Protease-catalyzed direct aldol reaction

Article abstract of DOI:10.1016/j.catcom.2010.12.003

A new function of BLAP (alkaline protease from Bacillus licheniformis) was first discovered to catalyze direct aldol reactions between aromatic aldehydes and cyclic ketones in an organic medium in the presence of water. The products were obtained in yield

Full text of DOI:10.1016/j.catcom.2010.12.003

Catalysis Communications 12 (2011) 580–582
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Catalysis Communications
journal homepage: www.elsevier.com/locate/catcom
Short Communication
Protease-catalyzed direct aldol reaction
Hai-Hong Li, Yan-Hong He, Zhi Guan ⁎
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:
A new function of BLAP (alkaline protease from Bacillus licheniformis) was first discovered to catalyze direct
aldol reactions between aromatic aldehydes and cyclic ketones in an organic medium in the presence of
water. The products were obtained in yields of 28–92% with 22–99% ee.
Received 7 September 2010
Received in revised form 1 December 2010
Accepted 5 December 2010
Available online 13 December 2010
©
2010 Elsevier B.V. All rights reserved.
Keywords:
Enzyme catalysis
Alkaline protease
Enantioselectivity
Aldol reaction
1
. Introduction
2. Results and discussion
Since enzymes were discovered to maintain their activities in
During screening new activities of existing enzymes in organic
solvents, we found that BLAP could catalyze the aldol reaction. Thus,
we further investigated this novel activity of the enzyme. Initial
studies were undertaken using 4-cyanobenzaldehyde and acetone as
a model reaction. A preliminary solvent screen was performed to find
out the optimal solvent for this biotransformation (see the supporting
information). We found that the reaction gave the best yield in DMSO.
organic solvents [1], enzymes as biocatalysts have been paid more and
more attention. Not only can enzymes work in organic media, but they
acquire remarkable properties such as enhanced stability, altered
substrate and enantiomeric specificity, molecular memory, and the
ability to catalyze unusual reactions which are impossible in aqueous
media [2–4]. Although some elegant works on enzyme catalysis in
organic solvents have been reported [5–17], more reaction types
catalyzed by each available enzyme are still greatly required in order
to utilize the enzymes in organic synthesis.
The direct aldol reaction is one of the most important C–C bond-
forming reactions in organic chemistry. Berglund and co-workers
used mutant CAL-B (lipase from Candida antarctica) to catalyze aldol
addition in cyclohexane in 2003 [18]. Wang and Yu reported the aldol
reaction catalyzed by lipase in “wet” acetone in 2008 [19], and they
also found lipase-catalyzed decarboxylative aldol reaction in acetoni-
trile in 2009 [20]. More recently, we found that nuclease p1 could
catalyze direct aldol reaction under solvent-free conditions [21].
However, to the best of our knowledge, protease-catalyzed aldol
addition has not been reported, so we decided to investigate this
biocatalytic transformation with respect to the synthetic potential and
stereochemistry. Herein, we wish to report the discovery that BLAP
However, in the other tested solvents, including DMF, CH
2 2
Cl , THF,
cyclohexane, TBME (tert-butyl methyl ether), CHCl , acetone and
3
toluene, the reaction gave lower yields. So DMSO was chosen as the
optimum solvent.
To demonstrate the specific catalytic effect of BLAP, control experi-
ments were performed (see the supporting information). Just as we
expected, the aldol reaction between 4-cyanobenzaldehyde and acetone
showed no adduct in the absence of enzyme in DMSO after 139 h, which
indicated that BLAP had a specific catalytic effect on the reaction.
Furthermore, in order to prove that the catalytic activity of BLAP for aldol
reaction did not arise from unspecific protein-derived activation, the
experiment catalyzed by the inhibited enzyme was conducted. A
complete inhibition of the catalytic activity of BLAP in aldol reaction
was observed by using serine protease inhibitor PMSF (phenylmethane-
sulfonyl fluoride). In addition, since BLAP is a Ca2 -dependent enzyme,
EDTA (ethylenediaminetetraacetic acid) was also used to denature the
enzyme. We found that the EDTA-denatured BLAP completely lost its
catalytic activity for aldol reaction, which further demonstrated that the
catalytic activity of BLAP for aldol reaction did not simply arise from the
amino acid sequence of the enzyme. Based on the results of control
experiments, we validated that BLAP catalyzed the direct aldol reaction.
It is well known that water plays a major role of “molecular
lubricant” in an enzyme resulting in conformational flexibility of the
+
(
alkaline protease from Bacillus licheniformis 2709) (EC 3.4.21.14, 200
U/mg) catalyzed direct aldol reactions of aromatic aldehydes with
some cyclic ketones in an organic medium in the presence of water.
⁎
Corresponding author. Tel.: +86 23 68254091; fax: +86 23 68254091.
E-mail address: guanzhi@swu.edu.cn (Z. Guan).
1566-7367/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.catcom.2010.12.003

