Candida antarctica lipase B-catalyzed synthesis of acetamides using [BMIm(PF6)] as a reaction medium

Article abstract of DOI:10.1016/j.tetlet.2009.03.170

An efficient protocol has been developed for synthesis of acetamides using Candida antarctica lipase B (CaL B) in [BMIm(PF₆)] as a greener reaction medium. The reaction is applicable to a wide variety of aliphatic esters/acetic acid and amines providing excellent yields of corresponding acetamide. The catalyst exhibits remarkable activity and is reusable for up to four consecutive cycles.

Full text of DOI:10.1016/j.tetlet.2009.03.170

Tetrahedron Letters 50 (2009) 2811–2814
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Tetrahedron Letters
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Candida antarctica lipase B-catalyzed synthesis of acetamides using
[BMIm(PF6)] as a reaction medium
Kishor P. Dhake a, Ziyauddin S. Qureshi a, Rekha S. Singhal b, Bhalchandra M. Bhanage a,
*
a Department of Chemistry, Institute of Chemical Technology, Matunga, Mumbai 400019, India
b Department of Food Engineering and Technology, Institute of Chemical Technology, Matunga, Mumbai 400019, 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 has been developed for synthesis of acetamides using Candida antarctica lipase B
(CaL B) in [BMIm(PF6)] as a greener reaction medium. The reaction is applicable to a wide variety of ali-
phatic esters/acetic acid and amines providing excellent yields of corresponding acetamide. The catalyst
exhibits remarkable activity and is reusable for up to four consecutive cycles.
Received 3 February 2009
Revised 19 March 2009
Accepted 26 March 2009
Available online 28 March 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Candida antarctica lipase B (CaL B)
Amidation
Ionic liquids
Acetamide-synthesis
Enzymatic synthesis
Biocatalysis in non-aqueous medium replacing the aqueous
ones has been widely discussed for more than two decades. How-
ever, use of the ionic liquids (ILs) as a reaction medium for bioca-
talysis has attracted great attention in recent years, as the
processes are green and ILs have several advantages compared to
conventional reaction media.1–4 Room temperature ionic liquids
(RTILs) are organic salts, which are liquids at room temperature.
Their unique properties such as non-volatility, non-flammability,
excellent chemical and thermal stability, high polarity, and recy-
clability, have made them a promising replacement for traditional
volatile organic solvents.5,6 By varying the cations and anions, the
physico-chemical properties of ILs such as hydrophobicity, density,
viscosity, melting point, polarity, and solvent properties may be
tailored in many ways and thus can be made compatible with var-
ious biocatalytic processes.6,7
which require an alternative non-conventional medium such as io-
nic liquids. Lipase-catalyzed reactions in ILs have been found to be
advantageous over those in conventional reaction medium, accom-
panied by increased activity, enantioselectivity and stability.10–12
After the first successful report by Erbeldinger et al.13 on thermol-
ysin-catalyzed synthesis of Z-aspartame in [BMIm(PF6)]/H2O, the
work from Sheldon and co-workers was the second publication
to demonstrate the potential use of ILs for enzymatic catalysis
and they were the first to use lipases.14
An exhaustive research work related to esterification and
transesterification catalyzed by lipase has been done but less
attention subsists toward amidation reactions. Though some re-
ports on Candida antarctica lipase-catalyzed amidation in organic
solvent exist,15 the area of amidation reaction using esters/acetic
acid as acyl donors remains unexplored. Considering this issue,
we have focused our attention toward the synthesis of acetamides.
Acetamides, being biologically active, have been widely used as
building blocks for pharmaceutical drugs,16 clinical applications,17
enzyme inhibitors,18 and herbicides.19
We, herein, report an efficient protocol for the synthesis of
acetamides from esters and acetic acid as acyl donors with ali-
phatic amines in [BMIm(PF6)] as a reaction medium using CaL B
as a biocatalyst. The catalyst showed remarkable activity and is
applicable to a wide variety of aliphatic esters/acetic acid and
amines to yield the corresponding acetamides (Scheme 1).
