Enzyme-Promoted Direct Asymmetric Michael Reaction by Using Protease from Streptomyces griseus

Article abstract of DOI:10.1007/s10562-017-2095-4

The direct asymmetric Michael addition of malonates and enones was promoted by protease from Streptomyces griseus for the first time. Yields of up to 84% with enantioselectivities of up to 98% enantiomeric excess (ee) were achieved under optimized conditi

Full text of DOI:10.1007/s10562-017-2095-4

Catal Lett
DOI 10.1007/s10562-017-2095-4
Enzyme-Promoted Direct Asymmetric Michael Reaction by Using
Protease from Streptomyces griseus
Ling-Ling Wu1 · Ling-Po Li1 · Yang Xiang1 · Zhi Guan1 · Yan-Hong He1
Received: 7 March 2017 / Accepted: 28 May 2017
© Springer Science+Business Media, LLC 2017
Abstract The direct asymmetric Michael addition of
malonates and enones was promoted by protease from
Streptomyces griseus for the first time. Yields of up to 84%
with enantioselectivities of up to 98% enantiomeric excess
(ee) were achieved under optimized conditions.
1 Introduction
The catalytic asymmetric Michael reaction of nucleophiles
to α,β-unsaturated carbonyl compounds is one of the most
powerful ways to construct carbon–carbon bonds in organic
synthesis [1–5]. Among the various stabilized carbanion
nucleophiles, malonate is considered to be an especially
useful Michael donor. Thus, considerable efforts have been
directed towards the development of catalytic asymmet-
ric Michael addition of malonates to enones. As a result,
various kinds of catalysts, such as organocatalysts [6–11],
chiral metal complexes [4, 12–14], phase-transfer catalysts
[15–17] and ionic liquids [18], have been used to catalyze
this type of asymmetric Michael addition.
Graphical Abstract Protease from Streptomyces griseus
(SGP) was used for the first time as a biocatalyst in asym-
metric Michael reaction of malonates.
O
O
COR2
COR1
SGP, 35 oC
MeOH/H2O
+
( )n
COR2
( )n
R1OC
Biocatalysis, as a green transformation tool in organic
synthesis, has attracted increased attention due to its
excellent selectivity and mild reaction conditions. A
growing number of enzymes have been found to be capa-
ble of catalyzing distinctly different chemical transfor-
mations from their natural activities which refers to the
term biocatalytic promiscuity [19–36]. This phenom-
enon largely expands the application of biocatalysts in
organic synthesis and has great potential of enriching
synthetic methodologies. A number of enzymes have
been found to be able to catalyze Michael reactions.
For instance, the lipase-catalyzed Michael additions
for the formation of C–N [37–40], C–S [41] and C–C
[42, 43] bonds and the D-aminoacylase-catalyzed C–C
Michael addition [44] have been reported. In 2011, our
group reported the C–C Michael addition of ketones to
nitroolefins by acidic proteinase from Aspergillus usamii
[45]. However, in all of these reactions, no enantiose-
lectivity was obtained for the Michael products. Up to
date, there are only few enzyme-catalyzed asymmetric
Michael reactions reported. In 1986, Kitaume group
11 examples, yields: 33-88%, ee up to 98%.
Keywords Asymmetric Michael reaction · Protease
from Streptomyces griseus · Enzyme · Enzyme-catalyzed
reaction · Biocatalytic promiscuity
Electronic supplementary material The online version of this
article (doi:10.1007/s10562-017-2095-4) contains supplementary
material, which is available to authorized users.
* Zhi Guan
guanzhi@swu.edu.cn
* Yan-Hong He
heyh@swu.edu.cn
1
Key Laboratory of Applied Chemistry of Chongqing
Municipality, School of Chemistry and Chemical
Engineering, Southwest University, Chongqing 400715,
People’s Republic of China
Vol.:(0123456789)
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L.-L. Wu et al.
reported the asymmetric C–O, C–N or C–S Michael
additions of 2-(trifluoromethyl)propenoic acid by hydro-
lytic enzymes for synthesis of optically active trifluori-
nated compounds [46]. In 1988, the same group reported
asymmetric C–S or C–N Michael additions of (E)-ethyl
3-(trifluoromethyl)propenate and ethyl 2-(trifluorome-
thyl)propenate by lipases in various kinds of organic sol-
vents [47]. In 2011, our group reported the asymmetric
C–C Michael addition of 1,3-dicarbonyl compounds or
cyclohexanone to aromatic and heteroaromatic nitroole-
fins or cyclohexenone by lipase from Thermomyces
lanuginosus [48]. Therefore, it is still highly desirable to
explore the enzyme-catalyzed asymmetric Michael reac-
tion. In this context, we screened some commercially
available enzymes and found that the protease type XIV
from Streptomyces griseus (SGP) has ability to catalyze
asymmetric Michael reaction. In our previous study on
enzyme-catalyzed multicomponent reactions which is
an area of particular interest in biocatalytic promiscuity
[22, 49], SGP has been used as a catalyst for the asym-
metric Mannich reaction [22]. Herein, we report our new
finding of SGP-catalyzed asymmetric Michael reaction.
This is the first successful example of enzyme-promoted
direct asymmetric Michael reaction of malonates and
enones.
2 Results and Discussion
Initially, the Michael reaction of dibenzyl malonate (1a)
with 2-cyclohexen-1-one (2a) was used as a model reac-
tion. A series of commercially available enzymes, includ-
ing proteases, lipases, and other enzymes, were screened as
catalysts (For details, please see the Supporting Informa-
tion Table S1). We found that SGP was able to promote the
model reaction in MeCN in the presence of water, which
gave the product in a yield of 15% with 43% ee. Since the
reaction medium plays a significant role in maintaining the
catalytic activity and stability of an enzyme [50], in particu-
lar, in enzyme enantioselectivity and regioselectivity, vari-
ous organic solvents were screened for the SGP-promoted
model Michael reaction in the presence of water (Table 1).
The Michael product could be obtained in a good yield of
75% with the best enantioselectivity of 87% ee in MeOH
(Table 1, entry 1). SGP exhibited better stereoselectivity
in protic solvents than in other tested solvents (Table 1,
entries 1–3). The reaction gave the highest yield of 84% but
with very low enantioselectivity of only 3% ee in DMSO
(Table 1, entry 8). No detectable reaction was observed in
toluene, CH2Cl2 and CHCl3 (Table 1, entries 9–11). In all
the tested solvents, no obvious side reaction was observed.
Thus, MeOH was confirmed as the optimal solvent for the
SGP-promoted Michael reaction.
Table 1 Effect of solvents on the SGP-promoted Michael reaction
Entry
Solvent
Yield (%)a
ee (%)b
1
2
3
4
5
6
7
8
MeOH
EtOH
i-PrOH
THF
75
73
48
19
36
25
15
84
–
87
80
62
57
54
53
43
3
DMF
1,4-dioxane
MeCN
DMSO
toluene
CH2Cl2
CHCl3
9
10
11
–
–
–
–
–
Reaction conditions: 1a (0.5 mmol), 2a (1.0 mmol) and SGP (125 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 analysis using Chiralpak AS-H
1 3

