Enzyme-catalyzed asymmetric domino aza-Michael/aldol reaction for the synthesis of 1,2-dihydroquinolines using pepsin from porcine gastric mucosa

Article abstract of DOI:10.1016/j.cclet.2016.02.013

An unprecedented enzyme-catalyzed asymmetric domino aza-Michael/aldol reaction of 2-aminobenzaldehyde and α,β-unsaturated aldehydes is achieved. Pepsin from porcine gastric mucosa provided mild and efficient access to diverse substituted 1,2-dihydroquinol

Full text of DOI:10.1016/j.cclet.2016.02.013

G Model
CCLET 3600 1–5
Chinese Chemical Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
1
2
Original article
3
4
5
Enzyme-catalyzed asymmetric domino aza-Michael/aldol reaction
for the synthesis of 1,2-dihydroquinolines using pepsin from porcine
gastric mucosa
* *
Q1 Xue-Dong Zhang, Na Gao, Zhi Guan , Yan-Hong He
6
7
8
Key Laboratory of Applied Chemistry of Chongqing Municipality, School of Chemistry and Chemical Engineering, Southwest University,
Chongqing 400715, China
A R T I C L E I N F O
A B S T R A C T
Article history:
An unprecedented enzyme-catalyzed asymmetric domino aza-Michael/aldol reaction of 2-aminoben-
Received 7 September 2015
Received in revised form 11 January 2016
Accepted 28 January 2016
Available online xxx
zaldehyde and
mild and efficient access to diverse substituted 1,2-dihydroquinolines in yields of 38–97% with 6–24%
enantiomeric excess (ee). This work not only provides novel method for the synthesis of
a,b-unsaturated aldehydes is achieved. Pepsin from porcine gastric mucosa provided
a
dihydroquinoline derivatives, but also promotes the development of enzyme catalytic promiscuity.
ß 2016 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
Keywords:
Pepsin
Biocatalysis
Domino aza-Michael/aldol reaction
1,2-Dihydroquinolines
Enzyme catalytic promiscuity
9
10
1. Introduction
using a chiral amine catalyst [22]. Subsequently, several similar 30
approaches for the synthesis of dihydroquinoline by chiral amine 31
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
The prevalent structure of quinoline derivatives is fundamental
in heterocyclic compounds, and these key structural units have
been shown to have pharmaceutical applications [1–5]. Especially
1,2-dihydroquinolines, which can be transformed to 1,2,3,4-
tetrahydroquinolines through the reduction reaction [6,7], were
widely synthesized due to their special biological activity and the
characteristics of the drug intermediates. To date, catalysts
including transition metals [8–16], Brønsted acids [17], Lewis acids
[18] and iodine [19], etc. have been reported for achieving 1,2-
dihydroquinoline derivatives. In 2001, Shibasaki and co-workers
first reported that a bifunctional Lewis acid was able to catalyze the
asymmetric addition of cyanide to various substituted quinolones
(isoquinoline) to give the corresponding Reissert compounds
[20]. In 2003, Hamada et al. used N-protected o-aminobenzalde-
catalysts or bifunctional thiourea catalysts were independently 32
reported [23–25]. Good yields and ee were reported utilizing 33
chemical catalysis. In consideration of the great significance of 1,2- 34
dihydroquinolidines, development of new methods with environ- 35
mentally friendly and sustainable catalysts to form this important 36
structure is still desired.
37
Enzymes, as a kind of green catalyst for modern organic 38
synthesis, have attracted increased attention. Enzyme catalytic 39
promiscuity is the functional property of an enzyme to catalyze 40
an otherwise unnatural reaction, using the same active site 41
responsible for its natural activity. Enzyme catalytic promiscuity 42
widens the scope of enzyme use in organic synthesis and allows 43
for the discovery of new synthetic methods [26,27]. Continuing 44
research has shown that many enzymes exhibit catalytic 45
promiscuity [28]. Some examples of the use of enzyme promis- 46
cuity, such as enzyme-catalyzed aldol [29–34], Henry [35–37], 47
Mannich [38–42], Povarov [43] and domino reactions [44,45], etc. 48
hydes and
a,b-unsaturatedcarbonyl compounds for the preparation
of1,2-dihydroquinolinesinthepresenceofaquaternaryammonium
´
salt [21]. In 2007, Cordova et al. demonstrated the asymmetric aza-
Michael/aldol reaction between 2-aminobenzaldehydes and
unsaturated aldehydes for the synthesis of 1,2-dihydroquinolidines
a,b-
have been reported.