In order to optimize the reaction conditions, reaction of benzyl
acetate with benzyl amine in presence of CaL B was chosen as a
model reaction.22 The type of C. antarctica lipase B enzyme used
Klibanov8 studied the utility of enzymes in organic solvents and
reported the non-aqueous ammonolysis reaction of methyl buty-
rate with butylamine using porcine pancreatic lipase. Though the
use of organic solvents for enzymatic transformation played an
important role, their use has raised a lot of questions regarding
the environmental hazards, safety, and health issues.9 When using
organic solvents at industrial scale, their post treatment in terms of
evaporation and recycling becomes more expensive and involves
tedious procedures. Also for many biocatalytic processes, sub-
strates and products have low solubilities in aqueous medium,
* Corresponding author. Tel.: +91 22 2414 5616; fax: +91 22 2414 5614.
E-mail address: bhalchandra_bhanage@yahoo.com (B.M. Bhanage).
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.03.170

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K. P. Dhake et al. / Tetrahedron Letters 50 (2009) 2811–2814
when molar ratio of benzyl acetate/benzyl amine (1.2:1) was used
O
whereas the equimolar concentration of substrates (1:1) yielded
81% of acetamides (Table 1, entry 11). The control reaction exper-
iments were also carried out in absence of the enzyme keeping
other reaction parameters constant, which demonstrated that
only the enzyme CaL B is responsible for the respective transfor-
mations (Table 1, entries 12 and 13). Hence, the optimum reac-
tion parameters were: ionic liquid [BMIm(PF6)] 0.5 ml,
temperature 60 °C, CaL B loading 20 mg, and time 24 h. Optimized
reaction conditions were subsequently applied for amidation of
various esters or acetic acid with different aliphatic and alicyclic
amines providing moderate to good yields of the desired products
(Table 2, entries 1–19).
It was observed that CaL B has the potential to catalyze the reac-
tions of four different esters as acyl donors with a comparative
high yield with butyl acetate (Table 2, entries 1–4). On the other
hand, CaL B was effective as catalyst only with aliphatic amines.
The reaction was smooth for simple aliphatic amines whereas yield
was very poor for the alicyclic amines. n-Butylamine and n-hexyl-
amine were found to react smoothly with benzyl acetate providing
good yields of the desired acetamides (Table 2, entries 5 and 6)
whereas with cyclohexylamine the yield had decreased (Table 2,
entry 7). The system also permits the reaction of benzyl acetate
with secondary amines such as piperidine with moderate yields
of the corresponding acetamide (Table 2, entry 8). The yield de-
creases with butylacetate as we move down from n-hexylamine
to cyclohexylamine (Table 2, entries 10–12). Vinyl acetate is the
most commonly used acyl donor for lipase-catalyzed acylation or
esterification reactions.3 Vinyl acetate has been observed to be an
efficient donor with benzyl amine as well as with long chain ali-
phatic dodecamine for acetamide-synthesis producing moderate
to good yields (Table 2, entries 4 and 13). Also ethyl acetate served
as a good acyl donor producing corresponding acetamide (Table 2,
entry 14).
Carboxylic acid has scarcely been used as acyl donor as it is sup-
posed that the spontaneous formation of unreactive salt with
amine would prevent the formation of the acyl–enzyme complex.
However, the salt formation is reversible reaction. Montet et al.24
demonstrated that the amide bond was synthesized by lipase in
hexane, though with a very low reaction rate of 60% conversion
of amine in 12 days. Also it is noteworthy to mention that while
using aliphatic acids25 with racemic 2-ethylhexyl amine, no amida-
tion reaction had occurred in organic solvents, i.e., diisopropyl
ether, tetrahydrofuran, and acetonitrile using CaL B and hence it
was carried out in bulk (without solvent) at 90 °C under vacuum.
In contrary, the use of acetic acid as an acyl donor has been found
to be effective for acetamide-synthesis in ionic liquid [BMIm(PF6)].
