Accepted Manuscript

Promiscuous enzyme-catalyzed cascade reaction in water: Synthesis of dicou-

marol derivatives

Yajie Fu, Zeping Lu, Ke Fang, Xinyi He, He Huang, Yi Hu

PII: S0960-894X(19)30137-4
DOI: https://doi.org/10.1016/j.bmcl.2019.03.007
Reference: BMCL 26321

To appear in: Bioorganic & Medicinal Chemistry Letters

Received Date: 4 January 2019

Revised Date: 2 March 2019
Accepted Date: 5 March 2019

Please cite this article as: Fu, Y., Lu, Z., Fang, K., He, X., Huang, H., Hu, Y., Promiscuous enzyme-catalyzed cascade
reaction in water: Synthesis of dicoumarol derivatives, Bioorganic & Medicinal Chemistry Letters (2019), doi:
https://doi.org/10.1016/j.bmcl.2019.03.007

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Promiscuous enzyme-catalyzed cascade reaction in water: Synthesis of dicoumarol
derivatives

Yajie Fu, Zeping Lu, Ke Fang, Xinyi He, He Huang, Yi Hu*

(State Key Laboratory of Materials-Oriented Chemical Engineering, School of Pharmaceutical

Sciences, Nanjing Tech University, Nanjing 210009, China)

Abstract

Lipase RMIM was firstly used as a promiscuous biocatalyst to catalyze the Knoevenagel-Michael
cascade reactions of 4-hydroxycoumarin with aromatic, heterocyclic or aliphatic aldehydes to
synthesize dicoumarol derivatives in water. Results showed that the adopted methodology could
offer many advantages, such as mild reaction conditions, pure aqueous reaction system, wide
substrate applicability, recyclable catalyst, excellent yields (81%-98%), operational simplicity, and
environmentally friendly reactions.

Keywords Enzymatic synthesis, cascade reactions, aromatic aldehyde, 4-hydroxycoumarin,

dicoumarol derivatives

Dicoumarol derivatives are widely used due to their prominent pharmacological and biological
activities. They can be used as a vitamin K antagonist to prevent blood clotting 1; an antipyretic2
due to their antibacterial activity on Staphylococcus aureus and Staphylococcus epidermidis; in
the treatment of HIV3, skin cancer4 and snake venom5; and also as an analytical reagent6.
Dicoumarol derivatives are generally obtained by Knoevenagel condensation-Michael addition of
4-hydroxycoumarin and aromatic aldehyde. Various catalysts for dicoumarol derivative synthesis
have been reported and classified as nanoparticle catalysts, which include the following: LTNPs7,
MgO- NPs8, BiVO4-NPs9, SiO2-OSO3H NPs10, P4VPy–CuO11, and magnetic nanoparticle catalyst
TrBr/[Fe3O4@SiO2@(CH2)3-ImSO3H]Cl12. Ionic liquid catalysts include: IL@CNTs13, Hnmp /
ZnCl314 [Dabco-H][AcO]15, [Et3NH][HSO4]16, and [TMG][Ac]17. Solid acid catalysts include:
RHA-SO3H18, KF-montmorillonite19, PS-Zn-anthra complex20, and Mn(pbdo)2Cl2/MCM-4 121.
However, these methods are usually restricted by their requirement for volatile organic solvents,
relatively high temperatures, high-cost catalysts, and difficult preparation, and occasional
moderate yields.
Enzymes have been widely used in many industries, such as chemical, pharmaceutical food, and
environmental industries. They offer specificity, high efficiency, mild reaction conditions,
environmentally friendly reactions, and catalytic activity22-24. Recently, enzymes with
promiscuous catalytic activity have been identified, for example in carbon–carbon bond–forming
reactions such as aldol condensations, Knoevenagel condensations, Michael additions, and
Mannich reactions 25-28. In our previous work, we reported that lipase LPL can catalyze the
Knoevenagel condensation reaction of aromatic aldehyde with active methylene compounds in
anhydrous DMSO, producing a target product with Z configuration28. Lipase PPL can also
catalyze the Knoevenagel condensation of aromatic aldehyde with the heterocyclic active
methylene compound indol-2-one at 45 ℃ in H2O-DMSO (20% aqueous solution) to generate
target products with E configuration and Z configuration29. Recently, we also reported that lipase
TLIM exhibits prominent promiscuity for Knoevenagel-Michael cascade reactions of 1,
3-diketones with aromatic aldehydes to synthesize xanthone derivatives30. In continuation of our
interest in lipase-catalyzed organic synthesis, we herein report the first evidence that lipase RMIM
acts as an efficient biocatalyst to synthesize dicoumarol derivatives by cascade reactions between
4-hydroxycoumarin and aromatic aldehydes in water.
Porcine pancreas lipase (PPL), Amano lipase PS from Burkholderia cepacia (BCL) and Candida
rugosa lipase (CRL) were purchased from Sigma. Lipase from Thermomyces lanuginosus (TLIM),
lipase from Rhizomucor miehei (RMIM), and Novozym 435 (lipase B from Candida antarctica,
immobilized on a macroporous acrylic resin) were purchased from Novo Nordisk. Lipase DF was
donated by Shanghai Guoguang. Bovine serum albumin (BSA) was purchased from Shanghai
Huixing. Other reagents are commercially available and were used without further purification.
Melting points were determined using a WRS-1B digital melting point instrument and were not
corrected. The 1H NMR and 13C NMR spectra were measured using a Bruker Advance 2B 400
MHz instrument with DMSO-d6 as solvent and TMS as internal standard. The HRMS were
measured using an Agilent LC/MS mass spectrometer. The progress of the reaction was monitored
by TLC using pre-coated Haiyang GF254 silica gel plates. HPLC data were obtained using a
Dionex liquid chromatography instrument (Amethyst C18-H (4.6×250 mm, 5 μm), formic acid
with 0.1% water/methanol (v:v=2/3, 30 min, equal gradient elution), 45 ℃, 254 nm).

