One-pot cascade synthesis of benzopyrans and dihydropyrano[c]chromenes catalyzed by lipase TLIM

Article abstract of DOI:10.1016/j.bioorg.2020.103888

Lipase TLIM was reported to be an efficient, commercially available and reusable catalyst for the Knoevenagel-Michael cascade reactions of aldehydes, malononitrile/ethyl cyanoacetate and 4-hydroxycoumarin/1, 3-cyclohexanedione/dimedone in aqueous DMSO. Th

Full text of DOI:10.1016/j.bioorg.2020.103888

Journal Pre-proofs
One-Pot Cascade Synthesis of Benzopyrans and Dihydropyrano[c]chromenes
Catalyzed by Lipase TLIM
Yajie Fu, Zeping Lu, Xiaolong Ma, Ke Fang, Xinyi He, Huajin Xu, Yi Hu
PII:
S0045-2068(19)32105-4
DOI:
https://doi.org/10.1016/j.bioorg.2020.103888
YBIOO 103888
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Bioorganic Chemistry
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Revised Date:
Accepted Date:
9 December 2019
23 April 2020
24 April 2020
Please cite this article as: Y. Fu, Z. Lu, X. Ma, K. Fang, X. He, H. Xu, Y. Hu, One-Pot Cascade Synthesis of
Benzopyrans and Dihydropyrano[c]chromenes Catalyzed by Lipase TLIM, Bioorganic Chemistry (2020), doi:
https://doi.org/10.1016/j.bioorg.2020.103888
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One-Pot Cascade Synthesis of Benzopyrans and Dihydropyrano[c]chromenes Catalyzed by
Lipase TLIM
Yajie Fu, Zeping Lu, Xiaolong Ma, Ke Fang, Xinyi He, Huajin Xu, Yi Hu*
(State Key Laboratory of Materials-Oriented Chemical Engineering, School of Pharmaceutical Sciences,
Nanjing Tech University, Nanjing 210009, China)
*Corresponding author, email: huyi@njtech.edu.cn
Abstract
Lipase TLIM was reported to be an efficient, commercially available and reusable catalyst for the
Knoevenagel-Michael cascade reactions of aldehydes, malononitrile / ethyl cyanoacetate and 4-
hydroxycoumarin/ 1, 3-cyclohexanedione/ dimedone in aqueous DMSO. This methodology presents
many superiorities such as simple procedure, mild reaction conditions, commercially available and
reusable catalyst, high substrate applicability, the ability to be scaled up, and good to excellent yields.
Keywords cascade reactions, enzymatic synthesis, benzopyrans, chromene, lipase
1 Introduction
Benzopyrans and dihydropyrano[c]chromenes have received more and more concerns due to their
prominent pharmacological and biological activities. They are able to inhibit the activities of
acetylcholinesterase and butyrylcholinesterase1, 2, which means that they play a role in the treatment of
neurodegenerative disorders such as Parkinson disease, Alzheimer’s, Huntington’s disease , etc. They
can act as xanthine oxidase inhibitors3, which means that they make contributions in reducing risks of
many diseases like gout, hyperuricemia, chronic kidney disease, hypertension, atherosclerosis, etc. They
also show notable cytotoxic and anti-tyrosinase activities4, which means that they work in the remedy of
Albinism. Benzopyrans and dihydropyrano[c]chromenes are usually synthesized by the three-component
tandem reaction of aldehydes, active methylene compounds and diverse enolizable C-H activated acidic
compounds employing various catalysts, such as IL@CNTs under ultrasonic radiation5, [DMAP-
PEG1000-DIL][BF4]6,
Fe3O4@SiO2-guanidine-PAA7,
Fe3O4@SiO2-imid-PMAn8,
10
{[HMIM]C(CN)3}NMS9, H5BW12O40 CTMAB-bentonite11, Ru@imine-Z12. Although the reported
,
protocols have their own advantages, they also have limits such as the use of expensive or troublesomely-
prepared catalysts, relative high temperature, and sometimes only moderate yields could be obtained.
Also, most of the reported procedures have limited substrate spectrum. Thence, it is necessary to develop
a green, convenient and efficient procedure to synthesize a wide range of benzopyrans and
dihydropyrano[c]chromenes.
Biocatalytic promiscuity is attracting more and more attention due to its wide application in C-C bond
forming reactions like Knoevenagel condensation, Aldol condensation, Henry reaction, Mannich
reaction, Michael addition, Baylis-Hillman reaction and so on13-16. In our previous studies, lipase LPL
was reported to catalyze the Knoevenagel condensation of aromatic aldehydes with active methylene

