Enantioselective ammonolysis of phenylglycine methyl ester with lipase-pluronic nanoconjugate in tertiary butanol

Article abstract of DOI:10.1007/s10562-014-1289-2

Asymmetrical ammonolysis of (R)- and (S)-phenylglycine methyl ester was carried out by using a lipase (CALB)-polymer (Pluronic) nanoconjugate as the catalyst, displaying a 11-fold increased catalytic rate compared to the free CALB in tertiary butanol. Graphical Abstract: The asymmetrical ammonolysis of (R)- and (S)-phenylglycine methyl ester was accomplished using a lipase-Pluronic nanoconjugate, displaying a 11-fold higher catalytic rate compared to the free lipase.[Figure not available: see fulltext.]

Full text of DOI:10.1007/s10562-014-1289-2

Catal Lett
DOI 10.1007/s10562-014-1289-2
Enantioselective Ammonolysis of Phenylglycine Methyl Ester
with Lipase–Pluronic Nanoconjugate in Tertiary Butanol
•
Xiaoling Wu Rui Wang Yifei Zhang
•
•
•
Jun Ge Zheng Liu
Received: 6 May 2014 / Accepted: 24 May 2014
Ó Springer Science+Business Media New York 2014
Abstract Asymmetrical ammonolysis of (R)- and (S)-
phenylglycine methyl ester was carried out by using a
lipase (CALB)–polymer (Pluronic) nanoconjugate as the
catalyst, displaying a 11-fold increased catalytic rate
compared to the free CALB in tertiary butanol.
apparent activity of enzyme in organic solvents, being one
to several orders of magnitude lower than that in aqueous
solution [6]. One major reason underpinned the low
apparent enzymatic activity is the poor solubility of
enzyme in the organic solvents, which leads to the aggre-
gation of enzyme into a rigid form that not only hinders the
conformational transition requested for the catalysis but
also reduces the accessibility to its substrate. On the other
hand, the ammonium salt as ammonia donor in the enzy-
matic ammonolysis reaction has very limited solubility in
organic solvents, which further hinders the contact and
subsequent uptake by the enzyme aggregates.
Keywords Enzymatic catalysis Á Nanoparticles Á
Enzymes
1 Introduction
R-Phenylglycine and its derivatives are key intermediates
in the synthesis of penicillin and cephalosporin antibiotic
[1, 2], which are produced by using the classical resolution
via diastereomeric salt crystallization and the enantiose-
lective hydrolysis of corresponding hydantion [3]. The
unsatisfactory selectivity and yield of above procedures
result in the implementation of laborious separation and
purification steps and hence the excess mass and energy
consumption and waste generation. While the enzymatic
ammonolysis is reported for the green synthesis of chiral
synthons [4, 5], the enzymatic ammonolysis of racemic
phenylglycine methyl esters for the production of R-
phenylglycine amide is challenged by the extremely low
Nanostructured enzyme catalysts [7–10] such as enzyme
nanoparticles [11, 12], enzyme nanogels [13–17], and
flower-like enzyme-inorganic hybrid crystals [18–22] have
shown novel ways to enhance enzymatic reactions in both
aqueous and non-aqueous media. For example, by conju-
gation with an amphiphilic polymer such as Pluronic, the
solubilization of enzyme catalysts in organic solvents is
realized. This facilitates the access of the substrate to the
active site of enzyme and elevates the apparent enzymatic
activity by several hundred folds compared to its native
counterpart [23]. This thus encourages us to explore the
potential of lipase–Pluronic conjugate for the enantiose-
lective ammonolysis of phenylglycine methyl ester in
organic media (Scheme 1).
Electronic supplementary material The online version of this
article (doi:10.1007/s10562-014-1289-2) contains supplementary
material, which is available to authorized users.
2 Experimental
X. Wu Á R. Wang Á Y. Zhang Á J. Ge (&) Á Z. Liu (&)
Department of Chemical Engineering, Tsinghua University,
Beijing, China
The Candida antarctica lipase B (CALB)–Pluronic con-
jugate was synthesized according to Ref. [23]. The
hydroxyl group of Pluronic F-127 was first oxidized to
aldehyde group by using Dess–Martin periodinane as the
oxidizing reagent. The obtained aldehyde-functionalized
e-mail: junge@mail.tsinghua.edu.cn
Z. Liu
e-mail: liuzheng@mail.tsinghua.edu.cn
123