H.-H. Li et al. / Catalysis Communications 12 (2011) 580–582
581
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
aldol product decreased evidently. All the results indicated that water
was obviously essential in the enzyme catalyzed aldol reaction in
organic solvents. Finally, we chose water/DMSO (0.15, v/v) as the
solvent system for aldol reaction.
The temperature is another important factor for the enzyme-catalyzed
reactions, due to its effects on the selectivity and rate of the reaction, and
also on the stability of the enzyme [24]. Thus, a temperature screen was
performed (Table 1). The yield of the enzymatic reaction could be
increased by raising the temperature from 15 °C to 35 °C. However, the
enantiomeric selectivity of the product reached the best ee of 51% at 20 °C,
and higher temperature caused an evident decrease of selectivity. Thus,
the optimum temperature for the reaction was 20 °C.
Finally, to test the substrate generality of BLAP-catalyzed aldol
reaction, various aldehydes and ketones were investigated under the
optimized conditions. The enzyme showed a wide range of substrate
specificity towards aromatic aldehydes and cyclic ketones (Table 2).
It can be seen that both electron-donating and electron-withdrawing
substituents of aromatic aldehydes could be accepted by the enzyme. In
general, when cyclohexanone was used as an aldol donor, the aromatic
aldehyde containing an electron-donating group gave inferior yield. For
instance, 4-methylbenzaldehyde provided the product only in yield of
Vwater /VDMSO
Vwater /VTBME
Vwater /Vcyclohexane
0
.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50
Water content (Vwater/Vorganic solvent
)
Fig. 1. The influence of water contents in organic solvents on the yield of BLAP-
catalyzed aldol reaction. Conditions: BLAP (200 mg), acetone (1.0 mL, 17.0 mmol),
-cyanobenzaldehyde (200 mg, 1.5 mmol) and solvent (5 mL) at 35 °C for 143 h.
Deionized water was added from 0 to 0.45 (water/solvent, v/v).
4
28% with moderate anti diastereoselectivity, but it gave the excellent
enzyme and the increased hydration leads to enhanced activity in
non-aqueous solvents. There appears to be a critical amount of water
necessary for enhancing activity [22,23]. So it is important to confirm
the optimal water content in the reaction system. Therefore, we
enantioselectivity (N99% ee) (Table 2, entry 1). In contrast, aromatic
aldehydes bearing electron-withdrawing substituents furnished aldol
products in good yields (65–92%) with low to good anti diastereoselec-
tivities (58:42–95:5) but low enantioselectivities (36–70% ee) (Table 2,
entries 2–11). Moreover, substituent positions on the benzene ring of
aromatic aldehydes had a great impact on the enantio- and diastereos-
electivity of the reaction. The substituent in the 4-position led to a higher
ee value than the same substituent in the 2- or 3-position (Table 2, entries
2 and 5). However, the substituent in the 2-position gave higher dr values
than the same substituent in the 3- or 4-position (Table 2, entries 3 and 6).
The best diastereoselectivity of 95:5 (anti/syn) was achieved by using
2,6-dichlorobenzaldehyde (Table 2, entry 9). In addition, when cyclo-
pentanone and cycloheptanone were used as the donors, the aldol
products were obtained in low yields with low enantioselectivities
(Table 2, entries 12 and 13). Interestingly, anti-isomers were received as
the major products by using cyclohexanone, but reversed diastereoselec-
tivities were observed by using cyclopentanone and cycloheptanone.
BLAP-catalyzed aldol reaction seemed to prefer cyclohexanone than
cyclopentanone and cycloheptanone. It was also worthy to note that this
enzyme showed different degrees of enantioselectivity for anti isomers,
but low or no enantioselectivity for syn isomers. Besides, we found that
BLAP did not catalyze the reactions between aliphatic aldehydes and
acetone. Also, the enzyme showed poor enantioselectivities for the
reactions of aromatic aldehydes and acetone. Maybe the catalytic site of
the BLAP had a specific selectivity for the aldol reaction.
2
analyzed the effects of the amount of H O on the reaction yields of
acetone with 4-cyanobenzaldehyde in three different solvents (Fig. 1).
It can be seen that the yield of the reaction could be evidently
affected by water content in an organic solvent. There were different
optimum water contents in different solvents for BLAP to show the best
activity in aldol reaction. The optimum water content in the water-
miscible hydrophilic solvent DMSO was 0.15 (Vwater/VDMSO). Mean-
while, the optimum water content for this reaction in the hydrophobic
water-immiscible solvent TBME was 0.10 (Vwater/VTBME), and in
cyclohexane was 0.25 (Vwater/Vcyclohexane). In the case of water/
DMSO, the enzyme was adequately suspended in the miscible solvent
system, which gave a good yield of 86% under the optimum water
content conditions. However, the water/TBME and water/cyclohexane
were biphasic water–water-immiscible organic solvent systems, and
the enzyme was distributed in the aqueous phase to form an emulsion.
The enzymatic aldol reaction in these two-phase systems only gave the
product in low yields, probably due to the insufficient contact between
the enzyme which was distributed in the aqueous phase and the
substrates which were mainly distributed in the organic phase. In
addition, once the concentration of water in different solvents
surpassed the corresponding optimum water content, the yields of
Table 1
a
The optimization of temperature.
O
O
OH
OHC
enzyme
+
DMSO/H O
2
CN
CN
Entry
Temp (°C)
Time [h]
Yield [%]b
Anti/Sync
ee (%) d (anti)
1
2
3
4
5
15
20
25
30
35
47
47
22
12
12
57
59
61
61
67
65:35
75:25
57:43
51:49
50:50
49
51
41
40
21
a
Reaction conditions: 4-cyanobenzaldehyde (1.0 mmol), cyclohexanone (5.0 mmol), BLAP (200 mg), H
Yield of the isolated product after chromatography on silica gel.
Determined by HNMR analysis.
2
O (0.15 mL), and DMSO (1.0 mL).
b
c
1
d
Enantiomeric excess was determined by HPLC analysis using a chiral column; absolute configurations of the products were determined by comparison with the known chiral
HPLC analysis.