For these particular transformations, water was produced instead
of alcohols and it had no adverse effects on the enzyme activity.
Acetic acid as acyl donor reacted smoothly with the aliphatic
amines producing the good yields of the corresponding acetamides
(Table 2, entries 15 and 16). However, as like with esters, the acetic
acid when reacted with secondary amines such as morpholine and
pyridine gave lower yields of 40% and 62%, respectively.(Table 2,
entries 17 and 18).
O
H
R2
CaL B
R2
H3C
N
N
[BMIm(PF6)], 60oC
+
H3C
OR
R1
R1
R= Benzyl, vinyl, ethyl, butyl, H.
R1, R2 = H,alkyl
Scheme 1. CaL B-catalyzed synthesis of acetamides in [BMIm(PF6)].
was lipase immobilized on acrylic resin from C. antarctica with
P10,000 U/g, recombinant, and expressed in Aspergillus oryzae.
Hence, the amount of enzyme equivalent units used for this study
is 200 U. The influence of several parameters such as solvent, tem-
perature, catalyst loading, time, and mole ratio of substrates has
been studied (Table 1, entries 1–13).
Lipase-catalyzed reactions are markedly influenced by the reac-
tion media. Recent studies,4,10,11 have also shown that the activity,
stability, and selectivity of the enzymes are considerably enhanced
by changing the reaction medium. As compared with organic sol-
vents and ionic liquids, high activity was observed with
[BMIm(PF6)] (Table 1, entries 1–4). Low yield with [BMIm(BF4)]
may be because it is prone to desorb water from the enzyme sur-
face and thus decrease the enzyme activity.20 Also the half-life per-
iod of [BMIm(PF6)] is greater than that of the other two ILs. The
effect of temperature on the activity of CaL B for acetamide synthe-
sis was studied in [BMIm(PF6)] by varying temperatures ranging
from 30 °C to 80 °C. It was observed that with increase in temper-
ature the yield of acetamide increases initially (Table 1, entries
5–7). However, at higher temperature, i.e., 80 °C the yield
decreases (Table 1, entry 7), reflecting that 60 °C is the optimum
temperature. In an effort to determine the optimum concentration
of the catalyst, various amounts of catalyst loadings were deliber-
ated as shown in Table 1 (entries 8 and 9). The optimum results
were observed with respect to yield 94% (Table 1, entry 3), dem-
onstrating that further increase in the catalyst concentration has
no significant effect on the yield of acetamides. A remarkable
increase in yield, that is, 94% (Table 1, entry 3) was obtained
Table 1
Effect of reaction parameters on amidation reaction catalyzed by CaL Ba
Entry
Solvent
Temp (°C)
Catalyst loading (mg)
Yieldb (%)
Influence of solvent
1
2
3
4
[BMIm(BF4)]
[BMIm(NTF2)]
[BMIm(PF6)]
60
60
60
60
20
20
20
20
76
80
94
66
Di-iso propyl ether
Influence of temperature
5
6
7
[BMIm(PF6)]
[BMIm(PF6)]
[BMIm(PF6)]
30
45
80
20
20
20
51
62
81
Influence of catalyst loading
The aromatic amines such as aniline were not compatible as
substrates for this particular protocol (Table 2, entries 9 and 19).
Basicity is one of the key factors differentiating the reactivity of ali-
phatic amines and aromatic amine, for example, aniline. It is well
known that aromatic amines are weak bases due to the aromatic-
ity. We think that it may be contributing to the results obtained in
present work. In case of acetic acid as an acyl donor in the reaction
with aniline, a low yield of 20% was obtained as compared with
benzyl acetate wherein the reaction did not proceed. Again this
may be due to the steric hindrance to N lone pair between these
two moieties.
