Lipase-catalyzed domino Michael-aldol reaction of 2-methyl-1,3-cycloalkanedione and methyl vinyl ketone for the synthesis of bicyclic compounds

Article abstract of DOI:10.1080/00397911.2015.1116584

Synthesis of bicyclic compounds was achieved via a lipase-catalyzed, stereoselective, domino Michael-aldol reaction of 2-methyl-1,3-cycloalkanedione and methyl vinyl ketone. Appropriate reaction conditions, including the type of enzyme, solvent, and temperature, were determined. In addition, the effects of solvent polarity and addtives were investigated. The reaction proceeded in the presence of lipase AS in a solution of 20% acetone in dimethylsulfoxide (DMSO) at 10 °C for 8 days, followed by the addition of p-toluenesulfonic acid (TsOH) to afford bicyclic compounds in 51-83% yields with moderate stereoselectivity. Although this domino Michael-aldol reaction showed only moderate stereoselectivity, even with the acid-supported enhancement of the reaction, these results represent potential new applications for lipase.

Full text of DOI:10.1080/00397911.2015.1116584

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
Chemistry
ISSN: 0039-7911 (Print) 1532-2432 (Online) Journal homepage: http://www.tandfonline.com/loi/lsyc20
Lipase-Catalyzed Domino Michael-Aldol Reaction
of 2-Methyl-1,3-Cycloalkanedione and Methyl Vinyl
Ketone for the Synthesis of Bicyclic Compounds
Kaoru Sano, Yoshihito Kohari, Hiroto Nakano, Chigusa Seki, Mitsuhiro
Takeshita, Micho Tokiwa, Yoshihiko Hirose & Koji Uwai
To cite this article: Kaoru Sano, Yoshihito Kohari, Hiroto Nakano, Chigusa Seki, Mitsuhiro
Takeshita, Micho Tokiwa, Yoshihiko Hirose & Koji Uwai (2015): Lipase-Catalyzed
Domino Michael-Aldol Reaction of 2-Methyl-1,3-Cycloalkanedione and Methyl Vinyl
Ketone for the Synthesis of Bicyclic Compounds, Synthetic Communications, DOI:
10.1080/00397911.2015.1116584
To link to this article: http://dx.doi.org/10.1080/00397911.2015.1116584
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Nov 2015.
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Date: 06 December 2015, At: 23:37

Lipase-catalyzed domino Michael-aldol reaction of 2-methyl-1,3-cycloalkanedione
and methyl vinyl ketone for the synthesis of bicyclic compounds
Kaoru Sano1, Yoshihito Kohari1, Hiroto Nakano1, Chigusa Seki1, Mitsuhiro Takeshita2,
Micho Tokiwa2, Yoshihiko Hirose3, Koji Uwai1,
1Division of Applied Sciences, Muroran Institute of Technology, Muroran, Japan
2Tokiwakai Group, Iwaki, Fukushima, Japan
3Amano Enzyme Inc. Gifu R&D Center, Kakamigahara-Shi, Gifu 509-0109, Japan
Corresponding author Koji Uwai, Division of Applied Sciences, Muroran Institute of
Technology, 27-1 Mizumoto-cho, Muroran 050-8585, Japan. E-mail:
uwai@mmm.muroran-it.ac.jp
Abstract
Synthesis of bicyclic compounds was achieved via a lipase-catalysed, stereoselective,
domino Michael-aldol reaction of 2-methyl-1,3-cycloalkanedione and methyl vinyl ketone.
Appropriate reaction conditions, including the type of enzyme, solvent, and temperature,
were determined. In addition, the effects of solvent polarity and addtives were investigated.
The reaction proceeded in the presence of lipase AS in a solution of 20% acetone in
dimethylsulfoxide (DMSO) at 10°C for 8 days, followed by the addition of
p-toluenesulfonic acid (TsOH) to afford bicyclic compounds in 51%–83% yields with
moderate stereoselectivity. Although this domino Michael-aldol reaction showed only
1

