Lipase-catalysed direct Mannich reaction in water: Utilization of biocatalytic promiscuity for C-C bond formation in a "one-pot" synthesis

Article abstract of DOI:10.1039/b817524a

A novel lipase-catalysed direct Mannich reaction has been developed under aqueous conditions in a "one-pot" strategy. Interestingly, these promiscuous reactions can be greatly promoted by water and generally require aromatic aldehydes.

Full text of DOI:10.1039/b817524a

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Lipase-catalysed direct Mannich reaction in water: utilization of
biocatalytic promiscuity for C–C bond formation in a “one-pot” synthesis†
Kun Li, Ting He, Chao Li, Xing-Wen Feng, Na Wang* and Xiao-Qi Yu*
Received 6th October 2008, Accepted 17th March 2009
First published as an Advance Article on the web 26th March 2009
DOI: 10.1039/b817524a
A novel lipase-catalysed direct Mannich reaction has
been developed under aqueous conditions in a “one-pot”
strategy. Interestingly, these promiscuous reactions can be
greatly promoted by water and generally require aromatic
aldehydes.
conditions, has never been reported. Herein, we described the
first lipase-catalysed direct, three-component Mannich reaction
with ketone, aldehyde and amine (under aqueous conditions)
to form b-amino-ketone compounds (Scheme 1). A series of
substrates have been explored, resulting in moderate to good
yields.
Enzymes have been widely used in modern organic synthesis
due to their simple processing requirements, high selectivity
and mild reaction conditions.1 In recent years, a new frontier,
known as biocatalytic promiscuity, has emerged and largely
extended the application of enzymes. Namely, it is the ability
of a enzyme to catalyse synthetic reactions which may vary from
its natural role.2 As one of the most rapidly growing areas in
enzymology, biocatalytic promiscuity not only highlights the
existing catalysts, but may provide novel and practical synthetic
pathways which are not currently available. Some elegant reports
address the importance and wide application of biocatalytic
promiscuity in organic synthesis.3 For example, lipase from
Candida antarctica (CAL-B), which possesses lipid hydrolysis as
its native activity, can also promiscuously catalyse various C–C,
C–N and C–S bond formations.4 Similarly, lipase from porcine
pancreas (PPL) was shown to catalyze an aldol reaction in our
previous study.5 Although a variety of two-component reactions
have been developed in the study of biocatalytic promiscuity,
three-component reactions, which can form multi-functional
compounds, have never been reported. This is possibly due to
the problem of side reactions caused by multi-reagents and the
strict range of substrates in enzymatic reactions.
Scheme 1 Lipase-catalysed direct Mannich reaction in water.
Water is the most abundant, inexpensive, and environmentally
friendly solvent, thus the development of organic reactions
in aqueous media is one of the practical trends in current
chemistry.9 Although some methods had realised the indirect
Mannich reaction in the presence of water, few examples of
direct three-component Mannich reactions in water have been
reported.10 Initially, we chose an acetone/water 1 : 1 (v/v)
system as the reaction medium, based on our previous research.
The substrates acetone, aniline and 4-nitrobenzadehyde were
catalysed by lipase in a one-pot synthesis to yield the Mannich
product. A wide variety of lipases had been investigated in
order to study the potential range of this enzyme’s promiscuous
catalytic activity (Table 1). To our surprise, some lipases did,
in fact, exhibit detectable catalytic activity in the experiments.
