The enzymatic asymmetric aldol reaction using acidic protease from Aspergillus usamii

Article abstract of DOI:10.1016/j.tet.2012.02.056

AUAP (acidic protease from Aspergillus usamii) could catalyze the direct aldol reactions between aromatic aldehydes and cyclic ketones in acetonitrile (MeCN) in the presence of water. The enantioselectivities of up to 88% ee and diastereoselectivities of

Full text of DOI:10.1016/j.tet.2012.02.056

Tetrahedron 68 (2012) 3160e3164
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The enzymatic asymmetric aldol reaction using acidic protease from Aspergillus
usamii
,
a,
*
Bang-Hua Xie a, Wei Li b, Yan Liu b, Hai-Hong Li a, Zhi Guan a , Yan-Hong He
*
a School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China
b School of Life Science, Southwest University, Chongqing 400715, China
a r t i c l e i n f o
a b s t r a c t
Article history:
AUAP (acidic protease from Aspergillus usamii) could catalyze the direct aldol reactions between aromatic
aldehydes and cyclic ketones in acetonitrile (MeCN) in the presence of water. The enantioselectivities of
up to 88% ee and diastereoselectivities of up to 97:3 (anti/syn) were achieved. This new activity of
protease expands the application of the biocatalyst and provides a novel example of enzymatic
promiscuity.
Received 7 September 2011
Received in revised form 19 February 2012
Accepted 24 February 2012
Available online 1 March 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Biocatalytic promiscuity
Acidic protease
Enantioselectivity
Aldol reaction
1. Introduction
enantioselectivity in recent years, the development of sustainable
and cost-efficient biocatalysts for the asymmetric aldol reaction still
New catalytic synthetic methods in organic chemistry that sat-
isfy increasingly stringent environmental constraints are in great
demand by the pharmaceutical and chemical industries. Enzyme-
catalyzed chemical transformations are now widely recognized as
practical alternatives to traditional (non-biological) organic syn-
thesis, and as convenient solutions to certain intractable synthetic
problems.1 Biocatalytic promiscuity, a new frontier extending the
use of enzymes in organic synthesis, has attracted much attention
and expanded rapidly in recent years.2,3 It means that enzymes were
capable of catalyzing not only their natural reactions but also one or
more alternative reactions. In the past few years, though some el-
egant works on enzymatic promiscuity have been reported,2,3a,4e7
exploiting more reaction types catalyzed by each available en-
zyme is still necessary, due to the high chemo-, regio-, and stereo-
selectivity and non-toxicity of enzymes in organic synthesis.8
The asymmetric aldol reaction is one of the most useful methods
for carbonecarbon bond formation in organic synthesis.9,10 It is
a useful approach for the preparation of pharmaceuticals, fine
chemicals and natural products, but is also considered a powerful
strategy for the preparation of complex molecules, since it allows
the linking of two units to obtain a more complex structure.
Although numerous successful organocatalysts for asymmetric
aldol reaction have been described with high efficiency and
remains a significant challenge. Wang and co-workers recently re-
ported the first enzyme-catalyzed asymmetric aldol addition be-
tween acetone and different aromatic aldehydes with strong
electron-withdrawing groups employing pig pancreas lipase (PPL)
as catalyst in wet reaction media in 2008,11 and they also found that
pepsin could catalyze aldol reactions in aqueous media in 2010.12
Very recently, our laboratory reported that nuclease p1 and alka-
line protease could catalyze direct asymmetric aldol reactions.13,14
As our continuous work on synthetic applications of enzymes,
herein, we wish to report the discovery that AUAP (acidic protease
from Aspergillus usamii No. 537) catalyzed direct asymmetric aldol
reactions of aromatic aldehydes with some cyclic ketones in organic
medium in the presence of water under mild reaction conditions.
2. Results and discussion
During the wide screening of promiscuous activities of enzymes,
we found that AUAP could catalyze direct asymmetric aldol re-
action. Thus, we further investigated this novel activity of AUAP. In
our initial studies, the aldol reaction of cyclohexanone and 4-
nitrobenzaldehyde was used as a model transformation. Since it
is known that enzymes not only can maintain their activities in
organic solvents, but also can acquire remarkable properties such
as enhanced stability, altered substrate and enantiomeric specific-
ities, and the ability to catalyze unusual reactions, which are im-
possible in aqueous media,15 therefore, some conventional organic
* Corresponding authors. Tel./fax: þ86 3 68254091; e-mail addresses: guanzhi@
swu.edu.cn (Z. Guan), heyh@swu.edu.cn (Y.-H. He).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2012.02.056

