Enzyme-Catalyzed Cascade Michael/Cyclization Reaction for the Synthesis of 3,4-Dihydropyran Derivatives by Using a Protease

Article abstract of DOI:10.1007/s10562-017-2275-2

Abstract: Protease from Streptomyces griseus (SGP) as a sustainable biocatalyst was successfully applied in the Michael/cyclization reaction between dimedone and aryl or alkyl substituted α,β-unsaturated ketones or ester for the synthesis of 3,4-dihydropyran derivatives. The products were obtained in moderate to excellent yields (46–95%) with certain enantioselectivities (up to 18% ee) for 27 examples. This process afforded a potential biocatalytic approach as alternative to chemical synthesis for 3,4-dihydropyran derivatives. Graphical Abstract: Protease from Streptomyces griseus (SGP) was used as a catalyst in the Michael addition/cyclization reaction for the synthesis of 3,4-dihydropyran derivatives.

Full text of DOI:10.1007/s10562-017-2275-2

Catalysis Letters
https://doi.org/10.1007/s10562-017-2275-2
Enzyme-Catalyzed Cascade Michael/Cyclization Reaction
for the Synthesis of 3,4-Dihydropyran Derivatives by Using a Protease
Xuan Ding1 · Xue‑Dong Zhang1 · Chun‑Lin Dong1 · Zhi Guan1 · Yan‑Hong He1
Received: 14 July 2017 / Accepted: 10 December 2017
© Springer Science+Business Media, LLC, part of Springer Nature 2017
Abstract
Protease from Streptomyces griseus (SGP) as a sustainable biocatalyst was successfully applied in the Michael/cyclization
reaction between dimedone and aryl or alkyl substituted α,β-unsaturated ketones or ester for the synthesis of 3,4-dihydro-
pyran derivatives. The products were obtained in moderate to excellent yields (46–95%) with certain enantioselectivities
(up to 18% ee) for 27 examples. This process afforded a potential biocatalytic approach as alternative to chemical synthesis
for 3,4-dihydropyran derivatives.
Graphical Abstract
Protease from Streptomyces griseus (SGP) was used as a catalyst in the Michael addition/cyclization reaction for the syn-
thesis of 3,4-dihydropyran derivatives
Keywords Enzyme catalysis · Protease · Enzymatic promiscuity · Michael/cyclization reaction · 3,4-Dihydropyran
derivatives
1 Introduction
Electronic supplementary material The online version of this
Enzymatic methods constitute a potential complement to the
chemical tools in the development of new methodologies
in organic synthesis. Enzymes as natural catalysts are safe,
non-toxic, renewable and degradable [1]. These advantages
make them superior to other chemical reagents in organic
synthesis [2]. It is believed that enzymes were generally
versatile in ancient times, and they have become more and
article (https://doi.org/10.1007/s10562-017-2275-2) contains
supplementary material, which is available to authorized users.
* Zhi Guan
guanzhi@swu.edu.cn
* Yan-Hong He
heyh@swu.edu.cn
Extended author information available on the last page of the article
Vol.:(0123456789)
1 3