Scheme 1. Reaction of p-chlorobenzaldehyde with 4-hydroxy dicoumarin catalyzed by lipase.

Screening of enzyme for Knoevenagel-Michael cascade reactions

The reaction of 4-hydroxycoumarin with p-chlorobenzaldehyde was used as the model reaction in
our initial research. In our previous study, lipase TLIM showed prominent promiscuity for the
Knoevenagel-Michael cascade reactions of 1, 3-diketones with aromatic aldehydes to synthesize
xanthone derivatives30. The same reaction conditions were not deemed suitable for
Knoevenagel-Michael cascade reactions of 4-hydroxycoumarin with p-chlorobenzaldehyde to
synthesize 3,3'-((4-chlorophenyl)methylene)bis(4-hydroxy-2H-chromen-2-one) (3a) (Table 1,
entry 10), as only 14% yield was obtained. Further research regarding biocatalyst screening of for
the model reaction was performed in pure aqueous phase (Table 1). Trace product was obtained
(Table 1, entry 1) in the absence of enzyme. Lipases DF, BCL, TLIM, CRL, PPL and Novozym
435 (Table 1, entries 2-7) showed low catalytic efficiency. And while RMIM (Table 1, entry 8)
showed the strongest catalytic efficiency, moderate yield was obtained. As expected, only low
yield was obtained when denatured RMIM (Table 1, entry 9) or BSA (Table 1, entry 12) was used.
The specific structure of lipase clearly imparts a significant effect on the catalytic performance for
this reaction. In addition, product was not detected in the absence of p-chlorobenzaldehyde (Table
1, entry 11), which indicates that the lactone structure of 4-hydroxycoumarin could not be
hydrolyzed by lipase under the reaction conditions.

Table 1. Effect of lipase source on the yield of 3a

Entry Enzymea Yield (%)b Entry Enzymea Yield (%)b

1 No enzyme trace 7 Novozym435 46
2 Lipase DF 15 8 RMIM 55
c
3 BCL 19 9 RMIM 27
d
4 TLIM 23 10 TLIM 14
e
5 CRL 31 11 RMIM ND
6 PPL 44 12 BSA 27
a
Reaction conditions: lipase (50 mg), 4-chlorobenzaldehyde (1 mmol), 4-hydroxycoumarin (2 mmol),
H2O (5 mL), 35 °C, 14 h.
b
HPLC yield.
c
Denatured RMIM was obtained by treating with acetone for 24 h.
d
Reaction conditions: lipase TLIM (50 mg), 4-chlorobenzaldehyde (1 mmol), 4-hydroxycoumarin (2
mmol), n-Hexane (5 mL), 35 °C, 14 h.
e
Reaction conditions: lipase (50 mg), 4-hydroxycoumarin (2 mmol), H2O (5 mL), 35 °C, 14 h.

