New view of acylase promiscuity: An extended study on the acylase-catalyzed Markovnikov addition

Article abstract of DOI:10.1016/j.molcatb.2011.08.002

Acylases have shown several promiscuities towards non-natural substrates. For example, acylases have been proved to be able to catalyze Markovnikov addition of N-heterocycles to vinyl esters recently. Some interesting and unexplainable observations drew our attention, and the acylase-catalyzed Markovnikov addition was investigated further in this paper. We detected an acylation product in the reaction and found that acetaldehyde was able to improve the reaction rate. Moreover, Markovnikov adduct could be formed using isopropenyl acetate or 2,2,2-trifluoro-ethyl acetate as substrates in the presence of acetaldehyde. Based on these results, it was proposed that the so-called acylase-catalyzed Markovnikov addition actually consisted of three steps identified as acylation, hemiaminal intermediate formation and transesterification. This discovery revealed that the promiscuity of acylase for Markovnikov addition was pseudo-promiscuity.

Full text of DOI:10.1016/j.molcatb.2011.08.002

Journal of Molecular Catalysis B: Enzymatic 73 (2011) 85–89
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
New view of acylase promiscuity: An extended study on the acylase-catalyzed
Markovnikov addition
Bo-Kai Liua,b, Qi Wua, De-Shui Lva, Xin-Zhi Chenb, Xian-Fu Lina,∗
a Department of Chemistry, Zhejiang University, Hangzhou 310027, PR China
b College of Material Chemistry and Chemical Engineering, Zhejiang University, Hangzhou, 310027, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 March 2011
Received in revised form 8 August 2011
Accepted 8 August 2011
Available online 12 August 2011
Acylases have shown several promiscuities towards non-natural substrates. For example, acylases have
been proved to be able to catalyze Markovnikov addition of N-heterocycles to vinyl esters recently. Some
interesting and unexplainable observations drew our attention, and the acylase-catalyzed Markovnikov
addition was investigated further in this paper. We detected an acylation product in the reaction and
found that acetaldehyde was able to improve the reaction rate. Moreover, Markovnikov adduct could
be formed using isopropenyl acetate or 2,2,2-trifluoro-ethyl acetate as substrates in the presence of
acetaldehyde. Based on these results, it was proposed that the so-called acylase-catalyzed Markovnikov
addition actually consisted of three steps identified as acylation, hemiaminal intermediate formation and
transesterification. This discovery revealed that the promiscuity of acylase for Markovnikov addition was
pseudo-promiscuity.
Keywords:
Acylase
Enzyme catalysis
Markovnikov addition
Promiscuity
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
although the reactivity of vinyl ether is higher than that of vinyl
ester. This unexpected result was hardly explained, which attracted
Enzymatic promiscuities in organic media cause enormous
interest, since the new catalytic activities of enzymes with non-
natural substrates provide useful methods in organic synthesis
[1,2]. The development of catalytic promiscuities expands the
application of enzymes in organic chemistry largely [3–7]. Inves-
tigation of enzymatic promiscuities offers several novel avenues
to understand enzymes. Hydrolases have been studied widely and
shown diverse functions [8–11]. Berglund revealed the mecha-
nism of CAL-B-catalyzed Michael addition and Aldol reaction by
calculation. And the catalytic activities of CAL-B towards these
two reactions were improved by mutation [12–15]. Acylases have
also been recognized as very promising biocatalysts. They could
promote Michael addition, which has been applied for the synthe-
sis of a series of N-heterocycles derivatives successfully [16–18].
Some studies demonstrated that acylases could also catalyze the
Markovnikov addition between N-heterocycles and vinyl esters
[19–21]. In the preliminary work, several control experiments by
using bovine serum albumin or deactivated acylases as catalysts
were carried out to prove that the active site of acylases was respon-
sible for the Markovnikov addition. Acylases could not catalyze
Markovnikov addition between N-heterocycles and vinyl ether,
our interest. Thus, some work needs to be done to go deep into this
reaction. Here, we explored the enzymatic process and reported
the new discovery of the acylase-catalyzed Markovnikov addi-
tion.