49
Pepsin, a kind of hydrolase, belongs to the family of aspartic acid 50
protease [46,47] and is present during chemical digestion of 51
protein. In the 1930s, Northrop crystallized swine pepsin supply- 52
ing convincing evidence for its identity as a protein. The purified 53
pepsin provided the needed evidence for confirming its peptide 54
*
Corresponding authors.
E-mail addresses: guanzhi@swu.edu.cn (Z. Guan), heyh@swu.edu.cn (Y.-H. He).
http://dx.doi.org/10.1016/j.cclet.2016.02.013
1001-8417/ß 2016 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: X.-D. Zhang, et al., Enzyme-catalyzed asymmetric domino aza-Michael/aldol reaction for the synthesis
of 1,2-dihydroquinolines using pepsin from porcine gastric mucosa, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/
j.cclet.2016.02.013

G Model
CCLET 3600 1–5
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X.-D. Zhang et al. / Chinese Chemical Letters xxx (2016) xxx–xxx
Table 1
55
56
57
58
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structure which is characteristic of proteins [48]. Pepsin-catalyzed
aldol reactions have been developed [32,33]. Herein, we report a
novel example of enzyme catalytic promiscuity using pepsin from
porcine gastric mucosa to catalyze the domino aza-Michael/aldol
reaction for the synthesis of 1,2-dihydroquinolidines.
Solvent screening and control experiments.a
O
CHO
CHO
Pepsin
H
Solvent/H2O, 30o C
N
H
NH2
2
1a
3a
60
2. Experimental
Entry
Solvent
Time (h)
Yield (%)b
ee (%)c
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
Pepsin from porcine gastric mucosa [EC 3.4.23.1, P7000-25g, Lot
#050M1304V, powder, 49.0% protein (UV), 920 units/mg protein,
and P7125-100g, Lot #SLBD7698V, powder, 18.0% protein (UV),
1
2
1,4-Dioxane
DMF
70
118
118
118
118
94
32
31
30
25
24
21
20
20
19
18
14
11
10
10
8
10
16
14
14
2
3
MeCN
721 units/mgprotein;oneunit willproduceaDA280 nmof 0.001per
4
DMSO
5
n-Butyl acetate
Toluene
EtOH
min at pH 2.0 at 37 8C, measured as TCA-soluble products using
hemoglobin as substrate. (Final vol. = 16 mL. Light path = 1 cm)]
werepurchasedfromSigma–Aldrich.Recombinantpepsinexpressed
in E. coli was purchased from Hangzhou Biosci Biotech Co., Ltd. Other
chemical reagents and solvents were purchased from commercial
vendors, and used without any further purification unless otherwise
stated.
Flash column chromatography was carried out using 200–300
mesh silica gel at increased pressure. The NMR spectra were
recorded with TMS as the internal standard in CDCl3 on a Bruker
Avance 600 Spectrometer (600 MHz 1H, 150 MHz 13C) at room
temperature. In each case, the enantiomeric excess was deter-
mined by chiral HPLC analysis on Chiralpak AD-H, IA-H and
Chiralcel OD-H in comparison with authentic racemates. High-
resolution mass spectra were obtained using an ESI ionization
source (Varian 7.0T FTICR-MS). All reactions were monitored by
thin-layer chromatography (TLC) with Haiyang GF254 silica gel
plates.
