Porcine pancreas lipase as a green catalyst for synthesis of bis-4-hydroxy coumarins

Article abstract of DOI:10.1016/j.bioorg.2020.104139

4-Hydroxycoumarins represents an important structural motif in life sciences molecules with remarkable biological properties. 4-Hydroxycoumarins are important precursors in the synthesis of organic compounds and critical pharmacophores in medicinal chemis

Full text of DOI:10.1016/j.bioorg.2020.104139

Journal Pre-proofs
Porcine pancreas lipase as a green catalyst for synthesis of bis-4-hydroxy
coumarins
Hossein Bavandi, Zohreh Habibi, Maryam Yousefi
PII:
S0045-2068(20)31436-X
DOI:
https://doi.org/10.1016/j.bioorg.2020.104139
YBIOO 104139
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
16 March 2020
9 June 2020
22 July 2020
Please cite this article as: H. Bavandi, Z. Habibi, M. Yousefi, Porcine pancreas lipase as a green catalyst for
synthesis of bis-4-hydroxy coumarins, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.
2020.104139
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Porcine pancreas lipase as a green catalyst for synthesis of bis-4-hydroxy coumarins
Hossein Bavandia, Zohreh Habibia*, Maryam Yousefib**
a Department of Pure Chemistry, Faculty of Chemistry, Shahid Beheshti University, G.C.,
Tehran, Iran
b Nanobiotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran
Abstract
4-Hydroxycoumarins represents an important structural motif in life sciences molecules with
remarkable biological properties. 4-Hydroxycoumarins are important precursors in the synthesis
of organic compounds and critical pharmacophores in medicinal chemistry. One of the
derivatives of this compound is biscoumarin, which has anticoagulant medicinal properties.
Porcine pancreas lipase (PPL) has been used as an efficient green biocatalyst for the synthesis of
bis-4-hydroxycoumarin compounds and expands the biocatalytic promiscuity of lipase in organic
synthesis. Some aromatic aldehydes have been used in this investigation for screening and
examining the ability of enzyme in knoevenagel condensation. Various parameters including
temperature, solvent, and the amount of biocatalyst were investigated, ethanol, 45 °C and 10 mg
of PPL gave rise to best yields. Bis-4-hydroxy coumarin compounds were synthesized under
mild reaction conditions with high conversion yields (81-88 %).
Key Words: Lipase, 4-Hydroxycoumarins, Knoevenagel condensation, Dicoumarol
*Corresponding author. Tel.: +982 129 903 110; fax: +982 122 431 663.
** Corresponding author Tel.: +982 122 432 020; fax: +982 122 432 021.
E-mail addresses: Z_habibi@sbu.ac.ir (Z. Habibi), M.yousefi@avicenna.ac.ir (M. Yousefi)
1

1. Introduction
Cancer is the second leading cause of death globally, and is responsible for an estimated 9.6
million deaths in 2018. Globally, about 1 in 6 deaths is due to cancer. Deaths from cancer
worldwide are projected to reach over 13 million in 2030. Scientists are looking for new ways to
fight the disease simultaneous the world's cancer rises. Chemists have also synthesized new
compounds that have anti-cancer properties [1-3].
Dicoumarol and derivatives of coumarines are a group of compounds that have many biological
and pharmacological activities, including anticancer, anticoagulant, antibacterial, antiviral,
antifungal, antitumor, anti-arthritis, anti-pyretic and antioxidant properties [4-9]. These
compounds are generally synthesized from the reaction of 4- hydroxy coumarin and aldehyde
derivatives in the presence of catalysts. Dicoumarol and warfarin are naturally occurring
anticoagulant drugs that depletes stores of vitamin K. they are also used in biochemical
experiments as an inhibitor of reductase. They are derivative of coumarin a substance made by
plants which are transformed in moldy feeds or silages by a number of species of fungi (Scheme
1) [10].
In recent years, various chemical catalysts have been used for the synthesis of dicoumarol such
as Ni Nano particle, o- Benzenedisulfonimide, starch–sulfuric acid, InCl3, RHA-SO3H, poly(4-
vinylpyridine), etc. but all of these methods have limitations including high environmental
hazards, complicated reaction process, long time or high temperatures [11-14]. So, another
environmentally catalyst is required for the synthesis of dicoumarols.
2