compounds in anhydrous DMSO and 76-81% yields of products were obtained 17, lipase TLIM
demonstrated noticeable promiscuity for the Knoevenagel-Michael cascade reactions of 1, 3-dicarbonyl
compounds with aldehydes to synthesize xanthone derivatives in anhydrous n-hexane and 80-97% yields
of products were obtained18, lipase RMIM was applied in the synthesis of dicoumarol derivatives in pure
water and 81-98% yields of products were achieved 19. Here we report a simple, efficient enzymatic
method for the multicomponent Knoevenagel-Michael cascade reactions of aldehydes, malononitrile /
ethyl cyanoacetate and 4-hydroxycoumarin/ 1, 3-cyclohexanedione/ dimedone catalyzed by lipase TLIM
in aqueous DMSO to synthesize benzopyrans and dihydropyrano[c]chromenes.
2. Materials and Methods
Porcine pancreas lipase (PPL), Amano lipase PS from burkholderia cepacia (BCL) and candida
rugosa lipase (CRL) were purchased from Sigma. Lipase from thermomyces lanuginosus immobilized
on particle silica gel (TLIM), lipase from rhizomucor miehei immobilized on anion exchange resin
(RMIM), lipase B from candida antarctica immobilized on macroporous acrylic resin (Novozym 435)
and papain were purchased from Novo Nordisk. Bovine serum albumin (BSA) was purchased from
Shanghai Huixing. Lipase XHlip-F was donated by XHSynbio. Lipase lipoprotein from aspergillus niger
(LPL) was donated by Ningxia Sunson group corporation. Lipase DF was donated by Amano Enzyme
China Ltd. Other reagents were commercially available and were used without further purification.
The melting points were determined on a WRS-1B digital melting point instrument and were not
1
corrected. The H NMR and 13C NMR spectra were measured on a Bruker Advance 2B 300 MHz
instrument with DMSO-d6 as solvent and TMS as internal standard. The HRMS were measured on
Agilent LC/MS mass spectrometer. The progress of the reaction was monitored by TLC using pre-coated
Haiyang GF254 silica gel plates. HPLC data was obtained using Dionex Liquid Chromatography
(diamonsil C18 (2) (4.6×250mm, 5μm), water with 0.1% formic acid /methanol (v/v=7/13,1mL/min,
20min, radiant elution), 40℃, 254nm).
2.1 General procedure for the synthesis of dihydropyrano[c]chromenes
A mixture of 4-hydroxycoumarin (1mmol), aldehydes (1mmol), malononitrile (1mmol)/ ethyl
cyanoacetate (1mmol), and TLIM (45mg) in 5mL solvent (DMSO/water, v/v=9/1) were stirred at 45℃.
The progress of the reaction was monitored by TLC (ethyl acetate / n-hexane = 2/1). Upon completion
of the reaction, lipase TLIM was recovered by centrifugation and could be reused in the next run directly.
The reaction solution was steamed to get crude product, which was then purified by recrystallization in
ethanol.
2.2 General procedure for the synthesis of benzopyrans
A mixture of 1, 3-cyclohexanedione (1mmol) / dimedone (1mmol), aldehydes (1mmol), malononitrile
(1mmol) / ethyl cyanoacetate (1mmol), and TLIM (45mg) in 5mL solvent (DMSO/water, v/v=9/1) were
stirred at 45℃. The progress of the reaction was monitored by TLC (ethyl acetate / n-hexane = 2/1).

