Promiscuous Candida antarctica lipase B-catalyzed synthesis of β-amino esters via aza-Michael addition of amines to acrylates

Article abstract of DOI:10.1016/j.tetlet.2010.06.089

An efficient protocol for the regioselective aza-Michael addition of amines with acrylates using CaL B as a biocatalyst at 60 °C has been developed. The reaction is applicable to a wide variety of primary and secondary amines with different acrylates to synthesize the corresponding β-amino esters with good yields. An alternative route for the synthesis of higher β-amino esters through the additional transesterification step is also studied and was found effective.

Full text of DOI:10.1016/j.tetlet.2010.06.089

Tetrahedron Letters 51 (2010) 4455–4458
Contents lists available at ScienceDirect
Tetrahedron Letters
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Promiscuous Candida antarctica lipase B-catalyzed synthesis of b-amino
esters via aza-Michael addition of amines to acrylates
Kishor P. Dhake a, Pawan J. Tambade a, Rekha S. Singhal b, Bhalchandra M. Bhanage a,
*
a Department of Chemistry, Institute of Chemical Technology (Autonomous), Matunga, Mumbai 400 019, India
b Department of Food Engineering and Technology, Institute of Chemical Technology, (Autonomous), Matunga, Mumbai 400 019, India
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient protocol for the regioselective aza-Michael addition of amines with acrylates using CaL B as a
biocatalyst at 60 °C has been developed. The reaction is applicable to a wide variety of primary and sec-
ondary amines with different acrylates to synthesize the corresponding b-amino esters with good yields.
An alternative route for the synthesis of higher b-amino esters through the additional transesterification
step is also studied and was found effective.
Received 19 May 2010
Revised 16 June 2010
Accepted 17 June 2010
Available online 20 June 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Biocatalysis has greatly gained importance in organic synthesis
as it provides a high selectivity with fewer by-products, thus mak-
ing it an environmentally benign alternative to chemical transfor-
mations.1 Hydrolases, especially lipases are the dominating
enzymes of the family being exploited for organic synthesis. Syn-
thesis of b-amino esters has immense importance as b-amino acid
moieties are biologically important molecules,2 and also contribute
as essential intermediates in the synthesis of b-lactams,2b,c and b-
peptides.2d The Mannich reaction is the most common method
used for the synthesis of b-amino esters.3a–c Also, Michael addition
has served as one of the fundamental reactions in the organic syn-
thesis of such important molecules. A variety of Lewis acids such as
transition metals, lanthanide halides,4a–c triflates,4d and silica gel4e
have been demonstrated as catalysts for the Michael addition reac-
tion. Heterogeneous solid catalysts like 5a Cu(acac)2 immobilized in
ionic liquid,5b quaternary ammonium salt/ionic liquid/H2O,5c boric
acid in H2O,5d b-cyclodextrin in H2O5e, and clay5f have also been
used for similar transformations.
Lipases are widely explored for esterification, transesterifica-
tion, and aminolysis type of reactions to synthesize various impor-
tant molecules related to pharmaceuticals, foods, and flavors.6a,b
Apart from their natural behavior to catalyze the specified reac-
tions, they are found to catalyze various unexpected reactions like
C–C,7a–c C–N,7d–g C–S7h which shows their promiscuous behavior
toward biocatalysis.7i Kitazume et al.8a were the first to reveal
the ability of the hydrolytic enzymes to catalyze aza-Michael addi-
reports on Candida antarctica lipase B-catalyzed Michael addition
in organic solvent exist, the area of b-amino esters synthesis using
diethyl amine and acrylate yet remain unexplored, considering
which we focused our attention toward b-amino esters synthesis.
In the present Letter, we report an efficient enzymatic protocol
for the b-amino esters synthesis via aza-Michael addition of the
primary and secondary amines to acrylates using CaL B as a biocat-
alyst (Scheme 1).
The catalyst exhibited remarkable activity and is applicable to a
wide variety of primary and secondary amines with different acry-
lates to synthesize the corresponding b-amino esters.
