Enzyme-Catalyzed Asymmetric Domino Thia-Michael/Aldol Condensation Using Pepsin

Article abstract of DOI:10.1021/acs.joc.6b01132

The novel catalytic promiscuity of pepsin from porcine gastric mucosa for the asymmetric catalysis of the domino thia-Michael/aldol condensation reaction in MeCN and buffer was discovered for the first time. Broad substrate specificity was tested, and a series of corresponding products were obtained with enantioselectivities of up to 84% ee. This specific catalysis was demonstrated by using recombinant pepsin and control experiments with denatured and inhibited pepsin. The reaction was also shown to occur in the active site by site-directed mutagenesis (the Asp32Ala mutant of pepsin), and a possible mechanism was proposed.

Full text of DOI:10.1021/acs.joc.6b01132

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Article
Enzyme-catalyzed asymmetric domino thia-
Michael/aldol condensation using pepsin
Yang Xiang, Jian Song, Yong Zhang, Da-Cheng Yang, Zhi Guan, and Yan-Hong He
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b01132 • Publication Date (Web): 27 Jun 2016
Downloaded from http://pubs.acs.org on July 6, 2016
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Enzyme-catalyzed asymmetric domino thia-Michael/aldol
condensation using pepsin
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Yang Xiang, Jian Song, Yong Zhang, Da-Cheng Yang, Zhi Guan*, Yan-Hong He*
Key Laboratory of Applied Chemistry of Chongqing Municipality, School of Chemistry and Chemical Engineering,
Southwest University, Chongqing 400715, PR China
E-mails: guanzhi@swu.edu.cn (for Z. Guan); heyh@swu.edu.cn (for Y.-H. He)
A table of contents entry
Abstract: The novel catalytic promiscuity of pepsin from porcine gastric mucosa for the
asymmetric catalysis of domino thia-Michael/aldol condensation reaction in MeCN/buffer was
discovered for the first time. Broad substrate specificity was tested and a series of corresponding
products were obtained with enantioselectivities of up to 84% ee. This specific catalysis was
demonstrated by using recombinant pepsin and the control experiments with denatured and
inhibited pepsin. The reaction was also proved to occur in the active site by site-directed
mutagenesis (the Asp32Ala mutant of pepsin) and a possible mechanism was proposed.
Key words: pepsin; enzyme catalytic promiscuity; domino reaction; asymmetric catalysis;
enzyme catalysis
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Introduction
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Enzymes, as green and efficient biocatalysts, have huge superiority in many important respects
such as mild reaction conditions, good stereoselectivity, easy processing, economically and
ecologically advantageous, etc.1 Enzymes have specific substrate recognition in metabolism,
which are optimized through evolution of a specific chemical transformation. Despite this, more
and more enzymes have been found to have the ability to catalyze reactions, or act on substrates,
other than those for which they evolved; this phenomenon is called enzyme promiscuity.2 Until
recently, the wider implications of the “darker” side of enzyme promiscuity were largely ignored.
Actually, promiscuous activities are not rare exceptions but are rather widely spread, inherent
features of enzymes and proteins in general.3 It is believed that promiscuous activities serve as
starting points for the divergence of new enzymes in natural evolution. Broad-specificity enzymes
acted as progenitors for today’s specialized enzymes.4 Thus, enzyme catalytic promiscuity is a key
factor in the evolution of new enzyme functions. Many examples of enzyme catalytic promiscuity
have been reported,2c,5 such as the aldol reactions,6 Henry reactions,7 Markovnikov additions,8
Michael additions,9 Mannich reactions,10 the asymmetric synthesis of α-aminonitrile amides,11
multi-component cascade or domino reactions,1a-c,12 etc. However, only very few mechanisms of
promiscuous functions have been proved by site-directed mutation of enzymes.6a,13 It has been
discovered that valuable insights regarding the catalytic mechanisms can be provided by a
systematic research of the hidden skills of enzymes.14 Thus, exploring more reaction types and
verification of catalytic mechanism are still greatly in demand.
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Dihydrothiophenes as valuable S-heterocycles have attracted great attention because of their
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special values in biological and medical properties,15 versatile synthetic intermediates16 and
material science.17 Thus, many efficient synthetic approaches have been devoted to the
development of these compounds.16c,18 However, only few successful procedures achieved the
enantioselective formation of dihydrothiophenes. In 2009, the De Risi19 group reported the
synthesis of chiral 4,5-dihydrothiophene; in 2010, the Xu20 group developed the enantioselective
formation of 2,5-dihydrothiophenes by domino thia-Michael/aldol condensation between
1,4-dithiane-2,5-diol and α,β-unsaturated aldehydes using chiral diphenylprolinol TMS ether as an
organocatalyst. Based on the work of Xu20 group, in 2015, De Risi21 group developed a one-pot,
four-step organocatalytic process catalyzed by (S)-diphenylprolinol TMS ether. This process
consists of the domino thia-Michael/aldol condensation between 1,4-dithiane-2,5-diol and
α,β-unsaturated aldehydes, and the derived chiral dihydrothiophene adducts subsequently react
with bromonitromethane via the domino Michael/α-alkylation reaction to obtain chiral
nitrocyclopropanes. Since the biological activities and pharmacological activities are often related
to the configurations of most natural products and pharmaceuticals, the enantioselective syntheses
of dihydrothiophenes is of great importance. Therefore, the development of novel catalysts that are
environmentally friendly, sustainable and cheap toward chiral synthesis of dihydrothiophenes is
still highly desired.