8
9
[BMIm(PF6)]
[BMIm(PF6)]
[BMIm(PF6)]
[BMIm(PF6)]
[BMIm(PF6)]
[BMIm(BF4)]
60
60
60
60
60
60
10
30
20
20
—
70
95
76
81
0
10c
11d
12e
13e
—
0
a
Reaction conditions: Benzyl acetate (0.3 mmol), benzyl amine (0.25 mmol),
solvent (0.5 ml), catalyst (CaL B lipase), molecular sieves 4 Å (35 mg), time = 24 h.
b
Yields based on GC analysis.
Time = 18 h.
Benzyl acetate/benzyl amine molar ratio is 1:1.
Without catalyst.
c
d
e

K. P. Dhake et al. / Tetrahedron Letters 50 (2009) 2811–2814
2813
Table 2
Reactions of esters/acetic acid with amines catalyzed by CaL B in [BMIm(PF6)]a
Table 2 (continued)
b
No.
Ester/Acid
Amine
Product
Yield (%)
b
No.
Ester/Acid
Amine
Product
Yield (%)
O
NH
2
NH2
2
O
O
O
O
O
16
84 (79)
40 (35)
NH2
NH2
NH2
OH
O
1
94 (90)
O
O
N
H
O
O
H
N
O
N
O
17
18
OH
2
3
97 (92)
82 (77)
N
H
O
H
N
O
O
O
N
62 (57)
20 (14)
N
H
OH
H
N
O
NH2
O
O
O
NH2
19
4
5
6
7
8
9
64 (61)
87 (82)
87 (84)
32 (27)
61 (57)
0
N
H
OH
O
a
Reaction conditions: Acetate/acetic acid (0.3 mmol), amine (0.25 mmol),
[BMIm(PF6)] (0.5 ml), CaL B (20 mg), molecular sieves 4 Å (35 mg), time = 24 h.
O
O
O
O
O
NH
NH2
b
Yields based on GC analysis, the yields given in the parentheses are the isolated
2
O
O
O
O
O
2
O
O
yields.
NH
NH2
NH2
4
The reaction mechanism involves covalently linked acyl–en-
zyme intermediate that deacylates via nucleophilic attack of
amine. Generally one of the possible reasons for lower yield with
alicyclic amines is that lipase, being a serine hydrolase, depends
on the accessibility of the nucleophile to attack and cleave the
acyl–serine bond inside the cavity of the active site. Lipase usually
discriminates against certain acyl chain length, degrees of unsatu-
ration and location of double bond in the chain. Any of these fac-
tors could affect the interaction between the acyl chain and the
active site. Also, the release of smaller alcohols may cause deacti-
vation of the enzyme to some extent; similar results were observed
with esterification using goat pregastric lipase where the rate of
formation of ethyl butyrate was found to be lower than that of bu-
tyl butyrate.21a Hence, the yield was more for butyl acetate than for
other acetates. Also, the alicyclic amines find it difficult to ap-
proach the catalytic site of the enzyme due to their size and thus
produce a poor yield.21b In addition, cyclic structures could pro-
duce steric hindrance thus reducing the product yield.
4
H
N
O
O
H
N
N
H
N
NH2
O
O
O
NH
NH2
10
11
91 (88)
64 (59)
4
4
O
In order to make the biocatalytic processes economical at large
scale, the recyclability of the enzyme in ionic liquids has to be ta-
ken into consideration.23 During this study, enzyme–ionic liquid
reaction system was recycled up to four cycles. There was no sig-
nificant decrease during first cycle whereas the yield declined up
to 65% after the completion of the fourth cycle as shown in Figure
O
O
O
H
N
N
H
N
NH2
O
92
100
80
60
40
20
0
88
12
35 (28)
78
O
O
65
O
O
NH
10
NH2
10
13
14
95 (90)
44 (39)
O
O
O
H
N
O
O
N
O
I
II
III
IV
O
Recycle No.
O
NH2
Figure 1. Reaction conditions: Benzyl amine (0.25 mmol), benzyl acetate
(0.3 mmol), 0.5 ml of [BMIm(PF6)], 20 mg CaL B, 35 mg molecular sieves 4 Å at
temp = 60 °C for time = 24 h.