In order to optimize the reaction conditions, the reaction of
diethyl amine with methyl acrylate in the presence of CaL B was
chosen as a model reaction12 and the influence of various reaction
parameters such as enzyme, solvent, temperature, time, catalyst
loading, and molar ratio of acrylate to amine was studied. Com-
mercially available lipases from various sources were screened
for their catalytic activity toward aza-Michael addition reaction
as shown in Table 1(entries 1–7). CaL B lipase (immobilized on ac-
rylic resin from C. antarctica with P10,000 U/g, recombinant, and
expressed in Aspergillus oryzae) was found to be the most efficient
lipase to catalyze the reaction while other lipases catalyzed the
reaction with a low yield ranging from 10% to 36% of the desired
1
R
R
O
O
tion in a buffer solution of Na2HPO4 and KH2PO4. Biomacromole-
1
R
R2
R2
CaL B
NH
8b
cules such as RNA,
antibody,8cand baker’s yeast8d were then
+
N
R
O
O
Toulene, 60 °C,
3.5 h
reported to catalyze the aza-Michael addition reaction. In the re-
cent decade, the promiscuity catalytic site has been discovered
and studied for various unexpected reactions.9a,b Although some
1
R, R = H, alkyl
R2= methyl, ethyl, butyl
* Corresponding author. Tel.: + 91 22 2414 5616; fax: +91 22 2414 5614.
E-mail address: bhalchandra_bhanage@yahoo.com (B.M. Bhanage).
Scheme 1. CaL B-catalyzed aza-Michael addition of amine to acrylate.
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.06.089

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K. P. Dhake et al. / Tetrahedron Letters 51 (2010) 4455–4458
Table 1
Table 3
Lipase screening for aza-Michael additiona
CaL B-catalyzed aza-Michael addition reactiona
Entry
Lipase
Enzyme activity (U/g)
Yieldb (%)
Entry Amine
Acrylate
CO2Me
Product
Yieldb (%)
1
2
3
4
5
6
7
8
Candida antarctica lipase B
Lipase PS 30
Lipase AYS
Aspergillus niger
Candida cylindracea
Hog pancreas
Rhizopus oryzae
No enzyme
P10,000
P30,000
P30,000
P12,000
P2,000
P15,000
P30,000
—
58
10
Traces
18
36
20
Traces
Traces
Et
O
Et
N
NH
89 (87, 83,78)d
OMe
O
1
Et
Et
1
n- But
CO2Me
CO2Me
n-But
N
n-But
OMe
NH
2
51
n-But
2
a
Reaction conditions: methyl acrylate (2 mmol), diethyl amine (1 mmol), tolu-
ene (3 ml), lipase (300 U), temperature (37 °C), time (3.5 h).
O
NH2
OMe
N
H
b
Yield based on GC analysis.
3
88
90
54
3
O
NH
CO2Me
CO2Me
product. It was observed that lipases studied led to a faster reac-
tion when compared with the reaction carried out in the absence
of a biocatalyst under the same reaction conditions.7d,f,g
O
OMe
OMe
N
4
5
O
4
NH2
H
N
An enzyme-catalyzed reaction in an organic solvent has numer-
ous potential applications which suggested us to study the effect of
solvents on enzyme activity.10 Overall five solvents with log P va-
lue ranging from À1.1 to 2.5 were screened for the standard reac-
tion, of which toluene was found to be the best solvent providing
89% yield of the desired product (Table 2, entry 5). CaL B was stud-
ied for its optimum temperature to obtain higher yields of the de-
sired product as shown in Table 2 (entries 5–8). It was observed
that the activity of enzyme increased as the temperature increased
up to 60 °C (Table 2, entry 5), but at a higher temperature, i.e.,
80 °C, the yield of the reaction had decreased to 75% (Table 2, entry
8) as the enzyme is believed to undergo a deactivation effect.
In an effort to evolve the best loading of a catalyst, reactions
with various catalysts concentration were carried out (Table 2, en-
tries 5, 9–12). The yield was observed to increase with the increase
in the catalyst loading ranging from 10 to 50 mg, whereas 30 mg of
CaL B was sufficient to catalyze the desired reaction under the
present reaction parameters with 89% of yield (Table 2, entry 5).