Enzyme-Promoted Direct Asymmetric Michael Reaction by Using Protease from Streptomyces…
Table 2 Control experiments for the SGP-promoted Michael reaction
Entry
Catalyst
Yield (%)a
ee (%)b
1
2
3
4
5
6
None
SGP (125 U)
albumin from chicken egg white (25 mg)
albumin from bovine serum (25 mg)
SGP (125 U) pretreated with 250 mM Cu2+ c
SGP (125 U) pretreated with PMSFd
–
75
–
–
–
–
87
–
–
–
3
21
Unless otherwise noted, reaction conditions: 1a (0.5 mmol), 2a (1.0 mmol) and catalyst in MeOH (0.9 mL) and deionized water (0.1 mL) at
30°C for 96 h
aIsolated yield
bEnantiomeric excess (ee) was determined by HPLC analysis using Chiralpak AS-H
cSGP (125 U) in 250 mM CuSO4 solution was stirred at 30°C for 24 h and then water was removed by lyophilization
dSGP (125 U) in 0.066 M PMSF solution (in THF) was stirred at 30°C for 24 h, and then THF was removed under reduced pressure
To verify the specific effect of SGP on the Michael reac-
tion, some control experiments were performed (Table 2).
In the absence of enzyme, no product was observed
(Table 2, entry 1), indicating that SGP indeed had an
effect on the model Michael reaction. Next, the albumins
from chicken egg white and bovine serum were used in the
reaction separately, which gave no product (Table 2, entries
3 and 4). These results confirmed that the catalysis was not
simply arisen by the amino acid residues on the surface of
the protein. Next, the Cu2+-denatured SGP was also used to
promote the model reaction and no product was observed
after 4 days (Table 2, entry 5). This suggested that the
Table 3 Effect of temperature on the SGP-promoted Michael reaction
Entry
Temperature (°C)
Yield (%)a
ee (%)b
1
2
3
4
5
6
7
8
15
20
25
30
35
37
40
50
48
58
76
77
84
79
74
65
64
73
85
95
98
96
95
88
Reaction conditions: 1a (0.5 mmol), 2a (1.0 mmol) and SGP (250 U) in MeOH (0.9 mL) and deionized water (0.1 mL) for 96 h
aIsolated yield
bEnantiomeric excess (ee) was determined by HPLC analysis using Chiralpak AS-H
1 3