Optimization of solvent for Knoevenagel-Michael cascade reactions

In non-aqueous enzymatic reactions, it was reported that the reaction medium LogP value could
influence catalytic activity31. It is generally believed that the catalytic activity of an enzyme
increases as the solvent LogP increases32. However, our results were not consistent with this
hypothesis (Table 2). Lipase RMIM showed stronger catalytic efficiency in isopropanol,
acetonitrile, DMF, water and DMSO (solutions with lower LogP value) (Table 2, entries 5-9) than
in n-Hexane, toluene, CH2Cl2 and THF (solutions with higher LogP value) (Table 2, entries 1-4).
In previous research, most enzymatic reactions were performed in aqueous-organic mixture
solvents29-31,33,34, while other enzymatic reactions were performed in organic solvents without
water32-35. Results in Table 2 illustrate that lipase RMIM in water displayed the noticeable priority
for this model reaction (Table 2, entry 8). As far as we know, this is the first evidence that
enzymatic Knoevenagel-Michael cascade reactions can be performed in pure water media.
Subsequently, we chose the combination of lipase RMIM and water to screen the following
conditions.
 Table 2. Effect of solvent source on the yield of 3a catalyzed by Lipase RMIM

Entry Solventa Yield (%)b Dielectric constant Log P

1 n-Hexane 18 1.9 3
2 Toluene 17 2.4 2.5
3 CH2Cl2 30 8.9 1
4 THF 8 7.5 0.4
5 Isopropanol 54 19.9 0.4
6 Acetonitrile 48 37.5 0.2
7 DMF 30 38.3 -0.6
8 H2O 55 78.5 -
9 DMSO 36 47.2 -1.5
a
Reaction conditions: lipase RMIM (50 mg), 4-chlorobenzaldehyde (1 mmol), 4-hydroxycoumarin (2
mmol), solvent (5 mL), 35 °C, 14 h.
b
HPLC yield.

Fig.1. Effect of temperature on the yield of 3a. Reaction conditions: RMIM (50 mg),
4-chlorobenzaldehyde (1 mmol), 4-hydroxycoumarin (2 mmol), water (5 mL), 14 h. HPLC yield.
Optimization of enzyme quantity, reaction time and temperature for Knoevenagel -Michael
cascade reactions

The reaction temperature exerted a vital influence on enzyme activity (Fig. 1). Based on the
experimental results, the optimal reaction temperature of lipase RMIM was selected as 45 °C.
Generally, the concentration of enzyme can exert significant effects on enzymatic reactions. It was
observed that 10 mg/mL RMIM was an appropriate dosage for this reaction. Ultimately, results
showed that the yield could be improved up to 93% when lipase RMIM loading was 10mg/mL
and reacted at 45 °C for 20 h in water (Table 3, entry 5).

Table3. Effects of enzyme amount and reaction time on the yield of 3a

Entrya Enzyme amount (mg) Time (h) Yield (%)b

1 30 14 58
2 70 14 76
3 50 14 79
4 50 18 91
5 50 20 93
6 50 24 92
a
Reaction conditions: lipase RMIM, 4-chlorobenzaldehyde (1 mmol), 4-hydroxycoumarin (2 mmol),
H2O (5mL), 45 °C.
b
HPLC yield.

