Efficient continuous kinetic resolution of racemic 2-aminobutanol over immobilized penicillin G acylase

Article abstract of DOI:10.1080/00397911.2016.1181763

In this paper, an efficient method was established for continuous kinetic resolution of racemic 2-aminobutanol by selective hydrolysis of N-phenylacetyl (±)-2-aminobutanol over immobilized penicillin G acylase (PGA) in a fixed-bed reactor. Several N-acylated derivatives of 2-aminobutanol were screened in batch experiments, and it was found that the hydrolysis of N-phenylacetyl (±)-2-aminobutanol proceeded smoothly in the presence of immobilized penicillin G acylase with satisfied enantioselectivity. Thus, the reaction parameters were optimized in a fixed-bed reactor. Under the optimized conditions, 39.3% conversion of N-phenylacetyl (±)-2-aminobutanol and 98.2% ee value of S-2-aminobutanol were obtained. This fixed-bed system was operated continuously for 40?h without significant decrease of enzyme activity. It has been demonstrated to be more efficient compared to the batch experiments.

Full text of DOI:10.1080/00397911.2016.1181763

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
Chemistry
ISSN: 0039-7911 (Print) 1532-2432 (Online) Journal homepage: http://www.tandfonline.com/loi/lsyc20
An Efficient Continuous Kinetic Resolution of
Racemic 2-aminobutanol over Immobilized
Penicillin G Acylase
Jianxin Wang, Na Liu, Xiaobo Cheng & Ligong Chen
To cite this article: Jianxin Wang, Na Liu, Xiaobo Cheng & Ligong Chen (2016): An Efficient
Continuous Kinetic Resolution of Racemic 2-aminobutanol over Immobilized Penicillin G
Acylase, Synthetic Communications, DOI: 10.1080/00397911.2016.1181763
To link to this article: http://dx.doi.org/10.1080/00397911.2016.1181763
View supplementary material
Accepted author version posted online: 28
Apr 2016.
Published online: 28 Apr 2016.
Submit your article to this journal
Article views: 6
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=lsyc20
Download by: [University of Florida]
Date: 31 May 2016, At: 18:06

An Efficient Continuous Kinetic Resolution of Racemic 2-aminobutanol over
Immobilized Penicillin G Acylase
1
,2
1
1
1,2
Jianxin Wang , Na Liu , Xiaobo Cheng , Ligong Chen
1
School of Chemical Engineering and Technology, Tianjin University, Tianjin, People's
Republic of China
2
Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin,
P. R. China
Abstract
In this paper, an efficient method was established for continuous kinetic resolution of
racemic 2-aminobutanol by selective hydrolysis of N-phenylacetyl (±)-2-aminobutanol
over immobilized penicillin G acylase (PGA) in a fixed-bed reactor. Several N-acylated
derivatives of 2-aminobutanol were screened in batch experiments, it was found that the
hydrolysis of N-phenylacetyl (±)-2-aminobutanol proceeded smoothly in the presence of
immobilized penicillin G acylase with satisfied enantioselectivity. Thus, the reaction
parameters were optimized in a fixed-bed reactor. Under the optimized conditions, 39.3%
conversion of N-phenylacetyl (±)-2-aminobutanol and 98.2% ee value of
S-2-aminobutanol were obtained. This fixed-bed system was operated continuously for 40
hours without significant decrease of enzyme activity. It has been demonstrated to be more
efficient compared to the batch experiments.
1

KEYWORDS: kinetic resolution; 2-aminobutanol; continuous; penicillin G acylase;
hydrolysis
INTRODUCTION
Optically pure amines are widely used as resolving reagents, chiral ligands and chiral
[
1,2]
synthetic building blocks for pharmaceutical and agrochemical industries . Since the
ever growing need of pharmaceutical and fine chemical industries for optically pure
amines, the approaches of preparing optically pure amines have been continuously
developed for the last two decades, including transition metal or organocatalyzed
2

