Pepsin-catalyzed direct asymmetric aldol reactions for the synthesis of vicinal diol compounds

Article abstract of DOI:10.1016/j.tet.2015.01.061

The catalytic promiscuity of pepsin from porcine gastric mucous was observed in catalysis of the direct asymmetric aldol reactions of aromatic aldehydes with acetones, which were substituted by hydroxy-, dihydroxy-, methoxy- and benzyloxy- for the synthes

Full text of DOI:10.1016/j.tet.2015.01.061

Tetrahedron 71 (2015) 1659e1667
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Pepsin-catalyzed direct asymmetric aldol reactions for the synthesis
of vicinal diol compounds
*
*
Ling-Yu Li, 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
a r t i c l e i n f o
a b s t r a c t
Article history:
The catalytic promiscuity of pepsin from porcine gastric mucous was observed in catalysis of the direct
asymmetric aldol reactions of aromatic aldehydes with acetones, which were substituted by hydroxy-,
dihydroxy-, methoxy- and benzyloxy- for the synthesis of diol compounds in acetonitrile. This bio-
catalysis was also applicable to the aldol reactions of cyclic or hetereocyclic ketones with aromatic al-
dehydes. Yields of up to 87%, diastereoselectivities of up to >99/1 dr and enantioselectivities of up to 75%
ee were achieved.
Received 20 October 2014
Received in revised form 19 January 2015
Accepted 27 January 2015
Available online 31 January 2015
Keywords:
Ó 2015 Elsevier Ltd. All rights reserved.
Porcine pepsin
Asymmetric aldol reaction
Diol compound
Biocatalysis
1. Introduction
aldol reactions of 1-hydroxypropan-2-one or 1,3-dihydroxypropan-
2-one to aldehydes are crucial for the biosynthesis of sugars and
Aldol reaction is one of the most classical and powerful methods
to construct CeC bonds in organic synthesis.1 Chiral 1,2-diols, which
are very important building blocks are found in a vast array of
natural and biologically active molecules (Fig. 1).2 1,2-Diols can be
formed via aldol reactions of 1-hydroxypropan-2-one or 1,3-
dihydroxypropan-2-one to aldehydes. However, these compounds
are still challenging in synthesis.2a,3 Especially, in asymmetric syn-
thesis, there still exist some limitations such as the need for pro-
tected hydroxyl and dihydroxy-acetones.2a On the other hand, the
their derivatives.4 Initially, the aldol condensations for the forma-
tion of carbohydrates were catalyzed by type I aldolase or type II
aldolase through different mechanism, respectively in nature
(Scheme 1).4 However, many of such reactions were only capable of
1,3-dihydroxypropan-2-one phosphate (DHAP) as a specific donor.5
This paper first report using hydrolase (pepsin from porcine gastric
mucous) to catalyze the aldol reactions with unprotected 1-
hydroxypropan-2-one and 1,3-dihydroxypropan-2-one as electron
donors.
Enzymes as practical catalysts and enzymatic methods as part of
green technologies for organic synthesis were considered to pos-
sess great potential, and they were exploited increasingly in
asymmetric synthesis.6 Originally, enzyme catalytic reactions had
a narrow scope of application since reactions were restricted to
natural substrates of enzyme and aqueous reaction media.6c,7 In
recent years, enzyme promiscuity including condition promiscuity,
substrate promiscuity and catalytic promiscuity has emerged as
a new frontier in bio-catalysis.8 The study of enzyme promiscuity
not only provided novel research methods and tools for organic
synthesis, but also formed a bond, which led to biology and organic
chemistry moving forward together.6d,9 As promiscuous enzymes,
hydrolases with high stability, multiple sources and a wide range of
substrates, undoubtedly played a pivotal role in organic synthe-
sis.3a,10 Some elegant works on hydrolase-catalyzed synthesis have
been reported.6c,10e11 Among them, hydrolase-catalyzed aldol re-
actions have been successfully achieved. In 2003, Berglund and
Fig. 1. 1,2-Diol units in natural products.
* Corresponding authors. Fax: þ86 23 68254091; e-mail addresses: guanzhi@
swu.edu.cn (Z. Guan), heyh@swu.edu.cn (Y.-H. He).
http://dx.doi.org/10.1016/j.tet.2015.01.061
0040-4020/Ó 2015 Elsevier Ltd. All rights reserved.

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L.-Y. Li et al. / Tetrahedron 71 (2015) 1659e1667
Scheme 1. Mechanism of type I and type II aldolases.
co-workers first used lipase from Candida Antarctica (CAL-B) to
catalyze the self-aldol additions of propanal and hexanal in cyclo-
hexane. They found that mutant CAL-B (mutants Ser105Ala and
Ser105Gly) exhibited increased reaction rate than that of wild-type
lipase.12 In 2008, Wang and co-workers reported the first lipase-
catalyzed asymmetric aldol reaction, and the best ee of 43.6% was
detected for the reaction of 4-nitrobenzaldehyde and acetone.13
Subsequently, some other asymmetric aldol reactions catalyzed
by lipases from porcine and bovine pancreas were described.14 In
addition, some proteases, such as pepsin15 and trypsin from porcine
pancreas,16 chymopapain,17 ficin,18 Alcalase-CLEAÒ,19 alkaline pro-
tease from Bacillus licheniformis,20 acidic protease from Aspergillus
usamii,21 and proteinase from Aspergillus melleus,22 were disclosed
to show the promiscuous behavior in catalysis of the asymmetric
aldol reactions of aromatic aldehydes with cyclic ketones, hetero-
cyclic ketones or acetone. Besides lipases and proteases, some other
hydrolases were also found to have the ability to catalyze asym-
metric aldol reactions. For example, nuclease P1 from Penicillium
citrinum could catalyze the asymmetric aldol reactions of aromatic
aldehdes with cyclic ketones, and isatin derivatives with cyclic
ketones;23 thermophilic esterase from the archaeon Aeropyrum
pernix K1 could promote the asymmetric aldol addition of 2-
butanone and 4-nitrobenzaldehyde.24 However, to date, no pro-
miscuous hydrolase-catalyzed aldol reaction for the synthesis of
vicinal diol compounds has been reported yet. Herein, we first re-
port that pepsin from porcine gastric mucous, a member of the
well-known hydrolases, can effectively catalyze the direct asym-
metric aldol reactions to afford syn-selective 1,2-diols using un-
protected 1-hydroxypropan-2-one as
a donor and various
substituted aromatic aldehydes as accepters.