moderate stereoselectivity, even with the acid-supported enhancement of the reaction,
these results represent potential new applications for lipase.
KEYWORDS: Biocatalyst, Lipase, Domino reaction, Michael-aldol reaction, Bicyclic
compound
1. INTRODUCTION
-Substitution reactions, including Michael and aldol reactions, are very important in
synthetic organic chemistry, because they are convenient for the formation of
carbon–carbon bonds. In the past, important compounds have been synthesised using this
type of reaction with metal[1,2] and organo catalysts. [3,4] For example, the Michael reaction
was used for the synthesis of the anti-influenza agent oseltamivir,[5] whereas an aldol
reaction was used for the synthesis of the anti-cancer agent rhizoxin D.[6] However, most of
these catalysts possess various disadvantages, such as expensive syntheses, commercially
2

unavailability, harsh reaction conditions and complicated design. In contrast, biocatalysts
have many advantages, including low cost, commercial availability in large quantities,
environmental friendliness, mild reaction conditions, and high stereoselectivity even when
they are used as received.
Lipases are biocatalysts known to catalyse the hydrolysis of triglycerides in vivo and are
used as catalysts for kinetic resolution in synthetic organic chemistry by utilising the
difference in reactivity of both enantiomers in hydrolysis.[7] In addition, lipases have been
found to be promiscuous during the catalysis of Michael[8,9] and aldol reactions. [10,11] We
also reported the Michael reaction of 4-hydroxycoumarin and benzylideneacetone, which
was catalysed by lipase AS in dimethylsulfoxide (DMSO) at 20°C for 8 days. This afforded
the well-known anti-coagulant agent warfarin in 85% and 45% enantiomeric excess (ee).
[13] This is the best stereoselectivity reported for the synthesis of warfarin catalysed by
lipases. [14]
Domino reactions are very effective in forming more than one bond in a single step via
several consecutive reactions. For example, the bicyclic compounds Hajos–Parrish ketone
(1a), Wieland–Miescher ketone (1b) and compound 1c were synthesised via a domino
3

Michael–aldol reaction of the corresponding diketones (2a-c) and methyl vinyl ketone (3)
(Scheme 1). These bicyclic compounds are useful synthetic intermediates for many
bioactive compounds such as the anti-angiogenic agent cortistatin A,[15] anti-cancer agent
paclitaxel,[16] and ant methicillin resistant staphylococcus aureus (MRSA) and anti
vancomycin-resistant enterococcus faecium (VREF) antibiotic guanacastepene A.[17]
Bicyclic compounds 1a–c were previously synthesised using organo catalysts such as
proline derivatives,[18,19] but their catalytic efficiencies and complicated synthetic methods
remain an issue. In contrast, only a few reports on promiscuous, single enzyme-catalysed,
multi-step or domino reactions have been described.[20-22] While a lipase-catalysed domino
reaction of Wieland–Miescher ketone was recently reported, no stereoselectivity was
observed. [23]
We proposed that the Michael reaction used in the synthesis of warfarin [13] could be
applied to the domino reaction, affording stereoselective products from the novel
lipase-catalyzed reaction that require less catalyst. Thus, we developed a lipase-catalysed,
stereoselective, domino Michael-aldol reaction for synthesis of bicyclic compounds (1a–c)
from 1,3-diketones (2a–c) and methyl vinyl ketone (3).
4

2. RESULTS AND DISCUSSION
To determine the appropriate reaction conditions, we investigated the synthesis of
Wieland–Miescher ketone (1b) via a lipase-catalyzed domino Michael-aldol reaction of
2-methyl-1,3-cyclohexanedione (2b) and methyl vinyl ketone (3). The initial reaction
conditions were 2b (0.14 mmol), 3 (0.42 mmol), lipase (28.4 mg) and anhydrous DMSO
(0.7 mL), in accordance with our previous work. [13]
First, a series of commercially available lipases were screened to determine a suitable
catalyst for the reaction (Table 1). Focusing simply on the compound 1b, Lipase AYS, PS,
AK, PL, Novozyme 435, Lipozyme RM IM, Lipazyme CAL-B, Lipozyme TL 100L,
Palatase 20000L, Stick Away, and Lipex 100L afforded low conversions with no
stereoselectivity (entries 2–5 and 11–17). In contrast, lipase AS, QLM, OF, SL, TL, MY-30,
PPL, F-AP-15 and immobilised lipase showed slight stereoselectivity (entries 1, 6–10 and
18–20). Lipase AS was determined to be the best catalyst for this domino Michael-aldol
reaction (entry 1, 28% yield, 9% ee). In the presence of BSA and the absence of lipase, the
product was not observed (entries 21 and 22). Thus, the domino Michael-aldol reaction
seemed to be catalysed by lipase. The stereochemistry of 1b obtained under these reaction
conditions was entirely S-form. Unfortunately, in these reactions, the intermediate Michael
5