The enzymes have many applications as biocatalysts in chemical transformation. These
biocatalysts as chemical catalysts have many advantages such as high selectivity, mild reaction
conditions, and low byproducts [15,16].
Lipases (EC 3.1.1.3) have emerged as a dominant class of enzymes in green chemistry [17–20].
They belong to the class of serine hydrolases and catalyze the hydrolysis of ester bonds at the
interface between an insoluble substrate phase and the aqueous phase. What makes lipases so
attractive and widely exploited class of enzymes is its structure and properties. Firstly, they
usually display excellent stereoselectivity, chemoselectivity, and regioselectivity [21]. Second is
their easy availability as many of them can be produced from microorganisms, namely, fungi and
bacteria [20]. Thirdly, the crystal structure exploration of many lipases has facilitated the design
of rational synthetic strategies with improved catalysis. Also they are free from cofactor
requirement. These properties make lipases prominent biocatalysts in organic chemistry [22].
Lipases are the most widely used class of enzymes in organic synthesis and they most often
constitute an environmentally friendly alternative to conventional chemical catalysts. Lipases are
known to promiscuity enzymes. Catalytic promiscuity is a chemical transformation that proceeds
through a different reaction mechanism from native transformation. In organic synthesis,
formation of the C-C bond is fundamental, which organic reactions like aldol condensation,
Hantzsch reaction, Cannizzaro reaction, Mannich reaction, Baylis-Hillman reaction,
Knoevenagel condensation, Michael addition, and other reactions are accomplished by the lipase
enzyme [23-26].
Knoevenagel condensation and Michael addition are the most fundamental reactions in organic
synthesis that involving the nucleophilic addition of an active hydrogen compound to an
3

aldehyde or ketone, and 1,4-addition of a carbon nucleophile to an α/β unsaturated carbonyl
compound respectively [27,28].
These reactions are commonly catalyzed by strong bases, Lewis acids. Nevertheless, most of
these catalytic strategies suffer from disadvantages such as the requirement of excess starting
materials, the formation of nonecofriendly and unavoidable byproducts, and sometimes only
moderate yields and enantioselectivity [28,29].
Here we report for the first-time use of lipase from porcine pancreas (PPL) to catalyze a
Knoevenagel condensation and synthesis of bis-coumarin compounds (Scheme 2). This method
of synthesis is simple, easy and does not require purification
2. Materials and Methods
Porcine pancreas lipase (PPL), Aspergillus niger lipase, Amano lipase A from Aspergillus niger,
used in this study were purchased from Sigma. Candida antarctica lipase B (CALB),
Thermomyces lanuginosa Lipase (TLL) were purchased from Novozyme. All the chemical
reagents were purchased from Merck. Commercially available reagents and solvents were used
without further purification.
2.1. Typical procedure for synthesis of 3,3'-(phenylmethylene) bis(4-hydroxy-2H-chromen-2-one)
In a 10 ml round-bottom flask 10mg of Porcine pancreas lipase (PPL) was mixed with, aldehyde
(0.2 mmol), 4-hydroxycoumarin (0.4 mmol) and organic solvent (2 mL). The mixture was stirred
at 45 °C (200 rpm) for the appropriate time (the reaction was monitored by TLC). Finally the
mixture was filtrated, and the residue was washed with water and ethanol to afford the pure
product. The structures of isolated products were elucidated by 1H NMR spectroscopy.
4

