Utilization of biocatalytic promiscuity for direct Mannich reaction

Article abstract of DOI:10.1016/j.molcatb.2010.08.004

A lipase-catalyzed direct Mannich reaction of arylamines with aromatic aldehydes and ketones including cyclohexanone, butanone and 1-hydroxy-2- propanone was developed under EtOH/H₂O system in a "one-pot" strategy. A series of experiments on the promiscuous activity of lipases were performed to optimize the biocatalytic process, and a wide scope of substrates was expanded with good yields.

Full text of DOI:10.1016/j.molcatb.2010.08.004

Journal of Molecular Catalysis B: Enzymatic 67 (2010) 189–194
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Utilization of biocatalytic promiscuity for direct Mannich reaction
Ting He, Kun Li∗, Ming-Yu Wu, Xing-Wen Feng, Na Wang, Hai-Yang Wang, Chao Li, Xiao-Qi Yu∗
College of Chemistry, Key Laboratory of Green Chemistry and Technology (Ministry of Education), Sichuan University, Chengdu 610064, 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 lipase-catalyzed direct Mannich reaction of arylamines with aromatic aldehydes and ketones including
cyclohexanone, butanone and 1-hydroxy-2-propanone was developed under EtOH/H2O system in a “one-
pot” strategy. A series of experiments on the promiscuous activity of lipases were performed to optimize
the biocatalytic process, and a wide scope of substrates was expanded with good yields.
© 2010 Elsevier B.V. All rights reserved.
Received 10 March 2010
Received in revised form 9 August 2010
Accepted 9 August 2010
Available online 13 August 2010
Keywords:
Biocatalytic promiscuity
Lipase
Mannich reaction
Water
1. Introduction
and can be used for different kinds of transformations with low
cost. Therefore, to find new promiscuous reaction of lipase is more
Nowadays, the usefulness of various biocatalysts (such as
enzymes and whole sells) for organic synthesis has become more
and more recognized [1–4]. Among them, the enzymatic promis-
cuity has been regarded as one of the most outstanding concepts
in biocatalysis [5–6]. This concept involves the abilities of a cer-
tain enzyme to catalyze different synthetic reactions, which maybe
more or less far from their natural role [3,7–10]. The importance
of the promiscuity concept in biocatalysis is noteworthy, since it
not only highlights the existing catalysts, but may provide novel
and practical synthetic pathways which are not currently avail-
able [11–17,31]. Within biocatalysis, the use of hydrolases has been
extensively documented and some excellent examples of the enzy-
matic promiscuity were presented. For example, Gotor group found
that lipase B from Candida antarctica (Lipase CAL-B) are able to
promiscuously catalyze Michael-type addition of secondary amines
to acrylonitrile [18]. Berglund and co-workers found that CAL-B not
only can catalyze the aldol reaction between adipic aldehydes, but
can catalyze direct epoxidation reaction of ␣,␤-unsaturated alde-
hydes [19,20]. Wu et al. reported that Penicillin G acylase, which is
widely used in biosynthesis of ␤-lactam antibiotics, is also capable
of Markovnikov addition of allopurinol to vinyl ester [16]. Although
the catalytic activities were quite low in some experiments, these
cases and other relevant reports indeed provide novel synthesis
pathways for organic synthesis [3]. Lipases are the most used and
well-known hydrolases, which have high stability and activity,
attractive and might provide novel process for industrialization.
On the other hand, in organic synthesis, the development of
efficient and environmentally friendly chemical processes for the
preparation of biologically active molecules remains a major chal-
lenge for chemists and biologists. Among them, Mannich reaction
is one of the most important C–C bond formations in organic
synthesis. They provide ␤-amino carbonyl compounds, which are
important intermediates for various pharmaceuticals and natural
products [21,22]. The preferable process is the use of a “one-pot”
three-component strategy that allows for a wide range of struc-
tural variations. However, most reaction methodology (referred
to as the indirect Mannich reaction) relies on the two-component
system using preformed electrophiles, such as imines, and stable
nucleophiles, such as enolates, enol ethers, and enamines [23–30].
Compared to direct Mannich reaction (carries out the reaction with
unmodified ketone donors), these methods need to firstly prepare
and isolate the intermediates, which is complicated and not atom
economic. Hence, to realize the direct Mannich reaction in “one
pot” strategy is currently concerned (Scheme 1), which is also the
trend for green chemistry.
Recently, we reported the lipase-catalyzed direct Mannich reac-
tion of aromatic aldehydes with arylamines and acetone in the
presence of water [32]. Although high yield was achieved in ace-
tone and water system (v/v = 1:1), the solvent was not environment
friendly because of the excessive acetone. Meanwhile this reac-
tion medium could hardly extend to other ketones due to the
competing reaction between of acetone and other ketones with
aldehydes and aniline. Therefore, to assess the generality of promis-
cuous lipase-catalyzed Mannich reaction, herein we present a more
∗
Corresponding author. Tel.: +86 28 85460576; fax: +86 28 85415886.
E-mail addresses: kli@scu.edu.cn (K. Li), xqyu@tfol.com (X.-Q. Yu).
1381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.08.004