2. Results and discussion
In our initial study, we chose the reaction of 4-
nitrophenylaldehyde and 1-hydroxypropan-2-one as a model re-
action. Originally, the model aldol reaction was performed in the
absence of enzyme, and no detectable product was obtained after
120 h (Table 1, entry 1). After that, we investigated some proteases
Table 1
The screening of proteases for the catalysis of aldol reactiona
Entry
Enzyme
Time (h)
Yield (%)b
dr (syn/anti)c
ee (syn) (%)d
1
2
3
4
5
6
7
8
None
120
120
120
96
96
120
96
n.d.e
76
47
79
9
23
13
7
33
36
60
56
d
d
35
26
18
18
17
17
13
10
6
Pepsin from porcine gastric mucous
Proteinase from Aspergillus melleus, type XXIII
Protease from Rhizopus sp.
Protease from Aspergillus saitoi, type XIII
Trypsin, from porcine pancreas
Protease from Bacillus sp.
Rennet from Mucor miehei, type II
Ficin from fig tree latex
Bromelain from pineapple stem
Papain from Carica papaya
63/37
63/37
61/39
65/35
62/38
63/37
67/33
61/39
55/45
51/49
64/36
96
9
120
120
96
10
11
12
0
0
Protease from Streptomyces griseus
96
a
The reactions were conducted using 4-nitrobenzaldehyde (0.5 mmol), 1-hydroxypropan-2-one (2.5 mmol), enzyme (50 mg, lyophilized powder), MeCN (0.9 mL),
deionized water (0.1 mL) at 25 ꢀC.
b
Yield of the isolated product after chromatography on silica gel.
c
Determined by HPLC analysis of the diastereomeric isomers.
d
Determined by HPLC analysis using a chiral column (AD-H); relative and absolute configurations of the products were determined by comparison with the known 1H NMR
and chiral HPLC analysis.
e
n.d.: no product was detected.

L.-Y. Li et al. / Tetrahedron 71 (2015) 1659e1667
1661
for the catalysis of the model aldol reaction (Table 1). The best yield
of 79% was achieved using protease from Rhizopus sp. as a catalyst,
but only 18% ee was obtained (Table 1, entry 4). Luckily, using pepsin
from porcine gastric mucous as a catalyst, we obtained a good yield
of 76%, moderate dr of 63/37 with the best ee of 35% (Table 1, entry
2). In addition, some other proteases investigated also exhibited
certain catalytic activity toward the model reaction, respectively
(Table 1, entries 3e12). In view of the above-mentioned results,
pepsin from porcine gastric mucous was chosen as the catalyst.
To confirm the specific catalytic effect of pepsin on the model
aldol reaction, some control experiments were performed (Table 2).
In the absence of enzyme, no reaction occurred (Table 2, entry 1).
The model aldol reaction with the pepsin preparation gave the
product in a good yield of 76% with 35% ee (Table 2, entry 2), which
indicated that pepsin preparation indeed catalyzed the reaction in
an asymmetric manner. To exclude the possibility that some non-
enzyme components catalyzed the reaction, the pepsin prepara-
tion was pretreated at high temperature (100 ꢀC) for 24 h, and then
used to catalyze the model reaction, which only gave the product in
a low yield of 13% with 22% ee (Table 2, entry 3). At the same time,
the natural activity of pepsin in hydrolyzing hemoglobin was also
tested, and it showed that high temperature treatment caused se-
rious denaturation of pepsin (the natural activity decreased from
451 U/mg protein to 41 U/mg protein after 100 ꢀC treatment). These
results indicated that the observed catalysis effect arose from the
enzyme itself instead of non-enzyme components, and the native
fold of the enzyme was not only responsible for the natural activity
but also for the promiscuous activity. To further confirm this spec-
ulation, metal ion Cu2þ was used as a denaturation agent to pretreat
the pepsin preparation at 25 ꢀC for 24 h, and the Cu2þ pretreated
pepsin was then used to catalyze the model aldol reaction, giving
the product in a low yield of 9% with 34% ee (Table 2, entry 4).
Meantime, a parallel experiment without Cu2þ was conducted,
which gave the product in a good yield of 78% with 37% ee (Table 2,
entry 5), indicating that the process itself (the treatment of pepsin
at 25 ꢀC for 24 h) did not influence the enzyme. Moreover, Cu2þ
alone was verified no effect on the model reaction (Table 2, entry 6).