[
3,4,5]
asymmetric synthesis of chiral amines
, chemo-enzymatic synthesis, chemical
[
6]
resolution through the formation of a diastereomeric salt , and enzymatic kinetic or
[
7,8,9]
dynamic resolution
. Among the above mentioned methods, enzymatic resolution has
been successfully applied in numerous stereoselective biotransformations by virtue of its
[
10,11,12]
excellent regio- and enantioselectivity
.
Recently, continuous-flow process represents an attractive trend applied in biotechnology
[
13]
and chemistry . Compared with batch process, it presents many advantages, such as
precise control over reaction conditions, including time, temperature and pressure;
improvement of productivity; safety; ease of product isolation; possibility of automation
[
14]
and ease of scale-up . Therefore, a few studies employed continuous-flow conditions to
[15,16,17]
realize the kinetic resolution of racemic compounds
.
S-2-aminobutanol is the key intermediate of ethambutol, which is an antibiotic for the
[7,18]
[19]
treatment of tuberculosis
, and also used as chiral auxiliaries . To the best of our
knowledge, few example of enzymatic resolution of racemic 2-aminobutanol under
continuous-flow conditions has been reported in the literatures. Thus, the continuous
kinetic resolution of racemic 2-aminobutanol catalyzed by penicillin G acylase from
Bacillus megaterium immobilized on polymethacrylate resin in a fixed-bed reactor is
summarized and reported here. Firstly, several N-acylated derivatives of racemic
2
-aminobutanol were synthesized by the nucleophilic substitution of carboxylic esters with
3

racemic 2-amino-butanol. Their hydrolysis behavior were then evaluated in the presence of
penicillin G acylase based on the principle of enantioselective hydrolysis of N-acylated
derivatives of racemic 2-aminobutanol over penicillin G acylase. Finally, it was found that
the S-enantiomer of N-acylated 2-aminobutaonl is selectively hydrolyzed to yield
S-2-aminobutanol over immobilized PGA.
RESULTS AND DISCUSSION
Initially, racemic 2-aminobutanol was acylated with several selected carboxylic esters (see
ESI † for further detail). Then, the hydrolysis performance of the obtained N-acylated
derivatives of 2-aminobutanol in the presence of immobilized PGA were examined and
screened in batch experiments, the obtained results were summarized in . It was found that
the hydrolysis of N-benzoyl-2-aminobutanol (3a) was not detected while the hydrolysis of
N-phenylacetyl-2-aminobutanols proceeded smoothly over the immobilized PGA，this is
possibly attributed to the conjugation of benzene ring and carbonyl group, decreasing the
activity of N-benzoyl-2-aminobutanol. So, N-benzoyl-2-aminobutanol can’t serve as the
resolution substrates of 2-aminobutanol.
As described in TableⅠ, S-2-aminobutanol was efficiently obtained in 99% ee at the 33.8%
conversion of 3b (entry 2), but with the increase of 3b conversion to 50.5%, the ee value of
S-2-aminobutanol dropped rapidly to 87.3%. It was found that excess hydrolysis of 3b
could lead to an obvious decrease of enantioselectivity. Then other derivatives were
4

examined (entry 3-4), it was found that the substituents at benzene ring in substrates also
had obvious influence on the reaction activity and enantioselectivity, both of 3c and 3d
displayed less reaction activity than 3b over immobilized PGA, but 3c presented better
enantioselectivity (E=96.6) than 3b (E=44.0) and 3d (E=17.2). However, 3c exhibits poor
solubility in water, so 3b was more suitable to be chosen as a resolution substrate for
S-2-aminobutanol.
In order to improve the productivity of S-2-aminobutanol and simplify the process, a
continuous system was established with fixed-bed reactor. Thus, 0.3g immobilized PGA
mixed with 3.0g silica sand was packed into a 2ml glass column, which was heated by a
water-bath, it was then fed with 0.1mol/L 3b in ammonia solution (pH=7.8). The effects of
temperature on the kinetic resolution of racemic 3b were examined in a range from 20℃ to
6
0℃ in the fixed-bed reactor at 0.04ml/min flow rate. The conversions of 3b at different
temperatures are shown in Figure 1. When the temperature increased from 20℃ to 40℃,
the conversion of 3b rapidly increased to 40%. With the temperature going on increasing,
the conversion of 3b didn’t increase obviously, mainly because the higher temperature can
destroy the enzymes. with consideration of the conversion of 3b and the lifetime of
immobilized PGA, 40℃ was chosen for the reaction temperature in further experiments.
Next, the effect of flow rate on the continuous kinetic resolution of racemic
2
-aminobutanol over immobilized PGAwas investigated and the results are shown in Table
5

