Catalytic promiscuity of Escherichia coli BioH esterase: Application in the synthesis of 3,4-dihydropyran derivatives

Article abstract of DOI:10.1016/j.procbio.2014.03.020

Enzymatic catalytic promiscuity has received increasing attention in the past decade. In this research, ten enzymes were investigated for the promiscuous activity in catalysis of the Michael addition-cyclization cascade reaction of p-nitrobenzalacetone with 1,3-cyclohexanedione to prepare 2-hydroxy-2-methyl-4- (4-nitrophenyl)-3,4,7,8-tetrahydro-2H-chromen-5(6H)-one in anhydrous media, and control experiments were conducted to exclude false positive results. The highest yield (46.1%) was observed with Escherichia coli BioH esterase and the optimal reaction condition was: 1 mmol α,β-unsaturated ketone, 1 mmol 1,3-dicarbonyl compound, 20 mg E. coli BioH esterase, 20 ml N,N-dimethylformamide at 37 °C for 120 h. To preliminarily investigate the mechanism, site-directed mutagenesis was performed on the hydrolysis catalytic triad of BioH, and the results indicated alternate-site enzyme promiscuity . When a series of substituted benzalacetones and 1,3-cyclic diketones were used as the reactants, yields of up to 76.3% were achieved. These results imply the potential industrial application of E. coli BioH in the preparation of dihydropyran derivatives.

Full text of DOI:10.1016/j.procbio.2014.03.020

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Short communication
Catalytic promiscuity of Escherichia coli BioH esterase:
Application in the synthesis of 3,4-dihydropyran derivatives
Ling Jianga, Bo Wangb, Rong-rong Lic, Sa Shenc, Hong-wei Yua, Li-dan Yea,∗
a Department of Chemical and Biological Engineering, Zhejiang University, 310027 Hangzhou, PR China
b College of Materials and Chemical Engineering, Hainan University, 570228 Haikou, PR China
c School of Chemical and Material Engineering, Jiangnan University, 214122 Wuxi, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Enzymatic catalytic promiscuity has received increasing attention in the past decade. In this
research, ten enzymes were investigated for the promiscuous activity in catalysis of the Michael
addition-cyclization cascade reaction of p-nitrobenzalacetone with 1,3-cyclohexanedione to prepare 2-
hydroxy-2-methyl-4-(4-nitrophenyl)-3,4,7,8-tetrahydro-2H-chromen-5(6H)-one in anhydrous media,
and control experiments were conducted to exclude false positive results. The highest yield (46.1%)
was observed with Escherichia coli BioH esterase and the optimal reaction condition was: 1 mmol
␣,␤-unsaturated ketone, 1 mmol 1,3-dicarbonyl compound, 20 mg E. coli BioH esterase, 20 ml N,N-
dimethylformamide at 37 ◦C for 120 h. To preliminarily investigate the mechanism, site-directed
mutagenesis was performed on the hydrolysis catalytic triad of BioH, and the results indicated “alternate-
site enzyme promiscuity”. When a series of substituted benzalacetones and 1,3-cyclic diketones were
used as the reactants, yields of up to 76.3% were achieved. These results imply the potential industrial
application of E. coli BioH in the preparation of dihydropyran derivatives.
Received 18 March 2014
Accepted 24 March 2014
Available online xxx
Keywords:
Catalytic promiscuity
Escherichia coli BioH esterase
Michael addition-cyclization cascade
reaction
Pyran derivatives
© 2014 Elsevier Ltd. All rights reserved.
1. Introduction
suffer from drawbacks such as high cost, harsh reaction conditions,
toxicity and laborious workup procedure. Therefore, the devel-
Dihydropyran derivatives have attracted significant atten-
tion due to their diverse biological activities [1]. Generally,
3,4-dihydropyran derivatives are prepared by inverse-electron-
demand hetero-Diels–Alder reactions of ␣,␤-unsaturated carbonyl
compounds with electron-rich alkenes [2]. In 2008, Franke
et al. used pyrrolidine derivatives as catalysts to prepare 3,4-
dihydropyrans [3]. After that, Yu et al. designed and utilized a fluo-
rinated diarylprolinol silyl ether to catalyze the reaction between
1,3-dicarbonyl compounds and ␣,␤-unsaturated aldehydes [4].
Then, Liu et al. applied chiral diamine catalysts to synthesize
3,4-dihydropyran derivatives using cyclic dimedone and ␣,␤-
unsaturated ketones as reactants [5]. Recently, Ray and coworkers
reported a facile method to synthesize 3,4-dihydronpyrans through
chiral pybox-diph-Zn(II) complex catalyzed Michael addition of
cyclic 1,3-dicarbonyls to 2-enoylpyridine N-oxides [6]. Despite the
great progresses made in the employment of organocatalysts, many
reported methods for the synthesis of 3,4-dihydropyran derivatives
opment of a new method with a simpler and more convenient
procedure using a cost-effective, eco-friendly catalyst is still in
demand.