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Pepsin is an aspartic protease that acts in food digestion in the mammal stomach. In recent years,
some promiscuous activities of pepsin from porcine gastric mucosa have been disclosed gradually.
In 2010, the Yu group reported the pepsin-catalyzed aldol reaction between acetone and
substituted benzaldehydes in which enantioselectivity of up to 44% ee was observed.22 Our group
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also has investigated the catalytic promiscuity of pepsin. In 2015, we found that pepsin can
catalyze direct asymmetric aldol reactions for the synthesis of vicinal diol compounds, and
products were obtained with the enantioselectivity of up to 75% ee.23 In 2016, we reported the
pepsin-catalyzed Morita–Baylis–Hillman (MBH) reaction between aromatic aldehydes and
2-cyclohexen-1-one or 2-cyclopenten-1-one, and the enantioselectivity of up to 38% ee was
achieved.24 Very recently, we found that pepsin can catalyze the domino
Knoevenagel/Michael/Michael reaction for the synthesis of spirooxindole derivatives with
diastereoselectivities of up to >99 : 1 dr without enantioselectivity.12b In view of the amazing
catalytic versatility of pepsin and its stereoselectivity toward some synthetically useful molecules,
it is necessary to gain more insights into its catalytic promiscuity. Herein, we report a novel
activity of pepsin for the asymmetric domino thia-Michael/aldol condensation reaction of
1,4-dithiane-2,5-diol with α,β-unsaturated aldehydes. Broad substrate specificity was tested and a
series of corresponding products were obtained with enantioselectivities of up to 84% ee. The
specific catalytic effect of pepsin was clearly proved to occur in the active site by site-directed
mutagenesis (the Asp32Ala mutant of pepsin). This is the first study utilizing site-directed
mutagenesis to confirm the promiscuous activity of pepsin. And a possible mechanism was
proposed.
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Results and discussion
The asymmetric domino thia-Michael/aldol condensation reaction of cinnamaldehyde (1a) and
1,4-dithiane-2,5-diol (2) was chosen as a model reaction, and pepsin from porcine gastric mucosa
was used as a catalyst. In this reaction 1,4-dithiane-2,5-diol (2), the mercaptoacetaldehyde dimer,
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was used as a convenient and efficient synthon to provide an in situ generated
mercaptoacetaldehyde (Scheme 1).25 It is known that reaction media plays an important role in
enzymatic reactions as the reaction media has a great effect on the stability and the catalytic
activity of an enzyme, particularly on enantioselectivity and regioselectivity.26 To optimize
experimental conditions, we first examined the effect of solvent on the model reaction (Table 1).
It could be seen that the catalytic effects of pepsin, especially the enantioselectivity, was obviously
influenced by different media. The enzyme showed the best enantioselectivity of 41% ee in MeCN
with a yield of 17% (Table 1, entry 1). Better yields of 20-21% were obtained in CHCl3, PhMe
and ClCH2CH2Cl, but with very low enantioselectivity (Table 1, entries 6-8). The reactions in the
other tested solvents gave either lower yields or lower ees, and even only trace amounts of the
product were observed in several solvents including water (Table 1, entries 2-5 and 9-16). These
results may be attributed to the solubility of substrates and specific interactions between the
solvent and pepsin. To get the best enantioselectivity, MeCN was chosen as a suitable solvent for
the reaction. The absolute configuration of the product 3a was determined as R-configuration by
comparing with the known chiral HPLC analysis.20
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Scheme 1 The model asymmetric domino thia-Michael/aldol condensation reaction
Table 1 Solvent screening for the model reaction a
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Entry
1
Solvent
MeCN
Yield (%)b
17c
ee (%)b
41
2
3
MeOH
THF
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35
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6
4
EtOAc
CH2Cl2
CHCl3
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5
17
6
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PhMe
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ClCH2CH2Cl
Et2O
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3
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3
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PhOMe
1,4-Dioxane
EtOH
Trace
Trace
Trace
Trace
Trace
Trace
DMF
DMSO
Xylene
16
H2O
Trace
a
Reaction conditions: a mixture of cinnamaldehyde 1a (0.5 mmol), 1,4-dithiane-2,5-diol 2 (0.35 mmol), solvent
(0.90 mL), deionized water (0.10 mL) and pepsin (6.5 kU) was stirred at 30 °C for 96 h.
b Determined by chiral HPLC using a chiralpak AS-H column.
c Yield of the isolated product after silica gel chromatography.
Since the pH value of the reaction medium significantly affects the stability and catalytic activity
of enzymes,27 phosphate buffer (NaH2PO4-Na2HPO4, 0.067 M, pH 4.7-8.7) was used to replace
the water in the reaction system (buffer/MeCN = 1/9, v/v) to obtain the optimum reaction
conditions (Table 2). It could be seen that addition of buffer obviously enhanced the
enantioselectivity of the reaction. Pepsin showed the best enantioselectivity in the presence of
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phosphate buffer (pH 6.5), giving the product in yield of 16% with 57% ee (Table 2, entry 4).
However, the addition of phosphate buffer was unable to increase the yield. Thus, the phosphate
buffer (pH 6.5, buffer/MeCN = 1/9, v/v) was selected as the optimum condition for the reaction.
