Catalytical promiscuity of α-amylase: Synthesis of 3-substituted 2H-chromene derivatives via biocatalytic domino oxa-Michael/aldol condensations

Article abstract of DOI:10.1016/j.molcatb.2013.02.001

An facile and green one-pot route has been developed for the synthesis of chromenes using salicylaldehyde and α,β-unsaturated ketones. α-Amylase from Bacillus subtilis shows excellent catalytic activity and exerts good adaptability to different substrates in the reaction. This promiscuous enzyme-catalyzed domino reaction not only extends the application of α-amylase from B. subtilis for new chemical transformations, but also provided an alternative synthetic method for 2H-chromene derivatives.

Full text of DOI:10.1016/j.molcatb.2013.02.001

Journal of Molecular Catalysis B: Enzymatic 91 (2013) 37–43
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Catalytical promiscuity of ␣-amylase: Synthesis of 3-substituted 2H-chromene
derivatives via biocatalytic domino oxa-Michael/aldol condensations
Long-Hua Zhou, Na Wang∗, Wei Zhang, Zong-Bo Xie, Xiao-Qi Yu∗
Key Laboratory of Green Chemistry and Technology, Ministry of Education, College of Chemistry, Sichuan University, Chengdu 610064, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
An facile and green one-pot route has been developed for the synthesis of chromenes using salicylal-
dehyde and ␣,␤-unsaturated ketones. ␣-Amylase from Bacillus subtilis shows excellent catalytic activity
and exerts good adaptability to different substrates in the reaction. This promiscuous enzyme-catalyzed
domino reaction not only extends the application of ␣-amylase from B. subtilis for new chemical trans-
formations, but also provided an alternative synthetic method for 2H-chromene derivatives.
© 2013 Elsevier B.V. All rights reserved.
Received 8 November 2012
Received in revised form 27 January 2013
Accepted 2 February 2013
Available online 26 February 2013
Keywords:
Chromenes
Biocatalytic promiscuity
␣-Amylase
Domino reaction
1. Introduction
extended the application of enzymes in organic synthesis. Some
promiscuous hydrolases-catalyzed carbon–carbon and carbon-
Chromene is an important core structural unit widely dis-
tributed in many natural products and as privileged precursors
heteroatom bond formations reactions have been done during
the past decade [13–17]. Although some promiscuous enzyme-
catalyzed single-step transformations have been reported, only
few of the promiscuous single enzyme-catalyzed multi-step or
domino reactions were described [18,19]. As a part of our contin-
uous efforts toward the development of biocatalytic promiscuity,
we have reported hydrolase-catalyzed aldol reaction [20], Man-
nich reaction [21,22] and Michael addition [23] etc. In the present
study, we are interested in designing a new domino reaction
based on the promiscuity of hydrolase. We serendipitously found
␣-amylase from Bacillus subtilis was able to catalyze domino oxa-
Michael/aldol condensations between salicylaldehyde and methyl
vinyl ketone efficiently. It provided a novel case of biocatalytic
promiscuity and more convenient synthetic method for green
chemistry.
to
a variety of bioactive molecules [1]. For instance, sub-
stituted (E)-3-styryl-2H-chromen-4-ones (A) exhibit excellent
anti-picornavirus activity (Fig. 1) [2]. 2-Amino-5-oxo-5,6,7,8-
tetrahydro-4H-chromenes (B) also show an important antibacterial
activity (Fig. 1) [3]. Moreover, some chromene derivatives have
attracted much attention due to their photochromic and ther-
mochromic properties such as some anthocyanidins [4]. Therefore,
it is of great significance to develop new efficient synthesis methods
for this “privileged” structural motif. Numerous organocatalysts
have been used for the construction of structurally diverse and
densely functionalized chromenes [5–7]. However, there are few
reports on biocatalysis method for the synthesis of 3-substituted
2H-chromene derivatives.
The potential of biocatalysis becomes increasingly recognized
as an efficient and green tool for modern organic synthesis [8–10].