Upon completion of the reaction, lipase TLIM was recovered by centrifugation and could be reused in
the next run directly. The reaction solution was steamed to get crude product, which was then purified
by recrystallization in ethanol.
3. Results and discussion
3.1 Optimization of enzyme sources for the synthesis of 1a
O
O
O
Cl
OH
O
O
lipase
H
NC
CN
+
+
solvent
O
Cl
CN
NH2
1a
Scheme 1 Enzymatic reaction of 4-chlorobenzaldehyde, 4-hydroxycoumarin and malononitrile
Table1. Effect of enzyme sources on the yield of 1a
Entry
Enzymea
PPLc
RMIMd
TLIMe
Yield (%)b
Entry
9
Enzymea
PPL
Yield (%)b
1
2
3
4
5
6
7
8
11
28
35
10
11
12
13
14
15
16
DF
37
31
LPL
40
No enzyme
ND
CRL
41
Novozym435 17
TLIM
Papain
BSA
46（44g）
Hlip-F
BCL
19
19
25
35
42
29
RMIM
TLIMf
a Reaction conditions: lipase (50 mg), 4-chlorobenzaldehyde (1mmol), 4-hydroxycoumarin (1mmol),
malononitrile (1mmol), n-hexane (5 mL), 45 °C, 24 h.
b HPLC yield.
c Reaction conditions: PPL (30 mg), 4-chlorobenzaldehyde (1mmol), 4-hydroxycoumarin (1mmol),
malononitrile (1mmol), solvent (5mL, ethanol /water, v/v=4/1), 35 °C, 1 h.
d Reaction conditions: RMIM (50 mg), 4-chlorobenzaldehyde (1mmol), 4-hydroxycoumarin (1mmol),
malononitrile (1mmol), water (5 mL), 45 °C, 20 h.
e Reaction conditions: TLIM (50 mg), 4-chlorobenzaldehyde (1mmol), 4-hydroxycoumarin (1mmol),
malononitrile (1mmol), n-hexane (5 mL), 35 °C, 12 h.
f Denatured TLIM was obtained by treating with acetone for 24 h.
g Isolated yield.

In the initial research, we chose the reaction between 4-chlorobenzaldehyde, 4-hydroxycoumarin and
malononitrile as the model reaction (Scheme 1). Based on the reported reaction conditions18-20, no
satisfactory result was obtained (Table 1, Entries1-3). Obviously, the previous methods are not suitable
for this three-component reactions. It is necessary to seek out better reaction conditions for the model
reaction. As can be seen in Table 1, no product could be detected in the blank control reaction (Table 1,
Entry 4). Lipases such as Novozym435, Hlip-F, BCL, RMIM, PPL, DF, LPL, CRL and TLIM could
promote the reaction (Table 1, Entries5-13), and TLIM showed the best catalytic activity among these
lipases to generate the target product with 46% yield (Table 1, Entry13). Other enzymes such as papain
and BSA could also promote the model reaction with yields of 35% and 42% separately (Table 1,
Entries14-15). Compared to the untreated TLIM, the denatured TLIM showed lower catalytic activity
and generated the target product with 29% yield. It can be supposed that enzyme active center is excluded
from reaction and only the enzyme functional groups catalyze the reaction.
3.2 Optimization of solvent and water content for the synthesis of 1a
Table2. Effect of solvent source and water content on the yield of 1a
Entry
Solventa
n-Hexane
Toluene
THF
Yield (%)b
Log P
3
1
46
50
4
2
2.5
0.4
0.4
0.2
-0.6
-
3
4
Isopropanol
Acetonitrile
DMF
28
5
5
6
38
26
97
99
48
39
7
H2O
8
DMSO
-1.5
-1.5
-1.5
-1.5
9
DMSOc
DMSOd
DMSOe
10
11
a
Reaction conditions: TLIM (50 mg), 4-chlorobenzaldehyde (1mmol), 4-hydroxycoumarin (1mmol),
malononitrile (1mmol), solvent (5 mL), 45 °C, 24 h.
b HPLC yield.
c
Reaction conditions: TLIM (50 mg), 4-chlorobenzaldehyde (1mmol), 4-hydroxycoumarin (1mmol),
malononitrile (1mmol), DMSO (4.5mL), water (0.5mL), 45 °C, 24 h.
d
Reaction conditions: TLIM (50 mg), 4-chlorobenzaldehyde (1mmol), 4-hydroxycoumarin (1mmol),
malononitrile (1mmol), DMSO (3.75mL), water (1.25mL), 45 °C, 24 h.
e
Reaction conditions: TLIM (50 mg), 4-chlorobenzaldehyde (1mmol), 4-hydroxycoumarin (1mmol),
malononitrile (1mmol), DMSO (2.5mL), water (2.5mL), 45 °C, 24 h.
Subsequently, we explored the effects of various solvents on the reactions to synthetize 1a using TLIM
as catalyst (Table 2, Entries1-8). As can be seen in Table 2, trace target product could be obtained in
THF (Table 2, Entry 3) and acetonitrile (Table 2, Entry 5). Low to moderate yield of target product could
be obtained in solvents like n-hexane (Table 2, Entry1), toluene (Table 2, Entry2), isopropanol (Table 2,
Entry4), DMF (Table 2, Entry6) and water (Table 2, Entry7). DMSO exhibited extraordinarily excellent

