanine. This could be
testified by the lower s/h ratio compared with DL-phenylalaine.
On the other hand, both conversion and ees were improved when
electron-withdrawing groups substituted at the p-position of ben-
zene ring (Table 3, entries 3–5). It should be noted that these enan-
tiomeric excess values could be improved after recrystallization
from water/ethanol. Thus, as shown in the last column of Table
xyl or amino group at
a position of phenylacetamide and methyl
phenylacetate (2e–h) caused an appreciable reduction of the reac-
tion rate and enantioselectivity (Table 2, entries 5–8). Similar re-
sults were also reported in Alessandra and co-workers’ work, in
which they indicated that this lack of activity might be ascribed
to unfavorable interactions between the polar amino group of
phenyglycine and aromatic side-chains linking the PGA hydropho-
bic pocket hosting the phenylacetic moiety.19 Another reason for
ineffective acylation of DL-phenylalanine when using (S)-methyl
mandelate (2f) and (S)-methyl phenylglycine (2h) as acyl donors
was the fast hydrolysis of acyl donors, involving self-hydrolysis
in alkaline condition and hydrolysis catalyzed by PGA. Interest-
ingly, when (S)-methyl mandelate (2f) and (S)-methyl phenylgly-
cine (2h) were replaced by their corresponding amides (2e and
2g), no hydrolysis products (namely (S)-mandelic acid and (S)-
phenyglycine) were detected, which meant neither acyl donors
nor acylation products were hydrolyzed by PGA in such conditions.
However, both 2e and 2g needed quite a long time to reach the
maximum conversion. Furthermore, the same as (S)-methyl phen-
ylglycine, (S)-phenylglycinamide can also be accepted as nucleo-
phile by PGA in aqueous medium thereby self-acylation was
observed when using them as acyl donors. So neither (S)-mandel-
amide nor (S)-phenylglycinamide was an ideal acyl donor for
3, D-amino acids were finally obtained with ee >91%, 4-chloro-D-
phenylalanine and 4-nitro-D-phenylalanine were obtained in
enantiopure.
In conclusion, we have developed a facile and efficient A. faecalis
PGA catalyzed enantioselective acylation method in aqueous med-
ium for obtaining pharmacologically interesting D-phenylalanine
and its p-substituted derivatives. This process has potential as an
alternative to other method in industrial applications.
Acknowledgments
This work was supported by the Fundamental Research Funds
for the Central Universities, the National Basic Research Program
(973 Program) of China (No. 2007CB714300) and the Specialized
Research Fund for the Doctoral Program (New Teachers) of Higher
Education (No. 20090074120015).
.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2011.08.056.

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X. Gong et al. / Tetrahedron Letters 52 (2011) 5398–5402
15. Cole, M. Biochem. J. 1969, 115, 747–756.
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