Enzyme as a practical and environmentally friendly alternative to
traditional metallo- and organocatalysts has been widely used in
chemical synthesis, which represents a remarkable opportunity
for the development of industrial chemical and pharmaceu-
tical processes [11,12]. Particularly, the developments in the
biocatalytic promiscuity have received considerable attention and
␣-Amylase (EC 3.2.1.1), as a kind of common hydrolases, which
is used to hydrolyze ␣-1,4-glycosidic bonds of starch in the lin-
ear amylase chain, generating glucose, maltose, and maltotriose
units [24]. Herein, we exploited ␣-amylase from B. subtilis for the
synthesis of 3-substituted 2H-chromene derivatives in organic sol-
vents (Scheme 1). To our knowledge, this is the first reported case
for the catalytical promiscuity of ␣-amylase. The oxa-Michael addi-
tion and aldol reaction could be performed in one-pot and a single
amylase catalyzing the domino biotransformation was constructed.
The influence of reaction conditions including solvents, temper-
ature, enzyme concentration and molar ratio of substrates were
investigated. A series of 2H-chromene derivatives were prepared
∗
Corresponding author. Tel.: +86 85415886; fax: +86 85415886.
E-mail addresses: wnchem@scu.edu.cn (N. Wang), xqyu@scu.edu.cn (X.-Q. Yu).
1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.02.001

38
L.-H. Zhou et al. / Journal of Molecular Catalysis B: Enzymatic 91 (2013) 37–43
Detector) using C18 column. Elution was performed with a mixture
of methanol/water (70/30, v/v) at 0.8 mL/min.
2.2. General procedure
A typical reaction mixture contained 1 (2 mmol), 2 (10 mmol),
50 mg ␣-amylase from B. subtilis, 500 ␮L H2O and 4.5 mL DMSO was
shaken at 50 ◦C and 200 rpm. Then, after the indicated time, the
reaction mixture was quenched with distilled water (20 mL) and
extracted with EtOAc (3× 10 mL). The organic layer was dried over
anhydrous Na2SO4 and evaporated. The crude product was puri-
fied by flash column chromatography with ethyl acetate/petroleum
ether (1:20–1:10) to obtain the product.
Fig. 1. Selected examples of chromenes embodied with biological and pharmaco-
logical activity.
2.2.1. 3-Acetyl-2H-chromene (3a)
Yellow crystal; 1H NMR (400 MHz, CDCl3): ı 7.31 (s, 1H),
7.26 (t, 1H), 7.17 (d, J = 7.2 Hz, 1H), 6.95 (t, J = 7.3 Hz, 1H), 6.86
(d, J = 8.0 Hz, 1H), 5.01 (s, 2H), 2.42 (s, 3H); 13C NMR (100 MHz,
CDCl3): 195.96, 155.59, 133.96, 132.48, 130.78, 129.17, 121.80),
120.75, 116.33, 64.26, 25.03. HRMS (ESI, m/z): [M+Na]+ = calcd. for
with moderate to high yields under the catalysis of ␣-amylase from
B. subtilis in DMSO successfully.
2. Experimental
C11H11O2, 175.0759; found 175.0759.
2.1. Materials and methods
2.2.2. 3-Formoxyl-2-methyl-2H-chromene (3b)
BSA (␣-amylase from B. subtilis, 50 U/mg), PPL (lipase from
porcine pancreas, 6.8 U/mg), MJL (amano lipase M from Mucor
javanicus, 10,000 U/mg), AMA (acylase I from Aspergillus melleus,
0.72 U/mg), HPA(␣-amylase from hog pancreas, 46.4 U/mg),
AOA(␣-amylase from Aspergillus oryzae, 28.7 U/mg) were pur-
chased from Sigma–Aldrich. BPL (lipase from bovine pancreas,
20 U/mg) was purchased from Aladdin. Unless otherwise men-
tioned, all reagents were obtained from commercial suppliers
and used without further purification. 1H NMR, 13C NMR spectra
were measured on Bruker AM400 NMR spectrometer (400 MHz or
100 MHz, respectively) with CDCl3 as solvent and recorded in ppm
relative to tetramethylsilane (TMS). Thin layer chromatography
(TLC) experiments were performed on glass-backed silica plates
and visualized with UV-detection. HPLC was carried on Waters
Associates equipment (Waters 2695 with 2998 Photodiode Array
Yellow oil; 1H NMR (400 MHz, CDCl3): ı 9.41 (s, 1H), 7.18 (t,
J = 7.8 Hz, 1H), 7.09 (d, J = 9.1 Hz, 1H), 7.06 (s, 1H), 6.82 (t, J = 7.0 Hz,
1H), 6.75 (s, 1H), 5.30 (q, J = 6.6 Hz, 1H), 1.24 (d, J = 6.6 Hz, 3H); 13
NMR (100 MHz, CDCl3): 190.00, 154.40, 140.22, 136.11, 133.39,
129.22, 121.59, 119.91, 117.29, 69.76, 19.88. HRMS (ESI, m/z):
[M+H]+ calcd. for C11H11O2, 175.0759; found 175.0756.