Further increase in enzyme concentration had no significant effect
on the yields. For aza-Michael addition reaction, molar ratio of sub-
strates has shown a promising effect on the yield of the desired
product. The optimum yield of 58% was obtained with 2:1 ratio
O
5
O
CO2Me
CO2Me
NH
OMe
N
6
7
65
94
6
7
O
NH
NH
OMe
N
N
Et
Et
O
CO2Et
CO2Et
Et
OEt
8
82
83
90
82
54
Et
8
O
NH2
N
H
OEt
9
9
NH2
H
N
CO2Et
OEt
10
11
12
O
10
O
NH
CO2Et
CO2Bu
O
OEt
N
O
11
12
Table 2
Optimization of reaction parameters for enzymatic aza-Michael addition reactiona
Et
NH
O
Et
OBu
O
N
Et
Yieldb (%)
Et
Entry
Influence of solvent
1
2
3
4
5
Solvent
Temp (°C)
Enzyme loading (mg)
1,4-Dioxane
Tetrahydrofuran
Diisopropyl ether
Chloroform
60
60
60
60
60
30
30
30
30
30
59
78
78
81
89
NH2
CO2Bu
OBu
N
H
13
14
15
16c
59
69
96
31
13
Toluene
NH2
H
CO2Bu
CO2Bu
Influence of temperature
N
OBu
6
7
8
Toluene
Toluene
Toluene
37
45
80
30
30
30
58
69
75
O
14
O
NH
Influence of catalyst loading
9
O
OBu
N
Toluene
Toluene
Toluene
Toluene
Toluene
Toluene
Toluene
60
60
60
60
37
37
37
10
20
40
50
30
30
—
45
75
91
93
45
46
Traces
O
10
11
12
13c
14d
15
15
NH2
H
CO2Me
N
OMe
O
16
a
a
Reaction conditions: methyl acrylate (2 mmol), diethyl amine (1 mmol), tolu-
Reaction conditions: acrylate (2 mmol), amine (1 mmol), toluene (3 ml), CaL B
ene (3 ml), lipase (CaL B), time (3.5 h).
(30 mg), temperature (60 °C), time (3.5 h).
b
b
Yields based on GC analysis.
Molar ratio of methyl acrylate/diethyl amine (1:1).
Yields based on GC analysis.
Time (42 h).
c
c
d
d
Time (2 h).
Recyclability study for three consecutive cycles.

K. P. Dhake et al. / Tetrahedron Letters 51 (2010) 4455–4458
4457
Table 4
of methyl acrylate to diethyl amine at 37 °C temperature (Table 2,
Alternative route for synthesis of (13)a
entry 6) while it was low, that is, 45% for 1:1 molar ratio (Table 2,
entry 13). During time study, the optimum yield was obtained in
3.5 h (Table 2, entry 5) whereas, lowering of time led to a decrease
in yield (Table 2, entry 14). Thus optimized reaction conditions are
solvent: toluene, temperature: 60 °C, time: 3.5 h, catalyst loading:
30 mg (300 U).
Entry
Reaction Mode
Acrylate
Yield (%)b (13)
1
2
3
4
Tandem
Stepwise
Tandem
Stepwise
Methyl acrylate
Methyl acrylate
Ethyl acrylate
Ethyl acrylate
34
58
42
61
a
In order to test the generality and efficiency of this system, the
optimized reaction conditions were then applied for aza-Michael
addition of various primary and secondary aliphatic amines, with
different acrylates (Table 3, entries 1–16). The reaction of diethyl
amine with methyl acrylate (Table 3, entry 1) was smooth but as
the chain length of the secondary amine increased, i.e., the reaction
of dibutylamine with methyl acrylate (Table 3, entry 2) was ham-
pered providing a comparatively low yield of 51%. A primary ali-
phatic amine such as benzylamine (Table 3, entry 3) also
provided a good yield of the desired product. The alicyclic amines
such as morpholine, cyclohexylamine, piperidine, and pyrrolidine
reacted effectively to provide good yields of the desired b-amino
esters (Table 3, entries 4–7). Other acrylates such as ethyl acrylate
and butyl acrylate were studied for aza-Michael addition where
the yield declined as the chain length increased from ethyl acrylate
to butyl acrylate (Table 3, entries 8–15) except for morpholine
where no significant effect of the chain length of the acrylate
was observed on the yields of the product (Table 3, entries 11
and 15). The protocol was further extended to study aromatic
amines as a substrate but the reaction was too sluggish. It was ob-
served that an aromatic amine like aniline was not compatible as a
substrate for this particular protocol as the yield was 31% with a
long reaction time of 42 h (Table 3, entry 16).
Alternatively, the synthesis of 13 can be done by transesterifica-
tion of 3 or 9 with n-butanol under the present conditions. This
transesterification method suggests that several other alcohols
can be used to synthesize the desired product starting with a single
type of acrylate available. The reaction was conducted in two
modes, tandem and sequential. In tandem, 1 mmol of n-butanol
was added directly after the completion of the first step whereas
in sequential, the 3 or 9 was isolated and to which 1 mmol of n-
butanol was then added under the mentioned reaction conditions
as shown in Scheme 2.