2. Materials and methods
2.1. Materials
d-Aminoacylase from Escherichia coli and lipase PS Amano were
purchased from Amano Enzyme Inc. Lipase immobilized on acrylic
resin from Candida antarctica was purchased from Sigma. Solvents
˚
were dried over 3 A molecular sieves for 24 h prior to use. All other
chemicals were of the highest purity commercially available.
2.2. Analytical methods
1H NMR spectra were recorded on a Bruker AMX-400 MHz
spectrometer, using CDCl3 as a solvent and TMS as internal refer-
ence. Analytical HPLC was performed using a SHIMADZU LC-10AVP
HPLC with a UV detector. IR spectra were measured with a Nico-
let Nexus FTIR 670 spectrophotometer. Mass spectra were obtained
by electrospray ionization (ESI) on a Bruker esquire 3000 plus mass
spectrometer. GC–MS was obtained by Agilent 5973.
∗
Corresponding author. Tel.: +86 571 87953001; fax: +86 571 87952618.
E-mail address: llc123@zju.edu.cn (X.-F. Lin).
1381-1177/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2011.08.002

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B.-K. Liu et al. / Journal of Molecular Catalysis B: Enzymatic 73 (2011) 85–89
2.3. General procedure
A mixture of dideuterated imidazole (1 mmol) and vinyl acetate
(4 mmol) was dissolved in 2 mL DMSO. The reaction was incubated
at 50 ◦C and 200 rpm and catalyzed by 25 mg d-amino acylase. After
96 h, the enzyme was filtered off to terminate the reaction. The
crude residue was purified by flash column chromatography on
silica gel using petroleum/ethyl acetate.
A mixture of 4-nitro-imidazole (1 mmol) and vinyl acetate
(3 mmol) was dissolved in 2 mL DMSO. Different amount of
acetaldehyde or water ranging from 0 to 100% (refer to the molar of
4-nitro-imidazole) was added to the reaction mixture. The reaction
was catalyzed by 25 mg d-amino acylase and incubated at 50 ◦C and
200 rpm. Eight samples of 20 ␮L were taken from the reaction mix-
ture after fixed intervals of time. Then samples were diluted to 1 mL
with methanol and analyzed by GC–MS or HPLC. The initial reaction
rate was calculated from the time curve plot of the concentration of
product versus time. The apparent turnover numbers (kcaaptp) were
calculated by dividing the number of millimoles of product by the
reaction time and number of millimoles of enzyme at low conver-
sion. The turnover numbers were apparent since they were only
measured at one concentration (500 mM).
1580
1600
1620
1640
1660
1680
1700
Fig. 1. Comparison of the second derivative spectra of d-amino acylase in DMSO
(line) and in H2O (dot).
78.8, 20.7; IR (neat, cm−1): 3583, 3136, 1761, 1547, 1495, 1348,
1278, 1213, 1025, 827, 735.
A
mixture of 4-nitro-imidazole (1 mmol), acetaldehyde
2.4.5. Acetic acid
(3 mmol) and isopropenyl acetate or 2,2,2-trifluoro-ethyl acetate
(3 mmol) was dissolved in 2 mL DMSO. The reaction was catalyzed
by 25 mg d-amino acylase, and incubated at 50 ◦C and 200 rpm.
After 96 h, samples were taken for GC–MS or ESI or HPLC detection.
(4-phenyl-phenyl)-(4-nitro-imidazol-1-yl)-methyl ester (1e)
Yellow oil; 1H NMR (CDCl3, 400 MHz, ı, ppm): 7.82 (s, 1H),
7.72–7.39 (m, 11H), 2.27 (s, 3H); 13C NMR (CDCl3, 100 MHz, ı, ppm):
169.1, 148.2, 143.6, 139.5, 135.9, 131.5, 128.9, 128.1, 127.1, 126.7,
118.2, 78.8, 20.7; IR (neat, cm−1): 3575, 1758, 1547, 1485, 1350,
1213, 1012, 824, 731.