Owing to the potential application of enzyme catalytic promis-
cuity in organic synthesis, considerable effort has been made over
the past decade [7]. For instance, enzymes have been used to
catalyze Aldol reaction [8], Baylis–Hillman reaction [9], Mannich
reaction [10], Michael addition [11] and Knoevenagel reaction [12].
This kind of catalytic promiscuity largely expands the application
scope of enzymes in organic synthesis.
In continuation of our work on enzyme catalytic promiscu-
ity [13], herein we describe another example of the catalytic
promiscuity of Escherichia coli BioH esterase and its application
in the synthesis of 3,4-dihydropyran derivatives via the Michael
addition-cyclization cascade reaction of ␣,␤-unsaturated ketones
and 1,3-dicarbonyl compounds (Fig. 1). The reaction reported here
employed the economical and eco-friendly E. coli BioH esterase as
the catalyst and successfully provided access to a diverse range of
3,4-dihydropyran derivatives. This protocol provides an interest-
ing insight into enzyme catalytic promiscuity and may have the
potential for industrial application.
∗
Corresponding author. Tel.: +86 571 87951873; fax: +86 571 87951873.
E-mail address: yelidan@zju.edu.cn (L.-d. Ye).
http://dx.doi.org/10.1016/j.procbio.2014.03.020
1359-5113/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Jiang L, et al. Catalytic promiscuity of Escherichia coli BioH esterase: Application in the synthesis of
3,4-dihydropyran derivatives. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.03.020

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R1
O
O
O
E. coli BioH
R2
R3
R1
+
DMF, 37 °C
R2
R3
O
HO
O
Fig. 1. E. coli BioH esterase-catalyzed synthesis of 3,4-dihydropyran derivatives via the Michael addition-cyclization cascade reaction between substituted benzalacetones
and 1,3-cyclic diketones.
dried. All the compounds were spectroscopically characterized (1H
NMR and 13C NMR).
2. Materials and methods
2.1. Materials
2.5. Analytical methods
“Amano” lipase AK, “Amano” lipase AS, “Amano” lipase AYS and
“Amano” lipase DF were purchased from Amano Enzyme Inc. Lipase
from Candida rugosa was purchased from Sigma–Aldrich. Lipozyme
RMIM and lipozyme TLIM were purchased from Beijing Gaoruisen
Technology Co. Ltd. Lipase from Rhizomucor miehei, esterase from
Rhodobacter sphaeroides (RspE) were expressed in E. coli [14,15].
Bovine serum albumin (BSA) was purchased from Aladdin Chem-
icals. Unless otherwise noted, all reagents were obtained from
commercial suppliers and used without further purification.
Analytical thin layer chromatography (TLC) was performed
on Haiyang precoated TLC plates (silica gel GF254), eluted with
petroleum ether/ethyl acetate (1/1, v/v). 1H NMR (400 MHz) and
13C NMR (100 MHz) spectra were recorded on a Bruker Advance
2B 400 instrument. Chemical shifts (ı) were quoted in ppm using
CDCl3 (1H NMR ı 7.26, 13C NMR ı 77.16) or DMSO-d6 (1H NMR ı
2.5, 13C NMR ı 39.52) as solvent and tetramethylsilane (TMS) as
an internal reference. The coupling constants (J) were quoted in
Hz. The structures of the products were confirmed by comparing
the 1H NMR and 13C NMR spectral data with those reported in the
literature [5].
2.2. Construction of mutants by site-directed mutagenesis
pET-30a plasmid harboring the recombinant BioH gene (E. coli
K12) was used as a template for polymerase chain reaction (PCR)-
based site directed mutagenesis using the QuikChangeTM method
(Stratagene, La Jolla, CA). The synthetic primers used to construct
the mutants are listed in Table S1 in Supplementary materials. The
mutations were confirmed by DNA sequencing.
3. Results and discussion
The reaction of p-nitrobenzalacetone 1a and 1,3-
cyclohexanedione 2a was chosen as a model reaction (Fig. 2).