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Table 2 The influence of pH conditions (phosphate buffer) on the model reaction a
Entry
pH of phosphate buffer
Yield (%)b
ee (%)b
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4.7
5.6
6.3
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a
Reaction conditions: a mixture of cinnamaldehyde 1a (0.5 mmol), 1,4-dithiane-2,5-diol 2 (0.35 mmol), MeCN
(0.90 mL), phosphate buffer (NaH2PO4-Na2HPO4, 0.067 M, pH 4.7-8.7, 0.10 mL) and pepsin (6.5 kU) was stirred
at 30 °C for 96 h.
b Determined by chiral HPLC using a chiralpak AS-H column.
The effects of substrate molar ratios on the model reaction were investigated (Table 3). The results
showed that the reaction was clearly influenced by changes in the molar ratio of substrates. As the
amount of cinnamaldehyde (1a) was increased, the enhanced yields were obtained but with
reduced ees (Table 3, entries 1-6). When increasing the amount of 1,4-dithiane-2,5-diol (2), a
slight improvement was observed both in yield and ee (Table 3, entries 1 and 7-10). The best ee of
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60% and yield of 19% were obtained at the molar ratio of 1a : 2 = 1 : 3 (Table 3, entry 8), which
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was chosen as the optimal ratio for further studies.
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Table 3 Effect of molar ratio of substrates on the model reaction a
Entry
Molar ratio (1a : 2)
Yield (%)b
ee (%)b
1
2
1 : 1
1.4 : 1
2 : 1
3 : 1
4 : 1
5 : 1
1 : 2
1 : 3
1 : 4
1 : 5
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19
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59
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3
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9
10
a
Reaction conditions: a mixture of cinnamaldehyde 1a (0.35-1.75 mmol), 1,4-dithiane-2,5-diol 2 (0.35-1.75
mmol), MeCN (0.90 mL), phosphate buffer (NaH2PO4-Na2HPO4, 0.067 M, pH 6.5, 0.10 mL) and pepsin (6.5 kU)
was stirred at 30 °C for 96 h.
b Determined by chiral HPLC using a chiralpak AS-H column.
Next, to further optimize the reaction conditions, the influence of phosphate buffer content in the
reaction system on the model reaction was investigated. The best yield of 24% with 59% ee was
received in the mixed solvent of phosphate buffer (pH 6.5) and MeCN (buffer/MeCN = 1/4, v/v),
which was chosen as the optimal condition for the reaction (for details, please see the Supporting
Information). Then the influence of the mixed solvent volume on the model reaction was also
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examined. To our delight, as the volume increased from 1.00 mL to 1.50 mL, the ee was raised
from 59% to 70% with a slight decrease in yield from 24% to 18%. Thus, to get better
enantioselectivity, a mixed solvent volume of 1.50 mL (buffer/MeCN = 1/4, v/v) was chosen as
the optimal reaction condition (for details, please see the Supporting Information).
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The influence of enzyme loading on the model reaction of cinnamaldehyde (0.35 mmol) and
1,4-dithiane-2,5-diol (1.05 mmol) was examined. The yield was visibly affected by enzyme
loading (Table 4). A great enhancement in yield was obtained when the enzyme loading was
increased from 3.9 kU to 16.9 kU (Table 4, entries 1-6) with ee remained about the same. Though
there was a slight enhancement in yield with further increases in the amount of enzyme from 16.9
kU to 26.0 kU, the ee began to decline (Table 4, entries 6-9). Thus, we chose an enzyme loading
of 16.9 kU (Table 4, entry 6) as the optimum condition for further studies.
Table 4 Effect of enzyme loading on the model reaction a
Entry
Enzyme loading (kU)
Yield (%)b
ee (%)b
1
2
3
4
5
6
7
8
9
3.9
6.5
11
18
28
30
37
46
46
53
55
70
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9.1
11.7
14.3
16.9
19.5
22.1
26.0
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a
Reaction conditions: a mixture of cinnamaldehyde 1a (0.35 mmol), 1,4-dithiane-2,5-diol 2 (1.05 mmol), MeCN
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(1.20 mL), phosphate buffer (NaH2PO4-Na2HPO4, 0.067 M, pH 6.5, 0.30 mL) and pepsin (3.9-26.0 kU) was stirred
at 30 °C for 96 h.
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b Determined by chiral HPLC using a chiralpak AS-H column.
Temperature also plays an important role in enzyme-catalyzed reactions due to its effects on
enzyme stability, as well as the rate and selectivity of the reaction.28 To further characterize the
activity and selectivity of pepsin in the model reaction, the influence of temperature was
investigated (Table 5). When raising the temperature from 20 °C to 35 °C, the yield improved
from 33% to 47% with ee staying at about the same level (Table 5, entries 1-4). However, once
the temperature surpassed 35 °C, the yield and ee began to decrease (Table 5, entries 5-8). When
the temperature was higher than 40 °C, the yield and ee sharply declined (Table 5, entries 7 and
8), which we believe to be due to high temperature causing denaturation of pepsin. Overall, a
relatively good result of 46% yield with 70% ee was exhibited at 30 °C (Table 5, entry 3). Thus,
carrying out the reaction at 30 °C was proven to be the optimal reaction condition.