L.-L. Wu et al.
tertiary structure of SGP was essential in the process of the
reaction. Phenylmethylsulfonyl fluoride (PMSF) is an irre-
versible inhibitor of serine proteases. The model Michael
reaction with PMSF-inhibited SGP only gave the product
in 3% yield after 4 days (Table 2, entry 6). The above con-
trol experiments indicated that the native structure of the
enzyme was responsible for this model Michael reaction.
In order to explore the optimal reaction conditions, vari-
ous reaction parameters including enzyme loading, water
content, mole ratio of substrates and time course were
investigated (For details, please see the Supporting Infor-
mation Tables S2, S3, Fig. 1). It seems that there is no
change in reactivity but the selectivity significantly varied
when increasing the enzyme loading from 125 to 250 U.
When 250 U of SGP was used, the reaction gave a yield of
77% with 95% ee; further increasing the enzyme loading
to 300 U did not get a better result. Taking into considera-
tion of the yield, selectivity and cost of the reaction, 250
U of enzyme loading, a mixed solvent of MeOH/H2O=9/1
(v/v), the molar ratio of 1a:2a=1:2 were selected as the
optimum reaction conditions. Temperature plays an impor-
tant role in enzyme-promoted reactions due to its signifi-
cant effect on enzyme stability and reaction rate [51]. Thus,
the effect of temperature on the SGP-promoted Michael
reaction was investigated (Table 3). The reaction exhibited
a good yield of 84% with the best enantioselectivity of 98%
ee at 35°C (Table 3, entry 5). Both the enantioselectivity
and yield were decreased at higher temperature (Table 3,
Table 4 Scope of the SGP-promoted Michael reaction
Entry
n
R1
R2
Time (h)
Product
Yield (%)a
ee (%)b
1
2
3
4
5
6
7
8
9
2
2
2
2
2
1
1
1
3
3
–OBn
–OMe
–OEt
–O (i-Pr)
Ph
–OMe
–OEt
–O (i-Pr)
–OEt
–OBn
–OMe
–OEt
–O (i-Pr)
–OEt
–OMe
–OEt
–O (i-Pr)
–OEt
96
84
96
96
72
48
12
48
96
84
3a
3b
3c
3d
3e
3f
84
88
63
73
75
62
33c
68
37
56
98
28
81
39
56
19
17
33
60
68
3g
3h
3i
10
–OBn
–OBn
3j
Reaction conditions: 1 (0.5 mmol), 2 (1.0 mmol) and SGP (250 U) in MeOH (0.9 mL) and deionized water (0.1 mL) at 35°C
aIsolated yield
bEnantiomeric excess (ee) was determined by HPLC analysis using a chiral column (for details, please see the Supporting Information)
cBesides 3g (33%), transesterification with solvent MeOH resulted in 3f (20%)
Scheme 1 The reaction of
4,4-dimethylcyclohex-2-en-
1-one and diethyl malonate
1 3