A
mixture of imidazole derivatives (1 mmol), isopropenyl
acetate (2 mmol) and aldehyde (2 mmol) was dissolved in 1 mL
organic solvent. The reaction was initiated by adding 20 mg lipases
and was incubated at 50 ◦C and 200 rpm. After 72 h, the enzyme
was filtered off to terminate the reaction. The crude residue
was purified by flash column chromatography on silica gel using
petroleum/ethyl acetate mixtures.
2.4.6. 1-(1-Imidazole)-propyl acetate (1f)
Yellow oil; 1H NMR (CDCl3, 400 MHz, ı, ppm): 7.73 (s, 1H,
N–CH N), 7.08 (s, 2H, N–CH CH–N), 6.43 (t, 1H, J = 7.6 Hz,
N–CH–O), 2.16–2.03 (m, 5H, CH3CO, CH2), 0.92 (t, 3H, J = 7.6 Hz,
CH3); 13C NMR (CDCl3, 100 MHz, ı, ppm): 169.5, 136.7, 129.5, 116.7,
79.7, 27.2, 20.7, 9.0; IR (neat, cm−1): 2977, 1749, 1495, 1216, 1046,
828, 740.
2.4. Characterization
2.4.1. Acetic acid 1-(4-nitro-imidazol-1-yl)-ethyl ester (1a)
White solid; 1H NMR (DMSO-d6, 400 MHz, ı, ppm): 8.68
(s, 1H, N CH–N), 8.11 (s, 1H, N–CH C), 6.77 (q, 1H, J = 6.2 Hz,
N–CH–O–C O), 2.05 (s, 3H, O C–CH3), 1.78 (d, 3H, J = 6.20 Hz,
–CH–CH3). 13C NMR (DMSO-d6, 125 MHz, ı, ppm): 169.8, 147.8,
137.2, 120.1, 77.3, 21.1, 20.2; IR (KBr, cm−1): 3445, 1748, 1511,
1213.
2.4.7. 1-(1-(2-Methyl-imidazole))-propyl acetate (1g)
Yellow oil; 1H NMR (CDCl3, 400 MHz, ı, ppm): 6.94 (s, 2H,
N–CH N, N–CH C), 6.37 (t, 1H, J = 7.6 Hz, N–CH–O), 2.47 (s, 3H,
CH3), 2.08–1.93 (m, 5H, CH3CO, CH2), 0.85 (t, 3H, J = 7.6 Hz, CH3);
13C NMR (CDCl3, 100 MHz, ı, ppm): 169.4, 145.2, 128.0, 152.3, 78.8,
28.0, 20.7, 13.1, 8.9; IR (neat, cm−1): 2969, 1755, 1490, 1218, 1044,
831, 736.
2.4.2. Acetic acid 4-nitro-imidazol-1-yl-methyl ester (1b)
White powder; 1H NMR (CDCl3, 400 MHz, ı, ppm): 8.47 (s, 1H),
8.01 (s, 1H), 5.99 (s, 2H), 2.07 (s, 3H); 13C NMR (CDCl3, 100 MHz, ı,
ppm): 170.2, 147.5, 138.6, 122.2, 69.0, 20.8; IR (neat, cm−1): 3448,
3084, 1753, 1543, 1491, 1349, 1217, 823.
2.4.8. 1-(1-(4-Methyl-imidazole))-propyl acetate (1h)
Yellow oil; 1H NMR (CDCl3, 400 MHz, ı, ppm): 7.58 (s, 1H,
N–CH N), 6.73 (s, 1H, N–CH C), 6.29 (t, 1H, J = 7.6 Hz, N–CH–O),
2.15 (s, 3H, CH3), 2.08–1.93 (m, 5H, CH3CO, CH2), 0.86 (t, 3H,
J = 7.6 Hz, CH3); 13C NMR (CDCl3, 100 MHz, ı, ppm): 169.5, 138.4,
136.1, 113.0, 79.7, 27.1, 20.7, 13.3, 9.0; IR (neat, cm−1): 2975, 1753,
1492, 1212, 1039, 822, 743.