adduct (4b) in addition to desired 1b were obtained. The results suggested that the
equilibrium of the second step in scheme 1 (between 4b and 5b) of the aldol reaction was in
favour of the Michael adduct (4b) as we discuss in detail below (around Table 4 and 5).
In general, the solvent for the enzyme-catalysed reaction affected not only chemical yield
but also stereoselectivity;[12] therefore, the choice of optimal reaction solvent is very
important. In the past, some conventional organic solvents were surveyed. The results
indicated that varying the solvent had significant effects on the activity and
stereoselectivity of the lipase AS-catalysed domino Michael-aldol reaction (Table 2).
While no product was observed with acetone (entry 10), cyclohexane, n-hexane, toluene,
1,4-dioxane, teterahydrofuran (THF), EtOAc, CHCl3, EtOH, H2O and methyl vinyl ketone
showed moderate chemical yields but no stereoselectivity (entries 1–3, 5–7, 9, 14, 16 and
17). In contrast, Et2O, CH2Cl2, dimethylformamide (DMF), DMSO, 2-PrOH and MeOH
demonstrated slight stereoselectivity (entries 4, 8, 11–13 and 15). The best result was
obtained in DMSO with product yield of 34% and 9% ee (entry 12).
In previous reports, the chemical yield and stereoselectivity of lipase-catalysed,
asymmetric aldol reactions were improved when a mixed solvent was used.[24] Therefore,
6

we investigated the use of a DMSO-based mixed solvent system. As a result (Table 3), 1 :
1 ratio of solvent/DMSO did not improve the results obtained with pure DMSO (entries
1–5). However, the reaction in a mixed solvent system of 20% acetone in DMSO afforded
the best result with a 44% yield and 11% ee (entry 7). This indicated that an aprotic polar
solvent was preferred for the lipase-catalyzed domino Michael-aldol reaction with the
polarity of the solvent being important factor.
The obtained results (Table 3, entry 7) suggested that the equilibrium of the second step
(between 4b and 5b) of the aldol reaction was in favour of the Michael adduct (4b). Thus,
to shift the equilibrium to the terminal product (1b), dehydration of the aldol adduct (5b)
was promoted through the addition of an acid to the reaction system. Various acids (0.5
mol%) were added after reacting for 3 days. When HCl, camphorsulfonic acid (CSA),
trifluoroacetic acid (TFA) and pyridinium p-toluenesulfonate (PPTS) were added,
chemical yield and stereoselectivity were nearly equal to those without additives. However,
the addition of p-toluenesulfonic acid (TsOH) improved the chemical yield and
stereoselectivity to 64% and 12% ee, respectively (Table 4, entry 2). Any products were
observed for the reaction in the presence of with TsOH and the absence of lipase (entry 4).
These results indicated that the addition of acid enhanced the dehydration reaction.
7

Following this, we investigated the amount of added TsOH (1.0–10.0 mol%) and reaction
time (0.5–12 h). The results indicated that the optimal amount of TsOH was 5.0 mol%
(entry 2). Furthermore, the chemical yield was increased upon extending the reaction time
to 2 h, resulting in 64% chemical yield and 12% ee (entry 6). From these results, we
established a lipase catalysed domino Michael-aldol reaction with good chemical yield.
The optimal conditions for the formation of 1b involved a mixed solvent system of 20%
acetone in DMSO in the presence of TsOH (64% yield, 12% ee, Table 5, entry 5). However,
as shown in Table 2, entry 10, no product was obtained when pure acetone was used as the
solvent. Thus, we proposed that the polarity of the solvent is important for the reactivity of
the lipase; therefore, the relationship between polarity values [ET(30)] and reactivity was
investigated (Figure 1). The examined solvent systems were acetone/DMSO and
DMF/DMSO. The more the ratio of DMSO increased (ratio of acetone (or DMSO)
decreased), the more ET (30) values increased. The reactivity in both system were had a
maximal value around ET(30) = 44.5 at which the components of the solvent system were
20% acetone in DMSO (44%yield, 11% ee) and 30% DMF in DMSO (40% yield, 10% ee).
This suggested that the solvent polarity was appropriate at least for this type of reaction.
8