Enzyme-Promoted Direct Asymmetric Michael Reaction by Using Protease from Streptomyces…
entries 6–8), probably due to the partial denaturation of the
enzyme caused by high temperature. No obvious side reac-
tion was observed under all the tested temperatures. Thus,
35°C was chosen as the optimal temperature.
The desired products were obtained with enantioselectivi-
ties of up to 98% ee. Results indicated that SGP has a cata-
lytic effect on the Michael reaction. This work provides a
new synthetic route for the asymmetric Michael reaction of
malonates to cyclic enones using a biocatalyst under mild
reaction conditions.
With the optimized conditions in hand, various
malonates and enones were examined to expand upon the
SGP-promoted Michael reaction (Table 4). Cyclic enones
including 2-cyclopenten-1-one, 2-cyclohexen-1-one and
2-cyclohepten-1-one could participate in the reaction
smoothly. Generally, 2-cyclohexen-1-one as a substrate
gave better yield and enantioselectivity than 2-cyclopenten-
1-one and 2-cyclohepten-1-one (Table 4, entries 1–4 and
6–10). The reaction with 2-cyclopenten-1-one gave low
selectivity (Table 4, entries 6–8). Different malonates
were also tested. When reacting with 2-cyclohexen-1-one,
the dibenzyl malonate gave an excellent enantioselectiv-
ity of 98% ee (Table 4, entry 1) and the diethyl malonate
also afforded a good ee of 81% (Table 4, entry 3). Compar-
ing with other malonates, dimethyl malonate as a substrate
afforded Michael adducts with low ee values (Table 4,
entries 2 and 6), probably due to the small size of the mol-
ecule which reduced the spatial effect of the enzyme on
it. When 2-cyclopenten-1-one and diethyl malonate were
used as substrates, the corresponding product 3g was suf-
fered largely from transesterification with solvent MeOH,
resulting in a yield of 3f (20%) and a low yield of 3g (33%)
(Table 4, entry 7). Besides malonates, ethyl benzoylacetate
was also applicable to this SGP-promoted Michael reac-
tion, giving the corresponding product with an acceptable
yield and moderate ee value (Table 4, entry 5). Addition-
ally, sterically hindered 4,4-dimethylcyclohex-2-enone can
also be used as a substrate in this biocatalytic reaction,
and a yield of 36% with 42% ee was obtained (Scheme 1).
For all the tested substrates, no obvious side reaction was
observed except the transesterification with solvent MeOH
for product 3g (Table 4, entry 7). All the products obtained
have only one asymmetric carbon atom (chiral center)
except product 3e which has two chiral centers. However,
there is a keto enol tautomerism equilibrium for 3e (for the
structure, please see the Supporting Information, 4 Char-
acterization of Michael products), so it actually has only
one stable chiral center. Thus, in this work, all the products
have been obtained in optically active form with 17–98%
ee (S).
4 Experimental Section
4.1 Materials
The protease type XIV from Streptomyces griseus
(SGP) was purchased from Sigma-Aldrich [P5147,
Lot#SLBF8736V. Tan powder; units/mg: 5.0. 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]. Unless
otherwise noted, all reagents were obtained from commer-
cial suppliers and used without further purification.
4.2 General Methods
NMR spectra were recorded on a 300 MHz spectrometer
(Bruker AVANCE DMX300). Melting points were taken
on a WPX-4 apparatus. Routine monitoring of reaction
was performed by TLC using pre-coated Haiyang GF254
silica gel TLC plates. All the column chromatography
separations were done by silica gel (200–300 mesh) at
increased pressure. Evaporation of solvents was performed
at reduced pressure. The crude products were purified by
column chromatography with petroleum ether/ethyl acetate
as eluent. The enantiomeric excess (ee) of aldol products
was determined by chiral HPLC analysis, performed using
Chiralcel OJ-H and Chiralpak AD-H, AS-H, IA-H col-
umns. All optical rotations [α]20 were obtained from Per-
D
kin Elmer Model 341 Polarimeter. Absolute configurations
of the products were determined by comparison with the
known 1H NMR, 13C NMR, chiral HPLC analysis and opti-
cal rotation.
4.3 General Procedure for the SGP-Promoted Michael
Reaction
SGP (250 U) was added to a round bottom flask contain-
ing malonate (0.5 mmol), enone (1.0 mmol), Methanol
(0.9 mL) and deionized water (0.1 mL). The resultant mix-
ture was stirred at 35°C and monitored by thin layer chro-
matography (TLC). The reaction was terminated by filter-
ing off the enzyme. The filter cake was washed with ethyl
acetate. Then the filtrate was concentrated under reduced
pressure. The residue was purified by flash column chro-
matography (ethyl acetate/petroleum ether: 6/1-3/1) to give
the product.
3 Conclusion
In summary, SGP was used for the first time as a biocata-
lyst in asymmetric Michael reaction of malonates to α,β-
unsaturated carbonyl compounds. The influence of several
factors including solvent, the mole ratio of substrates, water
content, temperature and enzyme loading were investigated.
1 3

L.-L. Wu et al.
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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 Chong-
qing (cstc2015jcyjBX0106).
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