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T. He et al. / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 189–194
Table 1
The catalytic activities of Mannich reaction by different lipases.a
.
Entry
Catalyst
Conversion (%)b
1
2
3
4
5
6
7
8
9
No enzyme
–
Lipase from Candida rugosa (CRL)
Lipase from Candida cylindracea (CCL)
Lipase from Pseudomonas fluorescens (PFL)
Lipase from Mucor miehei (MML)
Lipase from porcine pancreas, type II (PPL)
Lipase from Burkholderia cepacia (BCL)
Lipase from Candida antarctica (CAL-B)
Lipase from Mucor javanicus (MJL)
Bovine Serum Albumin (BSA)
52.0
43.6
41.3
27.4
25.1
27.8
23.8
5.6
Scheme 1. Lipase-catalyzed reactions utilizing of biocatalytic promiscuity
10
11
–
<5
Denatured CRLc
a
Reaction conditions: a solution of 4-nitrobenzaldehyde (1a, 0.1 mmol), aniline
benign reaction system (ethanol/water) for the direct Mannich
reaction. Meanwhile, more substrates were tested including cyclo-
hexanone, butanone and 1-hydroxy-2-propanone, which indicated
the wide application of the lipase-catalyzed biocatalytic promiscu-
ity in organic synthesis.
(2, 0.11 mmol), cyclohexanone (3a, 0.2 ml), 2 ml solvent (the water content is 10%
in volume) and 10 mg enzyme was shaken at 200 rpm at 30 ◦C for 24 h.
b
Conversion was calculated based on 1a detected by HPLC.
c
Denatured CRL was treated by urea at 100 ◦C for 24 h.
3. Results and discussion
2. Experimental
3.1. The catalytic activities of different lipases for the Mannich
reaction
2.1. General
Initially, we chose the reaction of 4-nitrobenzaldehyde (1a), ani-
line (2) and cyclohexanone (3a) with the formation of 4a as a model
transformation, which is a common process of Mannich reaction
(Table 1). Despite the diverse synthetic routes so far developed for
the Mannich reaction, only a few one-pot procedures using unmod-
ified aldehydes or ketones in water have been reported in the
literature [33–39]. Based on the previous results, we believe that
the water could accelerate the protonation of ketones and make it
easier to react with the Schiff base formed by arylamines and aro-
matic aldehydes. In our previous work, acetone/water system was
chosen as the reaction medium, in which acetone is one of reactants
of the reaction as well as the solvent to dissolve other reactants. To
test the generality of promiscuous lipase-catalyzed Mannich reac-
tion, we initially process our reaction using 4-nitrobenzaldehyde,
aniline and cyclohexanone in acetone/water 9:1 (v/v) medium, and
the result indicated that the major product was the Schiff base
transformed from aldehydes and aniline and further reaction could
not proceed to form Mannich product at 30 ◦C in 48 h, which indi-
cated that this reaction medium is only suitable for acetone and
could not extend to other ketones. Therefore, to develop a universal
