One-pot synthesis of Wieland-Miescher ketone by enzymes

Article abstract of DOI:10.1007/s11164-013-1512-6

We first report that lipase from porcine pancreas catalyzed Robinson annulation in the organic media to synthesize the Wieland-Miescher ketone. The promiscuous catalytic activity of lipase in the Robinson reaction is due to an important role played by lipase activity in both the Michael addition and Aldol reaction.

Full text of DOI:10.1007/s11164-013-1512-6

Res Chem Intermed
DOI 10.1007/s11164-013-1512-6
One-pot synthesis of Wieland–Miescher ketone
by enzymes
Yi-Feng Lai Peng-Fei Zhang
•
Received: 28 August 2013 / Accepted: 13 December 2013
Ó Springer Science+Business Media Dordrecht 2013
Abstract We first report that lipase from porcine pancreas catalyzed Robinson
annulation in the organic media to synthesize the Wieland–Miescher ketone. The
promiscuous catalytic activity of lipase in the Robinson reaction is due to an
important role played by lipase activity in both the Michael addition and Aldol
reaction.
Keywords Promiscuity Á Lipase Á Wieland–Miescher ketone Á
Robinson reaction
Introduction
The unprecedented work of Klibanov and his team in the early 1980s laid the
foundation for the application of enzymes for organic syntheses [1]. Here, we report
the use of lipase from porcine pancreas (PPL) to catalyze a Robinson annulation,
which forms Wieland–Miescher ketone in organic media. This has practical
significance for environmentally friendly syntheses and the evolution of biosyntheses.
Enzyme catalytic promiscuity has become a prominent research area in recent
years. Enzymes catalyzing accidental or induced new reactions are recognized as
Electronic supplementary material The online version of this article (doi:10.1007/s11164-013-1512-
6) contains supplementary material, which is available to authorized users.
Y.-F. Lai
Zhejiang University of Science and Technology, Hangzhou 310036, China
e-mail: gexincumt@163.com
P.-F. Zhang (&)
College of Material Chemistry and Chemical Engineering, Hangzhou Normal University,
Hangzhou 310036, China
e-mail: chxyzpf@hotmail.com
123

Y.-F. Lai, P.-F. Zhang
valuable research and synthesis tools [2]. Exploiting enzyme catalytic promiscuity
might lead to improvements in existing catalysts and provide novel synthesis
pathways. Some elegant work in this field has been done in recent decades. Hilvert
and co-workers reported that oxaloacetate went through decarboxylation and then
an aldol reaction with aldehydes in the presence of macrophomate synthase [3]. Lin
and his co-workers used alkaline protease from Bacillus subtilis to catalyze a
Michael-type addition of pyrimidine derivatives and imidazole to acrylates in the
organic solvent [4, 5]. In our previous work, we reported on lipase-catalyzed
Tandem Knoevenagel condensation and esterification with alcohol cosolvents [6]
and one-pot synthesis of spirooxindole derivatives catalyzed by lipase [7]. It has
been reported that lipases can catalyze a Michael addition and an aldol reaction
separately [8, 9]. These findings inspired us to seek enzymes that could catalyze
both a Michael addition and an aldol reaction in one-pot (Robinson annulations).
Experimental
An amount of 1 mmol dione was added to a solution containing 50 mg enzyme and
6
1
mmol methyl vinyl ketone, then incubated at 50 °C in an end-over-end rotator at
50 rpm. Samples were withdrawn from the reaction and directly analyzed using a
GC–MS (Agilent 5975C). Enantiomeric excesses (% ee) were determined by HPLC
Lab Alliance Model 500) analysis using Chiralcel OD-H. All the compounds were
spectroscopically characterized (1H, 13C NMR and MS).
(
Results and discussion
Wieland–Miescher (WM) ketone has proven to be a particularly useful synthesis for
the construction of a variety of biologically active compounds including steroids,
terpenoids and taxol [10–12]. In most instances, to synthesize WM ketone, both the
Michael addition and acyclodehydration step are required in sequence, first leading
to b then to d (shown in Scheme 1). Traditional chemical methods usually adopt a
metallic catalyst followed by a base to carry out the reaction. Due to the
disadvantages of pretreatments and the drawbacks caused by isolation and
purification, recent efforts have been devoted to performing the direct Robinson
reaction via organocatalysts [13, 14]. The goal of the research described here is to
explore the possibility of utilizing enzymes to achieve the entire Robinson
annulation sequence.
First, we used several commercially available hydrolytic enzymes to screen their
ability to catalyze the reaction. 2-Methylcyclohexane-1,3-dione (n = 2) and methyl
vinyl ketone were used as the model reactants. The reaction was monitored by TLC.
We catalyzed this reaction with the enzymes in DMSO at 37 °C. We eventually
found that the least expensive enzyme, PPL could catalyze the reaction to produce
WM ketone with a high yield (entry 1, Table 1). The other tested enzymes also
presented low activities for this reaction. As expected, the control experiment and
those using BSA showed poor activity. We then used denatured PPL to catalyze the
1
23

