Stereoselective synthesis of spiro[5.5]undecane derivatives via biocatalytic [5+1] double Michael additions

Article abstract of DOI:10.1016/j.molcatb.2013.07.012

A novel enzymatic, promiscuous protocol of d-aminoacylase (DA)-catalyzed [5+1] double Michael addition was developed herein, for the synthesis of (hetero)spiro[5.5]undecane derivatives in moderate yields. It is notable that almost only the cis isomers were obtained through this biocatalytic methodology in all the cases according to their ¹H and ¹³C NMR spectra. It is the first report on hydrolase-catalyzed double Michael addition in organic solvent.

Full text of DOI:10.1016/j.molcatb.2013.07.012

Journal of Molecular Catalysis B: Enzymatic 97 (2013) 18–22
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Stereoselective synthesis of spiro[5.5]undecane derivatives via
biocatalytic [5+1] double Michael additions
Xiao-Yang Chen, Yi-Ru Liang, Fang-Li Xu, Qi Wu∗, Xian-Fu Lin∗
Department of Chemistry, Zhejiang University, Hangzhou 310027, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel enzymatic, promiscuous protocol of d-aminoacylase (DA)-catalyzed [5+1] double Michael addi-
tion was developed herein, for the synthesis of (hetero)spiro[5.5]undecane derivatives in moderate yields.
It is notable that almost only the cis isomers were obtained through this biocatalytic methodology in all
the cases according to their 1H and 13C NMR spectra. It is the first report on hydrolase-catalyzed double
Michael addition in organic solvent.
Received 26 April 2013
Received in revised form 8 July 2013
Accepted 20 July 2013
Available online xxx
© 2013 Elsevier B.V. All rights reserved.
Keywords:
Double Michael addition
Spiro compounds
Enzyme
Stereoselectivity
1. Introduction
be one of the most basic and powerful methods for the con-
struction of carbon–carbon and carbon-hetero bonds, has been
The
synthesis
of
spiro[5.5]undecane
and
het-
frequently reported and widely used [18–33]. For instance, our
group has demonstrated that d-aminoacylase from Escherichia coli
(DA) could catalyze the C C bond formations via Michael additions
between ␣,␤-unsaturated carbonyl compounds and activated car-
bon nucleophiles such as acetylacetone and ethyl acetoacetate [29].
Inspired by this promiscuous behavior, we report a novel discovery
that the commercially available DA promotes a cascade [5+1] dou-
ble Michael addition to form cis-spiro[5.5]undecane derivatives in
the present work (Scheme 1), yet other types of biocatalyzed dou-
ble Michael addition has never been reported to the best of our
knowledge.
erospiro[5.5]undecane motifs has engrossed substantial attention
from organic chemists for many years, not only because of
their unique structural properties [1,2], but also because of
their presence in several natural products such as elatol (I),
isoobtusol (II) and (−)-sibirine (III) (Fig. 1) [3–8]. Of all the
construction methods of the spirocyclics, which can be roughly
categorized into alkylation, rearrangement, cycloaddition and
cleavage of bridged systems, the alkylation on the quaternary
carbon, especially 1,4-addition, is one of the most common
methods for the preparation for spiro[5.5]undecane derivatives.
However, the conventional methods usually involve bases or
Lewis acids as the catalyst under homogeneous conditions, which
encountered environmental problems [9–12]. Therefore, the inven-
tion and introduction of environmentally compatible catalysts
have always showed great importance and attracted enormous
attention.
2. Experimental
2.1. Materials
Biocatalysis is a powerful tool for organic synthesis due to
its high efficiency, good selectivity and great environmental
acceptability [13–16]. The recent progress in catalytic promis-
cuity of enzymes [17–20] has greatly expanded its application
scope. Among them, the Michael addition, widely considered to
Lipase from Candida antarctica (CALB) immobilized on acrylic
resin (≥10,000 U/g, recombinant, expressed in Aspergillus oryzae),
Lipase from hog pancreas (HPL) (2.4 U/mg, 1 U is the amount of
immobilized enzyme which forms 1% octyl laurate from 0.5 mmol
lauric acid and 1.0 mmol 1-octanol in 10 ml water-saturated isooc-
tane in 1 h at 20 ◦C) was purchased from Fluka (Switzerland).
d-Aminoacylase from E. coli (DA) (Not less than 5 MU/g, 1 U is
defined as enzyme quantity which produces 1 ␮mol of d-amino
acid per 30 min under the condition as below: 0.1 M N-acetyl-d-
methionine, pH8.0, 37 ◦C) was purchased from Amano Enzyme Inc
∗
Corresponding authors. Tel.: +86 571 87951588; fax: +86 571 87952618.
E-mail addresses: wuqi1000@yahoo.com.cn (Q. Wu), llc123@zju.edu.cn
(X.-F. Lin).
1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.07.012