biocatalytic process for the synthesis of ␤-amino-ketone com-
pounds under benign conditions, we primarily chose ethanol/water
9:1 (v/v) as the reaction medium.
The effect of various enzymes on model reaction was investi-
gated and the results are shown in Table 1. Lipase from C. rugosa
(CRL, Table 1, entry 2) was identified to be the best catalyst for this
direct Mannich reaction, CCL and PFL presented moderate activities
(Table 1, entries 3 and 4); while other tested lipases (Table 1, entries
5–8) resulted in low conversion and no product was detected in the
absence of enzymes. Therefore, CRL was chose as the catalyst in the
following experiments. Additionally, although Catalytic promiscu-
ity in enzymes is more common than generally appreciated [40,7],
to prove this direct Mannich reaction is not catalyzed by impurities
in enzymes, Bovine Serum Albumin (BSA) and denatured CRL were
investigated and the results indicated that denatured CRL showed
Lipase from C. antarctica (CAL-B, 10,000 u/g), lipase from porcine
pancreas (PPL,100,000–400,000 u/g), lipase from Candida cylin-
dracea (CCL, 7290 u/g), lipase from Candida rugosa (CRL, 700 u/mg)
and lipase from Mucor javanicus (MJL, 10,000 u/g) were pur-
chased from Sigma (St. Louis, USA). Lipase from Mucor miehei
(MML, 86.8 u/g) was purchased from Fluka (Buchs, Switzerland).
Lipase from Pseudomonas fluorescens (PFL, 10.2 u/g) and lipase from
Burkholderia cepacia (BCL, 23,000 u/g) were a gift from Amano
(Nagoya, Japan). For CRL, MML, MJL and PPL, one unit of enzyme
activity is defined as the amount of enzyme that hydrolyzes 1.0
microequivalent of fatty acid from a triglyceride in 1 h at pH 7.7 at
37 ◦C; while for CCL, PFL and CAL-B, one unit corresponds to the
amount of enzyme which liberates 1 mol oleic acid per minute at
pH 8.0 and 40 ◦C. All other chemicals and reagents were obtained
commercially and used as received.
1H NMR and 13C NMR spectra were recorded on a Bruker DMX
400. Chemical shifts are given in ı relative to tetramethylsilane
(TMS). HPLC was carried out using a Shimadzu organizer consisting
of a LC-2010A HT integrator, a UV/VIS detector. AD-H column was
used in the HPLC experiments with n-hexane/iso-propanol = 85:15
(v/v), 0.8 ml/min and UV = 254 nm, Rt (4a) = 14.5 min, 18.4 min,
21.7 min, 25.2 min. Electrospray ionization (ESI) mass spectrome-
try experiments were performed on Bruker Daltonics Bio TOF mass
spectrometer.
2.2. Substrate expansion
Reaction conditions: 100 mg CRL was added to a mixture of alde-
hyde (1 mmol), amine (1.1 mmol) and ketone (3 mmol) in 10 ml
ethanol/water medium. The mixture was maintained at 30 ◦C and
shaken at 200 rpm for 24 or 48 h. The residue was filtered off and
the solvent was evaporated. A single product was prepared by sil-
ica gel chromatography with an eluent consisting of petroleum
ether/ethyl acetate (4:1, v/v).