C
2.2.3. 2,3,4,4a-Tetrahydroxanthen-1-one (3c)
Yellow crystal; 1H NMR (400 MHz, CDCl3): ı 7.43 (s, 1H),
7.26–7.22 (m, J = 7.5 Hz, 2H), 6.96 (t, 1H), 6.88 (d, J = 8.1 Hz, 1H),
5.05–4.96 (m, 1H), 2.62–2.57 (m, J = 18.0, 1.9 Hz, 1H), 2.55–2.45
(m, 1H), 2.45–2.34 (m, 1H), 2.15–2.05 (m, 1H), 2.05–1.94 (m, 1H),
1.76–1.69 (m, J = 21.6, 8.0 Hz, 1H); 13C NMR (100 MHz, CDCl3):
197.51, 155.88, 132.05, 131.52, 130.43, 129.82, 122.14, 122.17,
Scheme 1. ␣-Amylase-catalyzed synthesis of 3-substituted 2H-chromene derivatives.

L.-H. Zhou et al. / Journal of Molecular Catalysis B: Enzymatic 91 (2013) 37–43
39
116.03, 74.64, 38.83, 29.69, 17.99. HRMS (ESI, m/z): [M+H]+ calcd.
for C13H13O2, 201.0916; found 201.0914.
Table 1
Solvent effect on the yield of 3-acetyl-2H-chromenea.
Entry
Solvent
Yield b (%)
2.2.4. 3-Acetyl-8-bromo-2H-chromene (3d)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
DMSO
DMF
36.5
2.5
8.4
12.6
2.5
Trace
Trace
Trace
10.7
Trace
8.1
12.3
10.6
5.4
Yellow crystal; 1H NMR (400 MHz, CDCl3): ı 7.33 (dd, J = 8.6,
2.4 Hz, 1H), 7.25 (d, J = 2.4 Hz, 1H), 7.21 (s, 1H), 6.74 (d, J = 8.6 Hz,
1H), 5.00 (s, J = 1.2 Hz, 2H), 2.41 (s, 3H); 13C NMR (100 MHz,
CDCl3): 195.71, 154.54, 134.82, 132.37, 131.66, 131.28, 122.54,
118.10, 113.63, 64.43, 25.10. HRMS (ESI, m/z): [M+Na]+ calcd. for
C11H9BrO2, 274.9684; found 274.9686.
Methanol
Ethanol
Isopropanol
1,4-Dioxane
CH3CN
THF
H2O
–
DMSO:CH3CN = 1:1
DMSO:THF = 1:1
DMSO:1,4-dioxane = 1:1
DMSO:EA = 1:1
2.2.5. 3-Formoxyl-2-methyl-8-bromo-2H-chromene (3e)
Yellow oil; 1H NMR (400 MHz, CDCl3): ı 9.48 (s, 1H), 7.31 (d,
J = 8.6, 2.3 Hz, 1H), 7.23 (d, 1H), 7.04 (s, 1H), 6.70 (d, J = 8.6 Hz, 1H),
5.34 (q, J = 6.6 Hz, 1H), 1.28 (d, J = 6.6 Hz, 3H); 13C NMR (100 MHz,
CDCl3): 189.76, 153.37, 138.46, 136.97, 135.72, 131.24, 121.63,
119.14, 113.44, 70.07, 19.86. HRMS (ESI, m/z): [M+Na]+ calcd. for
a
Reaction conditions: salicylaldehyde (0.2 mmol), methyl vinyl ketone
(0.5 mmol), ␣-amylase from B. subtilis (10 mg) were shaken at 200 rpm at 50 ◦C for
48 h.
C11H9BrO2, 274.9684; found 274.9685.
b
Determined by HPLC.
