Asymmetric Morita-Baylis-Hillman reaction catalyzed by pepsin

Article abstract of DOI:10.1016/j.molcatb.2015.12.002

Pepsin from porcine gastric mucous shown catalytic promiscuity was first discovered to catalyze the Morita-Baylis-Hillman (MBH) reaction between aromatic aldehydes with 2-cyclohexen-1-one or 2-cyclopenten-1-one in a two-phase medium of phosphate buffer/cy

Full text of DOI:10.1016/j.molcatb.2015.12.002

Accepted Manuscript
Title: Asymmetric Morita-Baylis-Hillman Reaction Catalyzed
by Pepsin
Author: Jing-Wen Xue Jian Song Ian C.K. Manion Yan-Hong
He Zhi Guan
PII:
S1381-1177(15)30122-3
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2015.12.002
MOLCAB 3292
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
24-9-2015
13-11-2015
2-12-2015
Please cite this article as: Jing-Wen Xue, Jian Song, Ian C.K.Manion, Yan-Hong He,
Zhi Guan, Asymmetric Morita-Baylis-Hillman Reaction Catalyzed by Pepsin, Journal
of Molecular Catalysis B: Enzymatic http://dx.doi.org/10.1016/j.molcatb.2015.12.002
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Asymmetric Morita-Baylis-Hillman Reaction Catalyzed by Pepsin
Jing-Wen Xue a, Jian Song a, Ian C. K. Manion b, Yan-Hong He*a, Zhi Guan*a
a Key Laboratory of Applied Chemistry of Chongqing Municipality, School of Chemistry and Chemical
Engineering, Southwest University, Chongqing, 400715, P. R. China
b Chemistry Department, College of Saint Benedict and Saint John's University, MN, 56374, USA
E-mails: heyh@swu.edu.cn (Y.-H. He); guanzhi@swu.edu.cn (Z. Guan)
1

Graphical abstract
Pepsin from porcine gastric mucous shown catalytic promiscuity was first used to catalyze
Morita-Baylis-Hillman reaction in combination with 1,4-diazabicyclo[2.2.2]octane (DABCO).
2

Highlights




Pepsin-catalyzed Morita-Baylis-Hillman (MBH) reaction was described.
14 examples of MBH products were obtained by this reaction.
The novel reaction also expands the field of organic synthesis.
This work promotes the development of enzyme catalytic promiscuity.
3

Abstract: Pepsin from porcine gastric mucous shown catalytic promiscuity was first discovered
to catalyze the Morita-Baylis-Hillman (MBH) reaction between aromatic aldehydes with
2-cyclohexen-1-one or 2-cyclopenten-1-one in
a
two-phase medium of phosphate
buffer/cyclohexane in the presence of 1,4-diazabicyclo[2.2.2]octane (DABCO). The best results of
the corresponding MBH products up to 77% yield and 38% ee were achieved.
Key words: Porcine pepsin; Asymmetric MBH reaction; Biocatalyst; Enzyme catalytic
promiscuity
4

1. Introduction
The Morita-Baylis-Hillman reaction (MBH reaction) is one of the most important
carbon-carbon bond-forming reactions and has drawn considerable attentions in the past decades
[1-8]. The MBH products can be used as valuable building blocks for complex natural products
containing functionalized allylic alcohols [9-12]. Since the first reported MBH reaction in 1972,
various kinds of catalysts and co-catalysts have been developed [13-31]. Hatakeyama et al.
achieved a significant breakthrough in the asymmetric MBH reaction; they reported
quinidine-derived chiral bases for the reaction of acrylates with aldehydes and got the MBH
product with ee values up to 99%, though unfortunately the substrate scope and the latter
transformation are very narrow [32]. Schaus et al. reported an asymmetric Brønsted acid catalyst
derived from BINOL in combination with PEt3 in 2003, giving ee values up to 96% and moderate
to good yields for various kinds of aldehydes in the reaction with cyclohexanone [33,34]. Connon
et al. disclosed the first application of (thio)urea catalysts for the MBH reaction but they did not
get the asymmetric MBH adducts [35]. In 2005, Wang et al. introduced a chiral bifunctional
catalyst, a chiral amine-thiourea compound derived from 1,1’-binaphthyl-2,2’-diamine, which has
shown highly efficient catalytic ability, and catalyzed asymmetric MBH reactions of different
aldehydes with 2-cyclohexen-1-one, achieving high yields and enantiomeric excesses [36].
Nagasawa et al. also proved bis(thio)ureas derived from chiral trans-1,2-diaminocyclohexane are
suitable catalysts for the asymmetric MBH reaction [37]. In 2006, Berkessel and co-workers
developed an isophoronediamine-derived bis(thio)urea organic catalyst in combination with a
novel base tested in the MBH reaction, and the desired products were obtained in 75% yield and
96% ee [38]. In 2008, Shi et al. disclosed a novel chiral bis(thio)urea, and they used the catalyst in
combination with 1,4-diazabicyclo[2.2.2]octane (DABCO) to catalyze the asymmetric MBH
5