Table 5 Influence of temperature on the model reaction a
Entry
T (°C)
Yield (%)b
ee (%)b
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2
3
4
20
25
30
35
33
44
46
47
69
69
70
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a
Reaction conditions: a mixture of cinnamaldehyde 1a (0.35 mmol), 1,4-dithiane-2,5-diol 2 (1.05 mmol), MeCN
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(1.20 mL), phosphate buffer (NaH2PO4-Na2HPO4, 0.067 M, pH 6.5, 0.30 mL) and pepsin (16.9 kU) was stirred at
20-60 °C for 96 h.
b Determined by chiral HPLC using a chiralpak AS-H column.
Time course of the model reaction was investigated under the aforementioned optimal conditions.
Generally, the yield increased as the reaction time went on at the early stage and finally kept at an
almost constant level. No significant change of ee was detected during the whole time course (for
details, please see the Supporting Information).
To verify the specific catalytic effect of pepsin on the domino reaction, some control experiments
were performed (Table 6). In the absence of pepsin, the reaction only gave a trace amount of the
product (Table 6, entry 2) which indicated that pepsin preparation had a catalytic effect on the
domino reaction. The catalytic site of pepsin consists of two aspartate residues, Asp32 and
Asp215. N,N'-carbonyldiimidazole (CDI) can be irreversibly covalently bound to carboxyl. Thus,
CDI was used to pretreat the pepsin, and the reaction with CDI pretreated pepsin only gave the
product in a low yield of 4% with 5% ee (Table 6, entry 3), which indicated that CDI strongly
inhibited enzyme activity in the domino reaction. At the same time, CDI alone was verified to
have no effect on the domino reaction (Table 6, entry 5). The results suggested that the enzymatic
process may proceed in the active site. Moreover, urea as a denaturing agent can change the
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conformational structure of enzymes, and ultimately denature the enzyme. Hence, urea was used
to denature pepsin which nearly made pepsin lose its catalytic activity completely (Table 6, entry
4). Meanwhile, the blank experiment showed that urea itself did not catalyze the domino reaction
(Table 6, entry 6). The above experiments indicated that the specific natural fold of pepsin was
responsible for its activity in the domino reaction.
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To further verify that the pepsin is truly the catalyst of the investigated domino reaction, we have
carried out a series of studies involving the cloning, expression, purification and activation of
recombinant porcine pepsinogen A,29 and the obtained recombinant pepsin was used to catalyze
the model domino reaction. A good yield of 60% with 43% ee was obtained from the recombinant
pepsin catalyzed reaction (Table 6, entry 7). The result clearly confirmed that pepsin indeed
catalyzed the domino reaction in an asymmetric manner.
Moreover, to validate if this observed promiscuous activity was arisen from the active site of the
pepsin, a site-directed mutagenesis was conducted. The catalytic site of pepsin is formed by two
aspartate residues, Asp32 and Asp215. We changed, by site-directed mutagenesis, pepsin active
site Asp32 to an Ala32.29 Only a trace amount of product was observed for the model domino
reaction with mutant pepsin (Asp32Ala) (Table 6, entry 8). It can be inferred that the enzymatic
process indeed proceeds in the active site, and the Asp residues located in the active centre are also
crucial for this enzymatic domino reaction.
Table 6 Control experiments for the model reaction a
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Entry
Catalyst
Yield (%)b
ee (%)b
1
2
3
4
5
6
7
8
Pepsin
46
Trace
4
70
-
None
Pepsin (pretreated with CDI)c
Pepsin (pretreated with urea)d
CDI (200 mg)
5
2
-
1
Trace
Trace
60
Urea (200 mg)
-
Recombinant pepsine
Mutant pepsin (Asp32Ala)e
43
-
Trace
a
Unless otherwise noted, reaction conditions: a mixture of cinnamaldehyde 1a (0.35 mmol), 1,4-dithiane-2,5-diol
2 (1.05 mmol), MeCN (1.20 mL), phosphate buffer (NaH2PO4-Na2HPO4, 0.067 M, pH 6.5, 0.30 mL) and pepsin
(16.9 kU) was stirred at 30 °C for 96 h.
b Determined by chiral HPLC using a chiralpak AS-H column.
c Pepsin (16.9 kU) in CDI solution (1.2 M in THF) (200 mg CDI in 1.0 mL THF) was stirred at rt for 4 h and THF
was removed under reduced pressure before use.
d Pepsin (16.9 kU) in urea solution (3.3 M) (200 mg urea in 1.0 mL deionized water) was stirred at rt for 24 h and
water was removed by lyophilization before use.
e
Reaction conditions: a mixture of cinnamaldehyde 1a (0.035 mmol), 1,4-dithiane-2,5-diol 2 (0.105 mmol),
MeCN (0.24 mL), phosphate buffer (NaH2PO4-Na2HPO4, 0.067 M, pH 6.5, 0.06 mL) and recombinant pepsin
(2.34 mg, 0.7 kU) (for entry 7) or mutant pepsin (Asp32Ala) (2.34 mg) (for entry 8) was stirred at 30 °C for 96 h.
