A novel trypsin-catalyzed three-component mannich reaction

Article abstract of DOI:10.1002/hlca.201000063

We found that the trypsin from hog pancreas displayed high activity to promote three-component Mannich reaction of aromatic aldehydes, aromatic amines, and acetone with moderate-to-excellent yields. The reaction tolerates a great range of substrates and e

Full text of DOI:10.1002/hlca.201000063

Helvetica Chimica Acta – Vol. 93 (2010)
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A Novel Trypsin-Catalyzed Three-Component Mannich Reaction
by She-Jie Chai, Yi-Feng Lai, Hui Zheng, and Peng-Fei Zhang*
College of Material, Chemistry and Chemical Engineering, Hangzhou Normal University, Hangzhou
310036, P. R. China (phone: þ 86-571-85996586; fax: þ 86-571-88484468; e-mail: pfzhang@hznu.edu.cn)
We found that the trypsin from hog pancreas displayed high activity to promote three-component
Mannich reaction of aromatic aldehydes, aromatic amines, and acetone with moderate-to-excellent
yields. The reaction tolerates a great range of substrates and extends the application of trypsin in organic
synthesis.
Introduction. – The Mannich reaction as a three-component reaction of an
aldehyde, a primary or secondary amine, and a ketone is one of the most powerful C,C
bond-forming reactions in organic synthesis [1]. It leads to b-amino carbonyl
compounds, which are useful for the syntheses of N-containing compounds, such as
natural products and medicinally relevant compounds [2]. The versatility and potential
of these compounds to introduce both functional and structural diversity using the
Mannich reaction have stimulated the creativity of chemists [3]. Most recently, directly
catalyzed Mannich-type reactions have been widely investigated, utilizing different
classes of catalysts, such as organometallic complexes, amino acids, and their
derivatives [1b][4]. Trypsin-catalyzed Mannich reactions, which can be performed
under mild and environment-friendly reaction conditions, have never been reported. In
this article, we describe new synthetic applications of biocatalysts. We have found that
some hydrolases are able to catalyze different reactions, such as Aldol reactions,
Michael additions, etc. Here, we report on the trypsin-catalyzed three-component
Mannich reaction of benzaldehydes 1 and anilines 2 in the presence of acetone (3) as
the third component and solvent (Scheme 1).
Results and Discussion. – Acetone was used as solvent instead of the commonly
used DMSO. Excellent yields were obtained in the reaction of benzaldehydes and p-
Scheme 1. Trypsin-Catalyzed Three-Component Mannich Reaction
ꢀ 2010 Verlag Helvetica Chimica Acta AG, Zꢁrich

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Helvetica Chimica Acta – Vol. 93 (2010)
anisidine (¼4-methoxybenzenamine). A series of commercially available hydrolytic
enzymes had been investigated in order to find the most suitable enzyme for the
envisaged one-pot Mannich reaction (Table 1). We found that several enzymes
displayed observable activities for this reaction, whereby trypsin from hog pancreas
showed an especially significant catalytic activity. Lipase from porcine pancreas (PPL),
amano lipase (AK) from Pseudomonas fluorescens, and a-amylase from hog pancreas
also showed moderate catalytic activities, while other tested enzymes presented very
low activities for this transformation. Considering these results, we performed some
additional experiments to test the catalytic activity in the absence of any biocatalyst
(enzyme). The reaction yielded no new product even after 96 h (Table 1, Entry 1).
Table 1. Screening the Different Enzyme Catalysts for Mannich Reactiona)
Entry
Catalyst
Yield [%]b)
1
2
3
4
5
No Enzyme
Trypsin from hog pancreas
a-Amylase from hog pancreas
Lipase acrylic resin from Candida antarctica
Lipase AY30
3c)
83
54
11
8
6
7
8
9
Lipase from porcine pancreas (PPL)
Amino lipase AK from Pseudomonas fluorescens
Amino lipase A from Aspergillus niger
Amino lipase M from Mucor javanicus
Cellulose (C0057)
46
17
20
28
6
10
a) Reaction conditions: a solution of 4-nitrobenzaldehyde (0.2 mmol), p-anisidine (0.2 mmol), acetone
(2 ml), and enzyme (20 mg) was shaken at 160 rpm at 308 for 12 h. b) Yield based on consumed p-
anisidine. c) Reaction for 96 h.
The reaction medium has been recognized to be one of the most important factors
influencing the enzymatic reaction. We screened the reaction in various organic
solvents (Table 2). We found that acetone as reactant and solvent, and acetone in
EtOH were the most efficient media to promote the Mannich reaction, and the
Mannich products were obtained in similar yields of 89 and 86%, respectively. Other
solvents, such as CH2Cl2, CCl4, and toluene did not promote the product formation
(Table 2, Entries 2 – 4), and other polar solvents (Table 2, Entries 6 – 9) led to moderate
yields.
In previous studies of enzymatic promiscuity, H2O has been considered as a very
important factor in enzymatic activity leading to an acceleration of the enzyme-
catalyzed reaction [5]. Therefore, we performed some experiments to optimize the
percentage of H2O in H2O/acetone binary mixtures for this reaction. The effect of H2O
concentration on the considered trypsin-catalyzed Mannich reaction is illustrated in the
Figure. Apparently, 25% of H2O added to the system was optimal for the trypsin-
catalyzed reaction. To our surprise, the yield of the reaction with 25% H2O in acetone is
even a little smaller than that in pure acetone. In other words, for optimal reaction
conditions, H2O is not necessary in this trypsin-catalyzed three-component Mannich
reaction.