Next, we applied this method to the synthesis of other bicyclic compounds (1a and 1c).
Diketone 2c was synthesized according to the procedure reported by Piekut et al.[25] As a
result (Table 6), bicyclic compounds (1a–c) were obtained from all diketones with
chemical yields being increased upon the expansion of the ring size, resulting in poor
stereoselectivities of 3% ee (1a), 4% ee (1c) and 12% ee (1b) (entries 1-3).
In general, chemical yield and stereoselectivity were affected by reaction temperature.
When the reaction temperature sets lower, the reaction rate becomes lower but the
stereoselectivity getting higher in the enzymatic reaction.[26] Therefore, the reaction
temperature was investigated to improve the stereoselectivity of the domino Michael-aldol
reaction (Figure 2a). The best chemical yields [66% (1a), 82% (1b) and 83% (1c)] with
low stereoselectivity were obtained at 50°C, while the best stereoselectivities [5% ee (1a),
18% ee (1b) and 8% ee (1c)] were obtained at 10°C. The chemical yield increased upon
increasing reaction temperature, while stereoselectivity increased with decreasing reaction
temperature. Next, the reaction time was investigated to improve the chemical yield at
10°C. As a result (Figure 2b), chemical yields were increased upon increasing reaction
time, while maintaining the stereoselectivity. The best results were 51% yield 5% ee (1a),
9

79% yield 18% ee (1b) and 83% yield 8% ee (1c) at 10°C for 8 days. No improvement in
chemical yield was observed after this time.
In general, lipase has an active site for histidine, aspartic acid and serine for hydrolysis.
Although the amino acid sequence of lipase AS has no yet been determined, we assumed
that these histidine, aspartic acid and oxyanion holes contributed to the mechanism of this
lipase-catalyzed domino Michael-aldol reaction similar to hydrolysis, leading to the
proposed aldol reaction[12] (Scheme 2). The negatively charged carboxylate anion of
aspartic acid was able to abstract the imidazole proton of histidine. The imidazole anion of
histidine acted as a base to abstract the carbonyl -proton of diketone 2b. Meanwhile, the
carbonyl oxygen of 3 can become trapped in the oxyanion hole activating the
, -unsaturated ketone. Diketone 2b acted as a Michael donor for 3, affording 4b. The
carbonyl oxygen of 4b may then be trapped by the oxyanion hole, abstracting its carbonyl
-proton as before. Negatively charged 4b underwent intramolecular nucleophilic attack
at the trapped carbonyl carbon in the oxyanion hole. The hydroxy group of the aldol adduct
(5b) was then activated for protonation by TsOH, affording 1b via dehydration.
Stereoselectivity may be affected by the conformation of the amino acid side chains of
lipase; however, the detailed conformation of lipase AS is still unclear.
10

3. CONCLUSION
In summary, we developed a stereoselective domino Michael-aldol reaction catalyzed by
lipase. The syntheses of bicyclic compounds (1a–c) using this lipase-catalyzed domino
Michael-aldol reaction were accomplished using lipase AS in a mixed solvent system of
20% acetone in DMSO at 10°C for 8 days, followed by the addition of TsOH at 10°C,
resulting in 51%–83% chemical yields and 5%–18% ee. However, for the practical use,
this reaction temperature and reaction time are might be slightly unuseful and the reaction
at 30°C may be more applicable even if they have some very low selectivity.
In the past, lipase-catalyzed domino Michael-aldol reactions were found to have no
stereoselectivity. Although this domino Michael-aldol reaction showed only moderate
stereoselectivity, even with the acid-supported enhancement of the reaction, these results
represent potential new applications for lipase. Further studies are now underway in our
laboratory, including the investigation of superior reaction conditions using mutant lipases,
as well as studies exploring the reaction mechanism.
4. EXPERIMENTAL
Typical Reaction Procedure
11