In our efforts to explore the application of this new area,
we became interested in the Mannich reaction, which is atom-
economic and a powerful synthetic method for generating
carbon–carbon bonds and nitrogenous compounds.6 Tradi-
tional chemical methods (referred to as the indirect Mannich
reaction), however, usually adopted imines or iminium salts and
preformed enolate equivalents to carry out the reaction. Due
to the disadvantages of the pretreatments and the drawbacks
caused by the isolation and purification, recent efforts have
been devoted to performing the direct Mannich reaction via
the metal catalyst systems and organocatalysts.7,8 Yet, to the
best of our knowledge, a hydrolase-catalysed Mannich reaction,
which could be performed under facile and eco-friendly reaction
Table 1 The catalytic activities of Mannich reaction by different
lipasesa
Entry
Catalyst
Yield (%)b
1
2
3
4
5
6
7
8
9
10
11
12
13
No Enzyme
3.2
3.7
8.2
16.1
3.5
14.9
4.5
16.7
12.0
10.1
48.3
72.2
8.2
Lipase from Penicillium camemberti
Lipase from Candida rugosa
Lipase from Pseudomonas fluorescens
Lipase from Penicillium roqueforti
Lipase from porcine pancreas, type II
Lipase from Mucor javanicus
Lipase from Burkholderia cepacia
Lipase from Rhizopus oryzae
Lipase from Candida cylindracea
Lipase from Candida antarctica
Lipase from Mucor miehei (MML)
Denatured MMLc
Key Laboratory of Green Chemistry and Technology, Ministry of
Education, College of Chemistry, Sichuan University, Chengdu, 610064,
P. R. China. E-mail: xqyu@tfol.com; Fax: +86 28 85415886; Tel: +86
28 85460576
† Electronic supplementary information (ESI) available: Further exper-
imental information. See DOI: 10.1039/b817524a
a Reaction conditions: a solution of 4-nitrobenzaldehyde (2, 0.1 mmol),
aniline (1, 0.11 mmol), acetone (3, 1 ml), water (1 ml) and 10 mg lipase
◦
was shaken at 200 rpm at 30 C for 24 h. b Yield was calculated based
on 2. c Pre-treated with urea at 60 ◦C for 12 h.
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Table 2 Direct Mannich reaction of other aromatic aldehydes and
Lipase from Mucor miehei (MML) was identified to be the
most effective catalyst for the direct Mannich reaction (Table 1,
Entry 12). CAL-B also showed moderate catalytic activity, while
other tested lipases presented very low activity in this reaction.
Additional experiments were also performed to confirm the
notable catalytic activities of MML in the Mannich reaction.
As expected, the control experiments and those using denatured
MML showed poor activity. All the results suggested that the
promiscuous catalytic activities of enzymes in the direct Man-
nich reaction largely depend on the special spatial conformation
of the natural lipases.
Solvents play an important role in the reaction process.
Therefore, we investigated the effect of different solvents on
the Mannich reaction (Fig. 1). In pure organic solvents such
as toluene, dichloromethane, DMF, THF, acetone, the reaction
most commonly observed was the formation of a Schiff base
(>90% yield), as opposed to the Mannich reaction. Interestingly,
substantial yields can be obtained in the mixture of water and
organic solvents in the same conditions, indicating that water can
obviously accelerate the MML-catalysed Mannich reaction.
aminesa
Entry
Time/h
R
Yield (%)b
1
2
3
4
5
6
7
48
48
24
24
48
48
48
4-NO2
3-NO2
H
4-OCH3
4-OH
4-CN
4-Cl
83.5
82.4
87.3
89.1
43.6
65.3
83.1
a Reaction conditions: a solution of aldehyde (2, 1 mmol), aniline (1,
1.1 mmol), acetone (3, 8 ml), 8 ml water and 50 mg MML was shaken
at 200 rpm at 30 ◦C for 24 or 48 h. b Isolated yield.
Fig.
2
The influence of water concentration on the MML-
catalysed Mannich reaction under these conditions: MML 10 mg, 4-
nitrobenzaldehyde (2) 0.1 mmol, aniline (1, 0.11 mmol) and 2 ml solvent
were shaken at 200 rpm at 30 ◦C, deionized water from 0% to 80%
[water/(water + acetone), v/v].
Based on the remarkable results obtained using the above
reaction conditions, some other aldehydes were used to expand
upon this MML-catalysed direct Mannich reaction to show
the generality and scope of this new biocatalytic promiscuity
(Table 2). It was found that a wide range of aromatic aldehydes
can effectively participate in the MML-catalysed Mannich
reaction. In addition, several aliphatic aldehydes (such as
acetaldehyde and propaldehyde) were also investigated, but no
products were detected using these reactants.
It is widely accepted that the hydrolysis active site in hydrolase
is also responsible for the promiscuous catalysis.11 Combining
that viewpoint with our observations, as described above,
we theorized the mechanism of the lipase-catalysed Mannich
reaction, summarized in Scheme 2. First, the aldehyde and
an amine can react to form the Schiff base quickly, and the
ketone is pre-activated by the lipase to form the enolate anion
at the same time. Second, the Schiff base can form complex
5, with the assistance of the His residue possessed by lipase.