6
0
7
117
70
16
4
8
CHCl3
9
MeOH
70
16
2
10
11
12
13
14
15
16
17
Isopropyl ether
CH2Cl2
94
94
4
Solvent-free
THF
48
3
70
2
Cyclohexane
H2O
70
2
118
118
118
0
DMF (no enzyme)
Albumin from chicken
egg white (30 mg)
DMF + pepsind
Trace
2
–
–
18
19
20
21
22
118
118
118
118
118
Trace
Trace
Trace
4
–
–
DMF + pepsine
DMF + pepsinf
–
DMF + pepsing
–
Pepsin (recombinant
as comparison)h
45
14
a
Unless otherwise noted, reaction conditions: cinnamaldehyde (0.26 mmol), 2-
aminobenzaldehyde (0.30 mmol), pepsin (13.5 kU), solvent (0.5 mL), deionized
water (0.1 mL) at 30 8C.
General procedure for the pepsin-catalyzed domino aza-
Michael/aldol reactions: To a mixture of 2-aminobenzaldehyde
b
Yield of the isolated product after silica gel chromatography.
c
Determined by chiral HPLC.
Pepsin (13.5 kU) in Ag+ solution (0.25 mol/L) [AgNO3 (42.5 mg) in deionized
(0.30 mmol),
a,b-unsaturated aldehyde (0.26 mmol), pepsin
d
(12.3 kU) and DMF (0.5 mL), deionized water (0.3 mL) was added.
The resultant mixture was stirred for the specified time at 40 8C,
and monitored by TLC analysis. The reaction was terminated
by filtering the enzyme. Ethyl acetate was employed to wash
the residue on the filter paper to assure that products obtained
were all dissolved in the filtrate. The filtrate was washed with
saturated brine three times, and the combined organic layers were
dried over anhydrous Na2SO4, and concentrated under vacuum.
The residue was purified by flash column chromatography on
silica gel using a mixture of petroleum ether and ethyl acetate
ratio 3:1–20:1 as eluent.
water (1.0 mL)] was stirred at 30 8C for 24 h, and then the water was removed by
lyophilization before use.
Pepsin (13.5 kU) in Cu2+ solution (0.25 mol/L) [CuSO4 (39.9 mg) in deionized
e
water (1.0 mL)] was stirred at 30 8C for 24 h, and then the water was removed by
lyophilization before use.
f
Pepsin (13.5 kU) in GuHCl solution (3.12 mol/L) [GuHCl (300 mg) in deionized
water (1.0 mL)] was stirred at 30 8C for 24 h, and then the water was removed by
lyophilization before use.
g
Pepsin (13.5 kU) in CDI (1.85 M) [CDI (300 mg) in CH2Cl2 (1.0 mL)] was stirred at
30 8C for 4 h, and then dialyzed against deionized water. The water was removed by
lyophilization before use.
h
Reaction conditions: cinnamaldehyde (0.052 mmol), 2-aminobenzaldehyde
(0.060 mmol), pepsin recombinant, expressed in E. coli (0.86 kU), DMF (0.1 mL),
deionized water (0.02 mL) at 30 8C. Yield determined by HPLC analysis.
97
3. Results and discussion
98
99
Through a large number of screenings, we found that pepsin
from porcine gastric mucosa could catalyze aza-Michael/aldol
reaction of 2-aminobenzaldehyde and cinnamaldehyde. Thus, this
reaction was used as a model to investigate the influence of
different parameters on the pepsin-catalyzed aza-Michael/aldol
reaction. In view of the fact that the reaction medium plays an
important role in the enzymatic reactions [49], different solvents
were screened (Table 1, entries 1–15). Based on the experimental
data, pepsin showed certain catalytic activity, not only in polar
solvents, but also in nonpolar solvents in the model reaction. The
highest yield of 32% was obtained in 1,4-dioxane (Table 1, entry 1).