A mixture of 2-methyl-1,3-cycloalkanedione (2a–c) (0.14 mmol) and lipase AS (28.4 mg,
0.81 mol, 0.6 mol%) in a solution of 20% acetone in DMSO (0.7 mL) was incubated
under an Ar atmosphere at 10°C for 5 min. Methyl vinyl ketone (3) (35 L, 0.42 mmol, 3.0
equiv.) was then added to the reaction mixture and incubated under Ar at 10oC for 8 days, at
which point TsOH was added and the reaction proceeded at 10°C for 2 h. The progress of
the reaction was monitored by TLC. The enzyme was filtered and partitioned between
EtOAc and H2O. The organic layer was dried over anhydrous MgSO4 and concentrated in
vacuo. Purification of the residual mixture by column chromatography on aluminum oxide
using hexane: EtOAc (3:1 then 1:1, v/v) as the eluent afforded (S)-bicyclic compounds
(1a–c).
ACKNOWLEGEMENT
Financial support from an MuIT Grant for Selected Research is gratefully acknowledged.
We thank Meito Sangyo Co, Novozymes A/S and Amano Enzyme Inc for their generous
donation of lipase.
SUPPLEMENTAL MATERIAL
12

Materials, Experimental Section Characterisation Data for Compounds for this article can
be accessed on the publisher’s website.
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15

Table 1. Domino Michael-aldol reaction of 2-methyl-1,3-cyclohexanedione (2b) and
methyl vinyl ketone (3) catalysed by various lipasesa)
Entry
Enzyme
4b yieldb) (%)
1b
yieldb) (%)
eec) (%)
1
AS
7
34
12
13
7
9
-
2
AYS
11
11
5
3
PS
-
4
AK
-
5
PL
5
5
-
6
QLM
8
9
4
2
4
4
2
-
7
OF
15
8
7
8
SL
13
5
9
TL
13
8
10
11
12
MY-30
4
Novozyme 435
Lipozyme RM
IM
1
23
10
n.d.d)
-
16

13
14
Lipozyme
CAL-B
4
18
7
-
-
Lipozyme TL
100L
11
15
16
17
18
19
20
Palatase 20000L
Stick Away
Lipex 100L
PPL
3
5
-
15
4
13
9
-
-
12
13
4
20
5
3
5
3
F-AP-15
Immobilised
lipase
2
21
22
BSA
n.d.d)
n.d.d)
n.d.d)
n.d.d)
-
-
Blank
a) Experimental conditions: 2-methyl-1,3-cyclohexanedione (0.14 mmol), methyl vinyl
ketone (0.42 mmol) and lipase (28.4 mg) in anhydrous DMSO (0.7 mL) were incubated at
30°C for 3 days in an Ar atmosphere. b) Isolated yield. c) Determined by chiral HPLC
analysis. d) Not detected.
17

Table 2. Domino Michael-aldol reaction of 2-methyl-1,3-cyclohexanedione (2b) and
methyl vinyl ketone (3) in various solventsa)
Entry
Solvent
4b yieldb)
1b
yieldb) (%)
eec) (%)
(%)
2
1
Cyclohexane
n-Hexane
Toluene
Et2O
3
-
2
3
4
-
3
12
3
5
-
4
10
3
2
-
5
1,4-Dioxane
THF
3
6
5
11
3
-
7
EtOAc
4
-
8
CH2Cl2
CHCl3
10
7
17
18
n.d.d)
23
34
13
3
-
9
10
11
12
13
Acetone
DMF
n.d.d)
9
-
5
9
2
DMSO
2-PrOH
7
5
18

14
EtOH
14
8
4
-
15
MeOH
7
4
-
16
H2O
15
24
13
27
17e)
Methyl vinyl ketone
-
a) Experimental conditions: 2-methyl-1,3-cyclohexanedione (0.14 mmol), methyl vinyl
ketone (0.42 mmol) and lipase AS (28.4 mg) in solvent (0.7 mL) were incubated at 30°C
for 3 days in an Ar atmosphere. b) Isolated yield. c) Determined by chiral HPLC analysis.
d) Not detected. e) No solvent.
19