X. Wu et al.
NH2
COOCH3
NH2COONH4
CALB-Pluronic conjugate
racemic phenylglycine methyl ester
NH2
NH2
CONH2
H
COOCH3
H
R-phenylglycine amide
S-phenylglycine methyl ester
Scheme 1 Ammonolysis of phenylglycine methyl ester in organic
media catalyzed by lipase–Pluronic nanoconjugate
Pluronic was dissolved in phosphate buffer (10 mM, pH
7.0) and mixed with aqueous solution of CALB (5–10 mg/
mL) (the molar ratio of the aldehyde group to the amine
group of protein is 1.1:1). After 2 h reaction, NaCNBH3
was added to the mixture to reduce the Schiff base, fol-
lowed by dialysis in phosphate buffer solution (10 mM, pH
7.0) to remove unreacted reagents. Finally, the powder of
CALB–Pluronic conjugate was obtained by lyophilization.
For transmission electron microscopy (TEM) measure-
ments, the dry powder of the lipase–Pluronic conjugate
obtained by lyophilization was dissolved in toluene (10 lg/
mL of protein concentration). Ten microlitre of this solu-
tion was placed on the carbon-coated grid. After toluene
evaporated, ten lL of sodium phosphotungstate aqueous
solution (1 %, pH 7.0) was applied to stain the sample. The
excess of the liquid was removed after 2 min and the
sample was dried for 24 h at room temperature before
making TEM measurements.
Fig. 1 a The activities of CALB–Pluronic conjugate compared to
native lipase in aqueous and organic media; b TEM image of the
CALB–Pluronic conjugate
conjugated protein, which resulted in the formation of the
self-assembled conjugates with sizes from 10 to 50 nm.
The catalytic performance of the CALB–Pluronic na-
noconjugate in the ammonolysis of phenylglycine methyl
ester was investigated in comparison with free CALB. In a
typical experiment, 66 mg of the racemic phenylglycine
methyl ester and 125 mg of ammonium carbamate as the
ammonia donor [2, 24] were added in 2 mL of tertiary
butanol (TBA). Then, 20 mg of the CALB–Pluronic na-
noconjugate (or free CALB containing the equivalent
amount of protein) was added to the mixture followed by
gentle shanking at 150 rpm, 30 °C. At intervals, samples
were taken for HPLC analysis to determine the conversion
of the substrate and the enantiomer excess (ee) value for
the enzymatic reaction.
3 Results and Discussion
The relative hydrolytic activity of the CALB–Pluronic
conjugate determined with 4-nitrophenyl butyrate (p-NPB)
as the substrate was *97 % with reference to free CALB.
In toluene, using hexanoic acid and n-butyl alcohol as the
substrates, the apparent esterification activity of the con-
jugate in toluene was increased by *67-fold in compari-
son to free CALB (Fig. 1a) at the same protein amount.
TEM image of the CALB–Pluronic conjugate showed that
it is presented in the form of nanospheres with diameters
from 10–50 nm (Fig. 1b). It is likely that the hydrophobic
PPO block of Pluronic extended into the organic phase, and
meanwhile the hydrophilic PEO blocks stayed around the
As shown in Fig. 2, at the same protein amount, the
reaction catalyzed by CALB–Pluronic nanoconjugate at
30 °C reached a conversion of *60 % in 30 h while that
catalyzed by free CALB below 20 %. Calculated from the
123