Helvetica Chimica Acta – Vol. 93 (2010)
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Table 2. Optimization of the Mannich Reaction in Various Solventsa)
Entry
Solvent
Time [h]
Yield [%]
1
2
3
4
5
6
7
8
9
Hexane
CCl4
Toluene
CH2Cl2
EtOH
THF
MeCN
DMF
DMSO
Acetone
Acetone
24
24
24
24
24
24
24
24
24
12
24
56
2
3
5
86
46
53
35
48
80
89
10
11
a) Reaction conditions: a solution of aldehyde (0.2 mmol), amine (0.2 mmol), acetone (2 ml), solvent
(3 ml), and trypsin (20 mg) was shaken at 160 rpm at 378 for 24 h.
Figure. Influence of H2O on the trypsin-catalyzed Mannich reaction. Conditions: 4-nitrobenzaldehyde
(0.2 mmol), p-anisidine (0.2 mmol), acetone (2 ml), and enzyme (20 mg) was stirred at 160 rpm at 378
for 48 h; deionized H2O from 0 to 70% (H2O/(H2O þ acetone) (v/v)). Yield of Mannich product was
determined by HPLC.
Other influencing factors such as temperature, concentration of catalyst, and
reaction time have also been investigated. The results showed that the desired products
were obtained in yields up to 93% when 20 mg of trypsin in 2 ml of acetone at 378/48 h
were used. Encouraged by this remarkable result and in order to expand the scope of

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Helvetica Chimica Acta – Vol. 93 (2010)
this new catalyst, we have subjected various aromatic aldehydes, aromatic amines, and
acetone to the three-component Mannich reaction (Table 3). Trypsin turned out to be
an excellent catalyst for all Mannich reactions. Excellent yields of b-amino ketones
were obtained from benzaldehydes with electron-acceptor substituents, specifically 4-
nitrobenzaldehyde. p-Anisaldehyde with MeO as electron-donor substituent gave a
lower yield. Fortunately, aromatic amines with MeO as electron-donating group
afforded the desired products in good yields.
Table 3. One-Pot, Three-Component Mannich Reactiona)
Entry
Product
R1
R2
Yield [%]b)
1
2
3
4
5
6
7
8
9
4a
4b
4c
4d
4e
4f
4g
4h
4i
4-NO2ꢀC6H4
4-MeOꢀC6H4
4-ClꢀC6H4
4-NO2ꢀC6H4
4-NO2ꢀC6H4
4-NO2ꢀC6H4
4-NO2ꢀC6H4
Ph
4-MeOꢀC6H4
4-MeOꢀC6H4
4-MeOꢀC6H4
2-MeOꢀC6H4
4-ClꢀC6H4
93
45
83
55
84
82
76
68
74
65
47
3-ClꢀC6H4
3-NO2ꢀC6H4
2-MeOꢀC6H4
4-ClꢀC6H4
Ph
Ph
Ph
10
11
4j
4k
3-ClꢀC6H4
3-NO2ꢀC6H4
a) Reaction conditions: a solution of the corresponding aldehyde (1 mmol), and amine (1 mmol),
acetone (5 ml), and trypsin (20 mg) was shaken at 160 rpm at 378 for 24 – 48 h. b) Yield of isolated
product.
Based on the mechanism of lipase-catalyzed [6] organic reaction which has been
widely accepted, we assume that a similar mechanism is involved in the trypsin-
catalyzed Mannich reaction (Scheme 2). The in situ generated Schiff base complexes
with the trypsin. At the same time, the enolate anion is stabilized by the oxyanion hole.
Then, the enolate anion attacks the Schiff base complexed with trypsin and a new CꢀC
bond is formed. H-atom transfer to the O-atom from acetone takes place in a concerted
process. Finally, the Mannich adduct is released from the oxyanion hole. We will study
this proposed mechanism further in detail.
In conclusion, we have found out that several enzymes, especially trypsin, have
remarkable catalytic activity to promote three-component Mannich reaction. This
reaction can be carried out under mild conditions both in organic and in aqueous
solvents, and provides b-amino carbonyl compounds in high yields. The trypsin-
catalyzed Mannich reaction covers a great range of substrates and further extends the
application of enzymes in the organic synthesis.
This work was supported by the National Science and Technology Ministry (No. 2007BAI34B05) and
the Natural Science Foundation of China (No. 20972037).