These results further confirmed that enzyme was responsible for
the observed catalytic effect, and once the enzyme denatured, its
catalytic ability in the aldol reaction nearly completely lost. In ad-
dition, pepstatin, a specific competitive peptide inhibitor of pep-
sin,25 was also used to inhibit the enzyme, and the decreases of 16%
in yield and of 3% in ee were detected in the presence of pepstatin
compared to the parallel experiment without pepstatin (Table 2,
entries 7 and 8), demonstrating that the promiscuous enzymatic
process may proceed in the active site. From the above control
experiments, it could be deduced that the specific fold of pepsin
played a decisive role for the asymmetric aldol reaction.
Reaction medium has been considered as one of the most im-
portant factors influencing catalytic activity and the stability of an
enzyme.7,26 Thus, we investigated the catalytic effects of pepsin in
differentsolvents(Table 3). Usingwaterasasolvent, apooryieldof7%
withenantioselectivityof21%eewasobtained (Table 3, entry12). The
reaction in toluene gave the product in a good yield of 87% but with
aloweevalueof26% (Table3, entry7). Amongthetestedsolvents, the
best product enantioselectivity of 35% ee in a moderate yield of 76%
was achieved in acetonitrile (Table 3, entry 1). Therefore, we chose
acetonitrile as the optimal solvent for the following studies.
As water content in an organic solvent affects both stereo-
selectivity and activity of the enzymatic reaction,16,17 we in-
vestigated the effects of different amounts of water addition in
acetonitrile on the pepsin-catalyzed model aldol reaction (Table 4).
Obviously, the water addition had a great influence on the selec-
tivity and activity of pepsin in the model aldol reaction. The best
yield of 76% with 63/37 dr and 35% ee was obtained at the water
addition of 10% [H2O/(H2OþMeCN), in vol.] (Table 4, entry 2), but
the best ee value of 42% with 70/30 dr and 70% yield was obtained
without adding water into the reaction system (Table 4, entry 1).
The acetonitrile employed was A.R. grade, and it was used directly
without drying treatment. Considering the selectivity of the
Table 2
Control experiments for the pepsin-catalyzed direct asymmetric aldol reactiona
Entry
Catalyst
Yield (%)b
dr (syn/anti)c
ee (syn) (%)d
1
2
3
4
5
6
7
8
No enzyme
Pepsin
n.d.k
76
13
d
d
63/37
57/43
61/39
60/40
d
35
22
34
37
d
Pepsin (pretreated at 100 ꢀC)e
Pepsin (pretreated with 0.25 M Cu2þ at 25 ꢀC)f
Pepsin (pretreated at 25 ꢀC)g
CuSO4 (39.9 mg)h
9
78
n.d.k
83l
67l
Pepsin (pretreated in DMSO with 10% H2O)i
Pepsin (pretreated with pepstatin in DMSO with 10% H2O)j
59/41
61/39
22
19
a
Unless otherwise noted, the reaction was conducted using 4-nitrobenzaldehyde (0.5 mmol), 1-hydroxypropan-2-one (2.5 mmol), pepsin (22.5 kU), MeCN (0.9 mL),
deionized water (0.1 mL) at 25 ꢀC for 120 h.
b
Yield of the isolated product after chromatography on silica gel.
Determined by HPLC analysis of the diastereomeric isomers.
c
d
Determined by HPLC analysis using a chiral column (AD-H).
e
Pepsin (22.5 kU) in deionized water (1.0 mL) was stirred at 100 ꢀC for 24 h, and then water was removed under reduced pressure before use.
f
Pepsin (22.5 kU) in Cu2þ solution (0.25 M, 39.9 mg CuSO4 in 1.0 mL deionized water) was stirred at 25 ꢀC for 24 h and then water was removed under reduced pressure
before use.
g
Pepsin (22.5 kU) in deionized water (1.0 mL) was stirred at 25 ꢀC for 24 h, and then water was removed under reduced pressure before use.
h
CuSO4 (39.9 mg) was used instead of pepsin.
i
Pepsin (2.25 kU) in the mixed solvents (0.09 mL DMSOþ0.01 mL H2O) was stirred at 25 ꢀC for 5 h, and then 4-nitrobenzaldehyde (0.05 mmol) and 1-hydroxypropan-2-one
(0.25 mmol) were added. The mixture was stirred at 25 ꢀC for another 120 h.
j
Pepsin (2.25 kU) and pepstatin (5 mg) in the mixed solvents (0.09 mL DMSOþ0.01 mL H2O) were stirred at 25 ꢀC for 5 h, and then 4-nitrobenzaldehyde (0.05 mmol) and 1-
hydroxypropan-2-one (0.25 mmol) was added. The mixture was stirred at 25 ꢀC for another 120 h.
k
n.d.: no reaction was detected.
l
Yield was determined by HPLC.

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L.-Y. Li et al. / Tetrahedron 71 (2015) 1659e1667
Table 3
The effect of solvents on the pepsin-catalyzed direct asymmetric aldol reactiona
Entry
Solvent
Yield (%)b
dr (syn/anti)c
ee (syn) (%)d
1
2
3
4
5
6
7
8
MeCN
EtOAc
EtOH
THF
Butyl acetate
CH2Cl2
Toluene
Cyclohexane
CHCl3
MTBE
Hexane
H2O
76
71
84
83
72
64
87
86
85
83
78
7
63/37
61/39
60/40
65/35
57/43
58/42
58/42
58/42
59/41
58/42
58/42
61/39
57/43
35
30
29
28
27
27
26
24
24
24
24
21
24
9
10
11
12
13
Organic solvent free
79
a
Unless otherwise noted, the reaction conditions were as follows: 4-nitrobenzaldehyde (0.5 mmol), 1-hydroxypropan-2-one (2.5 mmol), solvent (0.9 mL), deionized water
(0.1 mL) and pepsin (22.5 kU) at 25 ꢀC for 120 h.
b
Yield of the isolated product after silica gel chromatography.
c
Determined by 1H NMR and chiral HPLC analysis of the diastereomeric isomers.
d
Determined by HPLC analysis using a chiral column (AD-H).