A wide range of enzymes were rapidly screened for catalytic
activity in this Michael addition-cyclization cascade reaction
(Table 1). When the reactants were incubated without any protein
or with a non-catalytic protein such as bovine serum albumin
(BSA), no product was formed even after 120 h (entries 1–2,
Table 1). “Amano” Lipase Ak, “Amano” Lipase AS, “Amano” Lipase
AYS and “Amano” Lipase DF all showed very low activity toward
this cascade reaction (entries 3–6, Table 1). Lipase from R. miehei,
Lipozyme TLIM from Thermomyces lanuginosus, Lipozyme RMIM
from R. miehei and lipase from C. rugosa also showed low catalytic
activities in this reaction, giving product yields of only 2.6%, 3.7%,
4.5% and 3.4%, respectively (entries 7–10, Table 1). When the R.
sphaeroides esterase (RspE) was applied, the reaction proceeded
slowly and the target compound was obtained in a yield of
8.7% (entry 11, Table 1). E. coli BioH esterase, which contains
a classical Ser-His-Asp catalytic triad and belongs to the group
of carboxylesterases [16], was identified as the most effective
biocatalyst for this cascade reaction and the target compound was
obtained in a yield of 46.1% (entry 12, Table 1). In the past few
years, some studies found that the previously reported enzyme
promiscuous activity was actually not catalyzed by the enzymes
[17,18]. As a control experiment, the reactants were incubated
2.3. Protein expression and purification
The resulting recombinant cells were grown in 500 ml LB media
containing 50 ␮g/ml kanamycine at 37 ◦C. The culture was allowed
to reach an OD600 of 0.4–0.6 before induced with 0.5 ml isopropyl
␤-d-1-thiogalactopyranoside (0.1 mM) for 4 h at 37 ◦C. The cells
were then harvested by centrifugation (2150 × g, 10 min, 4 ◦C), and
washed twice with 100 ml phosphate-buffered saline (0.1 M, pH
7.4). Then the cells were resuspended in 50 ml Tris–HCl buffer
(50 mM, pH 8.0) and disrupted by sonication. The supernatants
were purified on a Nickel column as reported [16]. The purity of
enzyme was checked by SDS-PAGE (Supplementary materials, Fig.
S1).
2.4. Typical enzymatic procedure for the formation of
3,4-dihydropyran derivatives
A mixture of the 1,3-dicarbonyl compound (1 mmol), ␣,␤-
unsaturated ketone (1 mmol), pure enzyme powder of E. coli BioH
esterase (20 mg) in DMF (20 ml) was shaken at 200 rpm, 37 ◦C
for specified time. The reaction progress was monitored by thin
layer chromatography (TLC). After the reaction was completed, the
enzyme was filtered off (Whatman® qualitative medium flow filter
paper, Ø15 cm, ash ≤ 0.06%, pore size 11 ␮m) and the filtrate was
washed with water (20 ml) and extracted with dichloromethane
(2× 20 ml). The organic phase was combined, dried with MgSO4
and concentrated under reduced pressure. The crude residue was
purified by flash column chromatography (200–300 mesh silica gel)
with an eluent consisting of petroleum ether/ethyl acetate (2:1,
v/v). Product-containing fractions were pooled, concentrated and
Fig. 2. Method of biocatalyst screening for the Michael addition-cyclization cascade
reaction between p-nitrobenzalacetone and cyclohexane-1,3-dione.
Please cite this article in press as: Jiang L, et al. Catalytic promiscuity of Escherichia coli BioH esterase: Application in the synthesis of
3,4-dihydropyran derivatives. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.03.020

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Table 2
3
Table 1
Biocatalysts screened for the Michael addition-cyclization cascade reaction between
E. coli BioH-catalyzed Michael addition-cyclization cascade reaction between ␣,␤-
unsaturated ketones and 1,3-dicarbonyl compoundsa (please refer to Fig. 1 for the
reaction schematic).
p-nitrobenzalacetone and cyclohexane-1,3-dione and their product yields.a
Entry
Catalyst
Yield (%)b
Entry
R1
R2
R3
Time (h)
Yields (%)b
1
2
No enzyme
Bovine Serum Albumin
0
0
1
2
3
4
5
6
7
8
9
4-NO2
3-NO2
2-NO2
4-H
4-NO2
3-NO2
2-NO2
4-F
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
120
120
120
120
144
144
168
144
168
168
168
168
46.1
27.5
17.0
14.1
76.3
66.4
47.0
68.7
31.9
19.2
35.8
41.4
3
4
5
6
7
8
9
10
11
12
13
14
15
“Amano” Lipase Ak from Pseudomonas fluorescen
“Amano” Lipase AS from Aspergillus niger
“Amano” Lipase AYS from Candida rugosa
“Amano” Lipase DF from Rhizopus oryzae
Lipase from Rhizomucor miehei
Lipozyme TLIM from Thermomyces lanuginosus
Lipozyme RMIM from Rhizomucor miehei
Lipase from Candida rugosa
Esterase from Rhodobacter sphaeroides
BioH esterase from Escherichia coli
Denatured BioHc
BioH esterase Ser82Ala
BioH esterase His235Ala
<1
<1
<1
<1
2.6
3.7
4.5
3.4
8.7
46.1
0
4-Me
4-OMe
2-Cl
10
11
12
2-F
43.0
44.5
a
Experimental conditions: ␣,␤-unsaturated ketone (1 mmol), 1,3-dicarbonyl
compound (1 mmol), DMF (20 ml), pure enzyme powder of BioH esterase (20 mg),
shaken at 200 rpm, 37 ◦C.