2.4.3. Acetic acid 1-(4-nitro-imidazol-1-yl)-propyl ester (1c)
Yellow oil; 1H NMR (CDCl3, 400 MHz, ı, ppm): 1.92 (d, 1H,
J = 1.2 Hz, N CH–N), 7.68 (s, 1H, J = 1.2 Hz, N–CH C), 6.42 (t, 1H,
J = 7.6 Hz, N–CH–O–C O), 2.12–2.04 (m, 5H, –CH2–CH3, O C–CH3),
0.99 (t, 3H, J = 7.6 Hz). 13C NMR (CDCl3, 100 MHz, ı, ppm): 169.4,
148.4, 135.4, 117.0, 80.3, 27.3, 20.6, 9.0; IR (KBr, cm−1): 3442, 1745,
1510, 1209, 825.
3. Results and discussion
3.1. Characterization of d-amino acylase
Second derivative IR spectra were used as a particularly sensitive
probe of protein conformation [22,23]. The IR spectrum of d-amino
acylase in DMSO was almost the same as that in water (Fig. 1), which
indicated that the conformation of d-amino acylase was preserved
in DMSO. Thus, it can be concluded that d-amino acylase may keep
its natural activity in DMSO.
2.4.4. Acetic acid (4-nitro-imidazol-1-yl)-phenyl-methyl ester
(1d)
Yellow oil; 1H NMR (CDCl3, 400 MHz, ı, ppm): 7.78 (s, 1H), 7.67
(s, 1H), 7.63 (s, 1H), 7.52 (m, 5H), 2.27 (s, 3H); 13C NMR (CDCl3,
100 MHz, ı, ppm): 169.0, 135.9, 132.7, 130.6, 128.4, 126.2, 118.1,

B.-K. Liu et al. / Journal of Molecular Catalysis B: Enzymatic 73 (2011) 85–89
87
Table 1
D
D
O
O
The influence of acetaldehyde on the Markovnikov addition catalyzed by d-amino
D
acylase
DMSO
acylase in DMSO at 50 ◦C.a
+
N
N
N
N
O
O
app
cat
Entry
Aldehyde
Yieldb (%)
Vo (mM h−1
)
Vrc
k
(h−1
)
Scheme 1. Acylase-catalyzed Markovnikov addition of dideuterated imidazole with
vinyl acetate.
1
2
3
4
5
0
86
95
94
91
90
31.1
183
92.2
76.2
64.8
1
69.7
10%
20%
50%
100%
5.87
2.96
2.45
2.08
410
206
171
145
3.2. Isotope experiment
a
Reaction condition: 1 mmol 4-nitro-imidazole, 3 mmol vinyl acetate, 25 mg d-
An isotope labeling experiment in the process of enzymatic
Markovnikov addition was carried out by using dideuterated imida-
zole (Fig. S1) as nucleophile in the reaction (Scheme 1). According to
the Markovnikov addition rule, a methyl group containing one deu-
terium atom should be formed. However, a methyl group that did
not contain deuterium atoms appeared in the final product (Fig. S2).
This result indicated that the apparent Markovnikov adduct was not
formed from Markovnikov addition through one step.
amino acylase, 2 mL DMSO, 50 ◦C.
b
Determined by HPLC.