Helvetica Chimica Acta – Vol. 93 (2010)
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Scheme 2. Proposed Mechanism of the Trypsin-Catalyzed Mannich Reaction
Experimental Part
General. Commercial reagents were used without further purification, unless otherwise indicated.
All solvents were distilled prior to use. Reactions were performed in oven-dried glassware. Flash
chromatography (FC): silica gel 60 (SiO2, 230 – 400 mesh; Fluka). Anal. TLC: Merck precoated TLC
(silica gel 60 F 254) plates. C18 column was used in the HPLC experiments with MeOH/H2O 70 :30 (v/v),
0.8 ml/min; UV detection at 254 nm. M.p.: X4-Data microscopic melting-point apparatus; uncorrected.
1
IR Spectra: Nicolet 380 FT-IR spectrophotometer using KBr discs. H- and 13C-NMR spectra: Bruker
Avance 400 spectrometer in CDCl3 using TMS as internal standard. ESI-MS: Bruker Esquire 3000 plus
spectrometer.
General Procedure. 4-[(4-Methoxyphenyl)amino]-4-(4-nitrophenyl)butan-2-one (4a). A soln. of 4-
nitrobenzaldehyde (1 mmol), p-anisidine (1 mmol), acetone (5 ml), and trypsin (30 mg) was shaken at
160 rpm at 378 for 24 h. The residue was then filtered off, and the solvent was evaporated. A single
product was obtained by FC (petroleum ether (PE)/AcOEt 4 :1 (v/v)). 1H-NMR: 8.19 – 8.17 (m, 2 arom.
H); 7.57 – 7.55 (m, 2 arom. H); 6.71 – 6.67 (m, 2 arom. H); 6.48 – 6.46 (m, 2 arom. H); 4.86 (t, J ¼ 6.4,
CH2CH); 3.70 (s, MeO); 2.96 (d, J ¼ 9.6, CH2); 2.15 (s, Me). ESI-MS: 314 ([M þ Na]þ).
4-(4-Methoxyphenyl)-4-[(4-methoxyphenyl)amino]butan-2-one (4b). 1H-NMR: 7.26 – 7.24 (m, 2
arom. H); 6.84 – 6.82 (m, 2 arom. H); 6.89 – 6.87 (m, 2 arom. H); 6.51 – 6.49 (m, 2 arom. H); 4.70 (t,
J ¼ 6.4, CH2CH); 3.76 (s, MeO); 3.68 (s, MeO); 2.87 (d, J ¼ 6.4, CH2); 2.07 (s, Me). ESI-MS: 299 ([M þ
Na]þ).