The best enantioselectivity of 16% ee was observed in DMF, ethanol,
and methanol, respectively (Table 1, entries 2, 7 and 9). Among
them, the yield of 31% was obtained in DMF. Considering both yield
and selectivity, DMF was selected as a suitable solvent for further
investigation.
conducted and only a trace amount of the desired product observed
(Table 1, entry 16). To exclude the possibility that the catalytic
activity of the pepsin for the aza-Michael/aldol reaction could arise
from the catalysis of an unspecific amino acid residue on the surface
of the enzyme [50], albumin from chicken egg white, representing a
protein without an enzymatic function, was used as a catalyst in the
model reaction, and only gave the product in 2% yield without ee
(Table 1, entry 17). Therefore, it can be assumed that the protein
surface of pepsin is predominately catalytically inactive in the
process. Enzymes maintain their native tertiary structures mainly
through a combination of coordinated hydrogen bonding, hydro-
phobic, electrostatic, steric, and other interactions [51]. Heavy metal
ions, as common denaturation agents, can inactivate enzymes by
reacting with some structural groups (e.g., –SH groups) resulting in
irreversible damage, or interacting with some amino acid residues
causing changes in three-dimensional structure. Thus, metal ions
Ag+ and Cu2+ were employed to pretreat the pepsin, separately,
and then the pretreated pepsin was used to catalyze the model
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
Next, to confirm the specific catalytic effect of pepsin on the
aza-Michael/aldol reaction, some control experiments were
performed (Table 1, entries 16–21). The blank experiment was
Please cite this article in press as: X.-D. Zhang, et al., Enzyme-catalyzed asymmetric domino aza-Michael/aldol reaction for the synthesis
of 1,2-dihydroquinolines using pepsin from porcine gastric mucosa, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/
j.cclet.2016.02.013

G Model
CCLET 3600 1–5
X.-D. Zhang et al. / Chinese Chemical Letters xxx (2016) xxx–xxx
3
135
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147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
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reaction, which gave only trace amounts of product (Table 1, entries
18 and 19). Another denaturation agent, guanidine hydrochloride
(GuHCl), also can change the conformational structure of enzymes,
and ultimately denature the enzyme; the reaction with GuHCl
pretreated pepsin only gave a trace amount of product (Table 1,
entry 20). These control experiments indicated that the three-
dimensionalstructureoftheenzymewasresponsibleforitscatalytic
activity inthedominoreaction. Moreover, accordingtotheliterature
[52], the active site of pepsin from porcine gastric mucosa contains
Asp32 and Asp215 residues. Carbonyldiimidazole (CDI), an irrevers-
ible inhibitor of aspartic acid, which can bond covalently with the
carbonyl of aspartic acid was used to pretreat pepsin, and the
reaction with CDI pretreated pepsin only gave the product in a low
yield of 4% without ee (Table 1, entry 21). This result implied that the
enzyme-catalyzed domino aza-Michael/aldol reaction may occur
at the active site of the enzyme, or in close proximity. The above
experiments demonstrated that inhibition and denaturation of
pepsin caused a nearly complete disruption of the catalytic activity
of the enzyme. As a consequence, it can be concluded that pepsin has
the ability to catalyze the domino aza-Michael/aldol reaction with a
certain degree of enantioselectivity, and the native structure of the
enzyme is responsible for its activity and aspartic acids may play a
key role in this catalysis event.
To rule out the possibility of catalysis by the presence of some
impure proteins, we purchased the pepsin from porcine gastric
mucosa, recombinant, expressed in E. coli for comparison. The
purity of this protein was checked by SDS-PAGE (for the SDS-PAGE
image, please see the Supporting information), which showed a
clear pepsin band with a molecular weight of 35 kDa and that the
protein was quite pure. Then, recombinant pepsin was used to
catalyze the model domino reaction providing yields of 45% and
14% ee (Table 1, entry 22). This result clearly confirmed that pepsin
indeed has the ability to catalyze the reaction.
Hereto, our supply of the enzyme preparation (pepsin from por- 181
cine gastric mucosa, Sigma–Aldrich, P7000-25g, Lot #050M1304V) 182
used for the above investigations (Table 1 and Fig. 1) was exhausted. 183
We then continued the study using another enzyme preparation 184
(Pepsin from porcine gastric mucosa, Sigma–Aldrich, P7125-100g, 185
Lot #SLBD7698V) for the following investigation.