X. Ding et al.
more specific and selective in catalysis through long-time
divergence and evolution [3]. However, some promiscu-
ous activities still remain which are often hidden behind a
native activity [4–6]. In the recent decades, a good deal of
enzymatic promiscuities have been revealed [7–14], and this
special aspect of enzymes has attracted growing attention.
Dihydropyran derivatives are widely used in the phar-
maceutical and medical fields. Particularly, those with sub-
stituents are intriguing building blocks for construction of
new biologically active compounds [15, 16]. Thus, some
efforts have been devoted toward the development of effec-
tive methods for the construction of dihydropyran com-
pounds. In 2003, Jørgensen and co-workers developed the
first organocatalytic asymmetric Michael addition of cyclic
1,3-dicarbonyl compounds with α,β-unsaturated enones
for the synthesis of dihydropyran derivatives in the pres-
ence of an imidazolidine catalyst [17]. After that, chiral
bisoxazoline–copper(II) complexes [18], vicinal diamine
[19], primary amine derived from quinine [20], cinchona
alkaloid-derived pyrimidines [21], chiral diamine catalyst
[22], chiral metal–organic framework (MOF) [23], and
l-diphenylprolinol trimethylsilyl ether [24] were reported as
catalysts for the synthesis of dihydropyran derivatives from
similar substrates. Moreover, some other Michael reactions
were expanded by many research groups [25, 26]. In 2008,
Ma et al. reported diarylprolinol ether/HOAc-catalyzed
cascade Michael addition and cyclization of aldehydes and
α-keto-α,β-unsaturated esters in water to afford function-
alized dihydropyrones [27]. In 2010, Trofimov group suc-
cessfully applied an acid-catalyzed rearrangement between
ketones and acetylene for the synthesis of functionalized
3,4-dihydropyrans [28]. Other Michael/cyclization [29–31],
Friedel–Crafts alkylation/cyclization [32], and radical cycli-
zation [33] reactions were also reported.
compounds with moderate yields (14–76%) without enanti-
oselectivity [37]. In 2015, Yu group found that lipase from
Pseudomonas fluorescens can catalyze the Micheal addi-
tion/cyclization cascade reaction of 1,3-dicarbonyls and
α,β-unsaturated aldehydes for the synthesis of 2-hydroxyl-
dihydropyran derivatives with moderate yields of 31–75%
without enantioselectivity [38].
Herein, we report a cascade Michael/cyclization reac-
tion of dimedone to aryl or alkyl substituted α,β-unsaturated
ketones or esters catalyzed by protease from Streptomyces
griseus (SGP) for the synthesis of 3,4-dihydropyran deriva-
tives. The products were obtained in moderate to excellent
yields (46–95%) with certain enantioselectivities (up to 18%
ee) for 27 examples.
2 Results and Discussion
In our initial study, benzalacetone 1a and dimedone 2 were
selected as the model substrates. The SGP-catalyzed model
Michael/cyclization reaction was investigated. Because of
the diversity of enzyme composition and conformation,
enzymes display different catalytic activities in organic sol-
vents [39]. Thus, various solvents were screened (Table 1,
entries 1–13). It is worth mentioning that among the tested
solvents ether solvents promoted the selectivity of the reac-
tion (Table 1, entries 1–6), and alcohol solvents promoted
the reaction yield (Table 1, entries 9 and 10). Weighing the
two aspects of yield and selectivity, THF was selected as an
optimal solvent.
Then, with the purpose of verifying that the reaction was
catalyzed by SGP, some control experiments were performed
(Table 1, entries 14–22). In the absence of enzyme, no prod-
uct was observed for the blank reaction (Table 1, entry 14).
Since heavy metal ions usually disturb the three-dimensional
structure of enzymes [40], Cu2+ was used to pretreat the
SGP. It can be seen that Cu2+ dramatically decreased the
enzyme activity towards the model reaction (yield decreased
from 67 to 11%) (Table 1, entry 15). Phenylmethylsulfo-
nyl fluoride (PMSF) is commonly used as a serine protease
inhibitor, which binds specifically to the active site serine
residue in a serine protease [41], and does not bind to any
other serine residue in the protein [42]. Therefore, PMSF as
an irreversible inhibitor was used to pretreat the SGP, and
the model reaction catalyzed by the PMSF-pretreated SGP
only gave the product in a low yield of 9% (Table 1, entry
17). It indicated that PMSF strongly inhibited the activity of
SGP in the model reaction, and thus it can be speculated that
the natural active center of SGP may be responsible for its
activity in the model reaction. Carbonyl diimidazole (CDI)
is capable of irreversibly affecting the carboxyl of carbox-
ylic amino acid residues, and it usually used as an inhibitor
of enzyme. After pretreated by CDI, the SGP completely
Recently, several cascade reactions involving Michael/
cyclization reaction by enzymatic promiscuity have been
reported. In 2011, Lin group reported that acylase from
Aspergillus oryzae can catalyze the cascade Knoevenagel/
Michael/cyclization reaction for the synthesis of 3,4-dihy-
dropyridin-2-ones by condensation of aldehyde with
1,3-dicarbonyl compounds and cyanoacetamide [34]. In
2013, Yu group repored catalytic promiscuity of α-amylase
from Bacillus subtilis for the asymmetric catalysis of the
domino Michael/aldol condensation of salicylaldehyde and
α,β-unsaturated ketones [35]. In 2015, our group applied a
cascade Michael/cyclization reaction of 2-hydroxychalcones
with malononitrile to the synthesis of 2-amino-4H-chromene
derivatives by using bovine serum albumin as a catalyst
[36]. However, biocatalytic synthesis of dihydropyran com-
pounds is rarely reported. In 2014, Ye group reported that
the Escherichia coli BioH esterase can catalyze the Micheal
addition/cyclization cascade reaction of substituted benza-
lacetones and 1,3-cyclic diketones to prepare dihydropyran
1 3