Reaction conditions: acrylate (2 mmol), amine (1 mmol), toluene (3 ml), n-
butanol (1 mmol), CaL
B (30 mg), temperature (60 °C), time (3.5 h)/(24 h),
respectively.
b
Yield based on GC analysis.
acrylates providing good to excellent yield of the desired products.
Also, alternative transesterification route was introduced to syn-
thesize higher b-amino esters.
Acknowledgments
The authors are greatly thankful to UGC-SAP (University Grant
Commission, India) for providing fellowship. Also, authors express
their gratitude to Amano enzymes, Japan (Lipase PS, Lipase AYS)
and Advanced Enzyme Technologies Ltd, Mumbai, India (Lipase
Aspergillus niger) for providing lipases as generous gift samples
during this research work.
References and notes
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9045–9048.
The yield was lower for the tandem type than for the sequential
reaction mode as shown in Table 4 (entries 1–4). Also, the low yield
of 13 may be due to alcohol, released as a by-product during the
transesterification, and they are found to deactivate the enzymes.6b
During recyclability studies of the enzyme CaL B, it was ob-
served that the enzyme worked efficiently with no significant de-
crease in yield during the first cycle whereas the yield declined
up to 78% after completion of the third cycle (Table 3, entry 1).
However, this slight decrease might be due to the handling loss
of lipase during recycling.
6. (a) Reetz, M. T. Curr. Opin. Chem. Biol. 2002, 6, 145–150; (b) Dhake, K. P.;
Qureshi, Z. S.; Singhal, R. S.; Bhanage, B. M. Tetrahedron Lett. 2009, 50, 2811–
2814.
7. (a) Li, C.; Feng, X. W.; Wang, N.; Zhou, Y. J.; Yu, X. Q. Green Chem. 2008, 10, 616–
618; (b) Li, K.; He, T.; Li, C.; Feng, X. W.; Wang, N.; Yu, X. Q. Green Chem. 2009,
11, 777–779; (c) Branneby, C.; Carlqvist, P.; Magnusson, A.; Hult, K.; Brinck, T.;
Berglund, P. J. Am. Chem. Soc. 2003, 125, 874–875; (d) Wang, J. L.; Xu, J. M.; Wu,
Q.; Lv, D. S.; Lin, X. F. Tetrahedron 2009, 65, 2531–2536; (e) Cai, Y.; Wu, Q.; Xiao,
M. Y.; Lv, D. S.; Lin, X. F. J. Biotechnol. 2006, 121, 330–337; (f) De Souza, R. O. M.
A.; Matos, L. M. C.; Goncalves, K. M.; Costa, I. C. R.; Babics, I.; Leite, S. G. F.;
Oestreicher, E. G.; Antunes, O. A. C. Tetrahedron Lett. 2009, 50, 2017–2018; (g)
Torre, O.; Alfonso, I.; Gotor, V. Chem. Commun. 2004, 1724–1725; (h) Lou, F. W.;
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Hult, K.; Berglund, P. Trends Biotechnol. 2007, 25, 231–238.
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1984, 13, 1815–1818.
In conclusion, we have developed an efficient protocol for the
synthesis of aliphatic b-amino esters via aza-Michael addition be-
tween the primary and secondary amines with acrylates. The
developed methodology is applicable for a variety of amines and
O
CaL B, 60 °C,
Toluene, 3.5 h
R
O
NH
N
H
2
O
R
+
O
3 or 9
n-Butanol
CaL B, 60 °C
Toulene, 24 h
9. (a) Carlqvist, P.; Svedendahl, M.; Branneby, C.; Hult, K.; Brinck, T.; Berglund, P.
ChemBioChem 2005, 6, 331–336; (b) Strohmeier, G. A.; Sovic, T.; Steinkellner,
G.; Hartner, F. S.; Andryushkova, A.; Purkarthofer, T.; Glieder, A.; Gruber, K.;
Griengl, H. Tetrahedron 2009, 65, 5663–5668.
10. Laane, C.; Boeren, S.; Vos, K.; Veeger, C. Biotechnol. Bioeng. 1987, 81–87.
11. Southwick, C. J. Am. Chem. Soc. 1953, 75, 3413–3416.
ROH
(MeOH / EtOH)
O
O
O
NH
2
CaL B, 60 °C
Toluene, 3.5 h
O
N
H
+
13
12. In a typical experimental procedure, to 3 ml of toluene, 2 mmol of methyl
acrylate was added. The reaction was initiated by adding 30 mg of lipase which
Scheme 2. Alternative route for synthesis of (13).