With the optimal reaction conditions in hand, to investigate the generality and scope of the
pepsin-catalyzed domino reaction, various α,β-unsaturated aldehydes were investigated. As shown
in Table 7, aromatic α,β-unsaturated aldehydes bearing either electron-withdrawing or
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electron-donating substituents could participate in this domino reaction smoothly. Substituents on
the aromatic ring had a significant influence on the enantioselectivities. Generally, aromatic
α,β-unsaturated aldehydes with electron-donating groups provided higher enantioselectivities than
those with electron-withdrawing substituents (Table 7, entries 2-10). When the substituents on the
aromatic ring were halogen atoms, the position of the substituents had effects on the
enantioselectivity. Substituents at para-position gave higher ee values than those at ortho- or
meta-position (Table 7, entries 5-9). The heteroaromatic α,β-unsaturated aldehyde,
3-(2-furyl)acrylaldehyde, could participate in the reaction as well, giving the best yield of 53%
with 70% ee (Table 7, entry 11). Besides aromatic α,β-unsaturated aldehydes, aliphatic
α,β-unsaturated aldehydes were also applicable to the reaction. The straight chain aliphatic
α,β-unsaturated aldehyde gave a less efficient result of 35% yield and 40% ee (Table 7, entry 12),
while the branched aliphatic α,β-unsaturated aldehyde exhibited the best ee of 84% with 38%
yield (Table 7, entry 13). In general, the desired products were obtained with moderate to good
enantioselectivities, but yields were not so high. In all cases, no obvious by-products were
observed. Long reaction time was required in this investigated promiscuous activity of pepsin,
which indicated that the reaction rates with substrates that are not natural to an enzyme are usually
inefficiency and slower than those observed for native substrates.
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Table 7 Investigation of substrate scope for the pepsin-catalyzed domino reaction a
Entry
R
Product No.
Time (h)
Yield (%)b
ee (%)c
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Ph
96
46
70
3a
5
6
7
8
2
3
4-MeC6H4
2-OMeC6H4
4-OMeC6H4
4-ClC6H4
132
132
132
144
120
120
144
120
144
36
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40
72
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70
66
56
50
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3b
3c
3d
3e
3f
4
9
5
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6
3-ClC6H4
7
2-ClC6H4
3g
3h
3i
8
4-BrC6H4
2-BrC6H4
4-NO2C6H4
9
10
3j
11
12
13
2-furanyl
propyl
144
168
168
53
35
38
70
40
84
3k
3l
i-propyl
3m
a
Reaction conditions: a mixture of α,β-unsaturated aldehyde 1 (0.35 mmol), 1,4-dithiane-2,5-diol 2 (1.05 mmol),
MeCN (1.20 mL), phosphate buffer (NaH2PO4-Na2HPO4, 0.067 M, pH 6.5, 0.30 mL) and pepsin (16.9 kU) was
stirred at 30 °C.
b Yield of the isolated product after silica gel chromatography.
c Determined by chiral HPLC using a chiralpak AS-H or chiralcel OD-H column.
The control experiments with denatured and inhibited pepsin as well as the experiments with
recombinant and mutant enzyme clearly demonstrated that the domino reaction occurs in the
active site of pepsin, and the Asp residues located in the active centre are responsible for this
promiscuous activity. According to the literature,30 the catalytic site of pepsin is formed by two
aspartate residues, Asp32 and Asp215, one of which (Asp215) has to be protonated, and the other
(Asp32) deprotonated, for the protein to be active. Thus, based on the control experiments and
literature, we attempted to proposed a possible mechanism for the pepsin-catalyzed domino
reaction of α,β-unsaturated aldehydes and 1,4-dithiane-2,5-diol (Scheme 2). Firstly, Asp32 acts as
a base to take away a proton from the mercaptoacetaldehyde which is generated in situ from
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1,4-dithiane-2,5-diol. Secondly, the activated mercaptoacetaldehyde and α,β-unsaturated aldehyde
undergo the intermolecular thia-Michael addition, and the derived adduct accepts the proton from
Asp215 forming the enol. Thirdly, the tetrahydrothiophen scaffold is provided through a
subsequent intramolecular aldol reaction. Finally, the dehydration process occurs forming the
dihydrothiophen skeleton.
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Scheme 2 The proposed mechanism for the pepsin-catalyzed domino reaction
Conclusion
In summary, we have shown a novel promiscuous pepsin-catalyzed asymmetric domino
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thia-Michael/aldol condensation reaction of aromatic, heteroaromatic and aliphatic
α,β-unsaturated aldehydes with 1,4-dithiane-2,5-diol in MeCN/buffer for the first time. The
corresponding functionalized chiral dihydrothiophenes were obtained with 40-84% ee without
additive. The specific catalytic effect of pepsin was demonstrated by combining some control
experiments and site-directed mutagenesis. This is the first study utilizing site-directed
mutagenesis to confirm the promiscuous activity of pepsin. This work broadens the scope of
enzyme catalyzed transformations. The performance of pepsin in the present work as well as its
other promiscuous activities reported previously demonstrate that pepsin has the considerable
potential on broad catalytic promiscuity, which may enable organic chemists to rapidly develop
new synthetic applications of pepsin to expand the repertoire of synthetic organic methodologies.
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Experimental Section
Materials
Pepsin from porcine gastric mucosa [EC number 3.4.23.1, CAS number: 9001-75-6, product
number: P7125-100G, Lot # SLBD7698V, 721 units per mg of protein, 18% protein (UV). One
unit will produce a change in ΔA280nm of 0.001 per minute at pH 2.0 at 37°C measured as
TCA-soluble products using hemoglobin as substrate] was purchased from Sigma-Aldrich. Unless
otherwise noted, all reagents were purchased from commercial suppliers and used without further
purification.