1H-NMR Spectral analysis of compounds (3a-3i)
Product 3a:1H NMR (300 MHz, DMSO d-6) δ 6.30 (S, 1H, CH), 7.12- 7.89 (m, 13H, CH)
Product 3b:1H NMR (300 MHz, DMSO d-6) δ 6.29 (S, 1H, CH), 7.02- 7.90 (m, 12H, CH)
Product 3c:1H NMR (300 MHz, DMSO d-6) δ 3.13 (S, 3H, CH3), 6.30 (S, 1H, CH), 7.20- 7.90
(m, 12H, CH)
Product 3d: 1H NMR (300 MHz, DMSO d-6) δ 6.36 (S, 1H, CH), 7.24- 8.10 (m, 12H, CH)
Product 3e: 1H NMR (300 MHz, DMSO d-6) δ 6.27 (S, 1H, CH), 7.12- 7.87 (m, 12H, CH)
Product 3f: 1H NMR (300 MHz, DMSO d-6) δ 3.70 (S, 3H, CH3), 6.28 (S, 1H, CH), 6.77- 7.90
(m, 12H, CH)
Product 3g: 1H NMR (300 MHz, DMSO d-6) δ 3.57 (S, 3H, CH3), 6.24 (S, 1H, CH), 6.79- 7.88
(m, 12H, CH)
Product 3h:1H NMR (300 MHz, DMSO d-6) δ 3.52 (S, 3H, CH3), 3.62 (S, 3H, CH3), 6.23 (S,
1H, CH), 6.70- 7.91 (m, 11H, CH)
Product 3i:1H NMR (300 MHz, DMSO d-6) δ 6.40 (S, 1H, CH), 6.60- 7.86 (m, 11H, CH),
3. Results and discussion
In this work, synthesis of 3,3'-(phenylmethylene) bis(4-Hydroxy-2H-chromen-2-one) from the
reaction of benzaldehyde and 4-hydroxycoumarin was selected as the model reaction. Different
lipases (PPL, CALB, Aspergillus niger lipase, TLL, Amano lipase A) were used as biocatalyst
which among them only PPL was able to catalyze the reaction and afforded the highest yield
(85%) (Table 1).
5

3.1.
Effect of organic solvents
One of the important parameters affecting the enzyme reaction is the type of solvent used in the
reaction. The medium of reaction affects the activity of the enzyme. Some organic solvents
decrease activity or completely inactivate the enzyme. Organic solvents disrupt disulfide bonds
and amino acid residues on the surface of the enzyme, which play important role in the second
and tertiary structure of the enzyme [30,31]. PPL is generally used with diethyl ether as a solvent
[32]. Different solvents in this investigation were used for evaluating the effect of solvent on the
yield of reaction (Table 2). The results showed that protic solvents such as water and ethanol are
suitable solvents for this reaction. Based on the yield of the final product, ethanol was used as
the reaction solvent.
3.2.
Effect of temperature
Another examined parameter was temperature, which is very important in enzymatic reactions.
Enzymes are delicate and brittle to their external environment such as temperature. Each
particular enzyme has an optimal temperature and enzyme will be easily denatured at higher
temperature. Therefore, it is important to select the temperature which the enzyme has higher
activities [33,34].
As shown in Table 3, the maximum yield of reaction was observed at 45 and 55 °C. Since the
mild conditions are better based on an economical view, 45 °C was selected as the appropriate
temperature for the reaction. As can be seen in Table 3, the reaction yield decreases at 65 °C,
which may be due to the loss of enzyme activity at higher temperatures.
3.3.
Effect of the catalyst
6