Third, a proton is transferred from the Schiff base to the
enolate anion to form a new carbon–carbon bond, and then
the Mannich adduct is released from the oxyanion hole. This
proposed catalytic mechanism is based on the results reported
by the Berglund et al.4 Formation of the enolate anion and
complex 5 are expected to be the key steps in this enzymatic
Fig. 1 The influence of solvents on the Mannich reaction. (Reaction
conditions: a solution of 4-nitrobenzaldehyde (2, 0.1 mmol), aniline (1,
0.11 mmol), acetone (3, 1 ml), 2 ml solvent (the ratio of the mixture
solvent is 1 : 1 in volume) and 10 mg MML was shaken at 200 rpm at
30 ◦C for 48 h.)
Notably, it is important to confirm the optimal percentage
of water in order to enhance the biocatalytic promiscuity of
the direct Mannich reaction. Therefore, experiments had been
performed to ascertain the catalytic activity of MML with
different amounts of water in the reaction system. The results are
shown in Fig. 2. In general, the effect of the water concentration
on the catalytic activity could be described using a hill-shaped
curve. It is hard to observe any Mannich product if no water
is present in the reaction system. The rate of the enzymatic
reaction can be accelerated by increasing the concentration
of water used, and reaches the highest rate between 40–50%
water content. However, once the water content surpasses
60%, the yield of Mannich product drops down, due to the
insolubility of the substrates. All the results indicated that
water is obviously essential in the promiscuous biocatalysis of
the Mannich reaction. Further experiments were performed to
investigate the yield of the Mannich adduct during the time
course (Fig. S1†).
778 | Green Chem., 2009, 11, 777–779
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of catalytic promiscuity and might be a useful synthetic method
for bioorganic synthesis.
We gratefully acknowledge the National Natural Science
Foundation of China (no. 20725206, 20732004 and 20702034)
for financial support. We also thank the Sichuan University
Analytical & Testing Center for NMR analysis.
Notes and references
1 J. M. Woodley, Trends Biotechnol., 2008, 26, 321; F. Kopp and M. A.
Marahiel, Curr. Opin. Biotechnol., 2007, 18, 513; K. Hult and P.
Berglund, Curr. Opin. Biotechnol., 2003, 14, 395.
2 O. P. Ward and A. Singh, Curr. Opin. Biotechnol., 2000, 11, 520.
3 R. J. Kazlauskas, Curr. Opin. Chem. Biol., 2005, 9, 195; P. Berglund
and S. Park, Curr. Org. Chem., 2005, 9, 325; D. M. Z. Schmidt, E. C.
Mundorff, M. Dojka, E. Bermudez, J. E. Ness, S. Govindarajan, P. C.
Babbitt, J. Minshull and J. A. Gerlt, Biochemistry (Mosc.), 2003, 42,
8387; O. Khersonsky, C. Roodveldt and D. S. Tawfik, Curr. Opin.
Chem. Biol., 2006, 10, 498; G. H. Dijoux, M. M. Elenkov, J. H. L.
Spelberg, B. Hauer and D. B. Janssen, ChemBioChem, 2008, 9, 1048;
R. Kourist, S. Bartch, L. Fransson, K. Hult and U. T. Bornscheuer,
ChemBioChem, 2008, 9, 67; C. Li, M. Hassler and T. D. H. Bugg,
ChemBioChem, 2008, 9, 71; W. B. Wu, J. M. Xu, Q. Wu, D. S. Lv and
X. F. Lin, Adv. Synth. Catal., 2006, 348, 487; J. M. Xu, F. Zhang,
B. K. Liu, Q. Wu and X. F. Lin, Chem. Commun., 2007, 2078; W. B.
Wu, N. Wang, J. M. Xu, Q. Wu and X. F. Lin, Chem. Commun., 2005,
2348.