Enzyme-Catalyzed Cascade Michael/Cyclization Reaction for the Synthesis of 3,4-Dihydropyran…
Table 1 Solvent screening and control experiments
Ph
O
O
O
O
O
Ph
O
O
SGP
30 oC
solvent,
HO
O
Solvent
THF
1a
2
3a
Entry
Yield (%)a
ee (%)
(R for
3a)b
1
2
3
4
5
6
67
13
13
13
12
10
8
Diethyl ether
Isopropyl ether
Anisole
1,4-Dioxane
MTBE
60
38
4
31
54
7
8
9
DMF
62
45
77
72
57
28
Trace
0
11
Trace
9
Trace
Trace
Trace
18
4
2
0
0
0
0
–
–
0
–
2
–
–
–
2
–
Ethyl acetate
Isopropanol
Ethanol
MeCN
Toluene
10
11
12
13
14
15
16
17
18
19
20
21
22
Water
THF (no enzyme)
THF (SGP pretreated with 0.25 M Cu2+ c
)
THF [CuSO4 (39.9 mg)]
THF (SGP pretreated with 1.0 M PMSF)d
THF [PMSF (174 mg)]
THF (SGP pretreated with 1.85 M CDI)e
THF [CDI (300 mg)]
THF (SGP pretreated with 0.3 M DEPC)f
THF [DEPC (43.3 μL)]
Trace
Reaction conditions unless noted otherwise: benzalacetone (0.45 mmol), dimedone (0.30 mmol), and SGP (174 U) in organic solvent (0.9 mL)
and deionized water (0.1 mL) at 30 °C for 96 h
aIsolated yield
bEnantiomeric excess (ee) was determined by HPLC with Chiralpak IA column; absolute configuration of the products were determined by com-
parison with the known chiral HPLC analysis results [22] (for details, please see the Supporting Information)
cSGP (174 U) in Cu2+ solution (0.25 M) [CuSO4 (39.9 mg) in deionized water (1 mL)] was stirred at 30 °C for 6 h, and then the water was
removed by lyophilization
dSGP (174 U) in 1.0 M PMSF solution [PMSF (174 mg) in THF (1 mL)] was stirred at 30 °C for 6 h, and then THF was removed under reduced
pressure
eSGP (174 U) in 1.85 M CDI solution [CDI (300 mg) in THF (1 mL)] was stirred at 30 °C for 6 h, and then THF was removed under reduced
pressure
fSGP (174 U) in 0.3 mM DEPC solution [DEPC (43.3 μL) in deionized water (1 mL)] was stirred at 30 °C for 6 h, and then the water was
removed by lyophilization
lost its catalytic activity toward the model reaction (Table 1,
entry 19). Diethy pyrocarbonate (DEPC) is capable of bind-
ing with the imidazole ring of histidine as well as –NH, –SH,
or –OH group of protein to inhibit the activity of enzyme.
The model reaction catalyzed by DEPC-pretreated SGP only
gave the product in a low yield of 18% (Table 1, entry 21).
The above control experiments showed that the model reac-
tion was catalyzed by the enzyme, and the active site of SGP
may play a key role.
Water can affect the conformation of proteins as well
as polarity and stability of the active site of enzyme. Usu-
ally enzymes require a certain amount of water to maintain
1 3