Table 4
Influence of water addition on the pepsin-catalyzed direct asymmetric aldol reactiona
Entry
Water addition (%)
Yield (%)b
dr (syn/anti)c
ee (syn) (%)d
1
2
3
4
5
6
7
8
0
10
20
30
40
50
60
70
80
90
100
70
76
66
66
54
43
34
35
19
13
7
70/30
63/37
63/37
63/37
63/37
60/40
60/40
60/40
61/39
66/34
61/39
42
35
28
22
22
20
19
19
19
19
21
9
10
11
a
The reaction conditions were as follows: 4-nitrobenzaldehyde (0.5 mmol), 1-hydroxypropan-2-one (2.5 mmol), deionized water (0e1.0 mL), MeCN (1.0e0 mL) and pepsin
(22.5 kU) at 25 ꢀC for 120 h.
b
Yield of the isolated product after silica gel chromatography.
c
Determined by 1H NMR and chiral HPLC analysis of the diastereomeric isomers.
d
Determined by HPLC analysis using a chiral column (AD-H).
reaction, ultimately we selected acetonitrile (A.R.) as the optimized
solvent without addition of water for further studies.
Next, we surveyed the effect of enzyme loading on the pepsin-
catalyzed aldol reaction (Table 6). The yield and stereoselectivity
of the reaction were affected by the enzyme loading. A growing ee
value was observed with increasing enzyme loading from 4.5 kU to
22.5 kU. The best enantioselectivity of 48% ee was obtained with
a yield of 77% when the reaction was performed with an enzyme
loading of 22.5 kU (Table 6, entry 5). Thus, we chose the enzyme
loading of 22.5 kU to continue investigation.
Temperature is a crucial factor affecting stability of enzymes,
and stereoselectivity and reaction rate of enzymatic re-
actions.20,27 Hence, investigating the influence of temperature on
the pepsin-catalyzed aldol reaction was essential. We then con-
ducted the model reaction at several different temperatures
(Table 7). A relatively good result of 87% yield with 71/29 dr and
After that, we examined the effect of molar ratio of substrates on
the pepsin-catalyzed aldol reaction (Table 5). The results of the re-
action were obviously influenced by the molar ratio of substrates.
We fixed the amount of 4-nitrobenzaldehyde, and gradually in-
creased the amount of 1-hydroxypropan-2-one. The yield, dr and ee
values ascended with the increasing amounts of 1-hydroxypropan-
2-one. Among the performed experiments, the best ee value of 48%
withyield of 77% and dr value of 72/28 was obtained when the molar
ratio of 1-hydroxypropan-2-one to 4-nitrobenzaldehyde was 13:1
(Table 5, entry 6). Thus, the molar ratio of 13:1 (1-hydroxypropan-2-
one 6.5 mmol to 4-nitrophenylaldehyde 0.5 mmol) was identified as
the optimal ratio.

L.-Y. Li et al. / Tetrahedron 71 (2015) 1659e1667
1663
Table 5
The effect of mole ratio of substrates on the pepsin-catalyzed direct asymmetric aldol reactiona
Entry
Molar ratio (1a/2a)
Yield (%)b
dr (syn/anti)c
ee (syn) (%)d
1
2
3
4
5
6
7
8
1/1
1/3
1/5
1/7
1/10
1/13
1/15
1/20
25
69
70
70
74
77
75
80
70/30
70/30
70/30
69/31
73/27
72/28
72/28
71/29
23
40
42
46
47
48
48
48
a
The reaction conditions were as follows: 4-nitrobenzaldehyde (0.5 mmol), 1-hydroxypropan-2-one (0.5e10 mmol), MeCN (1.0 mL) and pepsin (22.5 kU) at 25 ꢀC for 120 h.
Yield of the isolated product after silica gel chromatography.
b
c
Determined by 1H NMR and chiral HPLC analysis of the diastereomeric isomers.
d
Determined by HPLC analysis using a chiral column (AD-H).
Table 6
Effect of enzyme loading on the pepsin-catalyzed direct asymmetric aldol reactiona
Entry
Enzyme loading (kU)
Yield (%)b
dr (syn/anti)c
ee (syn) (%)d
1
2
3
4
5
6
4.5
9.0
13.5
18.0
22.5
27
39
51
76
80
77
77
62/38
68/32
70/30
70/30
72/28
70/30
31
40
43
45
48
46
a
The reaction conditions were as follows: 4-nitrobenzaldehyde (0.5 mmol), 1-hydroxypropan-2-one (6.5 mmol), MeCN (1.0 mL) and pepsin (4.5e27 kU) at 25 ꢀC for 120 h.
Yield of the isolated product after silica gel chromatography.
b
c
Determined by 1H NMR and chiral HPLC analysis of the diastereomeric isomers.
d
Determined by HPLC analysis using a chiral column (AD-H).
Table 7
Effect of temperature on the pepsin-catalyzed direct asymmetric aldol reactiona
Entry
Temperature (ꢀC)
Yield (%)b
dr (syn/anti)c
ee (syn) (%)d
1
2
3
4
5
6
7
8
25
30
35
40
45
50
55
60
77
87
88
84
82
83
85
84
72/28
71/29
70/30
70/30
70/30
70/30
70/30
64/36
48
47
44
42
38
39
35
30
a
The reaction conditions were as follows: a mixture of 4-nitrobenzaldehyde (0.5 mmol), 1-hydroxypropan-2-one (6.5 mmol) and pepsin (22.5 kU) in MeCN (1.0 mL) was
stirring at 25e60 ꢀC for 120 h.
b
Yield of the isolated product after silica gel chromatography.
c
Determined by 1H NMR and chiral HPLC analysis of the diastereomeric isomers.
d
Determined by HPLC analysis using a chiral column (AD-H).