4 M. Svedendahl, K. Hult and P. Berglund, J. Am. Chem. Soc., 2005,
127, 17988; O. Torre, I. Alfonso and V. Gotor, Chem. Commun., 2004,
1724; C. Branneby, P. Carlqvist, A. Magnusson, K. Hult, T. Brinck
and P. Berglund, J. Am. Chem. Soc., 2003, 125, 874; P. Carlqvist,
M. Svedendahl, C. Branneby, K. Hult, T. Brinck and P. Berglund,
ChemBioChem, 2005, 6, 331.
Scheme 2 Proposed mechanism of lipase-catalysed Mannich reaction.
process. In the pure organic solvents, the enolate was difficult to
form and the Mannich reaction could not happen. Once water
(an accelerating agent of the enolate formation) is added into the
reaction system, the reaction could proceed. However, further
experiments are necessary to prove this hypothesis.
On the other hand, although three-component, direct Man-
nich reactions can be catalysed by metal complexes and
organocatalyst systems, almost all the systems need to first
mix the aldehyde and amine to form the Schiff base and avoid
side reactions, such as the aldol reaction. Interestingly, in our
biocatalytic system, the order of the reagent addition did not
affect the yield of the product, and the Mannich reaction can be
easily carried out in one vessel. Moreover, the recovery of metal
complexes and organocatalysts is difficult, while lipase MML
can be reused at least six times without a significant loss of
activity (Fig. S2†). Although various conditions were tested to
improve the enantioselectivity, no clear progress has been made
at this time. Thus, other methods such as directed evolution,
site-directed mutagenesis, and enzymatic resolution are being
investigated as potential solutions to this problem.
5 C. Li, X. W. Feng, N. Wang, Y. J. Zhou and X. Q. Yu, Green Chem.,
2008, 10, 616.
6 S. Kobayashi and H. Ishitani, Chem. Rev., 1999, 99, 1069; M. Arend,
B. Westermann and N. Risch, Angew. Chem., Int. Ed., 1998, 37,
1044; M. M. B. Marques, Angew. Chem., Int. Ed., 2006, 45, 348; A.
Co´rdova, Acc. Chem. Res., 2004, 37, 102.
7 B. M. Trost, J. Jaratjaroonphong and V. Reutrakul, J. Am. Chem.
Soc., 2006, 128, 2778; S. Matsunaga, T. Yoshida, H. Morimoto, N.
Kumagai and M. Shibasaki, J. Am. Chem. Soc., 2004, 126, 8777.
8 S. S. V. Ramasastry, H. Zhang, F. Tanaka and C. F. Barbas III, J. Am.
Chem. Soc., 2007, 129, 288; J. W. Yang and M. Stadler, Angew. Chem.,
Int. Ed., 2007, 46, 609; B. List, T. Kano, Y. Yamaguchi, O. Tokuda
and K. Maruoka, J. Am. Chem. Soc., 2005, 127, 16408; Q. X. Guo,
H. Liu, C. Guo, S. W. Luo, Y. Gu and L. Z. Gong, J. Am. Chem.
Soc., 2007, 129, 3790.
9 Organic Reactions in Water, Ed. U. M. Lindstro¨m, Blackwell
Publishing, Oxford, 2007; U. M. Lindstr
o¨m, Chem. Rev., 2002, 102,
2751; D. G. Blackmond, A. Armstrong, V. Coombe and A. Wells,
Angew. Chem., Int. Ed., 2007, 46, 3798.
In conclusion, we describe here the first lipase-catalysed,
three-component direct Mannich reactions. Lipase MML ef-
ficiently catalysed the Mannich reaction, while lipase CAL-B
showed moderate catalytic activity. Notably, these promiscuous
reactions can be greatly promoted by water. The “one-pot”
lipase-catalysed, direct Mannich reaction provides a novel case
10 T. Akiyama, K. Matsuda and K. Fuchibe, Synlett, 2005, 322; K.
Manabe, Y. Mori and S. Kobayashi, Tetrahedron, 2001, 57, 2537;
K. Manabe and S. Kobayashi, Org. Lett., 1999, 1, 1965; S. Iimura,
D. Nobutou, K. Manabe and S. Kobayashi, Chem. Commun., 2003,
1644.
11 C. Branneby, P. Carlqvist, K. Hult, T. Brinck and P. Berglund, J. Mol.
Catal. B: Enzym., 2004, 31, 123.
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The Royal Society of Chemistry 2009
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