15
96 (91)
N
H
OH

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K. P. Dhake et al. / Tetrahedron Letters 50 (2009) 2811–2814
Tetrahedron: Asymmetry 1997, 8, 2099–2100; (g) Gotor-Fernández, V.; Busto,
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Epilepsia 2001, 42, 825–830.
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1. This decrease in yield might be due to handling loss of the en-
zyme during recycles or inactivation of the enzyme as the cycles
are increased.
In conclusion, we have studied the behavior of CaL B in ionic
liquid for acetamide-synthesis. The ionic liquid has influenced
the stability and yield of the acetamides with considerable recy-
clability. These advantages thus suggest that ionic liquid would
be used as green alternative reaction medium for acetamide-
synthesis.
19. Jeffrey, A.; Bunting, F.; Simmons, W.; Wax, L. M. Weed Technol. 2003, 17, 291–
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T.; Maeno, S.; Takaya, Y.; Sakai, T.; Utaka, M. J. Org. Chem. 1996, 61, 8610–8616.
22. In a typical experimental procedure, to 0.5 ml of ionic liquid, 0.3 mmol of
benzyl acetate and 0.25 mmol of benzyl amine were added. In order to
maintain the water activity of enzyme 70 mg/ml of molecular sieves 4 Å was
added. The reaction was initiated by adding 20 mg of the enzyme and reaction
mixture was stirred at 60 °C for 24 h. After completion, 2–3 ml of di-isopropyl
ether was added and vigorously shaken to extract all the reactants and
products in the ether phase. The extraction procedure was repeated for about
3–4 times. The combined organic extracts were dried over Na2SO4 and the
solvent was evaporated in vacuo. The residue obtained was purified using
column chromatography (silica gel, mesh size 60–120) using pet ether/ethyl
acetate (90:10) as eluent to afford the pure products. The products were known
and were compared with authentic samples. The products are characterized by
1H NMR (JEOL FT-NMR, Model-AL300), GC–MS (Shimadzu QP 2010), FT-IR
(Perkin Elmer).
Acknowledgment
The author (K.P.D.) is greatly thankful to the UGC-SAP (Univer-
sity Grant Commission, India) for providing him the fellowship
during this research work.
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Spectral data for selected products:
Benzyl acetamide, Table 2, entry 1: 1H NMR (300 MHz, CDCl3, 25 °C, TMS):
d = 1.99 (s, 3H), 4.34 (s, 2H), 6.75 (br s, 1H), 7.24 (s, 5H) ppm. MS (70 eV, EI): m/
z (%): 149 (75) (M+), 106 (100), 91 (35). IR (KBr):
m = 1650 (CO), 3287 (NH).
n-Hexyl acetamide, Table 2, entry 6: 1H NMR (300 MHz, CDCl3, 25 °C, TMS):
d = 0.88 (s, 3H), 1.28 (s, 6H), 1.43–1.49 (t, 2H), 1.97 (s, 3H), 3.19–3.25 (q, 2H),
5.90 (br s, 1H) ppm. MS (70 eV, EI): m/z (%): 143 (20) (M+), 128 (15), 72 (90), 43
(100). IR (KBr):
m = 1652 (CO), 3274 (NH).
Cyclohexyl acetamide, Table 2, entry 7: 1H NMR (300 MHz, CDCl3, 25 °C, TMS):
d = 1.09–1.68 (m, 10H), 1.95 (s, 3H), 3.48 (s, 1H), 5.43 (br s, 1H) ppm. MS
(70 eV, EI): m/z (%): 141 (20) (M+), 98 (15), 60 (100), 43 (50). IR (KBr):
m = 1637
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´
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d = 2.10 (s, 3H), 3.45–3.70 (m, 8H) ppm. MS (70 eV, EI): m/z (%): 129 (35) (M+),
114 (20), 86 (50), 57 (100), 43 (85). IR (KBr):
m = 1630 (CO).
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