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L.-Y. Li et al. / Tetrahedron 71 (2015) 1659e1667
47% ee was exhibited at 30 ꢀC (Table 7, entry 2). Although tem-
perature was greatly elevated, the yield was not improved. On the
contrary, high temperature brought a decline in enantiose-
lectivity. Accordingly, carrying out the reaction at 30 ꢀC was
proven to be the best choice.
Subsequently, time course of the pepsin-catalyzed asymmetric
aldol reaction between4-nitrobenzaldehyde and 1-hydroxypropan-
2-one was tested at 30 ꢀC, 40 ꢀC and 50 ꢀC, respectively (Table 8).
Generally, the yield increased as the reaction time went on at the
early stage and finally kept almost constant. A slight growth of ee
value was detected at the early stage when the reaction was per-
formed at 30 ꢀC or 40 ꢀC (Table 8, entries 1e7), while no significant
change of dr was detected along the time course at each tempera-
ture. Obviously, increasing the temperature could accelerate the
reaction, but the lower stereoselectivities were received at higher
temperature.
To test the generality and application scope of catalytic pro-
miscuity embodied by pepsin, some other substrates including
various aldehydes and ketones were investigated to expand upon
the pepsin-catalyzed direct asymmetric aldol reaction. The results
were summarized in Table 9 and Table 10. All the experiments
showed that pepsin had the ability to tolerate different aromatic
aldehydes as acceptors and various ketones as donors such as
1-hydroxypropan-2-one, protected-1-hydroxypropan-2-one, 1,3-
dihydroxypropan-2-one, acetone and cyclic-ketones. In these
reactions, aromatic aldehydes exhibited excellent reactivity. The
interaction of aromatic aldehydes with acyclic ketones catalyzed by
pepsin mainly offered syn-products (Table 9), while anti-products
were obtained from the reactions of aromatic aldehydes with cyclic
ketones (Table 10). Substituents on the aromatic ring had a signifi-
cant impact on both the yields and enantioselectivities. Generally,
aromatic aldehydes with electron-withdrawing groups provided
higher yields than those with electron-donating substituents (Table
9, entries 1e11 and 13). Of course, different ketones as substrates
showed different effects on the aldol reaction. Acyclic ketones as
substrates reacting with various aromatic aldehydes provided low
to high yields and low to moderate enantioselectivities (Table 9).
In general, 1-hydroxypropan-2-one showed better reactivity
than dihydroxylacetone, acetone and protected 1-hydroxypropan-
2-one. For instance, the reaction of 4-nitrobenzaldehyde with
1-hydroxypropan-2-one provided product in a good yield of 87%
with 69/31 dr (syn/anti) and 47% ee (Table 9, entry 1). The best
enantioselectivity of 70% ee with 88/12 dr in a fair yield of 53%
was obtained when 1-hydroxypropan-2-one reacted with
1-naphthaldehyde (Table 9, entry 14). However, dihydroxylacetone
or acetone reacting with aromatic aldehydes only gave low yields of
11e16% with 30e52% ee (Table 9, entries 15, 16, 21 and 22). The
reactions of methoxyacetone with nitrobenzaldehydes produced
products in yields of 21e74% with 55/45->99/1 dr and 28e40% ee
(Table 9, entries 17 and 18). The reactions of 1-(benzyloxy) propan-
2-one with aromatic aldehydes gave products in yields of 24e58%
with 37e53% ee (Table 9, entries 19 and 20). Moreover, hetero-
aromatic aldehyde, 2-thenaldehyde, could also participate in the
reaction with 1-hydroxypropan-2-one giving the desired product
in a low yield of 7% with >99/1 dr and 30% ee (Table 9, entry 23). In
addition, we attempted to use aliphatic aldehydes as aldol accep-
tors, such as using isobutyraldehyde and (R)-2,2-dimethyl-1,3-
dioxolane-4-carbaldehyde to react with 1-hydroxypropan-2-one
and 1,3-dihydroxypropan-2-one, respectively, but no products
were detected after 96 h (Table 9, entries 24 and 25).
The reactions of cyclic ketones with aromatic aldehydes were
listed in Table 10. Pepsin showed good substrate adaptability to
different cyclic ketones including cyclohexanone, cyclopentanone
and heterocyclic ketones containing nitrogen, oxygen or sulfur. The
heterocyclic ketones as the aldol donors showed good reactivity.
The best yield of 85% was obtained with 77/23 dr and 66% ee for the
reaction of 4-nitrobenzaldehyde and tetrahydropyran-4-one (Table
10, entry 8). The best enantioselectivity of 75% ee with 80/20 dr and
40% yield was received when 3-nitrobenzaldehyde reacted with
cyclohexanone (Table 10, entry 2). Generally, the reactions of aro-
matic aldehydes with cyclopentanone or heterocyclic ketones gave
better yields than those with cyclohexanone.