4458
K. P. Dhake et al. / Tetrahedron Letters 51 (2010) 4455–4458
was stirred at 37 °C for 5 min. Then 1 mmol of diethyl amine was added and
(75 MHz, CDCl3) d = 173.3, 140.1, 128.5, 128.2, 127.1, 53.8, 51.7, 44.5, 34.6.
MS (70 eV, EI): m/z (%): 193 (15) (M+), 120 (35), 106 (65), 91 (100), 65
(20), 42 (15).
stirred for 3.5 h at 37 °C or 60 °C as specified. The progress of the reaction was
monitored by TLC analysis. After completion, the reaction mixture was filtered
and was quantitatively analyzed using gas chromatography (Chemito 1000).
The reaction mixture was then evaporated under vacuo. The residue obtained
was purified with column chromatography (silica gel, mesh size 60–120) using
chloroform/methanol (95:5) as an eluent to afford the pure products. All the
products are well known 5a,11 and were compared with the authentic samples.
The products are well characterized by 1H and 13C NMR spectra recorded on an
NMR spectrometer (Varian-300) using TMS as internal standard and mass
spectra by GC–MS (Shimadzu QP 2010) analysis.
3-Morpholin-4-yl-propionic acid methyl ester (4): 1H NMR (300 MHz, CDCl3,
25 °C, TMS) d = 2.44–2.51 (6H, m), 2.66–2.71 (2H, t, J = 7.14 Hz), 3.69–3.71
(7H, m). 13C NMR (75 MHz, CDCl3) d = 172.9, 66.9, 53.1, 53.4, 51.7, 31.9.
MS (70 eV, EI): m/z (%): 173 (15) (M+), 100 (100), 70 (15), 56 (40), 42
(30).
3-Benzylamino-propionic acid butyl ester (13): 1H NMR (300 MHz, CDCl3,
25 °C, TMS) d = 0.89–0.94 (3H, t), 1.35–1.42 (2H, m),1.55–1.62 (2H, m),
1.86 (1H, s), 2.50–2.54 (2H, t, J = 6.41 Hz), 2.87–2.91 (2H, t, J = 6.41), 3.79
(2H, s), 4.05–4.10 (2H, t, J = 6.77), 7.23–7.32 (5H, m). 13C NMR (75 MHz,
CDCl3) d = 172.9, 140.1, 128.4, 128, 126.9, 64.3, 53.8, 44.5, 34.7, 30.6, 19.1,
13.7. MS (70 eV, EI): m/z (%): 235 (10) (M+), 144 (15), 120 (45), 106 (95),
91 (100), 65 (15).
Spectral data for selected products:
3-Diethylamino-propionic acid methyl ester (1): 1H NMR (300 MHz, CDCl3, 25 °C,
TMS) d = 1.13–1.18 (6H, t, J = 7.14 Hz), 2.66–2.71 (2H, t, J = 7.69 Hz), 2.79–2.86
(4H, q, J = 7.20 Hz), 3.03–3.08 (2H, t, J = 7.51 Hz), 3.69 (3H, s). 13C NMR
(75 MHz, CDCl3) d = 172, 51.1, 46.8, 45.9, 30, 11.3. MS (70 eV, EI): m/z (%): 159
(15) (M+), 144 (25), 86 (100), 42 (20).
3-Phenylamino-propionic acid methyl ester (16): 1H NMR (300 MHz, CDCl3,
25 °C, TMS) d = 2.60 (2H, t, J = 6.42 Hz), 3.43 (2H, t, J = 6.42 Hz), 3.67 (3H,
s), 6.62 (2H, d), 6.73 (1H, t), 7.16 (2H, t). 13C NMR (75 MHz, CDCl3)
d = 172.8, 147.5, 129.2, 117.6, 112.9, 51.7, 39.3, 33.6. MS (70 eV, EI): m/z
(%): 179 (25) (M+), 106 (100), 77 (20), 65 (15), 51 (15).
3-Benzylamino-propionic acid methyl ester (3): 1H NMR (300 MHz, CDCl3,
25 °C, TMS) d = 1.94 (1H, s), 2.52–2.56 (2H, t, J = 6.41 Hz), 2.88–2.92 (2H, t,
J = 6.59 Hz), 3.68 (3H, s), 3.80 (2H, s), 7.26–7.32 (5H, m). 13C NMR