RMIM-catalyzed synthesis of dicoumarol derivatives

After the optimal reaction conditions were established, the substrate applicability of
Knoevenagel-Michael cascade reactions catalyzed by lipase RMIM was further investigated
(Table 4). TLC was used to monitor reactions. It was found that both aromatic aldehydes with
electron-withdrawing substituents and aromatic aldehydes with electrondonating substituents
could generate product 3 in excellent yields (86–98%) (Table 4, entries 1-12). It is also remarkable
that aromatic aldehydes with high steric hindrance, such as 2, 6-dichlorobenzaldehyde (Table 4,
entry 13) and 4-tertbutylbenzaldehyde (Table 4, entry 14) could generate product 3m and 3n in
high yields (85% and 88%, respectively) after reacting with 4-hydroxycoumarin for an appropriate
period of time. Moreover, satisfactory yield were also obtained for heterocyclic aromatic
aldehydes such as 2-thiophenealdehyde (Table 4, entry 15), pyridine-2-carbaldehyde (Table 4,
entry 16) and indole-3-carbaldehyde (Table 4, entry 17), and also aliphatic aldehyde like
butyraldehyde (Table 4, entry 18). Compared with other enzyme-catalyzed Knoevenagel or
Michael reactions31, 33, 35, 36, this procedure provides clear advantages, such as clean solvent,
broader substrate adaptability, shorter reaction times. Also, compared with chemically catalyzed
reactions8, 12, 14, 16, 18, 19, we obtained competitive yields under lower temperature using this
protocol.
Scheme 2. Reaction of aldehydes with 4-hydroxy dicoumarin in water catalyzed by lipase RMIM.

Table 4. Comparing the yield of 3a catalyzed by lipase RMIM

Entrya Ar Product Time/h Yield/%b Entrya Ar Product Time/h Yield/%b

1 4-ClC6H4 3a 20 93 11 2-OH-3-OMeC6H3 3k 20 86

2 2-NO2C6H4 3b 20 90 12 2-OHC6H4 3l 20 98

3 3-NO2C6H4 3c 20 93 13 2,6-Cl2C6H3 3m 24 85

4 4-NO2C6H4 3d 20 96 14 4-C4H9C6H4 3n 24 88

5 4-CNC6H4 3e 20 94 15 2-Thienyl 3o 20 81

6 4-FC6H4 3f 20 94 16 2-Pyridyl 3p 20 93

7 4-OHC6H4 3g 20 89 17 3-Indolyl 3q 48 81

8 C6H5 3h 20 89 18 Butyl 3r 48 87
c
9 4-MeC6H4 3i 20 88 19 4-Cl -- 48 ND

10 4-OMeC6H4 3j 20 96
a
Reaction conditions: RMIM (50 mg), aldehyde (1 mmol), 4-hydroxycoumarin (2 mmol), H2O (5 mL),
45 °C.
b
Isolated yield.
c
Reaction conditions: RMIM (50 mg), 4-chlorobenzaldehyde (1 mmol), coumarin (2 mmol), H2O (5
mL), 45 °C.

Fig.2. Reusability of the lipase RMIM for the synthesis of 3a

Reusability of lipase RMIM for the synthesis of dicoumarol derivatives

The reusability of lipase RMIM for this cascade reactions is shown in Fig.2. After five uses,
although the catalytic efficiency of lipase RMIM decreased to a certain extent, satisfactory yield
(81%) was obtained. Circular dichroism (CD) spectroscopy is a powerful method in structural
biology that has been used to examine proteins, polypeptides and peptide structures since the
1960s36. CD data analysis (Table 5) shows that compared with fresh lipase RMIM, the α-helix
content and β-sheet ratio decreased in lipase RMIM after five uses. The decrease in α-helix
content indicates that the two-dimensional structure of lipase RMIM was changed37, and the
decrease in β-sheet ratio indicates that the rigidity, activity, and stability were reduced to some
extent37,38,39. CD spectral analysis was consistent with the results of enzymatic assays in Fig.2.
The good reusability of this method greatly enhances its practical application potential.

Table 5 Secondary structure percentages of lipase RMIM calculated from CD spectra

run Alpha helix（%） Beta-sheet（%） Beta-turn（%） Random coil（%）

1 20.7 31.2 18.9 28.6
5 13.6 24 20.7 41.3

Possible mechanism for the synthesis of dicoumarol derivatives catalyzed by RMIM

In our method, enzymatic synthesis of dicoumarol derivatives was performed using Knoevenagel
condensation-Michael addition as a two-step tandem reaction. A possible mechanism is proposed
in Scheme 3: First, the His-Asp residue of lipase interacts with the hydroxyl group of
4-hydroxycoumarin to form (I), which causes Knoevenagel condensation with aromatic aldehyde,
and removes one molecule of water to form intermediate product (II). Secondly, the Michael
addition occurs between intermediate product (II) and structure (I) to generate the intermediate
product (III). Finally, the catalytic cycle is completed via an enolization process, therefore the
product is obtained. This speculation was verified by the reaction between coumarin and
4-chlorobenzaldehyde (Table 4, entry 19). As expected, no detectable product was formed, since
there is no hydroxyl group in benzene ring of coumarin.
 Scheme 3. Possible mechanism for the synthesis of dicoumarin derivatives catalyzed by RMIM