The amount of catalyst has a great effect on yield and rate of reaction. In order to optimize the
amount of biocatalyst, 5-15 mg PPL lipase were examined and the best result was obtained by
using 10 mg PPL. By increasing the amount of the enzyme, more active sites involved in the
reaction [35]. As the catalyst was further increased, little change was observed in the time and
yield of reaction (Table 4).
3.4.
Effect of various aldehydes
After optimizing the reaction conditions, various aromatic aldehydes were selected and reacted
under optimum conditions (ethanol, 45 °C and 10 mg PPL). The results showed that the reaction
is not electron-sensitive. Electron-donating or electron-withdrawing groups on the ring have no
effect on the yield of reaction (Table 5).
Based on the mechanism of lipase-promiscuity, the carbon-carbon bond formation has been
accepted [36-37]. We propose a mechanism for the synthesis of 3,3'-(phenylmethylene)bis(4-
Hydroxy-2H-chromen-2-one) derivative as shown in Scheme 3. First, lipase catalyzes the 4-
hydroxycoumarin and forms an enolate ion 2, second, aldehyde accepts the proton and
nucleophile (enolate) attacks to activated aldehyde , result in C-C bond formation in 4. After the
dehydration, a knoevenagel product 6 produce. Third, the Michael addition occurs between
intermediate product 6 and 5 to generate the intermediate product 7. Finally, the catalytic cycle is
completed via an enolization process; therefore the product 8 is obtained.
Some researchers had utilized lipase as catalysts for the construction of C-C bond by
knoevenagel and Michel reactions. For example, Xiang et al. in 2013 reported reaction between
indole and aldehyde in the synthesis of bis(indolyl)methane by PPL in Dioxane/H2O, 50 ºC and
7

48 h [38]. In 2014 Wan et al. developed a proficient pathway for Candida sp. lipase catalyzed
addition of 2-Hydroxy-1,4-naphthoquinone to aldehyde in ethanol/60 ºC in 2h. [35]
There are some other chemical catalysts for reaction of 4-hydroxycoumarin with benzaldehyde.
Table 6 shows some of those reported in the literature. As can be seen in Table 6, PPL besides
being a green catalyst, operate in mild conditions and low temperature compared with other
reported procedures.
Conclusion
In summary, we herein report for the first time using Porcine pancreas lipase to synthesize bis-4-
hydroxycoumarin with high yields (81-88 %). Reaction solvent, temperature and enzyme loading
had been investigated and finally ethanol as solvent reaction, 45 °C optimum temperature and 10
mg amount of PPL were chosed. The significant advantages of this methodology are simplicity
of reaction conditions, green and mild conditions, simple work-up procedure, easy handling of
the biocatalyst and environmental-friendly processes.
Table 1
Different lipase enzymes in synthesis of 3,3'-(phenylmethylene)bis(4-hydroxy-2H-chromen-2-one)a
Entr
y
Enzyme
Yield (%)b
1
Porcine Pancreas lipase(PPL)
Candida Antarctica lipase B (CALB)
Aspergillus niger lipase
85
2
3
4
5
trace
trace
trace
trace
Thermomyces lanuginosa lipase (TLL)
Aspergillus niger lipase (Amano lipase A)
a Reaction condition: 4-Hydroxycoumarin (0.4 mmol), Benzaldhyde (0.2 mmol), Ethanol (2 ml), PPL (10 mg), Temperature 45
°C, Time 6h, b Isolated yields
Table 2
Effect of organic solvent on the synthesis of 3,3'-(phenylmethylene)bis(4-hydroxy-2H-chromen-2-one) a
Entry
1
Solvent
Yield (%) b
40
Acetonitrile
8