2.2.6. 7-Bromo-2,3,4,4a-tetrahydroxanthen-1-one (3f)
123.32, 121.92, 71.08, 20.08. HRMS (ESI, m/z): [M+Na]+ calcd. for
Yellow crystal; 1H NMR (400 MHz, CDCl3): ı 7.38–7.28 (m, 3H),
6.77 (d, J = 9.3 Hz, 1H), 5.02–4.97 (m, 1H), 2.65–2.56 (m, 1H), 2.49
(dd, J = 7.4, 5.3 Hz, 1H), 2.44–2.33 (m, 1H), 2.15–2.06 (m, 1H), 2.00
(dd, J = 10.7, 2.7 Hz, 1H), 1.77–1.63 (m, 1H); 13C NMR (100 MHz,
CDCl3): 197.17, 154.78, 134.44, 131.83, 131.37, 129.99, 123.91,
117.83, 114.06, 74.83, 38.83, 29.60, 17.88. HRMS (ESI, m/z): [M+Na]+
calcd. for C13H11BrO2, 300.9840; found 300.9839.
C11H8Cl2O2, 264.9798; found 264.9799.
3. Results and discussion
3.1. Selection of biocatalysts
Initial efforts were undertaken using salicylaldehyde and
methyl vinyl ketone as a model reaction catalyzed by differ-
ent hydrolases. Several commercially available hydrolases were
screened in order to select the appropriate biocatalyst. We chose
DMSO as the reaction media based on the previous reports on
enzymatic domino reaction. For example, Guan et al. reported that
alkaline protease from Bacillus licheniformis could catalyze the one-
pot multiple reaction for synthesizing 2H-1-benzopyran-2-one
derivatives by domino Knoevenagel/intramolecular transesterifi-
cation reaction in DMSO recently [25]. The results showed that only
BSA is markedly preferred for this model reaction with 36% yield.
Other enzymes including PPL, AMA, MJL, BPL, OAA and HPA dis-
played very low catalytic activities toward the reaction, and only
trace product was detected by HPLC. Some controlled experiments
were performed to demonstrate the specific catalytic effect of BSA.
When reactions were incubated with no enzyme or denatured BSA
(pretreated with FeSO4 at 50 ◦C for 24 h), no product was detected.
All the results suggested that the specific catalytic site and spatial
conformation of ␣-amylase from B. subtilis are necessary to catalyze
the domino reaction.
2.2.7. 3-Acetyl-8-methoxy-2H-chromene (3g)
Yellow crystal; 1H NMR (400 MHz, CDCl3): ı 7.28 (s, 1H),
6.86–6.78 (m, 2H), 6.72 (d, J = 2.7 Hz, 1H), 4.95 (s, 2H), 3.78 (s, 3H),
2.41 (s, 3H); 13C NMR (100 MHz, CDCl3): 195.93, 154.35, 149.60,
133.99, 131.59, 121.34, 118.30, 116.99, 113.25, 64.20, 55.81, 25.07.
HRMS (ESI, m/z): [M+H]+ calcd. for C12H13O3, 205.0865; found
205.0860.
2.2.8. 3-Formoxyl-2-methyl-8-methoxy-2H-chromene (3h)
Yellow oil; 1H NMR (400 MHz, CDCl3): ı 9.42 (s, 1H), 7.03
(s, 1H), 6.80–6.68 (m, J = 26.1, 5.9 Hz, 2H), 6.63 (d, J = 2.9 Hz, 1H),
5.26 (q, J = 6.6 Hz, 1H), 3.68 (s, 3H), 1.23 (d, J = 6.6 Hz, 3H). 13C
NMR (100 MHz, CDCl3): 189.98, 154.17, 148.23, 140.20, 136.76,
120.28, 119.57, 118.03, 112.91, 69.50, 55.74, 19.41. HRMS (ESI, m/z):
[M+H]+ calcd. for C12H13O3, 205.0865; found 205.0859.