X.-Y. Chen et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 18–22
19
Fig. 1. Some natural products with (hetero)spiro[5.5]undecane.
Scheme 1. (a) DA-catalyzed mono-Michael additions (previous work). (b) DA-catalyzed double Michael additions (this work).
(Japan). All reagents used in the experiments were obtained from
commercial sources and used without further purification.
a Agilent 1100 series with a reversed-phase Shim-Pack VP-ODS
column (150 mm × 4.6 mm) and a UV detector (210 nm). All the
known products were characterized by comparing the 1H NMR
with those reported in the literature. IR spectra were measured
with a Nicolet Nexus FTIR 670 spectrophotometer.
2.2. Analytical methods
The process of reactions was monitored by TLC on silica with
Petroleum ether/EtOAc (6/1, v/v) as solvent. The 1H spectra were
recorded with TMS as internal standard using a Bruker AMX-
400 MHz spectrometer. Chemical shifts were expressed in ppm and
coupling constants (J) in Hz. Analytical HPLC was performed using
2.3. General procedure for the double Michael additions
1,3-Dione (0.25 mmol), (1E,4E)-1,5-diarylpenta-1,4-dien-3-one
(1 mmol), DA (20 mg), DMSO (0.9 ml) and water (0.1 ml) were taken
in a flask and the reaction mixture was incubated at 50 ◦C for 48
or 72 h. Enzyme was filtered off to stop the reaction. CH2Cl2 was
used to wash the filter paper to assure that products obtained were
all dissolved in the filtrate. Then 10 ml of water was added to the
filtrate, and the filtrate was extracted with CH2Cl2. The organic
Table 1
Double Michael additions catalyzed by various enzymes.a
Table 2
Screen of reaction conditions of DA-catalyzed double Michael addition of 1a and
2a.a
.
Entry
Solvent
Temp. (◦C)
Yield (%)
Entry
Catalyst
Yield (%)
1
2
3
4
5
6
7
8
9
DMSO
50
50
50
50
50
50
40
60
70
48
57
61
57
56
19
42
57
54
DMSO (5% water)
DMSO (10% water)
DMSO (15% water)
DMSO (20% water)
DMSO (30% water)
DMSO (10% water)
DMSO (10% water)
DMSO (10% water)
1
2
3
4
5
6
Blank
DA
N.R.
25 (48b)
Inhibited DAc
BSA
5
9
8
2
HPL
CALB
a
Experimental conditions: 0.25 mmol cyclohexane-1,3-dione (1a), 0.25 mmol
(1E,4E)-1,5-diphenylpenta-1,4-dien-3-one (2a), 20 mg Enzyme, 1 ml DMSO, 50 ◦C,
48 h. All yields were determined by HPLC. N.R. means no reaction.
a
Experimental conditions: 0.25 mmol cyclohexane-1,3-dione (1a), 1 mmol
(1E,4E)-1,5-diphenylpenta-1,4-dien-3-one (2a), 20 mg DA, 1 ml solvent, 48 h. All
yields were determined by HPLC.
b
1 mmol (1E,4E)-1,5-diphenylpenta-1,4-dien-3-one (2a) was used.
50 mM ZnCl2 was added to inhibit DA.
c

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X.-Y. Chen et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 18–22
Table 3
Substrate scope of the double Michael addition.a
.
Entry
1
1
R2
Product
Yield (%)
61
cis/transb
C6H5
>100/1
(2a)
(1a)
(3a)
2c
C6H5
36
>100/1
(2a)
(1b)
(1c)
(3b)
3
C6H5
41
>100/1
(2a)
(3c)
4c
4-F-C6H4
53
20/1
(1a)
(2b)
(3d)
5c
2-Thienyl
30
>100/1
(1a)
(2c)
(3e)
a
Reaction conditions: 0.25 mmol 1,3-dione (1a–1c), 1 mmol penta-1,4-dien-3-one (2a–2c), 20 mg DA, 0.1 ml water, 0.9 ml DMSO, 50 ◦C, 48 h. All yields were determined
by HPLC.
b
Determined by 1H NMR spectroscopy.
The reaction time was 72 h.
c
phase was dried over anhydrous Na2SO4, and the solvents were
removed under reduced pressure. The crude residue was purified
by silica gel column chromatography with an eluent consisting
of petroleum ether/EtOAc (6/1, v/v). Product-contained fractions
were combined, concentrated, and dried to afford the respective
product.
3. Results and discussion
First, we started our study with the reaction of cyclohexane-
1,3-dione (1a) and (1E,4E)-1,5-diphenylpenta-1,4-dien-3-one
(2a) in the presence of DA in DMSO at 50 ◦C according
to our previous work [29]. The double Michael product