4-(bis(4-hydroxy-2-oxo-2H-chromen-3-yl)methyl)benzonitrile(3e):

White solid; m.p.：281-282℃ ;1H NMR (400 MHz, DMSO-d6) δ 12.48 (s, 2H), 7.86 (d, J = 7.8 Hz,
2H), 7.66 (d, J = 8.3 Hz, 2H), 7.56 (t, J = 8.2 Hz, 2H), 7.33 (s, 2H), 7.31 (s, 2H), 7.27 (d, J = 7.4
Hz, 2H), 6.35 (s, 1H);13C NMR (100 MHz, DMSO-d6) δ 166.39 , 164.37 , 152.34 , 147.68 ,
131.79 , 131.56 , 127.80 , 123.95 , 123.34 , 119.07 , 118.57 , 115.73 , 108.01 , 103.11 , 36.62 ;
HRMS
(ESI-TOF) m/z [M+Na]+:460.0820, found: 460.0821.
3,3'-((4-(tert-butyl)phenyl)methylene)bis(4-hydroxy-2H-chromen-2-one)(3n):

White solid; m.p.:250-251℃;1H NMR (400 MHz, DMSO-d6) δ 10.81 (s, 2H), 7.90 (d, J = 7.9 Hz,
2H), 7.64 – 7.53 (m, 2H), 7.44 – 7.27 (m, 4H), 7.24 (d, J = 8.4 Hz, 2H), 7.06 (d, J = 8.1 Hz, 2H),
6.31 (s, 1H), 1.24 (s, 9H);13C NMR (100 MHz, DMSO-d6) δ 165.03 , 164.77 , 152.11 , 147.70 ,
136.57 , 131.82 , 126.31 , 124.75 , 123.82 , 123.67 , 117.78 , 115.88 , 104.13 , 35.48 , 33.90 ,
31.12 ; HRMS(ESI-TOF) m/z [M+Na]+:491.1529, found: 491.1533.

3,3'-(thiophen-2-ylmethylene)bis(4-hydroxy-2H-chromen-2-one)(3o):

Ching solid; m.p.:215-215.6℃;1H NMR (400 MHz, DMSO-d6) δ 12.48 (s, 2H), 7.88 (dd, J = 7.8,
1.2 Hz, 2H), 7.60 – 7.51 (m, 2H), 7.28 (td, J = 8.4, 2.2 Hz, 4H), 7.19 (d, J = 5.1 Hz, 1H), 6.82 (dd,
J = 5.0, 3.6 Hz, 1H), 6.65 (dd, J = 3.1, 1.5 Hz, 1H), 6.46 (s, 1H);13C NMR (100 MHz, DMSO-d6)
δ 166.17 , 164.19 , 152.22 , 146.07 , 131.66 , 126.26 , 124.02 , 123.60 , 123.43 , 123.22 , 118.46 ,
115.74 , 104.10 , 32.71 ; HRMS(ESI-TOF) m/z [M+Na]+:441.0411, found: 441.0401.

In summary, we have demonstrated an efficient enzymatic method for the synthesis of dicoumarol
derivatives using Knoevenagel-Michael cascade reactions of 4-hydroxycoumarin with aromatic
aldehyde in water. The use of green solvent, mild reaction conditions, recyclable biocatalyst,
generality high yields, and environmental friendliness are the important attributes of the present
protocol. The present methodology has extended the potential use of lipase in organic and
pharmaceutical synthesis applications.

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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We have demonstrated an efficient enzymatic method for the synthesis of dicoumarol
derivatives using Knoevenagel-Michael cascade reactions of 4-hydroxycoumarin with
aromatic aldehyde in water. The use of green solvent, mild reaction conditions,
recyclable biocatalyst, generality high yields and environmental friendliness are the
important attributes of the present protocol. The present methodology has extended
the potential use of lipase in organic and pharmaceutical synthesis.