Ⅱ. It was found that with the decrease in flow rate from 0.12ml/min to 0.02ml/min, the
conversion of 3b increased from 28.1% to 50.6%. The ee value of S-2-aminobutanol kept
more than 99% with the flow rate ranging from 0.12ml/min to 0.08ml/min, but it began to
decrease from 99% to 86.8%, when the flow rate went on decreasing to 0.02ml/min. There
is no doubt that the immobilized PGA could hydrolyze S-3b much faster than R-3b. But
when majority of S-3b was hydrolyzed, the concentration of S-3b decreased obviously, this
would give R-3b more chances to combine with the active site of the immobilized PGA,
then R-3b was hydrolyzed faster than before. Excess hydrolysis would decrease the ee
value of S-2-aminobutanol. So a suitable flow rate of 0.06ml/ml was chosen for the kinetic
resolution of racemic 2-aminobutanol in a fixed-bed reactor. Thus, under the optimized
resolution conditions, 39.3% conversion of 3b and 98.2% ee value of S-2-aminobutanol
were realized.
In addition, the service time of the immobilized PGA was also evaluated during the kinetic
resolution for S-2-aminobutanol using 3b as the substrate at 40 °C and 0.06ml/min flow
rate. As shown in Figure 2, the immobilized PGA catalytic activity didn’t decrease
obviously after 40 hours of operation. Samples were taken every 3 hours and analyzed by
HPLC.
Compared with the reported results, this continuous-flow catalytic system realized a faster
resolution of racemic 2-aminobutanol at the same conversion and better robustness. It is
6

mainly attributed to that the continuous-flow catalytic system is in favour of the
absorption- desorption balance of product on PGA. What is more, the continuous-flow
catalytic system can also avoid the mechanical damage of PGA caused by vigorously
stirring in batch experiments.
CONCLUSION
In summary, an efficient kinetic resolution of racemic 2-aminobutanol was successfully
realized by the selective hydrolysis of N-phenylacetyl-2-aminobutanol over PGA with
excellent enantioselectivity in a fixed-bed reactor. The resolution parameters, such as
temperature and flow rate, were optimized in a fixed-bed reactor. Under the optimized
conditions this continuous-flow procedure led to 39.3% conversion of
N-phenylacetyl-2-aminobutanol and 98.2% ee value of S-2-aminobutanol at 40℃ and
0
.06 mL/min, and no obvious decrease of immobilized PGA catalytic activity was detected
in 40h.
EXPERIMENTAL
Penicillin G acylase from Bacillus megaterium immobilized on polymethacrylate was
purchased from NovoCata (200U/g). all the other chemicals were purchased from Tianjin
Jiangtian Chemical Co., Ltd., Tianjin, China. NMR spectra were recorded on a Bruker 400
MHz spectrometer. Chiral HPLC was performed using a SP-086 Series HPLC (Baseline)
with a UV detector and Daicel Chiralpak AS-H column 5×250 mm.
7

Preparation Of N-Acylated Derivative Of Racemic 2-Aminobutanol -General Process
The carboxylic esters (0.08mol) were respectively heated with racemic 2-aminobutanol
(0.08 mol) in a 50ml round bottomed flask at 140°C. The reaction was followed by TLC.
After the reaction, the residue was then recrystallised from chloroform–hexane.
Batch Experiment – General Procedure
The racemic substrate 3b (0.08g, 0.4mmol) was dissolved in 4ml water by warming, the
pH was adjusted to 7.8 with ammonia and immobilized PGA (0.04g) was added. The
reaction mixture was stirred at 100 rpm with an overhead stirrer at 40°C. The conversion
during the hydrolysis was followed by HPLC.
Fixed-Bed Experiment- General Procedure
Immobilized PGA(0.3 g) mixed with silica sand(3.0g) was packed into a glass column with
a water-bath for maintaining temperature at 40 °C. it was then fed with 0.1mol/L 3b in
ammonia solution (pH=7.8) at various flow rates (0.12, 0.10, 0.08, 0.06, 0.04 and 0.02
mL/min),0.5mL of samples were collected for HPLC analysis.
N-Phenylacetyl-(±)-2-Aminobutanol (3b)
white powder; mp 62°C;IR (CHCl3): 3468, 1616 cm−1 ;1H NMR (CD3OD, 400MHz):
δ= 0.88 (t, 3H), 1.3–1.7 (m, 2H), 2.7 (br s,1H), 3.5–3.9 (m, 5H), 5.5 (br s, 1H), 7.4 (m,
8