B.-H. Xie et al. / Tetrahedron 68 (2012) 3160e3164
3161
solvents were screened for the AUAP catalyzed aldol reaction (Table
1). Among the solvents used, AUAP exhibited the best catalytic ac-
tivity and stereoselectivity in MeCN, which gave the corresponding
aldol product in good yield of 63% with the enantioselectivity of 82%
ee for anti isomer (anti/syn 83:17) (Table 1, entry 1). The reaction in
CH2Cl2 afforded the aldol product with good ee of 80%, but in low
yield of 33% (Table 1, entry 2). The other tested solvents gave the
product in lower enantioselectivities (Table 1, entries 3e9). The re-
actionwas also carried out under solvent-free conditions, which only
gave a low yield of 37% with moderate stereoselectivity of 63% ee
(Table 1, entry 10). Interestingly, when water was used as a solvent,
the reaction only gave a low yield of 19% with moderate ee of 60%
(Table 1, entry 11). The results clearly indicated that AUAP displayed
better reaction activity and selectivity in most organic solvents than
in water towards aldol reaction. So we chose MeCN as the optimum
solvent for the AUAP catalyzed direct asymmetric aldol reaction.
completely lost its catalytic activity for aldol reaction (Table 1, en-
tries 14 and 15), which excluded the possibility that the reaction
was caused by the catalysis simply arose from amino acids of the
protein. On the other hand, it suggested that the tertiary structure
of the enzyme is necessary to catalyze the reaction. In addition,
since the enzyme AUAP we used was purchased as an industrial
enzyme preparation, to further rule out the possibility of the ca-
talysis of some impure protein or other impurities, we purified the
enzyme (for the purification of enzyme, see Supplementary data),
and used the purified AUAP to catalyze the model aldol reaction in
MeCN. The purified AUAP showed high activity and good selectiv-
ity, and only 10 mg/ml of purified AUAP could give the product in
yield of 45% with 74% ee for anti isomer (anti/syn 80:20) (Table 1,
entry 16), which was almost 10 times more active than industrial
enzyme preparation of AUAP (as described above, 100 mg/ml of
industrial enzyme preparation of AUAP gave the product in yield
Table 1
Solvent screening and control experimentsa
O
OH
O
OHC
AUAP
+
solvent/H2O, 25 °C
NO2
NO2
3a
1
2a
Entry
Solvent
Yieldb [%]
anti/sync
eec [%]
(anti)
1
2
3
4
5
6
7
8
MeCN
CH2Cl2
DMSO
DMF
EtOH
THF
Toluene
Cyclohexane
1,4-Dioxane
Solvent-free
H2O
63
33
49
46
53
29
23
26
39
37
19
Trace
45
83:17
71:29
66:34
72:28
68:32
70:30
67:33
67:33
72:28
62:38
67:33
d
82
80
47
56
57
66
73
76
67
63
60
d
0
9
10
11
12
13
14
MeCN (no enzyme)
MeCN (bovine serum albumin)
MeCN (enzyme denatured
with NBSd)
49:51
d
Trace
d
15
MeCN (enzyme denatured
Trace
d
d
with ureae)
16
MeCN (purified AUAP)f
45
80:20
74
a
Reaction conditions: AUAP (100 mg), 1 (2.5 mmol), 2a (0.5 mmol), deionized water (0.10 mL) and solvent (0.9 mL) at 25 ꢀC for 144 h.
Yield of the isolated product after chromatography on silica gel.
b
c
d
e
f
Determined by chiral HPLC analysis (AD-H).
Pre-treated with NBS at 25 ꢀC for 24 h.
Pre-treated with urea at 100 ꢀC for 24 h.
Reaction conditions: purified AUAP (5 mg), 1 (1.5 mmol), 2a (0.3 mmol), deionized water (0.05 mL) and solvent (0.45 mL) at 25 ꢀC for 144 h.
In order to verify the specific catalytic effect of acidic protease
of 63% with 82% ee for anti isomer (anti/syn 83:17) (Table 1, entry
1)). The experiment with purified AUAP clearly confirmed that
AUAP indeed has the promiscuity for the catalysis of direct asym-
metric aldol reaction. Thus, we continued using the commercially
available industrial enzyme preparation of AUAP in the following
investigation.
In previous studies of enzymatic promiscuity, water has been
considered as a very important factor in enzymatic activity leading
to an acceleration of the enzyme-catalyzed reaction.16 Therefore, in
order to confirm the optimal water content in the AUAP catalyzed
aldol reaction, we decided to carry out the reaction with different
water content in MeCN. The range of water concentration from 0 to
40% (water/[waterþMeCN], v/v) was screened for the AUAP cata-
lyzed aldol reaction (Fig. 1). As shown in Fig. 1, the activity and
selectivity of the enzyme could be affected by the water content.
When the reaction was performed with 10% water content, AUAP
exhibited the highest activity and selectivity, and the best ee value
on the aldol reaction, we performed some control experiments
(Table 1, entries 12e16). The aldol reaction between 4-
nitrobenzaldehyde and cyclohexanone gave trace adduct in the
absence of enzyme in MeCN after 144 h, which indicated that AUAP
had a specific catalytic effect on the reaction (Table 1, entry 12).
Furthermore, in order to prove that the catalytic activity of AUAP
for aldol reaction did not arise from unspecific protein-derived
activation, bovine serum albumin (BSA) was used to catalyze the
model reaction in MeCN, which gave the product in yield of 45%,
but no enantioselectivity and almost no diastereoselectivity were
observed (Table 1, entry 13). The experiment indicated that the
non-enzyme protein BSA also had the ability to catalyze the aldol
reaction, but it did not display any selectivity. Besides, the experi-
ments catalyzed by the denatured enzyme were conducted. NBS
(N-bromosuccinimide) and urea were used to denature the en-
zyme. We found that the NBS-denatured and urea-denatured AUAP