activity to promote the model reaction to generate the target product in 97% yield (Table 2, Entry8),
which may result from its good solubility for substrates and its quality to keep the catalytic activity of
enzymes. It was reported that the LogP value of the reaction medium influenced the catalytic activity of
enzymes21. While no obvious correlation was observed in the TLIM catalyzed model reaction, which
18,
was consistent with literatures17,
22. In our previous researches, lipase LPL could catalyze the
Knoevenagel condensation reaction in anhydrous DMSO to generate target products efficiently17 and
lipase TLIM could catalyze the Knoevenagel-Michael cascade reaction in anhydrous n -hexane to give
target products in excellent yields18. As for this research, a gentle rise in the yield from 97% to 99% was
observed as the water content increased from 0 to 10% (Table 2, Entries8-9), and a sharp decrease in the
yield from 99% to 39% was detected as the water content increased from 10% to 50% (Table 2, Entries9-
11). This may be related to the fact that the right amount of water is useful in keeping the structural
stability and catalytic activity of lipase TLIM. Consequently, 10% of water content was selected as an
optimum condition for the synthesis of 1a.
3.3 Optimization of enzyme quantity, temperature and reaction time for the synthesis of 1a
Table3. Effects of enzyme quantity, temperature and reaction time on the yield of 1a
Entrya
Temperature(℃)
Enzyme quantity
Time (h)
Yield (%)b
(mg)
35
1
2
3
4
5
6
7
8
45
45
45
45
35
55
45
45
24
24
24
24
24
24
8
63
99
99
89
43
60
72
99
45
50
55
45
45
45
45
16
a
Reaction conditions: TLIM, 4-chlorobenzaldehyde (1mmol), 4-hydroxycoumarin (1mmol),
malononitrile (1mmol), DMSO (4.5mL), water (0.5mL).
b HPLC yield.
The effect of enzyme quantity on the yield of 1a was investigated (Table 3, Entries1-4). The yield of
1a kept increasing as the enzyme quantity increased from 35mg to 45 mg and tended to decline as the
enzyme quantity is 55mg. It was reported that temperature could affect the catalytic activity and substrate
specificity of enzymes23. We explored the influence of temperature on the yield of 1a (Table 3, Entry2
and Entries5-6), and the suitable temperature for this enzymatic reaction was 45℃. The yield of 1a kept
increasing as the reaction time increased from 8h to 16h (Table 3, Entries7-8) and tended to be stable
after 16h (Table 3, Entry2).

3.4 TLIM-catalyzed cascade synthesis of dihydropyrano[c]chromenes
O
O
O
X
TLIM, DMSO/water(v/v,9/1)
CN
NC
CN
NH2
OH
O
1a-1j
O
+
X
H
O
O
O
O
O
X
NC
O
O
NH2
O
TLIM, DMSO/water(v/v,9/1)
2a-2c
Scheme 2 TLIM-catalyzed synthesis of dihydropyrano[c]chromenes
Table 4. TLIM-catalyzed synthesis of dihydropyrano[c]chromenes
Entrya
Yield/%b
X
Product
Time/h
4-ClC6H5
1
2
1a
1b
16
16
99
98
4-NO2C6H5
4-CNC6H5
4-CH3C6H5
3
4
5
1c
1d
1e
16
24
24
98
93
91
4-OH-3-OCH3C6H5
4-OCH3C6H5
6
1f
24
91
4-OHC6H5
4-t-C4H9
2-thienyl
7
8
9
1g
1h
1i
24
48
16
80
75
91
10
11
12
13
14
butyl
1j
2a
2b
2c
1a
48
16
24
24
16
80
93
90
85
95c
4-CF3C6H5
4-CH3C6H5
2-thienyl
4-ClC6H5
a Reaction conditions: TLIM (45 mg), aldehyde (1mmol), 4-hydroxycoumarin (1mmol), malononitrile/
ethyl cyanoacetate (1mmol), DMSO (4.5mL), water (0.5mL), 45 °C.
b Isolated yield.
c Reaction conditions: TLIM (450 mg), 4-chlorobenzaldehyde (10mmol), 4-hydroxycoumarin
(10mmol), malononitrile (10mmol), DMSO (45mL), water (5mL), 45 °C.