5
H).13C NMR (CD3OD, 400 MHz):δ= 10.44, 23.61, 43.62, 57.05, 63.58,127.56, 129.14,
1
29.95, 135.60, 171.30. MS: m/z calcd for C12H17NO2+: 207.1320; found: 207.2715 [M
+
H] +.
ACKNOWLEDGMENT
We thank the institution Tianjin Engineering Research Center of Functional Fine
Chemicals and Tianjin Science and Technology Innovation Platform Program for their
supporting.
REFERENCE
[
[
[
[
[
[
[
[
[
[
[
1] Nugent, T. C.; El-Shazly, M. Adv. Synth. Catal. 2010, 352,753.
2] Verho, O.; Baeckvall, Jan-E. J. Am. Chem. Soc. 2015, 137, 3996.
3] Verkade, J. M. M.; Rutjes, P. F. P. Chem. Soc. Rev. 2008, 37, 29
4] Gautier, F.-M.; Jones, S.; Martin, S. J. Org. Biomol. Chem. 2011, 9, 7860.
5] Zhou, S.; Junge, F. K.; Beller, M. Angew. Chem. Int. Ed. 2011, 50, 5120.
6] Sakamoto, M.; Fujita, K.; Yagishita, F.; Chem. Commun. 2011, 47, 4267.
7] Fadnavis, N. W.; Sharfuddin, M. Tetrahedron: Asymmetry 1999, 10, 4495.
8] Shi, J.; Wang, Q.; Tang, Y. J. Catal. 2012, 291, 87.
9] Paivio, M.; Kanerva, L. T. Tetrahedron: Asymmetry 2012, 23, 230.
10] Munoz, L.; Guerrero, A. Org. Biomol. Chem. 2011, 9, 8171.
11] Ismail, H.; Sheldon, R. A. Adv. Synth. Catal. 2008, 350, 1511.
9

[
[
[
[
[
[
[
[
12] Ismail, H.; Lau, R. M.; Svedas, V. K. Green Chem. 2008, 10, 415.
13] Cancogni, D.; Lay, L. Synlett 2014, 25, 2873.
14] Junior, I. I.; Souza, R. O. M. A. J. Mol. Catal. B: Enzym. 2013, 85–86, 1.
15] Miranda, A. S.; Souza, R. O. M. A. Org. Biomol. Chem. 2013, 11, 3332.
16] Kim, Y.-J.; Choi, Y.-S.; Jeong, J.-H. Synlett 2015, 26, 1981.
17] Tomin, A.; Darvas, F.; Poppe, L. Process Biochem. 2010, 45, 859.
18] Zhang, Y. C.; Chen, L. C. J. Catal. 2005, 236, 176.
19] MacLaren, J. K.; Janiak, C. New. J. Chem. 2012, 36, 1596;
10

Table I. Screening of the N-acylated derivatives for the kinetic resolution for
a
S-2-aminobutanol
b
s
c
d
d
Entry
1
N-acylated
derivative
Time(h) ee (%) ee (%) conversion
E
p
(%)
-
20
-
-
-
2
3
6
50.6
89.0
99.0
87.3
33.8
50.5
>200
44.0
1
0
26
32
96.1
80.2
92.2
75.4
51.0
51.5
96.6
17.2
4
a
Reaction conditions: 0.4mmol substrate in 4ml of ammonia solution (pH=7.8) in the
presence of 0.04g immobilized PGA at 40℃ with 180 rpm.
11

b
The ee value of the substrate was determined by chiral HPLC on a chiralpak AS-H
column.
c
The ee value of the product was determined by chiral HPLC on a chiralpak AS-H column
after it was acylated by benzoyl chloride.
d
The conversion was calculated by C=ee /(ee +ee ), Enantiomeric ratios (E) were
s
s
p
calculated by E=ln[1-c(1+ee )]/ ln[1-c(1-ee )].
p
p
12

Table II. Effects of flow rate (residence time) on the kinetic resolution for
a
S-2-aminobutanol by immobilized PGA in a fixed-bed reactor
Entry Flow rate(residence
time)
ee (%)
ee (%)
conversion(%) E
s
p
1
2
3
4
5
0.12ml/min(6.7min)
0.10ml/min(8min)
0.08ml/min(10min)
38.7
48.9
57.4
>99
>99
>99
98.2
94.3
86.8
28.1
33.0
36.6
39.3
43.9
50.6
＞200
＞200
＞200
＞200
75.7
0.06ml/min(13.3min) 63.6
0.04ml/min(20min)
0.02ml/min(40min)
73.9
89
6
42.0
a
Reaction conditions: a fixed-bed reactor packed with 0.3g immobilized PGA(mixed with
3
.0g silica sand), 0.1mol/L 3b in ammonia solution(pH=7.8), 40℃.
b
The ee of the substrate was determined by chiral HPLC on a chiralpak AS-H column.
c
The ee of the product was determined by chiral HPLC after acylated by benzoyl
chloride.
d
The conversion was calculated by: C=ee /(ee +ee ) , Enantiomeric ratios (E) were
s
s
p
calculated by E=ln[1-c(1+ee )]/ ln[1-c(1-ee )].
p
p
13

Figure 1. Effects of temperature on the kinetic resolution for S-2-aminobutanol by
immobilized PGA in a fixed-bed reactor
14

Figure 2. Recyclability of the continuous-flow system
15