T. He et al. / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 189–194
191
Fig. 1. The influence of solvents on the direct Mannich reaction. Reaction conditions:
a solution of 4-nitrobenzaldehyde (1a, 0.1 mmol), aniline (2, 0.11 mmol), cyclohex-
anone (3a, 0.2 ml), 2 ml solvent (the water content is 10% in volume) and 10 mg CRL
was shaken at 200 rpm at 30 ◦C for 24 h.
Fig. 3. The effect of lipase activity. Reaction conditions:
a
solution of 4-
nitrobenzaldehyde (1a, 0.1 mmol), aniline (2, 0.11 mmol), cyclohexanone (3a,
0.2 ml), 2 ml solvent (the water content is 10% in volume) was shaken at 200 rpm
at 30 ◦C for 24 h. 2.5 mg/ml of CRL correspond to 1750 U activity in 1 ml solution.
Conversion was calculated based on 1a detected by HPLC.
poor activity (less than 5%, Table 1, entry 11), while BSA could not
catalyze this reaction which indicated that the protein could not
exhibit the catalytic activity. These results proved that the specific
structure of lipase is important to this reaction, and make us tend
to believe that the Mannich reaction is catalyzed by lipase rather
than impurities.
3.2. The influence of solvents on the direct Mannich reaction
Several other protic solvents in the presence of water were also
investigated (Fig. 1
). The results indicated that ethanol/water system is the best
reaction medium for the model Mannich reaction, and poor yields
were observed in other medium. Therefore, ethanol/water system
was applied in the following process.
3.3. The effect of the amount of ketone
We next surveyed the effect of the amount of the cyclohexanone
(Fig. 2). The yield can be improved by increasing the amount of the
cyclohexanone, and it got to the highest point at 30 equivalent. Fur-
ther increasing of ketone/aldehyde ratio failed to improve the yield.
Therefore, 30 equivalent cyclohexanone was used in the following
reaction.
Fig. 4. The influence of water content on the CRL catalyzed Mannich reaction.
Reaction conditions: a solution of 4-nitrobenzaldehyde (1a, 0.1 mmol), aniline (2,
0.11 mmol), cyclohexanone (3a, 0.3 ml), 2 ml solvent (the water content is from
0–60% in volume) and 20 mg CRL was shaken at 200 rpm at 30 ◦C for 24 h. Conversion
was calculated based on 1a detected by HPLC.
Fig. 2. Influence of molar equivalents of cyclohexanone on the direct Mannich reac-
tion. Reaction conditions: a solution of 4-nitrobenzaldehyde (1a, 0.1 mmol), aniline
(2, 0.11 mmol), 2 ml solvent (the water content is 10% in volume) and 10 mg CRL was
shaken at 200 rpm at 30 ◦C for 24 h. Conversion was calculated based on 1a detected
by HPLC.
Fig. 5. Time course of the lipase-catalyzed Mannich reaction. Reaction conditions: a
solution of aromatic aldehyde (1, 0.25 mmol), aniline (2, 0.28 mmol), cyclohexanone
(3a, 0.75 ml), 5 ml solvent (the water content is 5% in volume) and 50 mg CRL was
shaken at 200 rpm at 30 ◦C. Conversion was calculated based on 1 detected by HPLC.