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Helvetica Chimica Acta – Vol. 93 (2010)
4-(4-Chlorophenyl)-4-[(4-methoxyphenyl)amino]butan-2-one (4c). 1H-NMR: 7.13 – 7.11 (m, 2 arom.
H); 6.62 – 6.58 (m, 4 arom. H); 6.44 – 6.42 (m, 2 arom. H); 4.70 (q, J ¼ 5.2, CH2CH); 2.70 (m, J ¼ 6.4,
CH2); 2.05 (s, Me). ESI-MS: 303 ([M þ Na]þ).
1
4-[(2-Methoxyphenyl)amino]-4-(4-nitrophenyl)butan-2-one (4d). H-NMR: 8.16 – 8.13 (m, 2 arom.
H); 7.55 – 7.53 (m, 2 arom. H); 6.77 – 6.27 (m, 4 arom. H); 4.96 (t, J ¼ 6.4, CH2CH); 3.87 (s, MeO); 2.98
(m, J ¼ 9.6, CH2); 2.14 (s, Me). ESI-MS: 314 ([M þ Na]þ).
4-[(4-Chlorophenyl)amino]-4-(4-nitrophenyl)butan-2-one (4e). 1H-NMR: 8.18 – 8.16 (m, 2 arom.
H); 7.55 – 7.53 (m, 2 arom. H); 7.04 – 7.02 (m, 2 arom. H); 6.42 – 6.40 (m, 2 arom. H); 4.88 (t, J ¼ 6.0,
HꢀCꢀPh); 4.67 (s, NH); 2.98 (d, J ¼ 5.6, CH2); 2.15 (s, Me). ESI-MS: 318 ([M þ Na]þ).
4-[(3-Chlorophenyl)amino]-4-(4-nitrophenyl)butan-2-one (4f). 1H-NMR: 8.20 – 8.18 (m, 2 arom.
H); 7.56 – 7.54 (m, 2 arom. H); 7.02 – 6.98 (m, 1 arom. H); 6.67 – 6.36 (m, 3 arom. H); 4.90 (t, J ¼ 6.4,
CH2CH); 4.75 (s, NH); 2.99 (d, J ¼ 6.4, CH2); 2.15 (s, Me). ESI-MS: 318 ([M þ Na]þ).
4-(4-Nitrophenyl)-4-[(3-nitrophenyl)amino]butan-2-one (4g). 1H-NMR: 8.18 – 8.16 (m, 2 arom. H);
7.57 – 7.55 (m, 2 arom. H); 7.50 – 6.76 (m, 4 arom. H); 5.18 (s, NH); 4.98 (t, J ¼ 6.4, CH2CH); 3.03 (m, J ¼
6.4, CH2); 2.16 (s, Me). ESI-MS: 329 ([M þ Na]þ).
4-[(2-Methoxyphenyl)amino]-4-phenylbutan-2-one (4h). 1H-NMR: 7.38 – 7.21 (m, 5 arom. H); 6.77 –
6.42 (m, 4 arom. H); 4.88 (t, J ¼ 6.4, CH2CH); 4.80 (s, NH); 3.07 (s, MeO); 2.96 (m, J ¼ 9.2, CH2); 2.12 (s,
Me). ESI-MS: 269 ([M þ Na]þ).
1
4-[(4-Chlorophenyl)amino]-4-phenylbutan-2-one (4i). H-NMR: 7.34 – 7.27 (m, 5 arom. H); 7.04 –
7.02 (m, 2 arom. H); 6.47 – 6.46 (m, 2 arom. H); 4.79 (t, J ¼ 6.4, CH2CH); 4.57 (s, NH); 2.92 (d, J ¼
6.0, CH2); 2.11 (s, Me). ESI-MS: 273 ([M þ Na]þ).
1
4-[(3-Chlorophenyl)amino]-4-phenylbutan-2-one (4j). H-NMR: 7.34 – 7.24 (m, 5 arom. H); 7.01 –
6.40 (m, 4 arom. H); 4.81 (t, J ¼ 6.4, CH2CH); 4.61 (s, NH); 2.92 (d, J ¼ 6.4, CH2); 2.09 (s, Me). ESI-
MS: 273 ([M þ Na]þ).
4-[(3-Nitrophenyl)amino]-4-phenylbutan-2-one (4k). 1H-NMR: 7.40 – 7.33 (m, 5 arom. H); 7.28 – 7.16
(m, 3 arom. H); 6.82 – 6.80 (m, 1 arom. H); 4.87 (t, J ¼ 6.0, CH2CH); 2.96 (d, J ¼ 6.4, CH2); 2.11 (s, Me).
ESI-MS: 284 ([M þ Na]þ).
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Received February 11, 2010