With the optimal reaction conditions in hand, substrate applicability of the multicomponent cascade
reactions catalyzed by lipase TLIM was further explored (Scheme 2). As can be seen in Table 4, aromatic
aldehydes with electron-withdrawing substituents (Table 4, Entries1-3) and aromatic aldehydes with
electron-donating substituents (Table 4, Entries4-7) all reacted efficiently with 4-hydroxycoumarin and
malononitrile to generate target products in excellent yields (80-99%). It took relatively longer time for
aromatic aldehydes with electron-donating substituents to finish the multicomponent cascade reactions
than aromatic aldehydes with electron-withdrawing substituents. It was noteworthy that heterocyclic
aldehydes like thiophene-2-aldehyde could react readily with 4-hydroxycoumarin and malononitrile to
get the target product in 91% yield after 16h (Table 4, Entry 9). Unexpectedly, aromatic aldehydes having
large steric hindrance such as 4-tert-butylbenzaldehyde (Table 4, Entry 8) and aliphatic aldehydes like
butyraldehyde (Table 4, Entry 10) could also react smoothly with 4-hydroxycoumarin and malononitrile
to generate 1h and 1j respectively in good yields after 48h. Outstanding results could also be obtained
when ethyl cyanoacetate was used as the substrate instead of malononitrile (Table 4, Entries11-13). In
addition, the developed method was applied to a gram-scale synthesis and 95% yield of product was
obtained (Table 4, entry 14).
3.5 TLIM-catalyzed cascade synthesis of benzopyrans
O
X
TLIM, DMSO/water(v/v,9/1)
CN
R1
O
NH2
NC
CN
R1
O
3a-3s
O
+
R1
O
X
H
R1
O
X
O
O
NC
O
O
O
NH2
TLIM, DMSO/water(v/v,9/1)
4a-4d
R1=H, CH3
Scheme 3 TLIM-catalyzed synthesis of benzopyrans

Table 5. TLIM-catalyzed cascade synthesis of benzopyrans
Entrya
Yield/%b
99
X
R1
H
H
H
H
H
H
H
H
H
Product
3a
Time/h
12
12
12
12
12
12
12
24
12
36
12
12
12
12
12
12
12
12
36
12
12
12
24
4-ClC6H5
1
4-NO2C6H5
4-CNC6H5
2
3b
3c
98
3
99
4-CH3C6H5
4-OH-3-OCH3C6H5
4-OCH3C6H5
4-OHC6H5
4-t-C4H9
4
3d
3e
91
5
99
6
3f
99
7
3g
3h
3i
95
8
98
9
2-thienyl
92
10
11
12
13
14
15
16
17
18
19
20
21
22
23
butyl
H
3j
96
4-NO2C6H5
CH3
3k
3l
99
4-CNC6H5
4-CH3C6H5
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
98
3m
3n
3o
3p
3q
3r
99
4-OH-3-OCH3C6H5
4-OCH3C6H5
4-OHC6H5
98
96
99
4-t-C4H9
98
2-thienyl
butyl
98
3s
98
4-NO2C6H5
4-OH-3-OCH3C6H5
4-CF3C6H5
H
H
H
H
4a
90
4b
4c
82
95
2-thienyl
4d
84
a
Reaction conditions: TLIM (45 mg), aldehyde (1mmol), 1,3-diketones (1mmol), malononitrile/ ethyl
cyanoacetate (1mmol), DMSO (4.5mL), water (0.5mL), 45 °C.
b Isolated yield.
As can be seen in Table 5, the optimum conditions are suitable not only for the synthesis of
dihydropyrano[c]chromenes but also for the synthesis of benzopyrans. Aromatic aldehydes with
electron-withdrawing substituents (Table 5, Entries1-3) and aromatic aldehydes with electron-donating
substituents (Table 5, Entries4-7) all reacted smoothly with 1, 3-cyclohexanedione and malononitrile to
generate target products in excellent yields (91-99%). Excellent results could also be acquired for the
reactions of aromatic aldehydes having large steric hindrance (Table 5, Entry8)/heterocyclic aldehydes
(Table 5, Entry9)/aliphatic aldehydes (Table 5, Entry10) , 1, 3-cyclohexanedione and malononitrile.
Moreover, the reactions of dimedone, malononitrile and aromatic aldehydes (Table 5, Entries11-17)/
heterocyclic aldehydes (Table 5, Entry18)/ aliphatic aldehydes (Table 5, Entry19) could also go well to
generate target products in 96-99% yields. Zhang et al. reported that PPL could efficiently catalyze the