Relative initial reaction rate to the reaction in the absence of acetaldehyde.
c
acetaldehyde on the reaction became weaker and weaker as its con-
centration increasing (Entries 2–5, Table 1). It may be attributed to
the deactivation of enzyme caused by a large number of acetalde-
hyde [24]. Another control reaction between 4-nitro-imidazole and
vinyl acetate with adding 3 equiv acetaldehyde in the absence of
acylase was carried out. The reaction could not take place, even pro-
longing reaction time to 120 h. This result ruled out the possibility
that the transesterification could be spontaneous in the presence
of acetaldehyde without d-amino acylase. Moreover, it indicated
that the role of the acylase is not only to generate acetaldehyde. It
should be taken into account that the acetaldehyde was an aqueous
solution. Thus, an experiment by adding water was carried out. As
seen from Fig. 2, there was no evident difference between the yields
in the presence of water or without water. This result excluded the
influence of water on the reaction. Thus, it can be concluded that
acetaldehyde is very important in enzymatic Markovnikov addi-
tion.
3.3. Influence of the amount of vinyl ester on the enzymatic
Markovnikov addition
To identify the intermediate, the reaction between 4-nitro-
imidazole and vinyl acetate was monitored by GC–MS. To our
surprise, a small amount of 2a was detected in the reaction sys-
tem besides the corresponding adduct (Scheme 2 and Fig. S3). 2a
was also isolated from the reaction in large scale and the structure
of 2a was characterized (Fig. S4). It demonstrated that the acylation
catalyzed by d-amino acylase took place in DMSO. The concentra-
tion of 2a increased slightly at the beginning and then decreased,
but the amount of 2a still kept at a very low level in the whole pro-
cedure. Therefore, the yield may decrease as the amount of vinyl
acetate decreasing, because vinyl acetate could be consumed dur-
ing the acylation reaction. When the molar ratio of vinyl acetate
to 4-nitro-imidazole was 1, the maximum yield was 64%. It was
consistent with our hypothesis.
3.5. Influence of the leaving group in substrate on the enzymatic
Markovnikov addition
3.4. Influence of the amount of acetaldehyde on the enzymatic
Markovnikov addition
In order to distinguish from vinyl acetate, isopropenyl acetate
was employed as the substrate in the presence of acetaldehyde
(Scheme 3). The product 1a was obtained in 70% yield confirmed
by mass spectrum (Fig. S5) and HPLC analysis. This observation
indicated that the ester bond was cleaved and a new one was
formed during the process. However, 4-nitro-imidazole could not
react with isopropenyl acetate in the absence of acetaldehyde. It
may be ascribed to the low reactivity of acetone which generated
from isopropenyl acetate. In addition, when isopropenyl acetate
was replaced by 2,2,2-trifluoro-ethyl acetate, 1a could also be syn-
thesized (Fig. S6).
In the enzymatic Markovnikov addition reaction, the acylation
of 4-nitro-imidazole catalyzed by d-amino acylase would produce
acetaldehyde. The question whether the amount of acetaldehyde
could affect the reaction arose. Thus, the influence of the molar ratio
of acetaldehyde to 4-nitro-imidazole on the reaction was exam-
ined. The data from a plot of reaction rate versus concentration of
acetaldehyde implied that the reaction was dependent on the con-
centration of acetaldehyde. All the reaction rates were improved.
The rate was increased nearly six times in the presence of 0.1 equiv
acetaldehyde (Entries 1 and 2, Table 1). However, the effect of
O
O
NO2
acylase
DMSO
HN
NO2
NO2
+
N
+
N
O
O
O
N
N
N
1a
2a
Scheme 2. Acylase-catalyzed Markovnikov addition of 4-nitro-imidazole with vinyl acetate.
O
O
O
NO2
acylase
DMSO
HN
NO2
R
N
+
+
O
O
N
H
N
1a
CH3
R=
CH2CF3
CH2
,
C
Scheme 3. The reaction of 4-nitro-imidazole with ester in the presence of acetaldehyde catalyzed by d-amino acylase in DMSO at 50 ◦C.