reaction between arylaldehydes with 2-cyclohexen-1-one which got the MBH products in 79%
yield and 88% ee [39].
Enzymes as an efficient catalyst have attracted significant attention due to their
environmentally benign quality and mild reaction conditions [40-43]. In recent years, an increasing
number of enzymes have been shown to have promiscuous behavior [44]. In particular, hydrolases
are considered one of the most useful enzymes due to its high stability, wide range of substrate
suitability, and high efficiency to catalyze various kinds of reactions [45]. The investigation of
enzyme promiscuity not only widens the novelty of bioorganic synthesis but also highlights
effective cooperation between the study of biology and organic synthesis [46-48]. Several
significant works of catalytic promiscuity of hydrolase have been reported, including but not
limited to aldol reactions [49-65], Michael reactions [66-68], Mannich reactions [69,70],
Markovnikov additions [71,72], and Henry reactions [73,74]. In 2007, Reetz et al. first reported the
MBH reaction catalyzed by carrier proteins such as serum albumins or enzymes such as certain
lipases, but they only achieved conversion up to 35% and ee up to 19%, and did not show a good
substrate scope [75]. In 2014, Jiang and co-workers introduced the Baylis-Hillman reaction
catalyzed by a biotin esterase, they achieved the products only with yield up to 46% and they did
not achieve any asymmetric products [76]. Herein we report pepsin from porcine gastric mucosa, a
hydrolase, can effectively catalyze the direct asymmetric MBH reactions in combination with
DABCO to afford desirable products using 2-cyclohexen-1-one and various substituted aromatic
aldehydes. The effects of the reaction medium, temperature, and other variables on the enzymatic
MBH reaction were researched in detail, and the catalytic specificity of pepsin was proved by
control experiments. This asymmetric MBH reaction provided a novel case of unnatural activities
6

of existing enzymes.
2. Results and discussion
In our initial study, the reaction of 4-nitrobenzaldehyde and 2-cyclohexen-1-one was chosen
as a model reaction. It is well known that the MBH reaction is the addition of electron-deficient
alkenes to aldehydes, and a nucleophilic base can promote the reaction effectively. Among these
bases, DABCO was the most popular one [38,40-43,77]. Thus, the model MBH reaction was
performed in the presence of enzyme in MeCN and water at 25 ºC in combination with DABCO to
find the best catalytic enzyme. The results of these experiments are summarized in Table 1. Pepsin
from porcine gastric mucosa as a catalyst obtained the best yield (40%) and ee (26%) (Table 1,
entry 11).
Several control tests were performed to confirm the specific catalytic effect of pepsin on the
model MBH reaction (Table 2). These control experiments were carried out under the optimized
conditions [details of the optimizations were described hereafter in the paper (Table 3 to Table 9)].
In the absence of enzyme the reaction with DABCO only gave product with 16% yield which
indicated that pepsin indeed catalyzed the model reaction in an asymmetric manner (Table 2, entry
2). In the absence of DABCO the reaction with pepsin only gave a trace amount of MBH product
(Table 2, entry 3). In the absence of both pepsin and DABCO (the blank), no reaction was
observed (Table 2, entry 4). The results above confirmed that DABCO plays an important role to
promote the MBH reaction, and the combination of pepsin and DABCO is essential for catalysis
of the MBH reaction. The reaction with egg white albumin instead of pepsin gave 22% yield and
2% ee (Table 2, entry 5), indicating that the enzymatic properties of pepsin was responsible for
catalysis. To further prove the necessity of native structure of pepsin, experiments with urea
7