582
H.-H. Li et al. / Catalysis Communications 12 (2011) 580–582
Table 2
a
The scope of BLAP-catalyzed aldol reactions.
OH
O
O
O
BLAP
R
+
H
R
DMSO/H O, 20 °C
2
n
n
1
Entry
2
3
R
Product
n
Time [h]
Yield [%]b
Anti/Sync
ee [%]d (anti)
−
1
2
3
4
5
6
7
8
9
1
1
1
1
4–Me–C
6
H
4
3a
3b
3c
3d
3e
3f
3 g
3 h
3i
2
2
2
2
2
2
2
2
2
2
2
1
3
72
72
72
72
72
72
72
72
76
72
76
65
120
28
91
81
87
74
72
73
82
92
65
91
61
35
70:30
68:32
80:20
58:42
75:25
80:20
72:28
62:38
95:5
73:27
68:32
33:67
32:68
N99
70
44
51
55
46
50
51
36
53
−
4–NO
2–NO
2
–C
–C
6
H
H
4
−
2
6
4
−
3−NO
2
−C
6
H
4
−
4–Cl–C
2–Cl–C
3–Cl–C
6
6
6
H
H
H
4
−
4
−
4
−
2,4–Cl
2,6–Cl
2
–C
–C
6
H
3
−
2
6
H
3
−
0
1
2
3
4–Br–C
4–CF –C
4–CN–C
4–NO –C
6
H
4
3j
−
e
3
6
H
4
3 k
3 l
3 m
50/22
−
22/47f
24
6
H
4
−
2
6
H
4
a
b
c
2
Reaction conditions: aldehyde (1.0 mmol), ketone (5.0 mmol), BLAP (200 mg), H O (0.15 mL), and DMSO (1.0 mL) at 20 °C.
Yield of the isolated product (anti + syn) after chromatography on silica gel.
1
Determined by HNMR analysis.
d
Enantiomeric excess was determined by HPLC analysis using a chiral column; absolute configurations of the products were determined by comparison with the known chiral
HPLC analysis [25–29]. (See the supporting information).
e
Anti (50% ee), and syn (22% ee).
Anti (22% ee), and syn (47% ee).
f
3
. Conclusion
Appendix A. Supplementary data
We reported an enzyme-catalyzed direct aldol reaction in an
Supplementary data to this article can be found online at
doi:10.1016/j.catcom.2010.12.003
organic medium in the presence of water. BLAP as a novel biocatalyst
showed a wide range of substrate tolerance towards aldol reactions of
aromatic aldehydes and cyclic ketones. The products were obtained in
yields of 28–92% with 22–99% ee without using any additive. The
influence of reaction conditions including solvents, water content and
temperature was also investigated. The specific catalytic effect of BLAP
was demonstrated by some control experiments. This BLAP-catalyzed
direct aldol reaction provided a novel case of unnatural activities of
existing enzymes in an organic medium. Further studies focusing on
the improvement of enantioselectivity of this enzyme-catalyzed
transformation are currently under investigation.
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Acknowledgements
Financial support from the Natural Science Foundation Project of
CQ CSTC (2009BA5051) and the Selected Project in Scientific and
Technological Activities in 2007 for Returned Scholars by the State
Personnel Ministry is gratefully acknowledged.
[
[