one-pot multiple reaction to synthesize benzopyrans, but only moderate yields of target products could
be obtained when it came to ethyl cyanoacetate20. While ethyl cyanoacetate could react easily with 1, 3-
cyclohexanedione and aromatic aldehydes (Table 5, Entries20-22)/ heterocyclic aldehydes (Table 5,
Entry23) to give target products in excellent yields using our procedure.
3.6 Reusability of lipase TLIM for the synthesis of 1a
Fig.1. Reusability of lipase TLIM for the synthesis of 1a
Reaction conditions: TLIM (45 mg), 4-
chlorobenzaldehyde (1mmol), 4-hydroxycoumarin (1mmol), malononitrile (1mmol), DMSO (4.5mL),
water (0.5mL), 45 °C, 24h, HPLC yield.
Reusability of catalyst is a vital factor to inspect the economical and environmental performance of
reactions. We investigated the reusability of catalyst by using 4-chlorobenzaldehyde, 4-hydroxycoumarin
and malononitrile as model substrates. The immobilized lipase TLIM could be collected by
centrifugation after the reaction and used in the next run directly. As can be seen in Fig.1, the immobilized
lipase TLIM showed satisfactory reusability and 84% yield of target product could be obtained after five
cycles. Actually, after the fifth recycling, we measured that the hydrolytic activity of lipase TLIM
decreased from original 1161U/g to 668U/g, which was consistent with the decrease of yield. It is
noteworthy that excellent yields could be obtained for reactions in anhydrous DMSO (Table2, Entry8).
After the fifth recycling, the activity of lipase TLIM in anhydrous DMSO decreased from original
1161U/g to 20U/g and only 11% yield of the target product could be obtained in anhydrous DMSO.
As a result, the right amount of water is useful in keeping the structural stability and catalytic activity
of lipase TLIM.

3.7 Possible mechanism for the synthesis of 1a catalyzed by TLIM
Taking the model reaction (Scheme 1) as an example, we proposed the possible mechanism based on
20, 24
literatures9,
and experiments. We chose the reaction between 4-hydroxycoumarin, 4-
chlorobenzaldehyde and malononitrile as the model reaction (Scheme 1). Firstly, we obtained the product
(5a) based on 4-hydroxycoumarin and 4-chlorobenzaldehyde (Scheme 4, (1)). No products were detected
based on 5a and malononitrile (Scheme 4, (2)). Secondly, we obtained the product (6a) based on 4-
chlorobenzaldehyde and malononitrile (Scheme 4, (3)). 96% Yield of target product (1a) were obtained
based on 6a and 4-hydroxycoumarin (Scheme 4, (4)). What’s more, we detected the intermediate product
by TLC and obtained it by column chromatography. The intermediate product was turned out to be 6a.
As a result, we surmised that 4-chlorobenzaldehyde reacted firstly with malononitrile instead of 4-
hydroxycoumarin. We assumed that the Knoevenagel condensation reaction occurred between
malononitrile and 4-chlorobenzaldehyde to generate the intermediate compound (6a) and water firstly.
Then the Michael addition reaction occurred between 4-hydroxycoumarin and 6a to give 7a, which
finally turned to the target product (1a) by cyclization and tautomerization (Scheme 4, (5)).