pre-treated pepsin were conducted which only gave 26% and 18% yields without
enantioselectivity, also the natural activity of pepsin was decreased from 100 U·mg-1 to 33 and 26
U·mg-1 after urea treatment (Table 2, entries 6 and 7). These results suggested that the tertiary
structure of pepsin was essential in the reaction. Pepsin contains 385 residues according to the
literature [78,79]. The active site of pepsin from porcine gastric mucosa contains Asp32 and
Asp215 residues. Thus, N,N'-carbonyldiimidazole (CDI), an irreversible inhibitor of aspartic acid,
was used to pre-treat the pepsin. The reaction with the CDI pre-treated pepsin gave the product in
yields of 27% and 21% with 2% ee, indicating that CDI inhibited the enzyme activity strongly in
the reaction, and at same time the natural activity of pepsin was decreased from 100 U·mg-1 to 38
and 30 U·mg-1 (Table 2, entries 8 and 9). The results testified that the enzymatic process may
proceed in the active site. Thus, we confirmed that pepsin catalyzed the asymmetric MBH reaction
in combination with DABCO.
Since organic media has a great influence on the stability and activities of an enzyme [80,81],
the catalytic effects of pepsin in different solvents were surveyed (Table 3). It could be seen that
the catalytic activity of pepsin in the MBH reaction was obviously influenced by different media.
Using MeCN as a solvent, the best yield of 40% with 26% ee was obtained after 96 h; initial
reaction rate of 2.00 mM/h was observed (Table 3, entry 14). The highest initial reaction rate of
2.15 mM/h was received in hexane (Table 3, entry 13). However, the best ee value of 30% was
obtained in cyclohexane with a yield of 22% (Table 3, entry 9). In consideration of
enantioselectivity cyclohexane was chosen as the optimal solvent.
8

The ratio of water and an organic solvent also affects the enzymatic activity and
enantioselectivity. In the present study, cyclohexane was used as a solvent which is not miscible
with water and addition of water would generate a two-phase medium. Thus, influence of the ratio
of water and cyclohexane on the pepsin-catalyzed MBH reaction was investigated (Table 4). The
reaction in pure cyclohexane gave the lowest yield and ee value (Table 4, entry 1). Better results
were obtained in a two-phase medium of water and cyclohexane (Table 4, entries 2-10). It could
be seen that the ratio of water and cyclohexane obviously affected the activity and selectivity of
pepsin for the MBH reaction. The best yield of 29% with 26% ee was obtained in
water/cyclohexane (0.20/0.80, v/v) (Table 4, entry 5), but the best ee value 30% was obtained in
water/cyclohexane (0.10/0.90, v/v) and (0.15/0.85, v/v) (Table 4, entries 3 and 4). Considering the
yield and selectivity, the two-phase medium of water/cyclohexane (0.15/0.85, v/v) was chosen for
the pepsin-catalyzed MBH reaction.
The pH of a reaction medium is another important factor influencing catalytic activity and the
stability of an enzyme. Phosphate buffer was used to replace the water in the two-phase reaction
medium to test the influence of pH in the reaction system (Table 5). It could be seen that pH had
obvious effects on the pepsin-catalyzed MBH reaction. The reaction showed the best activity in
the presence of the phosphate buffer with a pH of 6.5, which gave the highest yield of 48% and
the highest ee of 32% (Table 5, entry 6). Although the addition of phosphate buffer increased the
enantioselectivity a little, it did enhance the yield. Therefore, the phosphate buffer (pH
6.5)/cyclohexane (0.15/0.85, v/v) as the optimum condition was chosen for the MBH reaction.
9