2.2.9. 7-Methoxy-2,3,4,4a-tetrahydroxanthen-1-one (3i)
Yellow crystal; 1H NMR (400 MHz, CDCl3): ı 7.40 (d, J = 2.2 Hz,
1H), 6.82 (d, J = 4.1 Hz, 2H), 6.78 (s, 1H), 4.90–4.95 (m, 1H), 3.77
(s, 3H), 2.59–2.57 (m, 1H), 2.52–2.44 (m, 1H), 2.44–2.33 (m, 2H),
2.08–1.98 (m, 2H), 1.72–1.68 (m, 1H); 13C NMR (100 MHz, CDCl3):
197.62, 154.51, 131.67, 131.02, 122.60, 118.27, 116.74, 114.82,
113.47, 74.63, 55.77, 38.84, 29.65, 14.12. HRMS (ESI, m/z): [M+Na]+
calcd. for C14H14O3, 253.0841; found 253.0842.
3.2. Effect of reaction media
Then, we investigated the effect of different reaction mediums
on the enzymatic domino reaction. The results were shown in
Table 1. It is noted that DMSO was found to promote this tandem
reaction and offered the maximum yield as compared with others.
The reaction of salicylaldehyde and methyl vinyl ketone in DMSO
could obtain the 36.5% yield at 50 ◦C (entry 1, Table 1). Thus we tried
some mixed solvents with DMSO (entries 11–14, Table 1), but all
these are leading to poorer yields. It seems that polar solvents favor
the reaction may be due to the stability of enzyme and the solubil-
ity of substrates. However, pure water (entry 11, Table 1) leads to
lower yield, which may be due to the poor solubility of substrates
in pure water. To our surprise, the alcohols (entries 3–5, Table 1)
also lead to inferior yields, this may be due to that proton solvents
could lower the nucleophilicity of the phenolate anion. While the
yield is just 2.5% in DMF, it may be attributed to specific interac-
tions between the solvent and ␣-amylase. The results revealed that
2.2.10. 3-Acetyl-6,8-dichloro-2H-chromene (3j)
Yellow crystal; 1H NMR (400 MHz, CDCl3): ı 7.31 (d, J = 2.4 Hz,
1H), 7.19 (s, 1H), 7.07 (d, J = 2.4 Hz, 1H), 5.11 (s, 2H), 2.42 (s, 3H);
13C NMR (100 MHz, CDCl3): 195.47, 149.82, 132.19, 131.94, 131.67,
126.88, 126.32, 122.80, 122.31, 65.21, 25.21. HRMS (ESI, m/z):
[M+Na]+ calcd. for C11H8Cl2O2, 264.9801; found 264.9799.
2.2.11. 3-Formoxyl-2-methyl-6,8-dichloro-2H-chromene (3k)
Yellow oil; 1H NMR (400 MHz, CDCl3): ı 9.52 (s, 1H), 7.34–7.25
(m, 1H), 7.18 (dd, J = 9.5, 4.8 Hz, 2H), 6.93 (t, J = 7.1 Hz, 2H), 5.41
(dd, J = 12.8, 6.3 Hz, 1H), 1.35 (d, J = 6.5 Hz, 3H); 13C NMR (100 MHz,
CDCl3): ı 189.47, 148.75, 137.63, 137.60, 132.68, 126.79, 126.21,

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L.-H. Zhou et al. / Journal of Molecular Catalysis B: Enzymatic 91 (2013) 37–43
5000000
4500000
4000000
3500000
3000000
2500000
2000000
1500000
1000000
500000
0
40
35
30
25
20
15
10
5
0%
10%
50%
100%
260
280
300
0
Wavelength (nm)
0
20
40
60
80
100
Water content %
Fig. 3. Fluorescence emission spectra of ␣-amylase from B. subtilis in different water
content under the fluorescence condition: excitation wavelength 344 nm and emis-
sion wavelengths from 250 nm to 330 nm with various slit widths.
Fig. 2. The effect of water content on the yield of 3-acetyl-2H-chromene under these
conditions: ␣-amylase from B. subtilis (10 mg), salicylaldehyde (0.2 mmol), methyl
vinyl ketone (2 mmol), 200 rpm, 50 ◦C, 48 h. All yields were determined by HPLC.
3.4. Effect of molar ratio
solvent shows great effect on the catalytic activity of ␣-amylase
from B. subtilis and the activation of substrates.