General procedure for the pepsin-catalyzed domino thia-Michael/aldol reaction
Pepsin (16.9 kU) was added to a 10 mL round-bottom flask containing α,β-unsaturated aldehyde
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(0.35 mmol), 1,4-dithiane-2,5-diol (1.05 mmol), MeCN (1.20 mL) and phosphate buffer
(NaH2PO4-Na2HPO4, 0.067 M, pH 6.5, 0.30 mL). The resultant mixture was stirred at 30 °C for
the specified reaction time and monitored by thin-layer chromatography (TLC). The reaction was
terminated by filtering the enzyme. The filter cake was washed with ethyl acetate (10 mL). Then,
the filtrate was concentrated in vacuo. The crude product was purified by flash column
chromatography with petroleum ether/ethyl acetate (4:1 – 8:1) as eluent.
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(R)-2-phenyl-2,5-dihydrothiophene-3-carbaldehyde (3a) (Table 7, Entry 1).20 (30.6 mg, 46%,
1
brown solid, mp 68-70 °C) H NMR (600 MHz, CDCl3): 9.71 (s, 1H), 7.28-7.27 (m, 4H),
7.22-7.19 (m, 1H), 7.01-7.00 (m, 1H), 5.50 (d, J = 5.6 Hz, 1H), 4.23-4.19 (m, 1H), 4.02-3.98 (m,
1H); 13C NMR (150 MHz, CDCl3): δ = 187.0, 148.8, 148.7, 142.3, 128.6, 127.5, 54.9, 38.8. HPLC
analysis: Chiralpak AS-H, n-hexane / i-PrOH 90 : 10, flow rate 1.0 mL / min, λ = 240 nm, tmajor
=
15.4 min, tminor = 19.3 min.
(R)-2-(p-tolyl)-2,5-dihydrothiophene-3-carbaldehyde (3b) (Table 7, Entry 2).20 (25.7 mg, 36%,
brown solid, mp 158-160 °C) 1H NMR (600 MHz, CDCl3): δ = 9.73 (s, 1H), 7.17 (d, J = 8.1 Hz,
2H), 7.09 (d, J = 7.9 Hz, 2H), 7.02-7.01 (m, 1H), 5.49 (d, J = 5.5 Hz, 1H), 4.24-4.20 (m, 1H),
4.03-3.99 (m, 1H), 2.30 (s, 3H); 13C NMR (150 MHz, CDCl3): δ = 187.1, 148.9, 148.5, 139.3,
137.2, 129.3, 127.3, 54.7, 38.6, 21.1. HPLC analysis: Chiralcel OD-H, n-hexane / i-PrOH 95 : 5,
flow rate 0.5 mL / min, λ = 244 nm, tmajor = 23.0 min, tminor = 25.8 min.
(R)-2-(2-methoxyphenyl)-2,5-dihydrothiophene-3-carbaldehyde (3c) (Table 7, Entry 3).20 (33.9 mg,
44%, brown solid, mp 152-154 °C) 1H NMR (600 MHz, CDCl3): δ = 9.80 (s, 1H), 7.20 (ddd, J =
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1.6, 1.1, 1.6 Hz, 1H), 7.12-7.10 (m, 1H), 6.97 (dd, J = 1.7, 1.5 Hz, 1H), 6.87-6.85 (m, 2H), 5.88 (d,
J = 5.6 Hz, 1H), 4.13-4.09 (m, 1H), 3.98-3.94 (m, 1H), 3.86 (s, 3H); 13C NMR (150 MHz, CDCl3):
δ = 187.2, 156.5, 150.1, 148.0, 130.5, 128.5, 127.0, 120.7, 111.0, 55.7, 47.9, 38.2. HPLC analysis:
Chiralcel OD-H, n-hexane / i-PrOH 90 : 10, flow rate 1.0 mL / min, λ = 220 nm, tmajor = 18.8 min,
tminor = 15.1 min.
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(R)-2-(4-methoxyphenyl)-2,5-dihydrothiophene-3-carbaldehyde (3d) (Table 7, Entry 4).20 (38.5 mg,
50%, brown solid, mp 134-136 °C) 1H NMR (600 MHz, CDCl3): δ = 9.73 (s, 1.0 Hz), 7.22-7.20
(m, 2H), 7.01-7.00 (m, 1H), 6.83-6.81 (m, 2H), 5.49 (d, J = 5.5 Hz, 1H), 4.24-4.20 (m, 1H),
4.03-4.00 (m, 1H), 3.77 (s, 3H); 13C NMR (150 MHz, CDCl3): δ = 187.1, 159.0, 148.9, 148.3,
134.4, 128.6, 114.0, 55.3, 54.5, 38.6. HPLC analysis: Chiralcel OD-H, n-hexane / i-PrOH 90 : 10,
flow rate 1.0 mL / min, λ = 244 nm, tmajor = 14.9 min, tminor = 19.8 min.
(R)-2-(4-chlorophenyl)-2,5-dihydrothiophene-3-carbaldehyde (3e) (Table 7, Entry 5).20 (33.0 mg,
1
42%, brown solid, mp 145-146 °C) H NMR (600 MHz, CDCl3): δ = 9.73 (s, 1H), 7.26-7.24 (m,
4H), 7.04-7.03 (m, 1H), 5.48-5.46 (m, 1H), 4.25-4.21 (m, 1H), 4.05-4.02 (m, 1H); 13C NMR (150
MHz, CDCl3): δ = 186.8, 148.9, 148.6, 140.8, 133.2, 128.9, 128.7, 54.3, 38.8. HPLC analysis:
Chiralcel OD-H, n-hexane / i-PrOH 90 : 10, flow rate 1.0 mL / min, λ = 240 nm, tmajor = 11.4 min,
tminor = 13.1 min.