Next, the effect of the molar ratio of substrates on the pepsin-catalyzed MBH reaction was
tested. The results were obviously influenced by the molar ratio of substrates (Table 6). First, we
fixed the amount of 4-nitrobenzaldehyde (1a), and increased the amount of 2-cyclohexen-1-one
(2a) gradually (Table 6, entries 1-5). The yield and ee values showed a certain increase with
increasing amounts of 2-cyclohexen-1-one. Then we fixed the amount of 2-cyclohexen-1-one, and
increased the amount of 4-nitrobenzaldehyde (Table 6, entries 6-8). The yield and ee values
drastically decreased with increases of 4-nitrobenzaldehyde. Among the performed reactions, the
best yield of 55% with ee value of 32% was obtained when the molar ratio of 4-nitrobenzaldehyde
to 2-cyclohexen-1-one was 1:5 (Table 6, entry 5). Thus, the molar ratio of 1:5
(4-nitrobenzaldehyde 0.3 mmol to 2-cyclohexen-1-one 1.5 mmol) was identified as the optimum
ratio.
Subsequently the enzyme loading on the pepsin-catalyzed MBH reaction was surveyed
(Table 7). The yield and selectivity were influenced by the enzyme loading. The yield grew
rapidly with increased enzyme loading from 4 kU to 10 kU (Table 7, entries 1-7) yet loading past
10 kU gave no more significant increase in yield or ee. The best enantioselectivity of 32% ee was
obtained with a yield of 64% when the reaction was performed with an enzyme loading of 10 kU
(Table 7, entry 7). Therefore, an enzyme loading of 10 kU was chosen for the optimum condition
for further study.
Temperature is also a significant factor that affects enzyme stability, stereoselectivity, and the
reaction time of enzymatic reactions [56]. To further characterize the activity and selectivity of
10

pepsin in the MBH reaction, the effect of temperature was surveyed (Table 8). The
pepsin-catalyzed MBH reaction exhibited the best yield and enantioselectivity at 25 ºC, which
gave a yield of 64%, with 32% ee (Table 8, entry 1). With the temperature elevated, the yield and
ee values both descended quickly (Table 8, entries 2-6), possibly due to the partial denaturation of
the enzyme caused by higher temperature. Hence, carrying out the reaction at 25 ºC was proven to
be the best choice for the following investigation.
Subsequently, the time course of the pepsin-catalyzed asymmetric MBH reaction between
4-nitrobenzaldehyde and 2-cyclohexen-1-one was tested (Table 9). The yield and ee values
increased with time before tapering off with no further changes in yield or ee observed. The
reaction time from 12 h to 96 h gave a clear growth of yield with a slight growth in ee (Table 9,
entries 1-8). After 96 h the yield and ee were almost unchanged (Table 9, entry 9).
With these optimized conditions in hand, the substrate scope and generality of the
pepsin-catalyzed MBH reaction were studied. Various aromatic aldehydes and 2-cyclohexen-1-one
were examined in a two-phase medium of cyclohexane and phosphate buffer at 25 ºC (Table 10).
In these reactions, aromatic aldehydes exhibited good reactivity and gave 35-77% yields and
14-38% enantiomeric excesses. Benzaldehydes with a substituent in the p-position gave higher
yields and ee values (Table 10, entries 1, 3, 8, 11) than those with a substituent in the o-position or
m-position (Table 10, entries 2, 4, 5, 9, 10, 12). The best result was achieved in the reaction of
4-bromobenzaldehyde with 2-cyclohexen-1-one giving the corresponding MBH adduct in 75%
yield and 38% ee (Table 10, entry 8). When 2,3-dichorobenzaldehyde and
11