Enzyme Nanobiocatalyst
Table 1 Comparison of the conversion and selectivity of CALB–
Pluronic conjugate with Novo 435 in different solvents
Solvent
Novo 435a
CALB–Pluronica
MTBE
Conversion (%)
ee (%)
53.7
54.9
53.9
50.7
TBA
Conversion (%)
ee (%)
41.8
42.9
62.9
64.8
a
The reaction condition 20 mg Novo 435 (or CALB–Pluronic con-
taining same amount of protein), 2 mL MTBE (or TBA), 66 mg
racemic phenylglycine methyl ester, 125 mg ammonium carbamate,
30 °C, 200 rpm
Fig. 2 Conversions of phenylglycine methyl ester in the ammonol-
ysis reaction in TBA catalyzed by CALB–Pluronic conjugate and free
CALB
To optimize the reaction catalyzed by CALB–Pluronic
nanoconjugate, the effects of the reaction parameters such
as the conjugate loading and temperature on the conversion
were examined.
As shown in Fig. 3a, the increase in the conjugate
concentration from 5 to 10 mg/mL elevated the conversion
from *10 to 28 % in 4 h. Further increase in the con-
centration contributed less to the conversion. Furthermore,
as shown in Fig. 3b, at which the conjugate concentration
was 10 mg/mL, the elevation of reaction temperature from
20 to 30 °C accelerated the reaction significantly while
further elevation to 40 °C contributed to a marginal
increase in the reaction rate. With the above mentioned
efforts, the optimal temperature and conjugate concentra-
tion was determined as 30 °C and 10 mg/mL, respectively.
Under this condition, the final conversion of the substrate
reached *60 % in 30 h. For the purpose of comparison,
the above reaction was also conducted using a commercial
immobilized CALB (Novo 435), which has excellent
activity and stability in organic media [25–27]. As shown
in Fig. 3b, the CALB–Pluronic nanoconjugate showed
similar catalytic activity as Novo 435.
The enantio-selectivity of CALB–Pluronic nanoconju-
gate in the ammonolysis of racemic phenylglycine methyl
ester in organic solvents was examined in comparison with
Novo 435 as well (Table 1). In TBA, the ee value of the
reaction catalyzed by the conjugate reached 64.8 % at a
final conversion of 62.9 %, which is higher than that with
Novo 435 (42.9 %). In principle, the theoretically highest
conversion of the substrate in an enatio-ammonolysis of
racemic substrate is 50 % at a 100 % selectivity. The
conversions above 50 % (Table 1) observed in both Novo
435 and CALB–Pluronic conjugate, indicated the non-
perfect selectivity of the conjugate and Novo 435, which
led to the ammonolysis of S-type substrate and thus gave
conversions over 50 %. In addition to TBA, methyl tertiary
butyl ether (MTBE) was also examined as the solvent for
the lipase-catalyzed ammonolysis reaction. In MTBE,
Fig. 3 a Conversions of the substrate catalyzed by the conjugate with
different concentrations, 4 h, 30 °C, in TBA. b Conversions of the
substrate catalyzed by the conjugate at different temperatures
initial reaction rate, the apparent activity of the conjugate
was about 11-fold higher than that of the free CALB in this
specific reaction.
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X. Wu et al.
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CALB–Pluronic nanoconjugate performed similarly as
Novo 435 in terms of both conversion and selectivity
(Table 1).
6. Klibanov AM (1997) Trends Biotechnol 15:97–101
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55:1070–1080
4 Conclusion
9. Kim J, Grate JW, Wang P (2006) Chem Eng Sci 61:1017–1026
10. Wang R, Zhang Y, Lu D, Ge J, Liu Z, Zare RN (2013) Wires
Nanomed Nanobiol 5:320–328
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In summary, we prepared a CALB–Pluronic nanoconjugate
for the ammonolysis of phenylglycine methyl ester in
organic media. For this reaction, the CALB–Pluronic
conjugate displayed a 11-fold higher apparent activity
compared to the free CALB in TBA. At an optimized
condition, the conversion and ee value of the reaction
catalyzed by the conjugate reached 62.9 and 64.8 %,
respectively. The high activity of CALB–Pluronic conju-
gate demonstrated by this study indicates its potential
applications in the lipase-catalyzed ammonlysis reactions
for the production of chiral amides which are important
intermediates for fine chemical and pharmaceutical
industry.
13. Ge J, Lu D, Wang J, Liu
10:1612–1618
Z (2009) Biomacromolecules
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112:14319–14324
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Chem 15:1155–1158
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Acknowledgment This work was supported by the National Natural
Science Foundation of China under the Grant Number of 21206082
and 21036003.
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