186
The influence of catalyst loading on the pepsin-catalyzed 187
domino reaction of 2-aminobenzaldehyde and cinnamaldehyde 188
was surveyed (Fig. 2). The best yield of 35% with 18% ee was 189
obtained when 12.3 kU of enzyme was used in the reaction system, 190
which was similar to the results obtained with the former enzyme 191
preparation (13.5 kU of enzyme, product yield 41% with 16% ee, 192
Fig. 2). On the other hand, these results demonstrated that this 193
procedure was not only applicable to a specific enzyme prepara- 194
tion, but also to other preparations of the same enzyme. Thus, 195
12.3 kU of enzyme was selected as a suitable catalyst loading for 196
the reaction system discussed.
197
Temperature has effects on the selectivity and rate of the 198
reaction, and also on the stability of the enzyme. Thus, the 199
influence of temperature on the pepsin-catalyzed model domino 200
reaction was examined (Fig. 3), demonstrating that a lower 201
temperature was beneficial to improve the enantioselectivity and 202
18% ee was obtained at 30–40 8C. A higher temperature was 203
beneficial to improve the yield, and a yield of 45% was received at 204
40–50 8C. Therefore, 40 8C was chosen as the optimal temperature 205
for the pepsin-catalyzed domino reaction.
206
40
35
30
25
20
15
10
5
Usually enzymes require water molecules to maintain their
optimum spatial structure via hydrogen bonding and other non-
covalentinteractions, thus, a smallamount ofwaterisoftenrequired
for enzymatic reactions in non-aqueous medium. Consequently, the
influence of water content on pepsin-catalyzed domino reaction in
DMF was investigated (Fig. 1). It can be seen that the yield of the
product tended to rise as the addition of water increased from 0 to
0.3 mL in DMF (0.5 mL), and further increases of water additions
caused a decrease in yield. The best yield of 41% with 16% ee was
obtained when 0.3 mL of water was added to the reaction system.
The addition of water had slight effects on the enantioselectivity of
the reaction. Thus0.3 mLof waterinDMF (0.5 mL) was chosenas the
optimal reaction medium for the following study.
Yield
ee
0
2
4
6
8
10
12
14
16
Amount of enzyme (kU)
Fig. 2. Influence of enzyme loading on the pepsin-catalyzed domino reaction.
45
50
45
Yield
40
ee
35
30
25
20
15
10
5
40
Yield
ee
35
30
25
20
15
10
5
0
0
0.0
0.1
0.2
0.3
0.4
0.5
15
20
25
30
35
40
45
50
55
Temperature (ºC)
Amount of water addition (mL)
Fig. 1. Influence of water addition on the pepsin-catalyzed domino reaction.
Fig. 3. Influence of temperature on the pepsin-catalyzed domino reaction.
Please cite this article in press as: X.-D. Zhang, et al., Enzyme-catalyzed asymmetric domino aza-Michael/aldol reaction for the synthesis
of 1,2-dihydroquinolines using pepsin from porcine gastric mucosa, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/
j.cclet.2016.02.013

G Model
CCLET 3600 1–5
4
X.-D. Zhang et al. / Chinese Chemical Letters xxx (2016) xxx–xxx
Table 2
to humans and the environment. As a proof of the concept, this
work provides a novel case of a promiscuous enzyme-catalyzed
enantioselective domino reaction. Meanwhile, this finding broad-
ens the application of pepsin in organic synthesis.
242
243
244
245
Substrate scope of the pepsin-catalyzed domino reactions.a
O
CHO
R
Pepsin
H
CHO
R
DMF/H2O, 40o C
N
H
NH2
Acknowledgment
246
1
2
3
This work was financially supported by the National Natural Q2 247
Science Foundation of China (Nos. 21276211 and 21472152).
Entry
R
Products
Time (h)
Yield (%)b
ee (%)c
248
249
1
2
C6H5
4-CH3C6H4
3a
3b
3c
3d
3e
3f
118
72
72
72
72
118
36
36
36
118
24
24
7
45
51
44
38
47
52
55
43
41
43
57
45
97
18
17
15
21
10
14
10
15
15
11
22
24
6
3
4-CH3OC6H4
4-FC6H4
Appendix A. Supplementary data
4
5
4-ClC6H4
4-BrC6H4
4-NO2C6H4
3-ClC6H4
2-CH3OC6H4
2-ClC6H4
n-Propyl
n-Butyl
6
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.cclet.2016.02.