2,4-dichorobenzaldehyde were used as substrates, 56% and 57% yields and 18% and 20% ee
values were achieved (Table 10, entries 6 and 7). The MBH reaction between the
4-nitrobenzaldehyde and 2-cyclopenten-1-one could also be carried out under the optimized
conditions giving the desired MBH product in 56% yield and 16% ee (Table 10, entry 15). In
these reactions, small amount of by-products could be monitored but too little to be isolated.
Unfortunately, the reaction with electron-donating substituted benzaldehydes only gave trace
amount of products observed on the thin-layer chromatography (TLC) plate. It should be noted
that all the corresponding products listed in Table 10 are the S configuration. The absolute
configuration was determined by comparing the chiral HPLC of the products with those reported
in literature previously [33,34,39].
3 Conclusion
We have developed a pepsin-catalyzed asymmetric MBH reaction. This biocatalysis with
DABCO showed good applicability to the MBH reaction involving the addition of
2-cyclohexen-1-one or 2-cyclopenten-1-one to various aromatic aldehydes. The desired adducts
were obtained with 35-77% yields and 14-38% enantiomeric excesses. Although the yields and
stereoselectivities were not as high as desired, to the best of our knowledge, we report the first use
of pepsin and DABCO together to catalyze the MBH reaction. As an example of enzymatic
promiscuity, this novel reaction expands the field of organic synthesis. Further efforts are
underway with a focus on improving the selectivity of these enzyme-catalyzed reactions.
4 Experimental Section
12

4.1 General procedure for the pepsin-catalyzed MBH reactions
A 10 mL round-bottom flask was charged with pepsin (10 kU), aldehyde (0.30 mmol), and
cyclohexane (0.85 mL), to which the 2-cyclohexen-1-one or 2-cyclopenten-1-one (1.50 mmol) and
phosphate buffer (0.067 M, pH 6.5, 0.15 mL) were added, and then DABCO (0.06 mmol) was
introduced. The resulting mixture was stirred at 25 ºC for the specified period of time and
monitored by TLC. The reaction was terminated by filtering the enzyme. Ethyl acetate was used to
wash the filter paper to assure that products obtained were all dissolved into the filtrate. The
filtrate was dried over anhydrous Na2SO4. The solvents were removed under reduced pressure.
The residue was purified by silica gel flash chromatography with petroleum ether/ethyl acetate
(3:1-5:1) as eluent to afford the products.
4.2 Materials
All enzymes were purchased from Sigma-Aldrich, Shanghai, China. Pepsin from porcine
gastric mucosa [P7125-100G, Lot# SLBD7698V, 130 units/mg solid. One unit will produce a
∆A280nm of 0.001 per minute at pH 2.0 at 37 ºC measured as TCA-soluble products using
hemoglobin as substrate (final volumes = 16 mL, light path = 1 cm); Papain from Carica pagaya
(76220-25G, Lot# BCBD3116V, 3.6 units/mg solid); Ficin, from fig tree latex (F4165-1KU, Lot#
030M1280V, 0.1 units/mg solid); Protease from Aspergillus saitoi, TypeⅩⅢ (P2143-5G, Lot#
074K0727, 0.6 units/mg solid ); α-Chymotrypsin from bovine pancreas (C4129-500MG, Lot#
060M7007V, 62 units/mg solid)；α-Amylase from hog pancreas (10080-25G, Lot# BCBK7223V,
50 units/mg solid); Protease from Streptomyces griseus, type XIV (P5147-1G, Lot# 040M1163V,
6.1 units/mg solid); Lipase from porcine pancreas, TypeⅡ (L3126-25G, Batch# :020M1589, 24
13

units/mg solid); Lipase from wheat germ Type I (L3001-1G, Lot# SLBG5523V, 5 units/mg solid);
Lipase B Candida antarctica immobilized on Immobead 150, recombinant from yeast (52583-10G,
Lot# BCBB5644, 20 units/mg solid); Trypsin, from porcine pancreas (93615-25G, Lot# 1434759V,
1460 units/mg solid). Unless otherwise noted, all reagents were purchased from commercial
suppliers and used without further purification.
4.3 Analytical Methods
Reactions were monitored by thin-layer chromatography (TLC) with Haiyang GF 254 silica
gel plates (Qingdao Haiyang chemical industry Co Ltd, Qingdao, China) using UV light and
vanillic aldehyde as visualizing agents. Flash column chromatography was performed using
1
200-300 mesh silica gel at increased pressure. H NMR spectra and 13C NMR spectra were
respectively recorded on 600 MHz and 150 MHz NMR spectrometers. Chemical shifts (δ) were
expressed in ppm with TMS as the internal standard, and coupling constants (J) were reported in
Hz. The enantiomeric excesses (ee) of MBH products were determined by chiral HPLC analysis
performed using chiralcel OJ-H and chiralcel OD-H columns (Daicel Chiral Technologies CO.,
LTD.; Shanghai China). Absolute configurations of the MBH products were determined by
comparisons with literature and all products are known compounds.
Acknowledgements
This work was financially supported by the National Natural Science Foundation of China
(No. 21276211 and No. 21472152).
14