3. Conclusion
In summary, the pepsin from porcine gastric mucous displayed
catalytic promiscuityin catalysisofdirectasymmetricaldol reactions
of aromatic aldehydes with acetones, which were substituted by
Table 8
The time course of the pepsin-catalyzed direct asymmetric aldol reaction at different temperaturesa
Entry
Time (h)
30 ꢀC
40 ꢀC
50 ꢀC
Yield (%)b
drc
ee (%)d
Yield (%)b
drc
ee (%)d
Yield (%)b
drc
ee (%)d
1
2
3
4
5
6
7
8
2
4
6
12
23
24
26
53
63
81
83
88
85
89
87
70/30
70/30
68/32
70/30
68/32
70/30
70/30
68/32
68/32
68/32
69/31
68/32
34
39
40
41
43
45
46
46
46
46
46
45
17
24
31
35
58
79
82
87
86
88
90
86
69/31
69/31
68/32
69/31
68/32
69/31
69/31
68/32
69/31
69/31
70/30
68/32
35
38
40
40
42
44
44
43
44
43
43
42
50
70
72
68
79
83
83
85
82
86
87
86
69/31
69/31
68/32
68/32
68/32
69/31
68/32
68/32
68/32
68/32
68/32
68/32
36
37
37
38
36
39
38
38
38
38
37
38
8
12
16
24
36
48
60
72
96
9
10
11
12
a
The reaction conditions were as follows: a mixture of 4-nitrobenzaldehyde (0.5 mmol), 1-hydroxypropan-2-one (6.5 mmol) and pepsin (22.5 kU) in MeCN (1.0 mL) was
stirred at 30 ꢀC, 40 ꢀC or 50 ꢀC for specified time.
b
Yield of the isolated product after silica gel chromatography.
c
dr of syn/anti determined by 1H NMR and chiral HPLC analysis of the diastereomeric isomers.
d
ee values of syn-isomer, determined by HPLC analysis using a chiral column (AD-H).

L.-Y. Li et al. / Tetrahedron 71 (2015) 1659e1667
1665
Table 9
Substrate scope of the pepsin-catalyzed direct asymmetric aldol reactions of aldehydes with acyclic ketonesa
Entry
R1
R2, R3
Prod.
Time
(h)
Yield (%)b
dr (syn/anti)c
ee (syn) (%)d
1
2
3
4
5
6
7
8
4-NO2C6H4
2-NO2C6H4
3-NO2C6H4
4-FC6H4
2-ClC6H4
4-ClC6H4
2,6-Cl2C6H3
3-BrC6H4
4-BrC6H4
4-CNC6H4
4-CF3C6H4
C6H5
4-MeC6H4
1-Naphthyl
2-NO2C6H4
4-NO2C6H4
2-NO2C6H4
4-NO2C6H4
4-NO2C6H4
4-ClC6H4
H, OH
H, OH
H, OH
H, OH
H, OH
H, OH
H, OH
H, OH
H, OH
H, OH
H, OH
H, OH
H, OH
H, OH
OH, OH
OH, OH
H, OMe
H, OMe
H, OBn
H, OBn
H, H
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
96
120
90
144
96
120
120
96
144
120
120
108
144
120
120
120
168
144
168
168
120
144
120
96
87
80
86
34
46
39
66
70
54
82
62
55
23
53
14
11
21
74
58
24
16
11
7
69/31
86/14
71/29
76/24
87/13
82/18
81/19
76/24
79/21
68/32
80/20
78/22
82/18
88/12
86/14
74/26
>99/1
55/45
60/40
74/26
d
47
64
53
49
59
45
24
54
44
11
44
66
59
70
52
30
40
28
37
53
32
46
30
d
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
3s
3t
4-NO2C6H4
4-BrC6H4
2-Thienyl
3u
3v
3w
3x
3y
H, H
d
H, OH
H, OH
OH, OH
>99/1
d
Isobutyl
n.d.e
n.d.e
2,2-Dimethyl-1,3-dioxolaneylf
96
d
d
a
Reaction conditions: a mixture of 1 (0.5 mmol), 2 (6.5 mmol) and pepsin (22.5 kU) in MeCN (1.0 mL) at 30 ꢀC.
Yield of isolated product after silica gel chromatography.
b
c
Determined by HPLC analysis of the diastereomeric isomers.
d
Determined by HPLC analysis using a chiral column; relative and absolute configurations of the products were determined by comparing with the known 1H NMR and
chiral HPLC analysis.
e
n.d.: no reaction was detected.
The 2,2-dimethyl-1,3-dioxolane-4-carbaldehyde is R configuration.
f
hydroxy-, dihydroxyl, methoxyl or benzyloxyl for the synthesis of
diol compounds in acetonitrile. This biocatalysis was also applicable
to the aldol reactions of cyclic ketones or hetereocyclic ketones with
aromatic aldehydes. Yields of up to 87%, diastereoselectivities of up
to >99/1 dr and enantioselectivities of up to 75% ee were achieved.
Although the yields and stereoselectivities were not thoroughly
satisfied, using hydrolase to catalyze the aldol reactions with un-
protected 1-hydroxypropan-2-one and 1,3-dihydroxypropan-2-one
as aldol donors to afford syn-selective 1,2-diols was first reported.
This finding not only provides a novel case for the application of
pepsin, but also offers an alternative method to prepare optically
active 1,2-diols.
(93615-25G, Lot#1434759V, 1460 units/mg solid); Protease from
Bacillus sp. (P0029-50G,119K1454,1.7 AU-NH/G); Rennetfrom Mucor
miehei, typeⅡ (R5876-10G, Lot#080M1388V); Ficin fromfig treelatex
(F4165e1KU, Lot#030M1280V, 0.1 units/mg solid); Bromelain from
pineapple stem (B4882-10G, Lot#SLBB8535V, 4 units/mg protein);
Papain from Carica papaya (76220-25G, Lot#BCBD3116V, 3.6 units/
mg solid); Protease from Streptomyces griseus, type XIV (P5147-1G,
Lot#040M1163V, 6.1 units/mg solid). Unless otherwise noted, all
reagents were purchased from commercial suppliers and used
without further purification.