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B.-H. Xie et al. / Tetrahedron 68 (2012) 3160e3164
To further improve the AUAP catalyzed aldol reaction, we then
yield
ee
80
70
60
50
40
30
20
10
0
examined the effects of molar ratio of substrates and enzyme con-
centration on the aldol reaction. As a result, 4-nitrobenzaldehyde/
cyclohexanone¼1:5 and enzyme concentration of 100 mg/ml were
chosen as the optimal conditions (for details see Supplementary
data).
Finally, we investigated the time course of the aldol reaction be-
tween 4-nitrobenzaldehyde and cyclohexanone catalyzed by AUAP
undertheoptimalconditions(Fig. 3). Itcouldbeseenthatthereaction
progressed at a nearly constant rate for the first 8 days, and after that
the yield did not increase. The best yield of 66% was observed.
Moreover, there was a slight increase of ee value for the first 2 days,
and after that the ee value almost kept invariably at about 80%.
100
yield
ee
90
0
10
20
30
40
80
70
60
50
40
30
20
10
Water content (%)
Fig. 1. Influence of water content on the AUAP catalyzed aldol reaction. Reaction
conditions: AUAP (100 mg), 4-nitrobenzaldehyde (0.5 mmol), cyclohexanone
(2.5 mmol), deionized water from
0
to 40% (water/[waterþMeCN], v/v, [water-
þMeCN]¼1 mL) at 25 ꢀC for 144 h; yield of the isolated product after chromatography
on silica gel; ee was determined by HPLC analysis on a Chiralpak AD-H column.
of 82% and yield of 63% were obtained. However, once the water
content exceeded 10%, the yield and ee value decreased, which
probably due to that excessive water leads to a serious decrease of
the solubility of substrates and change of the special conformation
of AUAP. Thus, 10% water content was chosen as the optimal con-
dition for the following studies.
0
2
4
6
8
10
12
Temperature plays an important role in enzyme-catalyzed re-
actions, due to its effects on the selectivity and rate of the reaction,
and the stability of the enzyme. Thus, we further investigated the
effect of temperature on the AUAP catalyzed aldol reaction (Fig. 2).
A noticeable increase of yield was obtained by raising the tem-
perature from 10 to 40 ꢀC, and AUAP exhibited the highest activity
at 40 ꢀC. However, the best enantioselectivity of the product was
obtained at 25 ꢀC, and higher temperature led to a remarkable
decrease of the ee value. In order to get the best enantioselectivity,
we chose 25 ꢀC as the optimal temperature for the AUAP catalyzed
aldol reaction.
Time (d)
Fig. 3. Time course of the AUAP catalyzed aldol reaction. Reaction conditions: AUAP
(100 mg), 4-nitrobenzaldehyde (0.5 mmol), cyclohexanone (2.5 mmol), deionized
water (0.10 mL) and MeCN (0.9 mL) at 25 ꢀC for 1e10 days; yield of the isolated
product after chromatography on silica gel; ee was determined by HPLC analysis on
a Chiralpak AD-H column.
Finally, with the optimal reaction conditions in hand, we further
studied the generality of the AUAP catalyzed direct asymmetric aldol
reaction (Table 2). It could be seen that the aldol reactions of cyclic
ketones to a number of aromatic and heteroaromatic aldehydes
proceeded smoothly to afford aldol adducts in moderate to good
enantioselectivities with good to excellent diastereoselectivities. In
general, the aromatic aldehydes bearing electron-withdrawing
substituents (Table 2, entries 1e11) were converted to the corre-
sponding aldol products in higher yields than the aromatic alde-
hydes containing electron-donating groups (Table 2, entry 13).
Moreover, the substituent positions on the benzene ring of aromatic
aldehydes had a great impact on the enantio- and diaster-
eoselectivity of the reaction. For example, the aldol reaction of m-
chlorobenzaldehyde with cyclohexanone generated the aldol prod-
uct with higher dr and ee values than o- and p-chlorobenzaldehydes
(Table 2, entries 3e5). To our surprise, the reaction appeared quite
tolerant with respect to the steric contribution of the substituents in
substituted benzaldehydes, and the most sterically hindered 2,6-
dichlorobenzaldehyde provided the product in an acceptable yield
of 59% with the best diastereoselectivity (97:3) and moderate
enantioselectivity (52% ee) (Table 2, entry 11). In contrast, the re-
action of the least sterically hindered benzaldehyde with cyclohex-
anone afforded the aldol product only in 26% yield with moderate
diastereoselectivity (72:28) and higher enantioselectivity (77% ee)
(Table 2, entry 12). Furthermore, heteroaromatic aldehydes 2-
furaldehyde and 2-thiophenaldehyde were also used as the sub-
strates, which gave the corresponding aldol products in low yields
100
90
80
70
60
50
40
30
20
10
yield
ee
10
20
30
40
50
60
o
T ( C)
Fig. 2. Influence of temperature on the AUAP catalyzed aldol reaction. Reaction con-
ditions: AUAP (100 mg), 4-nitrobenzaldehyde (0.5 mmol), cyclohexanone (2.5 mmol),
temperature (10e50 ꢀC), deionized water (0.10 mL) and MeCN (0.9 mL) for 144 h; yield
of the isolated product after chromatography on silica gel; ee was determined by HPLC
analysis on a Chiralpak AD-H column.