X.-Y. Chen et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 18–22
21
Fig. 2. (a) 1H NMR spectrum of trans-3d; (b) 1H NMR spectrum of cis-3d; (c) 13C NMR spectrum of trans-3d; (d) 13C NMR spectrum of cis-3d.
7,11-diphenylspiro[5.5]undecane-1,5,9-trione (3a) was obtained
in a 25% yield after 48 h (Table 1, entry 2). No product was detected
in the absence of enzyme (Table 1, entry 1). The DA is presumed to
be zinc-dependent and our previous work [29] has proposed that
the zinc atom is involved in DA-catalyzed additions. To demon-
strate the specific catalytic effect of DA in this reaction, control
reactions were run by adding 50 mM non-competitive inhibitor
ZnCl2 to inhibit DA. As the ligation of the inhibitory zinc ion by the
highly conserved residues Asp366, His67 and His69 in the active
site would lower their pKa values and/or hold the nucleophile to
perturb the proton shuttle and intermediate stabilization [34],
product 3a was only obtained in a low yield of 5% (Table 1, entry
3). The experiments using bovine serum albumin (BSA) and Lipase
from hog pancreas (HPL) gave the results similar to the inhibited
DA (Table 1, entries 4 and 5), implying that the active site of DA
and protein surface were both responsible for the double Michael
addition, while the active site played a dominant role. However,
surprisingly, CALB, which was widely applied in other Michael
type additions [24,27,28], did not catalyze this reaction smoothly
(Table 1, entry 6), which might be caused by the difficulty for
the bulky substrates entering into the active site of CALB. Several
recent publications demonstrated that earlier claimed promiscu-
ous enzyme reactions turned out not to be promiscuous, such as
Markovnikov addition [35], decarboxylative aldol reaction [36]
and Henry reaction [37], but doubt about the enzymatic Michael
reaction have never been raised to the best of our knowledge.
The biocatalyst’s concentration and the molar ratio of substrates
were examined first (Table S1, S2 in Support Information). Then the

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X.-Y. Chen et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 18–22
water content of the reaction media was investigated in detail with
the optimized catalyst. It was known that all enzymes need essen-
tially bound water, and some enzymatic activity in organic solvent
depends on water content [38]. Solvent mixtures containing 0–30%
water were screened in the DA-catalyzed reaction (Table 2, entries
1–6). The data showed that a water content of 10% attained a high-
est yield up to 61% after 48 h. The yield decreased evidently once
the water content surpassed 20%, maybe due to the low solubility
of substrates. Next, the influence of reaction temperature on this
enzymatic double Michael addition reaction was also considered
(Table 2 entries 3, 7–9). The highest yield was obtained at 50 ◦C.
Finally, we investigated the time course of the double Michael reac-
tion under the optimal conditions and the best yield was obtained
after 2 days (Fig. S1 in Support Information).In order to extend
the scope of this methodology, Meldrum’s acid and some other
(1E,4E)-1,5-diarylpenta-1,4-dien-3-ones were examined under the
optimized conditions (Table 3). When 5,5-dimethylcyclohexane-
1,3-dione or Meldrum’s acid were used as Michael donors, the
products were obtained in a lower yield (Table 3, entries 1 and 2)
due to the steric hindrance. (1E,4E)-1,5-Diphenylpenta-1,4-dien-
3-one with an electron-withdrawing group (4-F) furnished the
spirotrione 4d in 53% yield (Table 3 entry 4), but electron-donating
functionalities were not compatible (4-MeO and 4-Me were tested
with trace yields). In addition, (1E,4E)-1,5-di(thiophen-2-yl)penta-
1,4-dien-3-one (2c) could also be reactive, giving the spiro product
3e in 30% yields (Table 3, entry 5). Based on these results, this DA-
catalyzed double Michael reaction could be of great utility in the
generation of libraries of spirotriones 3 with moderate yields in a
stereospecific manner from simple substrates.
Foundation of Ministry of Education of China (20110101110008)
is gratefully acknowledged.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.molcatb.2013.07.012.
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Acknowledgements
The financial support from the National Natural Science Foun-
dation of China (No. 21072172, 21272208) and Ph.D. Programs