Encouraged by the above results, other influencing factors were
screened to obtain the higher yield. The influence of molar ratio
of substrates on the reaction between salicylaldehyde and methyl
vinyl ketone was investigated. As shown in Fig. 4, it was found that
the molar ratio had important influence on the yield of domino
reaction. When the molar ratio of salicylaldehyde to methyl vinyl
ketone varied from 1/1 to 1/10, the yield was improved from7%
to 45%. Then the yield would decline sharply at 2/1 and the
bell-shaped curves presented. This may be due to that excess salic-
ylaldehyde lead to some inactivation of the enzyme. However, no
obvious difference was observed in the yield as the molar ratio
continually increased from 1/5 to 1/20.
3.3. Effect of water content
Our previous studies of enzymatic promiscuity indicated
that the content of water was essential to the activity of
enzymes [20–23]. Experiments were performed to optimize the
water/DMSO ratio for this reaction, and the results were shown
in Fig. 2. It was found that the yield of the enzymatic reaction could
be enhanced by increasing the concentration of water slightly. The
desired product could be obtained in the highest yield of 39% in
DMSO at 10% (water/[water + DMSO], v/v) water content, suggest-
ing the activity of amylase is very sensitive to water. However, the
yield decreased with the increasing of water, which may be due to
the lower solubility of the substrates.
For the sake of further study the effect of water content, the dif-
ferent water contents on the conformation of ␣-amylase from B.
subtilis were investigated by means of fluorescence spectroscopy.
The amylase contains intrinsic fluorophores residues such as Phe,
Tyr or Trp, which highly sensitive to the polarity of microenvi-
ronment, thus structural variations are reflected by changes in
the maximal emission wavelength (ꢀmax) and the maximal inten-
sity of fluorescence (Imax) [26,27]. Generally, a red shift of ꢀmax
and incensement of fluorescent intensity corresponds to unfol-
ding of the enzyme, which enhances the exposure of fluorescent
residues to the system [28]. As shown in Fig. 3, the fluorescent
intensity reached a maximum when water content was 10%, and
the Imax decreased with the increase of water content (0–100%).
The increase of Imax can be attributed to conformational changes
of ␣-amylase, which is consistent with the yield of different
water contents. Moreover, ꢀmax were slightly blue-shifted with the
increase of water content (0–100%), this might be explained by the
fact the change of microenvironment and cause the unfolding of the
protein. Combining the water content experiments with the fluo-
rescence results, it is evident that the ability of ␣-amylase from B.
subtilis to adopt an appropriate conformation in 10% water content
is significant to its ability to catalyze oxa-Michael/aldol reactions.
Additionally, the fluorescence experiments provided important
evidence for a relationship between the promiscuous catalytic
activity of ␣-amylase from B. subtilis in the reaction and its spatial
conformation.
3.5. Effect of enzyme loading
We then examined the influence of enzyme loading on the reac-
tion. The results are shown in Fig. 5, it can be seen that the yields
of oxa-Michael/aldol reaction were significantly influenced by dif-
ferent enzyme loading, and the evident difference in the yields (9%
Fig. 4. The influence of molar ratio of salicylaldehyde to methyl vinyl ketone on the
yield of 3-acetyl-2H-chromene, experimental conditions: ␣-amylase from B. subtilis
(10 mg), 100 ␮L H2O, 900 ␮L DMSO, 200 rpm, 50 ◦C, 48 h. All yields were determined
by HPLC.

L.-H. Zhou et al. / Journal of Molecular Catalysis B: Enzymatic 91 (2013) 37–43
41
60
50
40
30
20
10
0
10
20
30
40
50
60
70
80
90
100
o
Temperatue( C)
Fig. 5. The effect of enzyme loading on the yield of 3-acetyl-2H-chromene under
these conditions: salicylaldehyde (0.2 mmol), methyl vinyl ketone (2 mmol), 100 ␮L
H2O, 900 ␮L DMSO, 200 rpm, 50 ◦C, 48 h. All yields were determined by HPLC.
Fig. 6. The effect of temperature on the yield of 3-acetyl-2H-chromene.
3.7. Substrate scope of the domino oxa-Micheal/aldol reactions
Finally, in order to extend the scope of the methodology, some
other salicylaldehyde derivatives and ␣,␤-unsaturated ketones or
aldehydes were tested in the presence of ␣-amylase from B. subtilis.
As shown in Table 2, a wide range of substrates could effectively
participate in this reaction and gave desired products. It can be
found that electronic effect of substituents on phenyl ring of salic-
ylaldehyde seemed to make a great difference on the yields of
the reaction. In general, salicylaldehyde with electron-donating
substituents (entries 7–9, Table 2) exhibited higher reactivity in
the enzymatic reaction as expected. In contrast, the substituted
salicylaldehyde with electron-withdrawing substituents (entry 12,
Table 2) showed inert to methyl vinyl ketone. The reason is
that electron-donating groups on phenyl ring of salicylaldehydes
enhance the nucleophilicity of the phenolate anion, while electron-
withdrawing groups make it less nucleophilic. Moreover, methyl
vinyl keton (entries 1, 4, 7, 10, Table 2) was more reactive than cro-
tonaldehyde and 2-cyclohexen-1-one. This is probably due to the
relatively smaller steric hinderance of methyl vinyl ketone.
and 56%) was observed from enzyme loading of 2–25 mg. There
was a slightly decline in yield when more enzyme was loaded. The
possible reason might be that more enzyme loading was unfavor-
able to the diffusion of the substrates. The results revealed that
25 mg ␣-amylase from B. subtilis was the optimum quantity for the
reaction.
3.6. Effect of temperature
Reaction temperature also plays an important role in enzymatic
reactions, owing to its effects on the catalytic properties and the
stability of enzyme. As shown in Fig. 6, higher temperature caused
higher yield in a wide range of temperature (20–80 ◦C). How-
ever, there was only a slight increase in yields between 50–80 ◦C,
and reached the best yield of 58% at 80 ◦C after 48 h. It has been
reported that the BSA could catalyze the hydrolysis of starch even
if the temperature exceeded 80 ◦C [29]. Although ␣-amylase from
B. subtilis is thermostable, the yield decreases when the reaction
temperature surpassed 80 ◦C. From the HPLC, it can be observed
that the yield slightly decreased due to the side-reactions. Con-
sequently, 50 ◦C was chosen as the optimum temperature for the
reaction.
3.8. Proposed reaction mechanism of enzymatic synthesis of
2H-chromenes
It is widely accepted that the hydrolysis site in hydrolase is also
the active site of its promiscuous catalysis [30]. Based on this, we
proposed a possible mechanism of the hydrolase-catalyzed domino
reaction. Firstly, the salicylaldehyde was activated by the amino
residue and the oxyanion of the amylase. Then, methyl vinyl ketone
Conditions ␣-amylase from B. subtilis (25 mg), salicylalde-
hyde (0.2 mmol), methyl vinyl ketone (2 mmol), 100 ␮L H2O,
900 ␮L DMSO, 200 rpm, 48 h. All yields were determined by
HPLC.
Scheme 2. The proposed mechanism for the ␣-amylase-catalyzed synthesis of 3-substituted 2H-chromene derivatives.

42
L.-H. Zhou et al. / Journal of Molecular Catalysis B: Enzymatic 91 (2013) 37–43
Table 2
Substrate scope of the domino oxa-Micheal/aldol reactiona.
.
Entry
1
2
Products
Yieldb (%)
1
2
61
48
3
48
4
5
63
50
6
51
7
8
70
58
9
51
10
11
44
18

L.-H. Zhou et al. / Journal of Molecular Catalysis B: Enzymatic 91 (2013) 37–43
43
Table 2 (Continued)
Entry
1
2
Products
Yieldb (%)
12
Trace
a
Conditions: ␣-amylase from B. subtilis (50 mg), salicylaldehyde or salicylaldehyde derivatives (1 mmol) ␣,␤-unsaturated ketones or aldehydes (5 mmol), 300 ␮L H2O,
2700 ␮L DMSO, 200 rpm, 50 ◦C, 48 h.
b
Yield of the isolated product after chromatography on silica gel.
was attacked by the activated salicylaldehyde, which leads to a new
O bond via oxa-Michael addition. Next, an intramolecular aldol
reaction proceeded with the forming of a carbon–carbon bond.
Finally, the dehydration of the resulted adduct took place, then the
desired product was released Scheme 2.
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C
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Acknowledgments
We gratefully acknowledge the National Natural Science Foun-
dation of China (nos. 21001077 and 20121001), Program for
Changjiang Scholars in China for financial support.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.molcatb.
2013.02.001.
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