B.-H. Xie et al. / Tetrahedron 68 (2012) 3160e3164
Table 2 (continued )
3163
Table 2
Scope of the AUAP catalyzed direct asymmetric aldol reaction under optimal
Entry
Product
No. Time Yieldb anti/
eec [%]
(anti)
conditionsa
[h]
[%]
sync
O
OH
O
O
O
OH
AUAP
MeCN/H2O, 25 oC
R
+
12
3l
168
26
72:28 77
H
R
2
3
1
O
OH
13
3m 168
11
86:14 82
Entry
Product
No. Time Yieldb anti/
eec [%]
(anti)
[h]
[%]
sync
CH3
O
OH
O
O
OH
OH
1
2
3a
3b
144
63
83:17 82
O
S
14
15
3n
3o
168
168
16
10
67:33 70
NO2
NO2
O
OH
168
168
61
20
74:26 85
75:25 75
O
OH
3
4
3c
82:18 76
OH
O
Cl
Cl
16
3p
144
78
53:47 60:40d
O
OH Cl
NO2
3d
168
48
87:13 62
a
Reaction conditions: AUAP (100 mg), ketone
(0.5 mmol), MeCN (0.9 mL) and deionized water (0.10 mL) at 25 ꢀC.
1 (2.5 mmol), aldehyde 2
b
Yield of the isolated product after chromatography on silica gel.
Determined by chiral HPLC analysis (AD-H, OD-H, AS-H).
anti (60% ee), and syn (40% ee).
c
O
O
OH
OH
d
5
6
3e
3f
168
168
29
20
92:8
88
with moderate diastereoselectivities and enantioselectivities (Table
2, entries 14 and 15). In addition, to further examine the generality of
this catalytic system, cyclopentanone was also used as the aldol
donor. The aldol reaction of cyclopentanone with 4-nitro-
benzaldehyde gave the corresponding product in good yield of 78%
but with low enantioselectivity (60% ee) and diastereoselectivity
(53:47) (Table 2, entry 16). It is worthy to note that the AUAP cata-
lyzed aldol reaction mainly provided anti-isomers as the major
products with moderate to high enantioselectivities, but low or no
enantioselectivity for syn-isomers. The yield of the aldol reaction
catalyzed by AUAP is still low and the reaction mechanism is unclear
at the moment. Further efforts to deal with these problems are
currently undertaken.
72:28 70
F
O
OH
OH
7
8
3g
3h
168
168
32
47
82:18 88
75:25 80
Br
O
CF3
CN
3. Conclusion
O
O
OH
OH
9
3i
3j
168
168
57
59
84:16 88
In summary, we described a novel example of biocatalytic pro-
miscuity, in which AUAP was used to catalyze the direct aldol re-
action of aromatic and heteroaromatic aldehydes with cyclic
ketones under mild reaction conditions. The reaction was carried
out in MeCN/H2O system, and the corresponding aldol products
were obtained in moderate to excellent diastereoselectivities (up to
97:3) with moderate to high enantioselectivities (up to 88% ee). The
influence of some parameters including solvents, water contents,
temperature, molar ratio of substrates and enzyme concentration
were also investigated. This AUAP catalyzed direct asymmetric al-
dol reaction expands the application of the biocatalyst in organic
synthesis.
10
79:21 74
CN
O
OH Cl
11
3k
168
59
97:3
52
Cl

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B.-H. Xie et al. / Tetrahedron 68 (2012) 3160e3164
4. Experimental section
References and notes
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