2
3
4
5
N,N-Dimethylformamide
Tetrahydrofuran
Water
63
trace
78
Ethanol
85
a Reaction condition: 4-Hydroxycoumarin (0.4 mmol), Benzaldhyde (0.2 mmol), Ethanol (2 ml), Lipase (PPL) (10 mg),
Temperature 45 °C, Time 6h, b Isolated yields
Table 3
Effect of temperature on the synthesis of 3,3'-(phenylmethylene)bis(4-hydroxy-2H-chromen-2-one) a
Entry
Temperature
Yield (%)b
1
2
3
4
r.t
35
85
88
71
45
55
66
a Reaction condition: 4-Hydroxycoumarin (0.4 mmol), Benzaldhyde (0.2 mmol), Ethanol (2 ml), Lipase (PPL) (10 mg), Time 6h,
b Isolated yields
Table 4
Effect of enzyme loading on the synthesis of 3,3'-(phenylmethylene)bis(4-hydroxy-2H-chromen-2-one) a
Entry
PPL (mg)
Yield (%)b
1
2
3
5
60
85
86
10
15
a Reaction condition: 4-Hydroxycoumarin (0.4 mmol), Benzaldhyde (0.2 mmol), Ethanol (2 ml), Lipase (PPL) (5-15 mg), Time
6h,
b Isolated yields
Table 5
Synthesis of 3,3'-(phenylmethylene)bis(4-hydroxy-2H-chromen-2-one) (3a-3i )with different aromatic aldehydes catalyzed by
lipase a
Entry Aldehyde
a
product
3a
Time
6h
Yield (%) b
85
O
H
O
b
3b
3c
3h
3h
86
88
H
O
c
H
N
9

O
d
e
f
3d
3e
3f
4h
4h
4h
3h
81
84
86
83
H
O2N
O
H
Cl
O
H
MeO
O
g
3g
H
OMe
O
h
i
3h
3i
3h
5h
84
82
MeO
S
H
OMe
O
H
a Reaction condition: 3,3'-(phenylmethylene) bis(4-hydroxy-2H-chromen-2-one) (0.4 mmol) aldehyde (0.2 mmol), ethanol (2 mL), PPL
b
(10 mg), 45 °C, Isolated yields.
Table 6
Comparison between PPL as biocatalyst with other conventional catalysts reported in the literature for the synthesis of product
(3a)
entry
1
Catalyst
o-
Condition
H2O, reflux
Time
30 min
Yield (%)
86
Reference
39
Benzenedisulfonimide
Iron(III) dodecyl
sulfate
2
H2O, 100 °C
7 min
79
40
3
4
I2
H2O, reflux
25 min
48 min
97
93
41
42
[P4VPy-
Toluene, 90 °C
BuSO3H]HSO4]
Fe3O4@B(HSO4)3
5
6
Solvent free 80
°C
15 min
6 hr
90
85
43
lipase (PPL)
Ethanol, 45 °C
This work
O
OH
OH
O
OH
O
O
O
O O
Dicoumarol
Scheme 1. Chemical structure of dicoumarol and warfarin
Warfarin
10

OH
O
OH
O
R
OH
O
O
+
O
PPL
Ethanol
45 °C, 3-6 h
R
H
O
O
1
3
2
R = Ph or Heterocycle
Scheme 2. Synthesis of 3,3'-(phenylmethylene) bis(4-hydroxy-2H-chromen-2-one)
O
O
O
His
His
Asp
O
Asp
O
His
H
H
Asp
O
N
H
N
N
N
N
N
H
H
H
O
O
O
O
O
O
O
O
H
R
R
H
O
O
O
2
O
1
3
O
O
His
His
Asp
Asp
O
H
H
N
N
N
N
H
H
O
OH
O
O
O
O
R
O
O
O
R
H
- H2O
H
O
O
O
4
O
5
6
OH
R
OH
O
O
O
R
OH
O
O
O
O
O
O
7
8
Scheme 3. Proposed mechanism of the lipase-catalyzed synthesis of 3,3'-(phenylmethylene) bis(4-hydroxy-2H-chromen-2-one)
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15

Highlights

Porcine pancreas lipase was used as biocatalyst for the synthesis of bis-4-
hydroxycoumarins


Aromatic aldehydes were used for knoevenagel condensation
Bis-4-hydroxy coumarin compounds were synthesized under mild reaction conditions
16

Synthesis of 3,3'-(phenylmethylene) bis(4-hydroxy-2H-chromen-2-one)
17