Cl
O
OH
Cl
Lipase TLIM
OH
O
OH
O
+
H
DMSO/Water (v/v, 9/1)
(1)
O
I
O
O
O
II
5a
Cl
Lipase TLIM
OH
OH
O
+
CN
No Reaction
(2)
CN
DMSO/Water (v/v, 9/1)
O
O
O
III
5a
Cl
NC
CN
H
Lipase TLIM
CN
CN
H
+
DMSO/Water (v/v, 9/1)
(3)
O
Cl
III
II
6a
NC
CN
H
O
O
Cl
OH
O
Lipase TLIM
(4)
+
DMSO/Water (v/v, 9/1)
O
O
Cl
CN
6a
NH
2
I
1a
N
N
Lipase TLIM
H O
2
Cl
NC
CN
H
DMSO/Water (v/v, 9/1)
CN
CN
H
+
Cl
H
Lipase TLIM
O
Cl
II
DMSO/Water (v/v, 9/1)
III
6a
H
OH
O
O
O
O
O
I
O
O
O
Cl
O
O
(5)
O
O
O
O
CN
OH
O
O
NH
H
NH
2
CN
N
CN
Cl
CN
H
1a
Cl
Cl
N
7a
Scheme 4. Possible mechanism for the synthesis of 1a catalyzed by TLIM
3.8 Comparison of the catalytic efficiency of TLIM with some reported catalysts
Table 6 Comparison of the catalytic efficiency of TLIM with some reported catalysts
Entry
Catalyst
Temperature/
Reusability of
catalyst
Yield/ Reference
%
℃

1
2
3
4
5
6
7
TLIM
45
Reusable
Non reusable
Reusable
99
80
91
90
88
70
85
This work
25
I2
(CTA)3[SiW12]-Li+-MMT
LiBr
120
26
27
28
29
30
Reflux
Reflux
Reflux
Reflux
80
Non reusable
Reusable
SiO2-Cu(Ⅱ)
Borax
Reusable
NH4Al(SO4)2·12H2O
Reusable
Furthermore, we took the reaction of dimedone, 4-hydroxybenzaldehyde and malononitrile as an
example and compared the catalytic efficiency of TLIM with other reported catalysts in terms of
temperature, reusability of catalyst and yields (Table6). As can be seen in Table6, this work used
commercially available and recyclable catalyst and much lower temperature was needed than other
reported protocols. Moreover, 99% yield of corresponding product (3p) could be obtained, which was
absolutely superior to other reported methods.
4 Spectroscopic data for representative products
Subsequently, TLIM-catalyzed condensation of malononitrile
/
ethyl cyanoacetate, 4-
hydroxycoumarin/ 1, 3-cyclohexanedione/ dimedone and a variety of aldehydes in aqueous DMSO was
investigated. All the products were characterized by 1H NMR, 13C NMR, HRMS and melting points，
and the data of known products are consistent with the literature(see SI）. Here is the spectroscopic data
for representative products.
2-amino-4-(4-chlorophenyl)-5-oxo-4H,5H-pyrano[3,2-c]chromene-3-carbonitrile（1a）:
Yield: 0.3472g (99%); white solid; m p 260℃-261℃.
1H NMR (300 MHz, DMSO-d6) δ 7.95 – 7.86 (m, 1H), 7.76 – 7.68 (m, 1H), 7.59 – 7.44 (m, 2H), 7.44
(d, J = 6.2 Hz, 2H), 7.37 (d, J = 8.5 Hz, 2H), 7.30 (d, J = 8.5 Hz, 2H), 4.49 (s, 1H).
HRMS (EI-TOF): m/z Calcd. for C19H11ClN2O3[M+Na]+: 350.0401, found 350.0404.
Ethyl 2-amino-5-oxo-4-(p-tolyl)-4H,5H-pyrano[3,2-c]chromene-3-carboxylate (2b)：
Yield: 0.3396g (90%); white solid; m p 118℃-119℃.
1H NMR (300 MHz, DMSO-d6) δ 8.47 (s, 3H), 7.90 (d, J = 7.8 Hz, 2H), 7.59 (t, J = 7.7 Hz, 2H), 7.45 –
7.21 (m, 4H), 7.03 (s, 4H), 6.31 (s, 1H), 2.24 (s, 3H).
HRMS (EI-TOF): m/z Calcd. for C22H19NO5[M+Na]+: 377.1212, found 377.1207.
2-amino-4-(4-chlorophenyl)-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (3a)：
Yield: 0.2977g (99%); white solid; m p 241℃-243℃.
1H NMR (400 MHz, DMSO-d6) δ 7.38 – 7.28 (m, 2H), 7.23 – 7.15 (m, 2H), 7.06 (s, 2H), 4.20 (s, 1H),
2.61 (q, J = 4.6, 4.0 Hz, 2H), 2.27 (dtq, J = 16.6, 11.1, 5.2 Hz, 2H), 1.92 (ddq, J = 23.8, 10.6, 5.8 Hz,
2H).
HRMS (EI-TOF): m/z Calcd. for C16H13ClN2O2[M+Na]+: 300.0603, found 300.0601.
2-amino-7,7-dimethyl-4-(4-nitrophenyl)-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile

(3k)：
Yield: 0.3360g (99%); white solid; m p 180℃-182℃.
1H NMR (300 MHz, DMSO-d6) δ 8.17 (d, J = 8.6 Hz, 2H), 7.45 (d, J = 8.6 Hz, 2H), 7.16 (s, 2H), 4.37
(s, 1H), 2.54 (s, 2H), 2.26 (d, J = 16.1 Hz, 1H), 2.11 (d, J = 16.1 Hz, 1H), 1.04 (s, 3H), 0.96 (s, 3H).
HRMS (EI-TOF): m/z Calcd. for C18H17N3O4[M+Na]+: 339.1109, found 339.1105.
Ethyl 2-amino-4-(4-nitrophenyl)-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carboxylate (4a)：
Yield: 0.3225g (90%); yellow solid; m p 181℃-182℃.
1H NMR (300 MHz, DMSO-d6) δ 8.09 (d, J = 8.6 Hz, 2H), 7.67 (s, 2H), 7.42 (d, J = 8.7 Hz, 2H), 4.63
(s, 1H), 3.94 (q, J = 7.0 Hz, 2H), 2.63 (t, J = 5.9 Hz, 2H), 2.39 – 2.18 (m, 2H), 2.04 – 1.90 (m, 1H), 1.85
(dd, J = 11.1, 5.0 Hz, 1H), 1.07 (t, J = 7.1 Hz, 3H).
HRMS (EI-TOF): m/z Calcd. for C18H18N2O6[M+Na]+: 358.1107, found 358.1110
5 Conclusion
In conclusion, TLIM is firstly reported to be a commercially available, reusable and efficient catalyst
in the Knoevenagel-Michael cascade reactions of aldehydes, malononitrile / ethyl cyanoacetate and 4-
hydroxycoumarin. Moreover, excellent yields could also be obtained when the substrate was 1, 3-
cyclohexanedione/ dimedone instead of 4-hydroxycoumarin. This methodology offers many
superiorities such as simple procedure, mild reaction conditions, high substrate applicability and the
ability to be scaled up.
Acknowledgments
This research was financially supported by the National Natural Science Foundation of China
(No.21676143), the self-owned research project from key laboratory of Materials-Oriented Chemical
Engineering (No. ZK201603), the Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture,
and the Qing Lan Project of Jiangsu Province.
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● An efficient enzymatic method for the synthesis of dihydropyrano[c]chromenes was developed
using three-component tandem reactions of aldehydes, malononitrile / ethyl cyanoacetate and 4-
hydroxycoumarin.
●An efficient method for the synthesis of benzopyrans was developed by one-
pot enzymatic cascade reactions of aldehydes, malononitrile / ethyl cyanoacetate and 1, 3-
cyclohexanedione/ dimedone.
● Simple procedure, mild reaction conditions, high substrate applicability, the ability to be scaled
up, and good to excellent yields, are the important attributes of the present protocol.

Declaration of interests
The authors declare that they have no known competing financial interests or personal
☒
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests: