Biocatalytic direct asymmetric aldol reaction using proteinase from Aspergillus melleus

Article abstract of DOI:10.1007/s11426-013-4874-0

The direct asymmetric aldol reaction of aromatic aldehydes with cyclic or acyclic ketones was catalyzed by proteinase from Aspergillus melleus (AMP) in acetonitrile in the presence of water. A wide range of substrates could be transformed into the corresp

Full text of DOI:10.1007/s11426-013-4874-0

SCIENCE CHINA
Chemistry
•
ARTICLES •
Ja nd uo ai :r y1 02 . 01 10 30 7/ sV1 1o 4l . 25 6-0 1N3 o- 4. 18 :7 14 –- 06
doi: 10.1007/s11426-013-4874-0
Biocatalytic direct asymmetric aldol reaction using proteinase
from Aspergillus melleus
*
*
YUAN Yi, GUAN Zhi & HE YanHong
School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China
Received November 26, 2013; accepted January 19, 2013
The direct asymmetric aldol reaction of aromatic aldehydes with cyclic or acyclic ketones was catalyzed by proteinase from
Aspergillus melleus (AMP) in acetonitrile in the presence of water. A wide range of substrates could be transformed into the
corresponding aldol products in yields up to 89%, enantioselectivities up to 91% ee and diastereoselectivities up to >99:1
(anti/syn). This work provided an example of enzyme catalytic promiscuity that widens the applicability of this biocatalyst in
organic synthesis without the need for additional cofactors or special equipment.
enzyme catalysis, aldol reaction, proteinase, promiscuity, asymmetric synthesis
1
Introduction
Consequently, their catalytic activity with unnatural sub-
strates in organic media is a realm attracting much attention
5]. Several examples with regard to the ability of hydrolyt-
ic enzymes to catalyze non-conventional synthetic organic
reactions, which are a long way from their natural scope
have recently been reported, such as Michael additions [6],
Markovnikov additions [7], direct Mannich reactions [8],
and Henry reactions [9].
Carbon-carbon bond formation is one of the most im-
portant keystone reactions in organic synthetic chemistry.
Among the available methods, the direct asymmetric aldol
reaction is a powerful strategy due to the concomitant crea-
tion and functionalization of stereogenic centers [10].
Thereby, developing catalysts for direct asymmetric aldol
reaction is in great demand. Catalysts like aldolases [11],
catalytic antibodies [12], and small molecules [13] have
been used in the aldol reactions. However, there are only a
few examples of aldol reactions catalyzed by hydrolases
[
Development of green catalytic methods for organic synthe-
sis is a topic of continuing interest. Biocatalysts often per-
form an economically feasible, ecologically advantageous
role which is more sustainable than current chemical tech-
nologies, and its high reaction selectivity, mild reaction
conditions and potential use of inexpensive regenerable
resources have often been proved [1]. Enzymes are efficient
and green biocatalysts in synthetic chemistry. Both “natu-
ral” reactions and other alternative reactions can be cata-
lyzed by enzymes [2]. The ability of enzymes to Catalyze
unnatural reactions is known as enzymatic promiscuity. As
one of the most outstanding concepts in biocatalysis, enzy-
matic promiscuity has caught researchers’ attention [3].
Hydrolases are a group of enzymes, which conventionally
catalyze bond cleavage using water as nucleophile. Due to
their high stability and activity with a broad range of sub-
strates, they have contributed significantly to the increasing
use of enzymes in the industrial manufacture of agrochemi-
cals, pharmaceuticals, and high value-added compounds [4].
[
14]. Recently, our group has reported some direct asym-
metric aldol reactions using nuclease p1 [15], alkaline pro-
tease [16], acidic protease [17], and chymopapain [18] as
catalysts. However, enzymatic promiscuity is still at the
exploratory stage, and no general methods are available to
profile enzyme catalytic promiscuity [19]. Therefore, it is
*
Corresponding authors (email: guanzhi@swu.edu.cn, heyh@swu.edu.cn)
©
Science China Press and Springer-Verlag Berlin Heidelberg 2013
chem.scichina.com www.springerlink.com

2
Yuan Y, et al. Sci China Chem January (2013) Vol.56 No.1
still a formidable but significant task to explore enzyme
catalyzed asymmetric aldol reactions and other reactions as
much as possible. In continuation of our interest in hydrolase-
catalyzed direct asymmetric aldol reaction, in this paper,
we established another new synthetic possibility of aldol
reaction by using a commercially available proteinase
from Aspergillus melleus, type XXIII (AMP) as a catalyst
without the need for additional cofactors or special
equipment.
reaction. Since the reaction medium plays a crucial role in
the maintenance of enzyme catalytic activity and stability
[20], the influence of some common solvents on the
AMP-catalyzed aldol reaction was investigated (Table 1).
Among the tested solvents, satisfactory stereoselectivities of
84% ee (with 85:15 dr, 53% yield) and 83% ee (with 83:17
dr, 28% yield) were obtained in MeCN and CHCl
3
, respec-
tively (Table 1, entries 1 and 2). Satisfactory yields of 80%
and 84% were achieved in ethanol and DMSO with low
enantioselectivities of 35% ee and 33% ee, respectively
(Table 1, entries 12 and 13). The reaction in other tested
2
Experimental
solvents including toluene, butyl acetate, THF, MTBE, cy-
clohexane, i-propyl ether and 1,4-dioxane gave moderate
yields and selectivities (Table 1, entries 3–5, and 7–10).
Moreover, it is noteworthy that AMP-catalyzed model aldol
reaction in water only gave product in a yield of 47% with a
low enantioselectivity of 48% ee (Table 1, entry 11). The
reaction under solvent-free conditions was also investigated,
which gave product in the best yield of 85% with 70% ee
(80:20 dr) (Table 1, entry 6). The results clearly indicated
that the catalytic activity and stereoselectivity of AMP were
significantly influenced by the reaction media. It seems that
the AMP-catalyzed model aldol reaction in high polar sol-
vents was in favour of higher yield, however, the reaction in
low to middle polar solvents appeared to give products in
higher selectivity. Thus, considering stereoselectivity of the
reaction, we chose MeCN as a solvent for the following
study.
Next, in order to verify the specific catalytic effect of
AMP on the aldol reaction, some control experiments were
performed in MeCN (Table 1, entries 14–19). Just as we
expected, the model aldol reaction in the absence of AMP
only afforded a trace amount of product after 90 h (Table 1,
entry 14). As comparison, a non-enzyme protein (bovine
serum albumin) was also used to catalyze the model reac-
tion, which gave the product in 47% yield without any en-
antioselectivity (Table 1, entry 19). This reaction suggested
that protein without enzyme function also has the ability to
catalyze the aldol reaction. Therefore, it is necessary to ver-
ify whether the catalytic activity of AMP for aldol reaction
arose from unspecific protein-derived activation of the rea-
gents by, for instance the surface of the enzyme. Thus, the
experiments with denatured and inhibited AMP were con-
ducted. The experiment with urea-denatured AMP only gave
product in 11% yield with 9% ee (Table 1, entry 15). The
experiment with phenylmethanesulfonyl fluoride (PMSF)-
inhibited AMP provided the product only in 6% yield with
6% ee (Table 1, entry 17). Meantime, the contrast experi-
ments were conducted using urea and PMSF to catalyze the
reaction, and only 4% and 5% yields were obtained, respec-
tively (Table 1, entries 16 and 18), which showed that urea or
PMSF alone only had tiny effect on the reaction. Therefore,
it was experimentally verified that inhibition and denatura-
tion of AMP caused an almost complete disruption of the
catalytic activity and selectivity of the enzyme. As a con-
2
.1 Material
Proteinase from Aspergillus melleus (Type XXIII, ≥3
units/mg solid; one unit will hydrolyze casein to produce
color equivalent to 1.0 mol (181 μg) of tyrosine per min at
pH 7.5 at 37 °C (color by Folin-Ciocalteu reagent)) was
purchased from Sigma-Aldrich Co. LLC. Unless otherwise
noted, all reagents were obtained from commercial suppliers
and were used without further purification.
2
.2 General procedure for preparation of products 3a–t
by aldol reaction
A 10 mL round-bottomed flask was charged with AMP (50
mg), aldehyde (0.5 mmol), ketone (2.5 mmol), MeCN (0.90
mL) and deionized water (0.10 mL). The resulting mixture
was stirred for the specified period of time at 30 °C. The
reaction was terminated by filtering the enzyme. Ethyl ace-
tate was used to wash the filter paper to assure that products
obtained were all dissolved in the filtrate. Saturated brine
(
15 mL) was then added to the filtrate, and the filtrate was
extracted three times with ethyl acetate (15 mL). The com-
bined extracts were dried over anhydrous Na
SO , and the
2
4
solvents were then removed under reduced pressure. The
crude products were purified by column chromatography
with petroleum ether/ethyl acetate as the eluent.
2
.3 General method
All NMR spectra were recorded on Bruker-AM 300 (300
MHz) spectrometer. Routine monitoring of reactions was
performed by TLC using precoated Haiyang GF254 silica
gel plates. Flash column chromatography was performed
using silica gel (100–200 mesh) at increased pressure. The
ee values of products were determined by chiral HPLC,
with Chiralpak AD-H, AS-H and Chiralcel OD-H, OJ-H
columns. Aldol adducts 3a–t are all known compounds.
3
Results and discussion
In our initial investigation, the direct aldol reaction of cy-
clohexanone and 4-nitrobenzaldehyde was used as a model

Yuan Y, et al. Sci China Chem January (2013) Vol.56 No.1
3
a)
Table 1 Effect of solvents on the AMP-catalyzed asymmetric direct aldol reaction
b)
c)
c)
Entry
Solvent
Yield (%)
dr (anti:syn)
85:15
ee (%)(anti)
1
2
MeCN
CHCl
53
28
84
83
3
83:17
3
4
5
6
7
8
9
0
1
2
3
4
5
6
toluene
butyl acetate
THF
solvent-free
MTBE
cyclohexane
i-propyl ether
1,4-dioxane
H O
2
EtOH
DMSO
MeCN (no enzyme)
31
38
74
85
32
37
43
70
47
80
84
trace
11
4
72:28
75:25
82:18
80:20
76:24
73:27
68:32
76:24
67:33
70:30
60:40

70
70
70
70
68
67
61
56
48
35
33

d)
1
1
1
1
1
1
1
e)
MeCN (urea-denatured AMP)
MeCN (urea)
58:42
37:63
9
f)

6

0
g)
1
1
1
7
8
9
MeCN (PMSF-inhibited AMP)
6
5
50:50
47:53
48:52
h)
MeCN (PMSF)
i)
MeCN (bovine serum albumin)
47
a) Reaction conditions: AMP (50 mg), 4-nitrobenzaldehyde (0.5 mmol), cyclohexanone (5 equiv), deionized water (0.10 mL) and solvent (0.90 mL) at
0 °C for 90 h; b) yield of the isolated product after silica gel chromatography; c) determined by chiral HPLC analysis; d) conditions: AMP (50 mg),
-nitrobenzaldehyde (0.5 mmol), cyclohexanone (5 equiv), deionized water (0.10 mL) at 30 °C for 90 h; e) AMP (50 mg) was pre-treated with urea (50 mg)
3
4
in deionized water (3.0 mL) at 100 °C for 48 h, and then water was removed under reduced pressure before reaction; f) urea (50 mg) was used instead of
AMP; g) AMP (50 mg) was pre-treated with PMSF (200 mg) in dry THF (2.0 mL) at 30 °C for 48 h, and then THF was removed under reduced pressure
before reaction; h) PMSF (200 mg) was used instead of AMP; i) bovine serum albumin (50 mg) was used instead of AMP.
Table 2 Effect of water content on the AMP-catalyzed asymmetric direct
sequence, it could be concluded that the native structure of
AMP is responsible for its catalytic activity and selectivity
for the aldol reaction. Thus, we validated that AMP cata-
lyzed the asymmetric direct aldol reaction.
Water concentration in organic solvent has been consid-
ered as an important factor in enzymatic reactions [21].
Thus, we carried out a series of reactions in MeCN with
different water content. The results are shown in Table 2.
AMP exhibited the highest diastereoselectivity and enanti-
a)
aldol reaction
b)
c)
c)
Entry
H
2
O (%)
0
Yield (%)
dr (anti:syn)
56:44
ee (%)(anti)
1
2
3
4
5
6
7
8
15
22
60
80
82
83
70
68
24
65
84
77
70
57
31
27
5
79:21
10
15
20
30
40
50
85:15
85:15
82:18
78:22
80:20
oselectivity at water content of 10% [H
2 2
O/(H O+MeCN),
v/v]. Under this condition, the enzymatic reaction gave the
product in 60% yield with 84% ee (85:15 dr) (Table 2, entry
77:23
a) Conditions: AMP (50 mg), 4-nitrobenzaldehyde (0.5 mmol), cyclo-
hexanone (5 equiv), deionized water (0–50%, H O/(H O+MeCN), v/v) and
vH2O+MeCN = 1.0 mL at 30 °C for 96 h; b) yield of the isolated product after
2
2
3
3
). However, the best yield was obtained at water content of
0% which gave product in 83% yield (57% ee, 78:22 dr)
silica gel chromatography; c) determined by chiral HPLC analysis.
(
Table 2, entry 6). It can be seen that water content in or-
ganic solvent has an apparent influence on the enzymatic
reaction. Therefore, to obtain the best enantioselectivity, we
chose the water content of 10% as the optimal condition for
the AMP-catalyzed aldol reaction.
Next, the effect of the molar ratio between two substrates
was tested (Table 3). The increase of molar ratio of cyclo-
hexanone to 4-nitrobenzaldehyde from 1:1 to 5:1 led to a
remarkable increase of the yield (Table 3, entries 1 and 2).
Further increase of the molar equivalents of cyclohexanone
could not improve the yield evidently. Moreover, the molar
ratio of the substrate did not show apparent effect on the
selectivity of the reaction. Thus, in consideration of atom
Table 3 Influence of molar equivalents of cyclohexanone on the model
aldol reaction
a)
b)
c)
c)
Entry
1a:2a
1:1
5:1
10:1
15:1
20:1
30:1
Yield (%)
dr (anti:syn)
87:13
ee (%)(anti)
1
2
3
4
5
6
15
70
75
78
77
75
82
84
83
82
81
81
88:12
88:12
88:12
87:13
88:12
a) Reaction conditions: AMP (50 mg), 4-nitrobenzaldehyde (0.5 mmol),
cyclohexanone (1–30 equiv), deionized water (0.10 mL) and MeCN (0.90
mL) at 30 °C for 100 h; b) yield of the isolated product after silica gel
chromatography; c) determined by chiral HPLC analysis.

4
Yuan Y, et al. Sci China Chem January (2013) Vol.56 No.1
Table 6 Time curve in the process of AMP catalyzed asymmetric direct
aldol reaction
economy, the 5:1 molar ratio of ketone to aldehyde was
chosen as the optimal ratio for further studies.
a)
b)
c)
c)
To further optimize the experimental conditions, we in-
vestigated the effect of enzyme concentration on the
AMP-catalyzed aldol reaction (Table 4). It was found that
both yield and ee value had an increase when the enzyme
concentration increased from 25 mg/mL to 50 mg/mL. Fur-
ther increase of enzyme concentration could not give better
results. Therefore, we chose 50 mg/mL as the optimal en-
zyme concentration for further study.
Entry
Time (h)
6
Yield (%)
dr (anti:syn)
86:14
ee (%)(anti)
1
2
3
4
5
6
7
8
9
7
13
25
37
51
68
80
86
86
84
84
85
84
84
84
84
85
84
12
89:11
24
88:12
48
88:12
72
90:10
96
90:10
120
144
168
89:11
88:12
Then we examined the effect of temperature on the
AMP-catalyzed aldol reaction (Table 5). The yield in-
creased apparently with the temperature ranging from 15 to
88:12
a) Conditions: 4-nitrobenzaldehyde (0.5 mmol), cyclohexanone (5
equiv), AMP (50 mg), deionized water (0.10 mL) and MeCN (0.90 mL) at
3
0 °C (Table 5, entries 1–4). Once the temperature sur-
3
0 °C; b) yield of the isolated product after silica gel chromatography; c)
passed 30 °C, the yield increased slightly but the ee value
decreased. Thus, 30 °C was chosen as the optimal tempera-
ture for the reaction.
determined by chiral HPLC analysis.
Time course of the AMP-catalyzed model aldol reaction
under optimal conditions was also tested. As shown in Ta-
ble 6, the selectivity was nearly constant while the yield had
been increasing to 86% and plateaued at 144 h.
With the optimized general procedure in hand, we ex-
plored the substrate scope and the generality of the
AMP-catalyzed asymmetric direct aldol reaction. Various
ketones and substituted benzaldehydes were investigated
general, AMP showed better diastereoselectivity and enan-
tioselectivity with cyclohexanone (Table 7, entries 1–16)
than with cyclopentanone, cycloheptanone and acetone (Ta-
ble 7, entries 17–20). The best diastereoselectivity of >99:1
(anti/syn) (Table 7, entry 12) and the best enantioselectivity
of 91% ee (Table 7, entry 3) were achieved. Aromatic al-
dehydes with electron-withdrawing substituents generally
gave higher yields than aromatic aldehydes with electron-
donating substituents. Meanwhile, the effect of steric hin-
drance of substituents on benzaldehydes also had a great
impact on the diastereoselectivity and yield of the reaction.
For instance, when reacting with cyclohexanone, 4-nitro-
benzaldehyde gave the highest yield but the lowest dr and
ee value among 2-, 3-, and 4-nitrobenzaldehyde. By contrast,
(
Table 7). A wide range of substrates could participate in
the reaction. Five-, six- and seven-membered cyclic ketones
and acetone as aldol donors could be accepted by AMP. In
Table 4 Effect of enzyme concentration on the AMP-catalyzed aldol
a)
reaction
2
-nitrobenzaldehyde gave the best dr and ee value but the
Enzyme concentra-
tion (mg/mL)
b)
c)
c)
Entry
Yield (%)
dr (anti:syn) ee (%)(anti)
lowest yield (Table 7, entries 1–3). The major products
were obtained as anti-isomers when the aldol donor was
cyclohexanone. However, the diastereoselectivity was
hardly observed by using cyclopentanone and cyclohep-
tanone as donors (Table 7, entries 17–19). Besides, acetone
was also accepted by AMP as a substrate, which gave a low
yield with low enantioselectivity (Table 7, entry 20). We
also attempted to use some aliphatic aldehyde as the aldol
acceptors, but nearly no desired products were obtained.
The results illustrated that AMP had a specific substrate
selectivity and stereoselectivity in direct asymmetry aldol
reaction.
1
2
3
4
5
25
50
51
68
66
69
70
88:12
88:12
87:13
86:14
86:14
82
84
83
82
82
75
100
200
a) Reaction conditions: AMP (25–200 mg), 4-nitrobenzaldehyde (0.5
mmol), cyclohexanone (5 equiv), deionized water (0.10 mL) and MeCN
(
0.90 mL) at 30 °C for 96 h; b) yield of the isolated product after silica gel
chromatography; c) determined by chiral HPLC analysis.
a)
Table 5 Effect of temperature on the AMP-catalyzed aldol reaction
Finally, we compared the performance of different pro-
teases which our group has reported on asymmetric aldol
reactions (Table 8). It elucidates that AMP-catalyzed aldol
reaction could obtain higher yields than that of acidic pro-
tease from Aspergillus usamii (AUAP) and chymopapain.
AMP also showed better diastereoselectivities and enanti-
oselectivities than alkaline protease from Bcaillus licheni-
formis (BALP) and chymopapain. Generally, AMP is ap-
parently superior than other reported proteases in activity
and selectivity towards asymmetric aldol reactions between
aromatic aldehydes and cyclic ketones.
b)
c)
c)
Entry
T (°C)
15
Yield (%)
dr (anti:syn)
86:14
ee (%)(anti)
1
2
3
4
5
6
28
35
77
88
90
91
78
80
78
84
77
70
20
85:15
25
85:15
30
85:15
35
78:22
40
71:29
a) Reaction conditions: AMP (50 mg), 4-nitrobenzaldehyde (0.5 mmol),
cyclohexanone (5 equiv), deionized water (0.10 mL), and MeCN (0.90 mL)
for 144 h; b) yield of the isolated product after silica gel chromatography;
c) determined by chiral HPLC analysis.

Yuan Y, et al. Sci China Chem January (2013) Vol.56 No.1
5
a)
Table 7 Scope of the AMP-catalyzed aldol reaction
b)
c)
c)
Entry
R
1
R
2
R
3
No.
3a
3b
3c
3d
3e
3f
Time (h)
143
143
143
130
130
192
192
192
192
180
192
192
216
216
204
204
96
Yield (%)
87
dr (anti:syn)
83:17
90:10
92:8
ee (%)(anti)
1
2
3
4
5
6
7
8
9
-(CH
-(CH
-(CH
-(CH
-(CH
-(CH
-(CH
-(CH
-(CH
-(CH
-(CH
-(CH
-(CH
-(CH
-(CH
-(CH
-(CH
-(CH
-(CH
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
4
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
4-NO
2
C
C
C
6
6
6
H
H
H
4
4
4
84
90
91
89
90
88
86
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
3
3
5
3-NO
2-NO
2
79
2
52
4-CNC
3-CNC
6
H
4
74
84:16
91:9
6
H
4
53
4-FC
6
H
4
27
84:16
84:16
72:28
86:14
80:20
93:7
4-ClC
3-ClC
6
H
4
3g
3h
3i
57
6
H
4
84
86 (anti)/55 (syn)
4-BrC
4-CF
6
H
4
57
88
1
1
1
1
1
1
1
1
1
1
2
0
1
2
3
4
5
6
7
8
9
0
3
C
6
H
4
3j
73
87
2,4-Cl
2,6-Cl
2
C
6
H
H
3
3k
3l
70
89
2
C
6
3
89
> 99:1
86:14
90:10
87:13
93:7
53
4-MeC
6
H
4
3m
3n
3o
3p
3q
3r
3s
24
87
4-MeOC
3-MeOC
2-MeOC
6
6
6
H
4
4
4
11
87
H
H
40
84
38
57
4-NO
3-NO
4-NO
4-NO
2
2
2
2
C
C
C
C
6
6
6
6
H
H
H
H
4
78
47:53
53:47
55:45

73 (anti)/53 (syn)
4
4
4
96
84
80 (anti)/59 (syn)
210
192
23
40
32
Me, H
3t
20
a) Reaction conditions: AMP (50 mg), aldehyde (0.5 mmol), ketone (5 equiv), MeCN (0.90 mL) and deionized water (0.10 mL) at 30 °C; b) yield of the
isolated product after silica gel chromatography; c) determined by chiral HPLC analysis, and absolute configuration was assigned by comparison with litera-
ture (for details, see the Supporting Information).
Table 8 The comparison of some reported proteases with AMP on the performance in asymmetric aldol reactions
Yield (%)
dr (anti:syn)
ee (%) (anti)
Entry
R
Chymo-
papain
Chymo-
papain
Chymo-
papain
a)[17]
b)[16]
AMP AUAP
BLAP
c)[18]
AMP
AUAP
BLAP
AMP
AUAP BLAP
1
2
3
4
5
6
7
4-NO
3-NO
2
87
79
74
57
84
57
89
63
61
59
20
29
32
59
91
87
59
74
73
65
92
69
83:17
90:10
84:16
84:16
72:28
84:16
>99:1
83:17
74:26
79:21
82:18
92:8
68:32
58:42
75:25
75:25
72:28
73:27
95:5
63:37
87:13
72:28
69:31
65:35
81:19
>99:1
84
90
89
86
86
88
53
82
85
74
76
88
88
52
70
51
51
55
50
53
36
78
86
76
75
93
84
58
2
41
4-CN
4-Cl
60
47
3-Cl
32
4-Br
29
82:18
97:3
2,6-Cl
2
39
a) AUAP: acidic protease from Aspergillus usamii; b) BLAP: alkaline protease from Bcaillus licheniformis; c) chymopapain is in the latex of the unripe
fruit of Carica papaya.
4
Conclusions
ing in moderate to high yields, with moderate to excellent
diastereoselectivity and enantioselectivity under mild reac-
tion conditions, and not any additional cofactors or special
equipment were required. These features are expected to
make AMP as an attractive tool in synthesis. This activity of
AMP greatly expands the application of this biocatalyst.
In summary, we showed that AMP had the ability to cata-
lyze asymmetric direct aldol reaction in organic media. The
influences of reaction conditions including solvent, water
content, molar ratio of substrates, enzyme concentration,
and temperature were investigated. The AMP can catalyze
direct aldol reaction with a wide range of substrates result-
Supporting Information Details of aldol products 3a–3t,
1
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and copies of the H and C NMR, as well as HPLC spectra

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