X. Ding et al.
100
90
80
70
60
50
40
30
20
10
0
30
25
20
15
10
5
optimize the reaction conditions. The optimal parameters
regarding to these aspects were found as the following:
0.75 mL of reaction medium (THF/H2O), 203 U of SGP
and 1.8:1 of molar ratio (1a:2) were selected for the model
reaction on the scale of 0.30 mmol. (For details, please see
the Supporting Information)
Yield (%)
ee (%)
Temperature is a crucial factor affecting the stability of
enzymes. Rising temperature can accelerate the chemical
reaction rate, but the high temperature can also inactivate
the enzyme through protein denaturation [43]. To obtain
the optimum temperature, the effect of temperature on the
model reaction in 0.75 mL of THF/H2O for 96 h was imple-
mented (Fig. 2). Rising temperature from 20 to 30 °C led to
a remarkable increase in the yield from 40 to 84%. Higher
temperature caused a decrease in the yield, and a rapid
drop in the yield was observed when the temperature was
above 50 °C. The temperature also showed obvious influ-
ence on the enantioselectivity. A significant decrease of ee
was observed once the temperature surpassed 50 °C, and
a better ee was obtained at 30 °C than other temperatures.
Thus, 30 °C was chosen as the optimum temperature for the
reaction.
0
0
10
20
30
40
50
60
water content (%)
Fig. 1 Influence of water content on the model reaction. Reaction
conditions: benzalacetone (0.45 mmol), dimedone (0.30 mmol), and
SGP (174 U) in THF (1.0–0.4 mL) and deionized water (0–0.6 mL)
at 30 °C for 96 h. Isolated yield. Enantiomeric excess (ee) (R for 3a)
was determined by HPLC with Chiralpak IA column
their three-dimensional conformation in an organic solvent.
Hence, effect of water contents on the model reaction was
investigated (Fig. 1). A rapid rise in the yield from 29 to 69%
was observed as the water content increased from 0 to 10%
[water/(water + THF), v/v]. Higher water content led to a
decrease in the yield. The same change trend was found for
the enantioselectivity as the water content was increasing.
Therefore, 10% of water content was selected as an optimum
condition for the reaction.
Finally, with the optimal reaction conditions in hand, the
generality of the SGP-catalyzed cascade Michael/cyclization
reaction for the synthesis of 3,4-dihydropyran derivatives
was evaluated using a variety of α,β-unsaturated enones 1
to react with dimedone 2. Both aryl and alkyl-substituted
α,β-unsaturated enones can participate in the reaction
well (Table 2, entries 1–27). For aromatic α,β-unsaturated
ketones, those with an electron-withdrawing substituent at
the para position of benzene ring exhibited higher reactivity
than those with an electron-donating substituent at the same
position (Table 2, entries 2–9). Among them, 4-benzyloxy
and 4-biphenyl substituted aromatic α,β-unsaturated ketones
showed much lower reaction efficiency probably due to the
dual influences of electron-donating and steric hindrance
(Table 2, entries 8 and 9). The electron-withdrawing or elec-
tron-donating substituent at the meta position of benzene
ring had no apparent effect on the yield (Table 2, entries
10–17). Aromatic α,β-unsaturated ketones with a substitu-
ent at the ortho position or with two substituents at ortho
and meta positions of benzene ring also can react smoothly
with dimedone 2 (Table 2, entries 18–22). Naphthyl or furyl
substituted α,β-unsaturated enones performed well in the
reaction (Table 2, entries 23 and 24). Furthermore, alkyl-
substituted α,β-unsaturated enones were also applicable to
the reaction (Table 2, entries 25 and 26). Finally, the α,β-
unsaturated ketones which bears a substituent of ester moiety
could react with dimedone very well giving the correspond-
ing product in a good yield (Table 2, entry 27). In general,
this SGP-catalyzed cascade Michael/cyclization reaction
displayed good tolerance to structural and electronic vari-
ation of the α,β-unsaturated enones 1 to access a variety of
Next, the volume of reaction medium, the loading of SGP
and the molar ratio of substrates were screened to further
100
80
60
40
20
0
30
25
20
15
10
5
Yield (%)
ee (%)
0
20
30
40
50
60
Temperature(ć)
Fig. 2 Influence of temperature on the model reaction. Reaction con-
ditions: benzalacetone (0.54 mmol), dimedone (0.30 mmol), and SGP
(203 U) in THF (0.675 mL) and deionized water (0.075 mL) for 96 h.
Isolated yield. Enantiomeric excess (ee) (R for 3a) was determined by
HPLC with Chiralpak IA column
1 3