013.
250
251
252
7
3g
3h
3i
8
9
10
11
12
13
3j
3k
3l
References
253
CO2Et
3m
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a
Reaction conditions:
a,b-unsaturated aldehyde (0.26 mmol), 2-aminobenzal-
dehyde (0.30 mmol), pepsin (12.3 kU), DMF (0.5 mL), deionized water (0.3 mL) at
40 8C.
b
Yield of the isolated product after chromatography on silica gel.
c
The ee was determined by chiral HPLC analyses; absolute configurations of the
products were determined by comparison with the known chiral HPLC analysis
results [22]. (For details, please see the Supporting information.)
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
After having established the optimal reaction conditions,
the scope generality of this pepsin-catalyzed domino aza-
Michael/aldol reaction was explored (Table 2). As documented
in the table, a variety of aliphatic and aromatic substituents in
the
a,b-unsaturated aldehydes were well tolerated, leading to
the corresponding products in moderate to good yields with low
enantioselectivity (Table 2, entries 1–13). Different electron-
donating and electron-withdrawing aromatic
aldehydes were used to test the influence of electronic effects of
substituents on the reaction (Table 2, entries 2–7), and some
aromatic
different position of the benzene ring were also investigated
to examine the steric effects of substituents on the reaction
(Table 2, entries 5, 8 and 10). Both electronic and steric effects of
aromatic
the yield and enantioselectivity of the reaction. The reactions
with aliphatic -unsaturated aldehydes, trans-2-hexenal and
trans-2-heptenal, gave the products in yields of 57% and 45% with
22% and 24% ee, respectively (Table 2, entries 11 and 12). It was
notable that trans-4-oxo-2-butenoate was successfully utilized
in the reaction providing the desired product in 97% yield with 6%
ee (Table 2, entry 13).
[7] J.V. Johnson, B.S. Rauckman, D.P. Baccanari, et al., X-ray analyses of aspartic
proteinases: II. Three-dimensional structure of the hexagonal crystal form of
a,b-unsaturated
˚
porcine pepsin at 2.3 A resolution, J. Med. Chem. 32 (1989) 1942–1949.
[8] J. Barluenga, M. Fan˜ana´s-Mastral, F. Aznar, et al., [1,5]-Hydride transfer/cycliza-
tions on alkynyl Fischer carbene complexes: synthesis of 1,2-dihydroquinolinyl
carbene complexes and cascade reactions, Angew. Chem. Int. Ed. 47 (2008)
6594–6597.
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a,b-unsaturated aldehydes had no obvious impact on
a,b
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4. Conclusion
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In summary, we developed a biocatalytic strategy to synthesize
1,2-dihydroquinoline derivatives. Pepsin from porcine gastric
mucosa has the ability to promote the enantioselective domino
aza-Michael/aldol reaction of
a,b-unsaturated aldehydes and
2-aminobenzaldehyde. The desired products were obtained in
yields of 38–97% with 6–24% ee. Although the yields and
stereoselectivities were not thoroughly satisfactory in comparison
with those reported by chemical catalysis, this is the first reported
study utilizing pepsin to catalyze the aza-Michael/aldol reaction to
afford 1,2-dihydroquinoline derivatives. The enzyme-catalyzed
domino reaction showed the comprehensive advantages of
biocatalysis, such as mild reaction conditions and reduced toxicity
Please cite this article in press as: X.-D. Zhang, et al., Enzyme-catalyzed asymmetric domino aza-Michael/aldol reaction for the synthesis
of 1,2-dihydroquinolines using pepsin from porcine gastric mucosa, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/
j.cclet.2016.02.013

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Please cite this article in press as: X.-D. Zhang, et al., Enzyme-catalyzed asymmetric domino aza-Michael/aldol reaction for the synthesis
of 1,2-dihydroquinolines using pepsin from porcine gastric mucosa, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/
j.cclet.2016.02.013