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17

Table 1. The screening of enzymes for the catalysis of the MBH reaction.a
Entry Type of enzyme
Enzyme
Yield (%)b
15
ee (%)c
0
1
2
Amylase
Lipase
α-Amylase from hog pancreas (1.5 kU)d
Lipase
B
Candida antarctica immobilized on
trace
--
Immobead 150, recombinant from yeast (0.1 kU)e
Lipase from porcine pancreas, Type II (0.1 kU)e
Lipase from wheat germ Type I (0.1 kU)e
Trypsin from porcine pancreas (8 kU)f
3
4
Lipase
Lipase
2
0
8
12
5
5
Protease
Protease
Protease
Protease
Protease
Protease
Protease
0
6
α-Chymotrypsin from bovine pancreas (8 kU)f
Papain from Carica pagaya (8 kU)f
6
0
7
10
17
18
23
40
2
8
Protease from Streptomyces griseus (8 kU)f
Protease from Aspergillus saitoi, Type XIII (8 kU)f
Ficin from fig tree latex (8 kU)f
0
9
2
10
11
0
Pepsin from porcine gastric mucous (8 kU)f
26
a Reaction conditions: a mixture of 4-nitrobenzaldehyde (0.30 mmol), 2-cyclohexen-1-one (0.45 mmol), deionized
water (0.10 mL), MeCN (0.90 mL), enzyme and DABCO (0.06 mmol) was stirred at 25 ºC for 96 h.
b Yield of the isolated product after chromatography on silica gel.
c ee determined by chiral HPLC using a chiralcel OJ-H column.
d Unit definition: 1 U corresponds to the amount of enzyme which liberates 1 umol maltose per minute at pH 6.9 at
25 ºC (starch acc. to Zulkowsky, Fluka NO. 85642, as substrate).
o
e Unit definition: 1 U will hydrolyze 1.0 micro equivalent of fatty acid from triacetin in 1 h at pH 7.4 at 37 C
(activity using triacetin substrate).
f
o
Unit definition: 1 U will produce a change in ∆A280nm of 0.001 per minute at pH 2.0 at 37 C, measured as
TCA-soluble products using hemoglobin as substrate (final volume = 16 mL. light path = 1 cm.).
18

Table 2. Control experiments for the pepsin-catalyzed MBH reaction.a
Natural activity
(U·mg-1 solid)d
Entry
Catalyst
Yield (%)b
ee (%)c
1
2
3
4
5
6
7
8
9
pepsin
64
16
32
0
100
--
no enzyme
no DABCO
trace
0
--
--
2
--
blank (no enzyme, no DABCO)
egg white albumin
--
22
--
pepsin pre-treated with urea for 2 he
pepsin pre-treated with urea for 12 he
pepsin pre-treated with CDI for 2 hf
pepsin pre-treated with CDI for 12 hf
26
0
33
26
38
30
18
0
27
2
21
2
a
Unless otherwise noted, the reactions were conducted using 4-nitrobenzaldehyde (0.30 mmol),
2-cyclohexen-1-one (1.50 mmol), phosphate buffer (0.067 M, pH 6.5, 0.15 mL), cyclohexane (0.85 mL), pepsin
(10 kU) and DABCO (0.06 mmol) at 25 ºC for 96 h.
b Yield of the isolated product after chromatography on silica gel.
c ee determined by chiral HPLC using a chiralcel OJ-H column.
d Unit definition (U·mg-1 solid): one unit will produce a change in ∆A280 nm of 0.001 per minute at pH 2.0 at 37
ºC measured as TCA-soluble products using hemoglobin as substrate (final volumes = 16 mL, light path = 1 cm).
e Pepsin (10 kU) in urea solution (8 M, in water) was stirred at 25 ºC for 2 h (for entry 6) or 12 h (for entry 7) and
then water was removed by lyophilization before use.
f Pepsin (10 kU) in CDI solution (1.2 M, in water) was stirred at 25 ºC for 2 h (for entry 8) or 12 h (for entry 9) and
then water was removed by lyophilization before use.
19