Enzyme-Catalyzed Cascade Michael/Cyclization Reaction for the Synthesis of 3,4-Dihydropyran…
Table 2 Substrate scope for the SGP-catalyzed synthesis of 3,4-dihydropyran derivatives
R1
O
O
R1
O
O
O
O
SGP
R2
30 oC
R2
R1
R2
THF/H
2O,
O
O
HO
1
2
3
Entry
R1
Ph
R2
Product
Time (h)
Yield (%)a
ee (%)b
1
2
3
4
5
6
7
8
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
COOMe
3a
3b
3c
3d
3e
3f
3g
3h
3i
96
96
96
96
96
96
96
108
144
96
108
96
108
108
96
108
96
96
96
88
91
66
85
81
91
60
49
46
66
75
77
79
77
82
62
70
71
69
68
62
58
53
95
82
53
94
15
16
8
16
13
15
7
4-F–C6H4
4-Cl–C6H4
4-Br–C6H4
4-CN–C6H4
4-NO2–C6H4
4-OMe–C6H4
4-BnO–C6H4
4-Ph–C6H4
3-F–C6H4
3-Cl–C6H4
3-Br–C6H4
3-CF3–C6H4
3-NO2–C6H4
3-MeO–C6H4
3-Me–C6H4
3-OH–C6H4
2-F–C6H4
8
9
15
12
16
10
18
3
10
11
11
10
13
14
14
10
13
12
15
5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
2-Cl–C6H4
2-Br–C6H4
2-MeO–C6H4
3t
96
3u
3v
3w
3x
3y
3z
3aa
108
108
144
96
108
108
48
2,3-(MeO)2–C6H3
2-Np
2-Furyl
n-Pr
i-Pr
Ph
4
Reaction conditions: 1 (0.54 mmol), 2 (0.30 mmol), and SGP (203 U) in THF (0.675 mL) and deionized water (0.075 mL) at 30 °C.
aIsolated yield
bEnantiomeric excess (ee) was determined by chiral HPLC; absolute configuration of the products were determined by comparison with the
known chiral HPLC analysis
complex 3,4-dihydropyrans 3a–3aa in moderate to excellent
yields of 46–95% (Table 2, entries 1–27). Unfortunately,
very low enantioselectivity was obtained for all the tested
substrates at the moment. The absolute configurations
were determined by comparing the chiral HPLC with lit-
erature [22, 38, 44] (for details, please see the Supporting
Information).
3 Conclusion
In summary, a new and convenient enzymatic transforma-
tion for the synthesis of 3,4-dihydropyran derivatives via
a successive Michael addition/cyclization reaction in a
“one-pot” protocol has been developed. A wide range of
aryl or alkyl substituted α,β-unsaturated ketones or ester can
react with dimedone to afford corresponding products in
1 3

X. Ding et al.
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moderate to excellent yields. Easily available SGP was used
as an environmentally friendly biocatalyst, and the reaction
was performed under mild reaction conditions. While this
work provides a useful alternative synthesis of 3,4-dihydro-
pyran derivatives, it extends the application of enzymatic
promiscuity.
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Protease from S. griseus was purchased from Sigma-Aldrich.
[P5174, Lot#SLBL3111V, powder, 5.8 U/mg. One unit will
hydrolyze casein to produce color equivalent to 1.0 μmole
(181 μg) of tyrosine per min at pH 7.5 at 37 °C (color by
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4.2 General Procedure for the SGP‑Catalyzed
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A mixture of α,β-unsaturated ketone or ester 1 (0.54 mmol),
dimedone 2 (0.30 mmol), SGP (203 U), THF (0.675 mL)
and deionized water (0.075 mL) was stirred for the speci-
fied time at 30 °C, and monitored by TLC analysis. After
completion, the enzyme was filtered out; ethyl acetate was
used to wash the residue on the filter paper. The filtrate was
concentrated under vacuum. The residue was purified by
flash column chromatography on silica gel using a mixture
of petroleum ether and ethyl acetate (petroleum ether/ethyl
acetate=3/1–8/1, v/v) as eluent to give the products.
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Acknowledgements This work was financially supported by the
National Natural Science Foundation of China (Nos. 21472152 and
21672174), and the Basic and Frontier Research Project of Chongqing
(cstc2015jcyjBX0106).
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15(4):743–759
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Enzyme-Catalyzed Cascade Michael/Cyclization Reaction for the Synthesis of 3,4-Dihydropyran…
Affiliations
Xuan Ding1 · Xue‑Dong Zhang1 · Chun‑Lin Dong1 · Zhi Guan1 · Yan‑Hong He1
1
Key Laboratory of Applied Chemistry of Chongqing
Municipality, School of Chemistry and Chemical
Engineering, Southwest University, Chongqing 400715,
People’s Republic of China
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