Table 3. Domino Michael-aldol reaction of 2-methyl-1,3-cyclohexanedione (2b) and
methyl vinyl ketone (3) in various DMSO-based various mixed solvent systemsa)
Entry
Solvent/DMSO
4b yieldb)
1b
yieldb) (%)
eec) (%)
(%)
5
1
2
3
4
5
6
7
8
50% 2-PrOH
50% MVK
21
29
21
20
5
5
3
17
10
10
4
50% CH2Cl2
50% DMF
5
7
50% Acetone
30% Acetone
20% Acetone
10% Acetone
3
13
15
8
25
44
35
4
11
9
a) Experimental conditions: 2-methyl-1,3-cyclohexanedione (0.14 mmol), methyl vinyl
ketone (0.42 mmol) and lipase AS (28.4 mg) in a mixed solvent DMSO system (0.7 mL)
were incubated at 30°C for 3 days in an Ar atmosphere. b) Isolated yield. c) Determined by
chiral HPLC analysis.
20

Table 4. Effect of the amount of TsOH (1–10 mol%) and reaction time (0.5–12 h) on the
reaction of 2-methyl-1,3-cyclohexanedione (2b) and methyl vinyl ketone (3)a)
Entry
TsOH (mol%) Time (h)
4b yieldb)
1b
yieldb)
(%)
55
eec) (%)
(%)
1
2
3
4
5
6
7
8
1.0
1.0
1.0
1.0
2
12
12
11
-
5.0
3
64
10.0
2
58
0 (no lipase)
1.0
n.d.d)
n.d.d)
52
5.0
5.0
5.0
5.0
0.5
2.0
6.0
12.0
9
3
2
2
11
12
11
13
64
62
63
a) Experimental conditions: 2-methyl-1,3-cyclohexanedione (0.14 mmol), methyl vinyl
ketone (0.42 mmol) and lipase AS (28.4 mg) in a solution of 20% acetone in DMSO (0.7
mL) were incubated at 30°C for 3 days in an Ar atmosphere, followed by the addition of
TsOH (1.0–10.0 mol%) and incubation for additional 0.5–12.0 h. b) Isolated yield. c)
Determined by chiral HPLC analysis.
21

Table 5. Syntheses of bicyclic compounds (1a–c) via the domino Michael-aldol reaction of
diketones (2a–c) and methyl vinyl ketone (3)a)
Entry
n
4a-c yieldb)
1a-c
yieldb) (%)
eec) (%)
(%)
5
1
2
3
1
2
3
45
64
67
3
3
12
4
4
a) Experimental conditions: 2-methyl-1,3-cyclohexanedione (0.14 mmol), methyl vinyl
ketone (0.42 mmol) and lipase AS (28.4 mg) in a solution of 20% acetone in DMSO (0.7
mL) were incubated at 30°C for 3 days in an Ar atmosphere, followed by the addition of
TsOH (0.5 mol%) and incubation for additional 2 h. b) Isolated yield. c) Determined by
chiral HPLC analysis.
22

Figure 1. Effect of the polarity value [ET(30)] of the mixed solvent system on the domino
Michael-aldol reaction (acetone/DMSO yield: ●, ee: ■, DMF/DMSO yield: ○, ee: □).
Reaction conditions: 2-methyl-1,3-cyclohexanedione (2b, 0.14 mmol), methyl vinyl
ketone (3, 0.42 mmol), and lipase AS (28.4 mg) in a mixed solvent (0.7 mL) at 30°C for 3
days, followed by the addition of TsOH (0.5 mol%) and incubation at 30oC for 2 h.
23

Figure 2. Effect of (a) reaction temperature and (b) reaction time on the domino
Michael-aldol reaction (yield: 1a ●, 1b ■, 1c ▲, ee: 1a ○, 1b □, 1c △). Reaction
conditions: 2-methyl-1,3-cycloalkanedione (2a-c, 0.14 mmol), methyl vinyl ketone (3,
0.42 mmol), lipase AS (28.4 mg) in a solution of 20% acetone in DMSO (0.7 mL).
24

Scheme 1. Syntheses of various bicyclic compounds (a: n = 1, b: n = 2, c: n = 3)
25

Scheme 2. Proposed mechanism of lipase AS-catalysed domino Michael-aldol reaction of
2-methyl-1,3-cyclohexanedione (2b) with methyl vinyl ketone (3)
26

Scheme 3. Optimal conditions for the lipase catalysed domino Michael-aldol reaction
27