88
B.-K. Liu et al. / Journal of Molecular Catalysis B: Enzymatic 73 (2011) 85–89
O
NO
O
2
N
2a
N
O
NO
2
NO
HN
2
HN
+
N
O
N
acylase
H
Step 1
Step 2
O
NO
N
2
acylase
OH
NO
2
N
O
N
N
1
a
HI
Step 3
oxyanion hole
O
O
Scheme 4. Proposed mechanism for enzymatic Markovnikov addition.
R2
N
H
O
O
N
Lipase
Organic
Solvent
R1
+
+
O
N
R1
O
R3
H
N
R2
O
R3
1a-h
Scheme 5. Lipase-catalyzed “apparent” Markovnikov addition.
3.6. The process of enzymatic Markovnikov addition
could decrease the reactivity of substrates (Table 2, Entries 3 and
6). In addition, steric hindrance showed negative effect on the reac-
tion and slight decrease in yields was observed (Table 2, Entries
4–8). To our disappointment, no induction of stereoselectivity was
Based on the above results, a hypothesis for the process of
enzymatic Markovnikov addition was proposed as Scheme 4. Acy-
lation of 4-nitro-imidazole with vinyl acetate was first catalyzed
by acylase and acetaldehyde generated simultaneously. The N-
heterocycle as nucleophile attacked the acetaldehyde and formed
the hemiaminal intermediate (HI). HI was supposed to be stabilized
by hydrogen bond in the catalytic site. Then, HI attacked the car-
bonyl group of vinyl ester which bonded with oxyanion hole in the
catalytic site. Next, transesterification was accomplished by acylase
and the final product was obtained. This hypothesis may provide
an explanation for no reaction between N-heterocycles and vinyl
ether.
80
60
40
20
0
3.7. Lipase-catalyzed “apparent” Markovnikov addition
According to our proposed mechanism, it could be envisioned
that lipases could catalyze this addition. The results in Table 2
showed that two lipases exhibited the ability to catalyze this reac-
tion involving other aldehydes bearing the alkyl or aryl group
and other imidazole derivatives (Scheme 5). The expected prod-
ucts were synthesized successfully. This reaction was favored
by aliphatic aldehyde, while aromatic aldehydes exhibited lower
reactivity (Table 2, Entries 1 and 5). Examination of substituted
groups on imidazole revealed that electron-withdrawing group
0
10
20
30
40
50
time (h)
Fig. 2. The influence of water on the Markovnikov addition catalyzed by d-amino
acylase in DMSO at 50 ◦C: (a) Blank (filled squares); (b) 10% (filled circles); (c) 20%
(filled triangles); (d) 50% (filled inverted-triangles); (e) 100% (filled diamonds).

B.-K. Liu et al. / Journal of Molecular Catalysis B: Enzymatic 73 (2011) 85–89
89
Table 2
Lipase-catalyzed “apparent” Markovnikov addition.a
Entry
Nucleophile
Aldehyde
Ester
Solvent
Enzyme
Yieldb
%
H
N
O
H
O
N
O2N
O2N
O2N
O2N
O
O
O
O
1
2
3
4
DMSO
DMSO
DMSO
DMSO
PSL
PSL
PSL
PSL
1a (36)
1b (48)
1c (26)
1d (13)
H
N
O
O
O
O
N
H
H
H
N
O
N
H
H
H
N
O
N
O
H
N
H
O
N
O2N
O
5
DMSO
PSL
1e (8)
H
N
O
O
O
O
N
H
H
O
O
6
7
MTBE
MTBE
CAL-B
CAL-B
1f (53)
1g (36)
H
N
N
H
N
O
O
N
H
O
8
MTBE
CAL-B
1h (40)
a
All reactions were run on a 0.5 mmol scale of imidazole derivatives with 2 equiv of aldehydes and isopropenyl acetate in 1 mL of organic solvent catalyzed by lipases at
50 ◦C, 72 h.
b
Detected by HPLC.
observed. Other study on the stereoselectivity of this reaction was
still under way.
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Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.molcatb.2011.08.002.