(R)-2-(3-chlorophenyl)-2,5-dihydrothiophene-3-carbaldehyde (3f) (Table 7, Entry 6).20 (32.2 mg,
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41%, brown solid, mp 140-142 °C) H NMR (600 MHz, CDCl3): δ = 9.74 (s, 1H), 7.26 (s, 1H),
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7.22-7.17 (m, 3H), 7.07-7.05 (m, 1H), 5.45 (d, J = 5.4 Hz, 1H), 4.26-4.22 (m, 1H), 4.06-4.02 (m,
1H); 13C NMR (150 MHz, CDCl3): δ = 186.8, 149.2, 148.4, 144.3, 134.4, 129.8, 127.7, 127.6,
125.9, 54.4, 38.9. HPLC analysis: Chiralcel OD-H, n-hexane / i-PrOH 90 : 10, flow rate 1.0 mL /
min, λ = 240 nm, tmajor = 11.3 min, tminor = 12.9 min.
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(S)-2-(2-chlorophenyl)-2,5-dihydrothiophene-3-carbaldehyde (3g) (Table 7, Entry 7).20 (31.5 mg,
1
40%, brown solid, mp 179-182 °C) H NMR (600 MHz, CDCl3): δ = 9.74 (s, 1H), 7.26 (s, 1H),
7.22-7.17 (m, 3H), 7.07-7.05 (m, 1H), 5.45 (d, J = 5.3 Hz, 1H), 4.26-4.22 (m, 1H), 4.06-4.02 (m,
1H); 13C NMR (150 MHz, CDCl3): δ = 186.8, 149.2, 148.4, 144.3, 134.4, 129.8, 127.7, 127.6,
125.9, 54.4, 38.9. HPLC analysis: Chiralcel OD-H, n-hexane / i-PrOH 90 : 10, flow rate 1.0 mL /
min, λ = 228 nm, tmajor = 15.7 min, tminor = 19.2 min.
(R)-2-(4-bromophenyl)-2,5-dihydrothiophene-3-carbaldehyde (3h) (Table 7, Entry 8).20 (36.7 mg,
1
39%, brown solid, mp 107-110 °C) H NMR (600 MHz, CDCl3): δ = 9.73 (s, 1H), 7.41-7.40 (m,
2H), 7.18-7.16 (m, 2H), 7.04-7.03 (m, 1H), 5.45 (d, J = 5.5 Hz, 1H), 4.25-4.20 (m, 1H), 4.05-4.01
(m, 1H); 13C NMR (150 MHz, CDCl3): δ = 186.8, 149.0, 148.5, 141.3, 131.7, 129.3, 121.3, 54.4,
38.8. HPLC analysis: Chiralcel OD-H, n-hexane / i-PrOH 90 : 10, flow rate 1.0 mL / min, λ = 236
nm, tmajor = 12.5 min, tminor = 13.9 min.
(S)-2-(2-bromophenyl)-2,5-dihydrothiophene-3-carbaldehyde (3i) (Table 7, Entry 9).20 (33.9 mg,
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36%, brown solid, mp 152-154 °C) H NMR (600 MHz, CDCl3): δ = 9.81 (s, 1H), 7.55 (d, J =
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10.0 Hz, 1H), 7.24-7.20 (m, 2H), 7.09-7.05 (m, 2H), 5.93 (d, J = 5.5 Hz, 1H), 4.17-4.12 (m, 1H),
4.04-4.00 (m, 1H); 13C NMR (150 MHz, CDCl3): δ = 186.8, 150.6, 147.6, 141.4, 133.1, 128.8,
127.8, 123.8, 53.9, 38.3. HPLC analysis: Chiralcel OD-H, n-hexane / i-PrOH 90 : 10, flow rate 1.0
mL / min, λ = 236 nm, tmajor = 19.0 min, tminor = 23.5 min.
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(R)-2-(4-nitrophenyl)-2,5-dihydrothiophene-3-carbaldehyde (3j) (Table 7, Entry 10).20 (32.9 mg,
40%, brown solid, mp 136-137 °C) 1H NMR (600 MHz, CDCl3): δ = 9.75 (s, 1H), 8.15 (d, J = 8.7
Hz, 2H), 7.46 (d, J = 8.6 Hz, 2H), 7.13 (s, 1H), 5.55 (d, J = 5.8 Hz, 1H), 4.31-4.26 (m, 1H),
4.13-4.09 (m, 1H); 13C NMR (150 MHz, CDCl3): δ = 186.7, 149.8, 149.5, 148.0, 128.5, 123.9,
54.2, 39.1. HPLC analysis: Chiralcel OD-H, n-hexane / i-PrOH 90 : 10, flow rate 1.0 mL / min, λ
= 220 nm, tmajor = 31.0 min, tminor = 33.9 min.