Table 3. Influence of solvents on the pepsin-catalyzed MBH reaction.a
Entry
1
Solvent
PhOMe
Yield (%)b
trace
ee (%)c
--
Initial reaction rate (mM/h)d
--
2
3
DMSO
1,4-Dioxane
Et2O
trace
trace
5
--
--
--
--
4
2
0.53
0.75
0.88
0.93
1.03
1.13
1.25
1.65
1.55
2.15
2.00
5
THF
8
10
10
18
22
30
16
16
20
24
26
6
Isopropanol
TCM
12
18
20
22
24
26
31
35
40
7
8
1,2-DiCl-ethane
Cyclohexane
DCM
9
10
11
12
13
14
MTBE
H2O
Hexane
MeCN
a
Reaction conditions: a mixture of 4-nitrobenzaldehyde (0.30 mmol), 2-cyclohexen-1-one (0.45 mmol), deionized
water (0.10 mL), solvent (0.90 mL), pepsin (8 kU) and DABCO (0.06 mmol) at 25 ºC for 96 h (for determination
of the yields and ee values).
b Yield of the isolated product after chromatography on silica gel.
c ee determined by chiral HPLC using a chiralcel OJ-H column.
d The initial reaction rate refers to the increase of product concentration (mM) per hour, which was detected by
HPLC analysis.
20

Table 4. Influence of the ratio of water and cyclohexane on the pepsin-catalyzed MBH reaction.a
Ratio of water and cyclohexane
Entry
1
Yield (%)b
16
ee (%)c
16
[Vwater/Vcyclohexane (mL/mL)]
0/1.00
2
3
4
5
0.05/0.95
0.10/0.90
0.15/0.85
0.20/0.80
22
22
26
29
20
30
30
26
6
7
0.25/0.75
0.30/0.70
0.40/0.60
0.50/0.50
0.60/0.40
25
24
27
21
16
26
25
18
20
19
8
9
10
a Reaction conditions: a mixture of 4-nitrobenzaldehyde (0.30 mmol), 2-cyclohexen-1-one (0.45 mmol), deionized
water (0-0.60 mL), cyclohexane (1.00-0.40 mL), pepsin (8 kU) and DABCO (0.06 mmol) at 25 ºC for 96 h.
b Yield of the isolated product after chromatography on silica gel.
c ee determined by chiral HPLC using a chiralcel OJ-H column.
21

Table 5. Effect of pH on the pepsin-catalyzed MBH reaction.a
Entry
Buffer pH
Yield (%)b
ee (%)c
1
2
4.7
5.0
5.6
5.9
6.3
6.5
7.0
7.4
8.0
8.3
30
35
37
40
45
48
28
26
25
28
22
28
28
30
32
32
30
30
28
27
3
4
5
6
7
8
9
10
a Reaction conditions: a mixture of 4-nitrobenzaldehyde (0.30 mmol), 2-cyclohexen-1-one (0.45 mmol), phosphate
buffer (NaH2PO4-Na2HPO4, 0.067 M, pH 4.7-8.3, 0.15 mL), cyclohexane (0.85 mL), pepsin (8 kU) and DABCO
(0.06 mmol) at 25 ºC for 96 h.
b Yield of the isolated product after chromatography on silica gel.
c ee determined by chiral HPLC using a chiralcel OJ-H column.
22

Table 6. Effect of molar ratio of substrates on the pepsin-catalyzed MBH reaction.a
Entry
Molar ratio (1a:2a)
Yield (%)b
ee (%)c
1
2
3
4
5
6
7
8
1:1
1:2
1:3
1:4
1:5
2:1
3:1
4:1
43
46
50
53
55
50
38
23
20
20
24
25
32
20
18
18
a Reaction conditions: a mixture of 4-nitrobenzaldehyde (0.30-1.20 mmol), 2-cyclohexen-1-one (0.30-1.50 mmol),
phosphate buffer (0.067 M, pH 6.5, 0.15 mL), cyclohexane (0.85 mL), pepsin (8 kU) and DABCO (0.06 mmol) at
25 ºC for 96 h.
b Yield of the isolated product after chromatography on silica gel.
c ee determined by chiral HPLC using a chiralcel OJ-H column.
23