One-pot synthesis of Wieland–Miescher ketone by enzymes
O
O
O
O
O
O
+
n
n
n
n
O
O
O
O
OH
c
a
d
b
n=1,2
Lipase
Organic solvent
Scheme 1 Robinson annulations catalyzed by lipase from porcine pancreas
Table 1 The catalytic activities of Robinson annulation between 2-methylcyclohexane-1,3-dione with
methyl vinyl ketone
a
Entry
Catalyst
Yield (%)
1
2
3
4
5
6
7
8
9
Lipase from porcine pancreas (PPL)
Lipase A, from Aspergillus niger
Lipase M, from Mucor javanicus
Lipase AK, from Pseudomonas fluore
Lipase AY30, from Candida rugosa
Lipase recombinant, from Candida antarctica
Bovine serum albumin (BSA)
Denatured lipase from porcine pancreas
Trypsase
61
21
27
Trace
Trace
Trace
Trace
Trace
Trace
Trace
Trace
1
1
0
1
Pepsin
Control
Reaction conditions: 50 mg enzyme, 1 mmol 2-methylcyclohexane-1,3-dione, 6 mmol methyl vinyl
ketone, 5 ml DMSO, and 0.5 ml water, 30 °C, 40 h, 150 rpm
All the enzymes were purchased from J&K Chemical
a
Isolated yields
reaction, and the result was the same as the control. Encouraged by this result, we
decided to study PPL further.
In order to achieve a higher conversion rate, conditions for the Robinson
annulation were optimized using 2-methylcyclohexane-1,3-dione (a n = 1) and
methyl vinyl ketone as the model reactants. The reaction media is one of the most
important factors influencing the enzymatic reaction, so we conducted the reaction
in various organic solvents (Table 2). The results suggest that ethanol and methanol
are favorable for this reaction (entries 2 and 3, Table 2). It should be noted that,
although the reactants and its products have low solubility in n-hexane, the yield of
7
9 % was obtained (entry 7, Table 2). DMF and THF only gave the lower yields
entries 1, 5, Table 2). Other solvents including CHCl and water cannot promote
(
3
this reaction (entries 6, 8, Table 2). In addition to the solvent effect, other
influencing factors, such as temperature, concentration of catalysts, water content,
123

Y.-F. Lai, P.-F. Zhang
Table 2 PPL-catalyzed Robinson annulation between 2-methylcyclohexane-1,3-dione with methyl vinyl
ketone in different solvents
a
Entry
Solvent
Yield (%)
1
2
3
4
5
6
7
8
DMF
31
Methanol
Ethanol
DMSO
THF
95
82
61
16
CHCl
3
Trace
79
n-Hexane
Water
Trace
Reaction conditions: 50 mg PPL, 1 mmol 2-methylcyclohexane-1,3-dione, 6 mmol methyl vinyl ketone,
5
ml sovlent, and 0.5 ml water, 30 °C for 40 h, 150 rpm
Isolated yields
a
Oxyanion
hole
His264
O
His264
N
N
Asp177
O
H
H
N
N
O
Asp177
O
O
O
H
O
H
O
O
H
O
O
O
N
O
His264
Asp177
Oxyanion
hole
N
H
Oxyanion
hole
O
Oxyanion
hole
O
His264
O
O
O
O
N
N
O
Asp177
H
H
O
O
O
b
Oxyanion
hole
O
O
O
O
O
O
OH
H
O
OH
His264
N
c
H
His264
N
N
O
Asp177
N
H
H
O
O
O
O
Asp177
-
H O
2
O
d
Scheme 2 The proposed mechanism for the PPL-catalyzed Robinson annulation
1
23

One-pot synthesis of Wieland–Miescher ketone by enzymes
and reaction time, were also optimized. When PPL-catalyzed Robinson annulations
were carried out for 40 h at 30 °C in methanol solvent and 10 % water content, we
produced the desired product yield with 95 %. Although WM ketone was applied to
measure the enantioselectivity, there is no obvious optical activity, and we will
undertake related further research to improve it.
A generally-accepted catalytic mechanism for lipases is that the active site for
hydrolysis also contributes to promiscuous catalysis [15]. PPL is composed of a
single polypeptide chain of 450 amino acid residues [16–19]. Based on the reported
architecture and catalytic mechanism of PPL, we hypothesized the mechanism of
the PPL-catalyzed Robinson annulation (Scheme 2). The proposed mechanism
would start with Michael reaction in the active site (catalytic triad). a (n = 2) was
deprotonated by His264–Asp177 pair to form an enolate nucleophile. Under the
interaction of the carbonyl with the oxyanion hole, the electrophilic ability of
methyl vinyl ketone was increased. The conjugated addition of the enolate
nucleophile led to an intermediate. Then, the proton was transferred to the a carbon
by the His264–Asp177 pair catalyzed, which formed the compound b (n = 2).
b (n = 2) was deprotonated by His264–Asp177 pair to form an enolate anion,
which was established under the influence of the oxyanion hole. The addition of
carbonyl with enolate anion formed a C–C bond to give the aldol adduct c (n = 2)
by the proton transference and the oxyanion hole released. The aldol adduct
c (n = 2) underwent dehydration to afford the desired product d (n = 2).
Conclusion
In conclusion, we have described that several enzymes, especially PPL, can catalyze
a Robinson reaction and produce WM ketone with high yields. As the active site of
lipase for hydrolysis also contributes to promiscuous catalysis, we proposed the
mechanism of the PPL-catalyzed Robinson annulation. Moreover, because PPL was
easily acquired and normally not soluble in organic media, our results are of
practical significance in many applications. The enzyme-catalyzed Robinson
reaction provides a novel case of catalytic promiscuity and a potential synthetic
method for organic chemistry.
Acknowledgment This work was supported by the Natural Science Foundation of Zhejiang Province
(No. R406378).
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