(S)-2-(furan-2-yl)-2,5-dihydrothiophene-3-carbaldehyde (3k) (Table 7, Entry 11).20 (33.4 mg, 53%,
brown oil) 1H NMR (600 MHz, CDCl3): δ = 9.79 (s, 1H), 7.31 (d, J = 1.1 Hz, 1H), 7.08-7.06 (m,
1H), 6.30-6.29 (m, 1H), 6.15 (d, J = 3.2 Hz, 1H), 5.60 (d, J = 5.3 Hz, 1H), 4.23-4.19 (m, 1H),
4.00-3.95 (m, 1H); 13C NMR (150 MHz, CDCl3): δ = 186.8, 153.4, 149.8, 146.0, 142.1, 110.7,
106.6, 47.3, 38.3. HPLC analysis: Chiralcel OD-H, n-hexane / i-PrOH 90 : 10, flow rate 1.0 mL /
min, λ = 232 nm, tmajor = 16.2 min, tminor = 14.1 min.
(R)-2-propyl-2,5-dihydrothiophene-3-carbaldehyde (3l) (Table 7, Entry 12).20 (19.1 mg, 35%,
1
brown oil) H NMR (600 MHz, CDCl3): δ = 9.75 (s, 1H), 6.85 (dd, J = 4.1, 2.9 Hz, 1H), 3.97 – 3.93
(m, 1H), 3.85 – 3.81 (m, 1H), 2.01 – 1.95 (m, 1H), 1.57 – 1.51 (m, 2H), 1.43 – 1.35 (m, 2H), 0.90 (t, J
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= 7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3): δ = 187.9, 149.6, 149.4, 52.1, 38.3, 37.5, 20.5, 13.7.
HPLC analysis: Chiralpak AS-H, n-hexane / i-PrOH 90 : 10, flow rate 1.0 mL / min, λ = 236 nm,
tmajor = 6.6 min, tminor = 7.4 min.
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(R)-2-isopropyl-2,5-dihydrothiophene-3-carbaldehyde (3m) (Table 7, Entry 13).20 (20.7 mg, 38%,
brown oil) 1H NMR (600 MHz, CDCl3) δ 9.76 (s, 1H), 6.92 – 6.90 (m, 1H), 4.54 – 4.53 (m, 1H), 3.83
– 3.80 (m, 2H), 2.42 – 2.39 (m, 1H), 0.95 (d, J = 6.8 Hz, 3H), 0.77 (d, J = 6.7 Hz, 3H); 13C NMR (150
MHz, CDCl3): δ = 187.8, 150.5, 148.6, 59.5, 37.7, 30.3, 21.8, 15.2. HPLC analysis: Chiralpak AS-H,
n-hexane / i-PrOH 90 : 10, flow rate 1.0 mL / min, λ = 244 nm, tmajor = 7.0 min, tminor = 7.9 min.
Construction of the expression vector for the production of pepsinogen A and Asp91Ala
mutant (See the Supporting Information for details.)
The cDNA for swine pepsinogen A precursor (accession number GI: 164603) was synthesized
from GenScript Corporation (Nanjing, China). The fragment of pepsinogen A which coding
sequence begins at amino acid position L16 (and thereby lacking the N-terminal signal peptide)
was
generated
by
PCR
amplification,
by
using
primers
and
5’-GGAATTCCATATGTTGGTAAAAGTTCCATTA-3’
5’-CCCAAGCTTAGCGACTGGGGCTAAA-3’ to introduce NdeI and HindIII restriction sites
(underlined sequence) at the 5’ and 3’ ends, respectively. The PCR products were cloned into the
pET28a (+) vector. The Asp91Ala point mutant construct was generated by QuikChange™
Site-Directed Mutagenesis Kit (Stratagene). All constructs were confirmed by DNA sequencing.
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Expression and purification of proteins (See the Supporting Information for details.)
Wild type pepsinogen A and Asp91Ala mutant were produced in E. coli BL21 (DE3) by using the
pET28a (+) expression system. Protein expression was overnight induced at 30 °C with 0.5 mM
isopropyl-β-D-thiogalactopyranoside (IPTG) after OD600 reached 0.6-0.8. A cell pellet was
harvested by centrifugation, resuspended and lysed by high-pressure homogenization, and soluble
protein was fractionated from insoluble material by centrifugation. The targeted protein was
purified by Ni-NTA affinity chromatography. 25 mg purified wild type pepsinogen A and 20 mg
purified Asp91Ala mutant were obtained from one liter cell culture, respectively.
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Activation of the pepsinogen A and Asp91Ala mutant
The pepsinogen A and Asp91Ala mutant were activated according to the literature.29 After
activation, the 60-385 amino acid residues were retained to get the active pepsin and Asp32Ala
mutant (numbering for porcine pepsin A). Enzyme assay was conducted according to the
reference.31 The activity of the pepsin was 299 U/mg protein (Unit definition: one unit will
produce a change in ΔA280nm of 0.001 per minute at pH 2.0 at 37°C measured as TCA-soluble
products using hemoglobin as substrate); no activity was detected for Asp32Ala mutant pepsin.
Acknowledgements
This work was financially supported by the National Natural Science Foundation of China (No.
21276211, No. 21472152 and No. 21542014) and the Basic and Frontier Research Project of
Chongqing (cstc2015jcyjBX0106). We thank professor Hong-Tao Li from School of Life Science,
Southwest University for kind help with manuscript preparation.
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Supporting Information:
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General methods, Extra information for optimization of the reaction conditions, The detailed
information for construction of the expression vector for the production of swine pepsinogen A
and Asp91Ala mutant, and expression and purification of proteins, 1H NMR and 13C NMR spectra
of the products, HPLC charts of the products.
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