Table 7. Effect of enzyme loading on the pepsin-catalyzed MBH reaction.a
Entry
Enzyme loading (kU)
Yield (%)b
ee (%)c
1
2
4
5
20
22
28
40
55
54
64
57
61
62
27
26
28
30
32
32
32
32
29
29
3
6
4
7
5
8
6
9
7
10
11
12
13
8
9
10
a
Reaction conditions: a mixture of 4-nitrobenzaldehyde (0.30 mmol), 2-cyclohexen-1-one (1.50 mmol), phosphate
buffer (0.067 M, pH 6.5, 0.15 mL), cyclohexane (0.85 mL), pepsin (4-13 kU) and DABCO (0.06 mmol) at 25 ºC
for 96 h.
b Yield of the isolated product after chromatography on silica gel.
c ee determined by chiral HPLC using a chiralcel OJ-H column.
24

Table 8. Influence of temperature on the pepsin-catalyzed MBH reaction.a
Entry
Temperature (°C)
Yield (%)b
ee (%)c
1
2
3
4
5
6
25
30
35
40
45
50
64
54
35
30
17
10
32
30
28
16
18
2
a Reaction conditions: a mixture of 4-nitrobenzaldehyde (0.30 mmol), 2-cyclohexen-1-one (1.50 mmol), phosphate
buffer (0.067 M, pH 6.5, 0.15 mL), cyclohexane (0.85 mL), pepsin (10 kU) and DABCO (0.06 mmol) at 25-50 ºC
for 96 h.
b Yield of the isolated product after chromatography on silica gel.
c ee determined by chiral HPLC using a chiralcel OJ-H column.
25

Table 9. Time course of the pepsin-catalyzed MBH reaction.a
Entry
Time (h)
Yield (%)b
ee (%)c
1
2
3
4
5
6
7
8
9
12
24
36
48
60
72
84
96
120
8
14
28
30
28
30
32
31
32
32
14
20
35
42
56
59
64
64
a Reaction conditions: a mixture of 4-nitrobenzaldehyde (0.30 mmol), 2-cyclohexen-1-one (1.50 mmol), phosphate
buffer (0.067 M, pH 6.5, 0.15 mL), cyclohexane (0.85 mL), pepsin (10 kU) and DABCO (0.06 mmol) at 25 ºC.
b Yield of the isolated product after chromatography on silica gel.
c ee determined by chiral HPLC using a chiralcel OJ-H column.
26

Table 10. Substrate scope of the pepsin-catalyzed MBH reaction.a
Entry
R
n
Product No.
Time (h)
Yield (%)b
ee (%)c
1
2
4-NO2
3-NO2
4-Cl
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
96
96
84
84
84
72
72
96
96
96
96
96
108
72
96
64
67
77
68
57
56
57
75
69
75
76
54
35
60
56
32
18
32
18
14
18
20
38
28
14
26
24
16
26
16
3a
3b
3c
3d
3e
3f
3
4
2-Cl
5
3-Cl
6
2,3-DiCl
2,4-DiCl
4-Br
7
3g
3h
3i
8
9
2-Br
10
11
12
13
14
15
3-Br
3j
4-F
3k
3l
3-F
3-CF3
H
3m
3n
3o
4-NO2
a Reaction conditions: A mixture of 1 (0.30 mmol), 2 (1.50 mmol), phosphate buffer (0.067 M, pH 6.5, 0.15 mL),
cyclohexane (0.85 mL), pepsin (10 kU) and DABCO (0.06 mmol) was stirred at 25 ºC.
b Yield of the isolated product after chromatography on silica gel.
c ee determined by chiral HPLC using a chiralcel OJ-H or a chiralcel OD-H column.
27