4.2. Analytical methods
4. Experimental
4.1. Materials
Reactions were monitored by thin-layer chromatography (TLC)
with Haiyang GF254 silica gel plates (Qingdao Haiyang chemical
industry Co Ltd, Qingdao, China) using UV light and vanillic alde-
hyde as visualizing agents. Flash column chromatography was
performed using 100e200 mesh silica gel at increased pressure. 1H
NMR and 13C NMR spectra were recorded on Bruker-AM 300
(300 MHz) (Bruker BioSpin AG Ltd., Beijing, China) and Bruker-AM
400 (400 MHz) (Bruker BioSpin AG Ltd., Beijing, China). Chemical
shifts were reported in ppm from TMS with the solvent resonance
as the internal standard. Data were reported as follows: chemical
All enzymes were purchased from SigmaeAldrich, Shanghai,
China. Pepsin from porcine gastric mucous [P-7000-25G,
Lot#050M1304V, 920 units/mg protein (used for Tables 1e7
and 9); Lot#SLBC4920V, 420 units/mg solid (used for Tables 8
and 10). One unit will produce DA280nm of 0.001 per minute at pH
2.0 at 37 ꢀC measured as TCA-soluble products using hemoglobin as
substrate (Final volume¼16 mL, Light path¼1 cm)]; Proteinase from
A. melleus, type XXIII (P4032-25G, Lot#080M1456V, 4 units/mg
solid); Protease from Aspergillus saitoi, type XIII (P2143-5G,
Lot#074K0727V, 1.0 units/mg solid); Trypsin, from porcine pancreas
shifts (d) in ppm, coupling constants (J) in Hz, and solvent (CD3OD
and CDCl3). The enantiomeric excesses (ee) of aldol products were
determined by chiral HPLC analysis performed using Chiralcel AD-
H, Chiralpak AS-H and OD-H columns (Daicel Chiral Technologies

1666
L.-Y. Li et al. / Tetrahedron 71 (2015) 1659e1667
Table 10
Substrate scope of the pepsin-catalyzed direct asymmetric aldol reactions of aromatic aldehydes with cyclic ketonesa
Entry
1
Ar
Cyclic ketone
Prod.
Time (h)
120
Yield (%)b
19
dr (anti/syn)c
>99/1
ee (anti) (%)d
2-NO2C6H4
5a
73
2
3
4
5
6
7
8
3-NO2C6H4
4-NO2C6H4
4-ClC6H4
5b
5c
5d
5e
5f
120
120
120
96
40
38
21
72
66
60
85
80/20
65/35
88/12
67/33
64/36
67/33
77/23
75
58
15
70
63
70
66
2-NO2C6H4
4-NO2C6H4
4-ClC6H4
72
5g
5h
72
4-NO2C6H4
120
9
4-NO2C6H4
4-NO2C6H4
5i
5j
120
120
46
69
93/7
70
56
10
76/24
a
Reaction conditions: a mixture of 1 (0.5 mmol), 4 (6.5 mmol) and pepsin (22.5 kU) in MeCN (1.0 mL) at 30 ꢀC.
Yield of isolated product after silica gel chromatography.
b
c
Determined by HPLC analysis of the diastereomeric isomers.
d
Determined by HPLC analysis using a chiral column; relative and absolute configurations of the products were determined by comparing with the known 1H NMR and
chiral HPLC analysis.
CO., LTD.; Shanghai, China). Relative and absolute configurations of
the products were determined by comparing with the known 1H
NMR, 13C NMR and chiral HPLC analysis. All the aldol products are
known compounds (for details about references, please see the
Supplementary data).
purified by silica gel flash column chromatography with petroleum
ether/ethyl acetate as eluent to afford the product.
Acknowledgements
This work was financially supported by the National Natural
Science Foundation of China (No. 21276211 and No. 21472152).
4.3. General procedure for the pepsin-catalyzed aldol
reactions
Supplementary data
A round-bottom flask was charged with pepsin (22.5 kU), al-
dehyde (0.5 mmol), and ketone (6.5 mmol), to which MeCN
(1.0 mL) was introduced. The resultant mixture was stirred at 30 ꢀC
for the specified reaction time and monitored by TLC. The reaction
was terminated by filtering out the enzyme (with 40 mm Buchner
funnel and qualitative filter paper), and mixed solvents (CH3OH/
CH2Cl2, 1:4) was employed to wash the filter paper and the residue
to assure that the products were dissolved in the filtrate. The sol-
vents were then removed under reduced pressure. The residue was
Supplementary data (Characterization of all the aldol products,
and 1H NMR, 13C NMR and Chiral HPLC spectra for the aldol prod-
ucts are available as Supplementary data) related to this article can
be found at http://dx.doi.org/10.1016/j.tet.2015.01.061.
References and notes
1. (a) Machajewski, T. D.; Wong, C. H. Angew. Chem., Int. Ed. 2000, 39, 1352; (b)
Mestres, R. Green Chem. 2004, 6, 583.

L.-Y. Li et al. / Tetrahedron 71 (2015) 1659e1667
1667
2. (a) Liu, L. Y.; Wang, B.; Zhu, Y.; Chang, W. Y.; Li, J. Tetrahedron: Asymmetry 2013,
24, 533; (b) Enders, D.; Voith, M.; Lenzen, A. Angew. Chem., Int. Ed. 2005, 44,
1304.