a
Experimental conditions: p-nitrobenzalacetone (200 mg, 1 mmol), cyclohexane-
1,3-dione (110 mg, 1 mmol), DMF (20 ml) and enzyme (20 mg) was shaken at
200 rpm at 37 ◦C for 120 h.
b
Isolated yields.
b
Determined by HPLC.
c
Pre-treated with urea at 100 ◦C for 8 h.
4. Conclusions
In conclusion, we have developed a novel E. coli BioH esterase-
catalyzed method to synthesize 3,4-dihydropyran derivatives with
moderate to high yields via the Michael addition-cyclization cas-
cade reaction. The two mutants BioH esterase Ser82Ala and BioH
esterase His235Ala showed a catalytic activity similar to that of the
wide-type, confirming the view proposed by Reetz and co-workers
called “alternate-site enzyme promiscuity”. In addition, because
this enzyme could be prepared by large scale fermentation of E. coli
with a low cost, this protocol could provide an economic route to
preparation of dihydropyran derivatives and might be potentially
useful for industrialization. Further studies on improving the activ-
ity and enantioselectivity of this enzyme by both directed evolution
and rational design are in progress.
with the urea-denatured E. coli BioH esterase, where no conversion
was observed (entry 13, Table 1), confirming that the catalysis was
indeed mediated by the bioactive enzyme. However, it remains
unknown whether the hydrolysis site in E. coli BioH esterase is
also responsible for its promiscuous activity, as believed for other
hydrolases [19]. In order to clarify this question, site-directed
mutagenesis studies were performed on the Ser-His-Asp cat-
alytic triad. The two mutants “BioH esterase Ser82Ala” and “BioH
esterase His235Ala” showed catalytic activities similar to that
of the wide-type (entries 14–15, Table 1), indicating an active
site other than this catalytic triad. In 2007, Reetz and co-workers
proposed a viewpoint called “alternate-site enzyme promiscuity”,
suggesting that the reaction catalyzed by promiscuous enzyme
neither involves any of the catalytic amino acids of the natural
enzymatic process, nor appears to occur in the natural binding
pocket [20]. To some extent, our study confirms this view. These
results implied that perhaps the tertiary structure and the special
spatial conformation rather than the hydrolysis site of the BioH
esterase are responsible for this cascade reaction. Other reaction
conditions such as enzyme loading and temperature were also
investigated. The results showed that yields of up to 46.1% were
achieved when 20 mg of pure enzyme powder of BioH esterase
were used in 20 ml of DMF at 37 ◦C for 120 h.
Acknowledgements
This work was financially supported by the Outstanding
Young Scholar Grant of Zhejiang University (No. R4110092), the
National Natural Science Foundation of China (No. 21176215) and
the Program for Zhejiang leading team of S&T Innovation (No.
2011R50007). We thank all of the constructive comments and sug-
gestions from the editors and reviewers.
Appendix A. Supplementary data
Encouraged by our initial study results, some more ␣,␤-
unsaturated ketones and 1,3-dicarbonyl compounds were tested
in order to investigate the generality and the substrate spectrum
of this E. coli BioH-catalyzed Michael addition-cyclization cas-
cade reaction (Table 2). It was noted that the reaction activity of
1,3-cyclohexanedione was higher than that of 5,5-dimethyl-1,3-
cyclohexanedione (entries 1–3 versus entries 5–7, Table 2). This
was probably due to the steric hinderance of methyl. In the case
of ␣,␤-unsaturated ketones, the electronic properties and sub-
stituent position at the aromatic ring played an important role
in the product yields. For example, ␣,␤-unsaturated ketones with
electron-withdrawing groups could enhance the reactivity of the
substrate (entries 5–8, entries 11–12, Table 2), and the substituent
in the 4-position of benzalacetone led to a higher yield than the
same substituent in the 2- or 3-position (entry 1 versus entries
2–3, entry 5 versus entries 6–7, entry 8 versus entry 12, Table 2).
This might be due to the stablilization of electron-withdrawing
groups on the phenyl ring of the benzalacetones by the amino acid
residues of the E. coli BioH via hydrogen bond, which was in favor
of nucleophilic attack by carbanion.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.procbio.2014.03.020.
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3,4-dihydropyran derivatives. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.03.020