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T. He et al. / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 189–194
Table 2
Direct Mannich reaction of various aryl aldehydes and ketones with aniline.a
.
Entry
R1
Ketone
Product
Yield (%)b
Dr.c
1
2
–NO2
91
74
2:1
–OCH3
1.6:1
3
4
–CN
62
82
2.4:1
1.7:1
–H
5
–Cl
94
1:1
6
7
8
–CH3
90
20
21
1.4:1
–NO2
n.d.
–OCH3
n.d.

T. He et al. / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 189–194
193
Table 2 (Continued )
Entry
R1
Ketone
Product
Yield (%)b
Dr.c
9
–H
32
n.d.
10
11
–NO2
81d
1.5:1
–H
90d
2:1
12
–OCH3
76d
3:1
a
Reaction conditions: a solution of aromatic aldehyde (1, 1.0 mmol), aniline (2, 1.1 mmol), ketone (3, 30 mmol), 10 ml ethanol/water medium (the water content is 5% in
volume) and 100 mg CRL was shaken at 200 rpm at 30 ◦C for 24 h or 48 h (entry 7–9).
b
Isolated yield.
c
Determined by HPLC (etntry 1 and entry 4) or 1H NMR spectroscopy of the crude products.
d
1-Hydroxy-2-propanone (3c, 5 mmol).
3.4. The effect of the enzyme concentration on Mannich reaction
4-nitrobenzaldehyde showed a higher activity for the experimental
conditions applied.
The effect of the enzyme concentration on Mannich reaction
was also investigated. As shown in Fig. 3, the yields were improved
with the increase of enzyme concentration within the range of
2.5–10 mg/ml (3500–21,000 U). However, continuing to increase
the amount of enzyme loading, the yield increased only a little.
Thus, the subsequent experiments were performed with an enzyme
concentration of 10 mg/ml (14,000 U).
3.7. The generality of the lipase promiscuous catalyzed Mannich
reaction on other substrates
Based on the above results obtained we extend the reac-
tion with a series of aldehydes bearing electron-withdrawing and
electron-donating groups and ketones including cyclohexanone,
butanone and 1-hydroxy-2-propanone in order to ascertain the
generality and scope of this one-pot Mannich reaction. It was
found that a wide range of aromatic aldehydes could effectively
participate in CRL catalyzed Mannich reaction to give the corre-
sponding ␤-amino carbonyl compounds. The reaction was favored
by electron-withdrawing substituents of aldehyde. The regioselec-
tivity of this lipase catalyzed Mannich reaction was investigated,
but poor regioselectivity was found. Both C1 and C3 site product of
butanone were observed, while only C3 site product was found for
the 1-hydroxy-2-propanone, because C3-H exhibits much higher
reactivity than C1-H. The highest diastereomeric ratio (Dr.) for 1-
hydroxy-2-propanone is 3:1, which we believe this is due to the
promiscuity of lipase for the Mannich reaction and hard to improve
through traditional methods. Meanwhile, several aliphatic aldehy-
des were also tested, but no products were detected under the same
conditions (Table 2).
3.5. The influence of water concentration on the Mannich
reaction
Based on previous results, water plays an important role in the
reaction process, it is necessary to confirm the optical percent-
age of water in ethanol/water system. Therefore, experiments were
performed to ascertain the catalytic activity of CRL with different
amounts of water in the reaction system (Fig. 4). Interestingly, 5%
water content in ethanol shows the best catalytic activity, which is
different from the MML-catalyzed reaction in acetone/water sys-
tem [32].
3.6. Time course of the reaction
The effects of time and substituent group of aldehydes on
the yield of Mannich reaction by CRL were assessed by using
three different aromatic aldehydes with electron-withdrawing and
electron-donating groups at the same reaction conditions. The
results are shown in Fig. 5. In general, the substituent group
showed significant influences on the initial rate of the reaction
and the reaction achieved equilibrium after 30 h. From this com-
parison, the conclusion can be drawn that the reaction with
4. Conclusion
In summary, we have demonstrated the scope and utility of
lipase-catalyzed direct Mannich reaction utilization of biocatalytic
promiscuity under ethanol/water medium in a “one-pot” strategy.
Lipase from C. rugosa (CRL) was identified as best catalyst for this

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T. He et al. / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 189–194
direct Mannich reaction among the tested lipases. A wide scope of
substrates was expanded to assess the generality of the promis-
cuity. This lipase-catalyzed C–C bond formation would not only
largely expand the application of hydrolases in biocatalysis, but be a
potential method as a practical strategy for green organic synthesis.
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Acknowledgements
We gratefully acknowledge the National Natural Science Foun-
dation of China (no. 20725206 and 20732004) for financial support;
K.L. thanks the Youth Scientific Fund of Sichuan University (no.
2008117); T.H. also thanks the sustentation of Student Innovation
Fund of Sichuan University. We also thank the Sichuan University
Analytical & Testing Center for NMR analysis.
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