14. (a) Guan, Z.; Fu, J. P.; He, Y. H. Tetrahedron Lett. 2012, 53, 4959; (b) Xie, Z. B.;
Wang, N.; Jiang, G. F.; Yu, X. Q. Tetrahedron Lett. 2013, 54, 945; (c) Xie, Z. B.;
Wang, N.; Zhou, L. H.; Wan, F.; He, T.; Le, Z. G.; Yu, X. Q. ChemCatChem 2013, 5,
1935.
15. Li, C.; Zhou, Y. J.; Wang, N.; Feng, X. W.; Li, K.; Yu, X. Q. J. Biotechnol. 2010, 150,
539.
3. (a) Larionova, N. A.; Kucherenko, A. S.; Siyutkin, D. E.; Zlotin, S. G. Tetrahedron
ꢀ
2011, 67, 1948; (b) Brovetto, M.; Gamenara, D.; Saenz Mendez, P.; Seoane, G. A.
Chem. Rev. 2011, 111, 4346.
4. Paradowska, J.; Pasternak, M.; Gut, B.; Gryz1o, B.; Mlynarski, J. J. Org. Chem. 2011,
77, 173.
5. (a) Iturrate, L.; García-Junceda, E. In Multi-Step Enzyme Catalysis; Wiley-VCH
GmbH KGaA: 2008; p 61; (b) Sugiyama, M.; Hong, Z.; Greenberg, W. A.; Wong,
C. H. In Biocatalysis and Biomolecular Engineering; John Wiley & Sons: 2010;
p 405.
16. Chen, Y. L.; Li, W.; Liu, Y.; Guan, Z.; He, Y. H. J. Mol. Catal. B: Enzym. 2013, 87, 83.
17. He, Y. H.; Li, H. H.; Chen, Y. L.; Xue, Y.; Yuan, Y.; Guan, Z. Adv. Synth. Catal. 2012,
354, 712.
18. Fu, J. P.; Gao, N.; Yang, Y.; Guan, Z.; He, Y. H. J. Mol. Catal. B: Enzym. 2013, 97, 1.
ꢀ
ꢀ
19. Lopez-Iglesias, M.; Busto, E.; Gotor, V.; Gotor-Fernandez, V. Adv. Synth. Catal.
2011, 353, 2345.
6. (a) Markert, M.; Mulzer, M.; Schetter, B.; Mahrwald, R. J. Am. Chem. Soc. 2007,
129, 7258; (b) Muller, M. Adv. Synth. Catal. 2012, 354, 3161; (c) Humble, M. S.;
Berglund, P. Eur. J. Org. Chem. 2011, 2011, 3391; (d) Koeller, K. M.; Wong, C. H.
Nature 2001, 409, 232.
20. Li, H. H.; He, Y. H.; Guan, Z. Catal. Commun. 2011, 12, 580.
21. Xie, B. H.; Li, W.; Liu, Y.; Li, H. H.; Guan, Z.; He, Y. H. Tetrahedron 2012, 68, 3160.
22. Yuan, Y.; Guan, Z.; He, Y. H. Sci. China Chem. 2013, 56, 939.
23. (a) Li, H. H.; He, Y. H.; Yuan, Y.; Guan, Z. Green Chem. 2011, 13, 185; (b) Liu, Z. Q.;
Xiang, Z. W.; Shen, Z.; Wu, Q.; Lin, X. F. Biochimie 2014, 101, 156.
24. Wang, H. R.; Wang, Z.; Zhang, H.; Chen, G.; Yue, H.; Wang, L. Green Chem. Lett.
Rev. 2014, 7, 145.
€
7. Klibanov, A. M. Nature 2001, 409, 241.
8. Hult, K.; Berglund, P. Trends Biotechnol. 2007, 25, 231.
ꢀ
9. (a) Almas¸ i, D.; Alonso, D. A.; Balaguer, A. N.; Najera, C. Adv. Synth. Catal. 2009,
351, 1123; (b) Wu, C.; Fu, X.; Li, S. Tetrahedron: Asymmetry 2011, 22, 1063.
10. (a) Busto, E.; Gotor-Fernandez, V.; Gotor, V. Chem. Soc. Rev. 2010, 39, 4504; (b)
Kapoor, M.; Gupta, M. N. Process Biochem. 2012, 47, 555.
11. Wu, Q.; Liu, B. K.; Lin, X. F. Curr. Org. Chem. 2010, 14, 1966.
12. Branneby, C.; Carlqvist, P.; Magnusson, A.; Hult, K.; Brinck, T.; Berglund, P. J. Am.
Chem. Soc. 2002, 125, 874.
25. (a) Marcinkeviciene, J.; Kopcho, L. M.; Yang, T.; Copeland, R. A.; Glass, B. M.;
Combs, A. P.; Falahatpisheh, N.; Thompson, L. J. Biol.Chem. 2002, 277, 28677; (b)
~
ꢀ
Cofrades, S.; Banon, S.; Carballo, J.; Jimenez Colmenero, F. Food Chem. 2003, 80,
241; (c) Yonezawa, H.; Uchikoba, T.; Kaneda, M. J. Biochem. 1997, 122, 294; (d)
Setsuko, K.; Takaaki, A.; Hajime, M.; Tomio, T.; Hamao, U. J. Antibiot.1972, 25, 251.
26. Wescott, C. R.; Klibanov, A. M. Biochim. Biophys. Acta 1994, 1206, 1.
27. Zaks, A.; Klibanov, A. Science 1984, 224, 1249.
13. Li, C.; Feng, X. W.; Wang, N.; Zhou, Y. J.; Yu, X. Q. Green Chem. 2008, 10, 616.