Investigation of the lipase-catalysed reaction of aliphatic amines with ethyl propiolate as a route to N-substituted propiolamides

Article abstract of DOI:10.1016/j.tet.2013.04.093

The lipase-catalysed reaction of aliphatic amine with ethyl propiolate was investigated using benzylamine as reference amine. The conditions were optimised to favour the 1,2-addition, i.e., formation of N-benzylprop-2-ynamide, versus the 1,4-addition. Immobilised Candida antarctica lipase (CALB) was found to be the most efficient enzyme, and the reactions were performed in solvents, such as tBME, dioxane or toluene. The methods were used to prepare propiolamides from aliphatic amines in good to excellent yields. The reactivity of O- and S-nucleophiles was compared in the same conditions.

Full text of DOI:10.1016/j.tet.2013.04.093

Tetrahedron 69 (2013) 5495e5500
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Investigation of the lipase-catalysed reaction of aliphatic amines
with ethyl propiolate as a route to N-substituted propiolamides
,
y
Simon Bonte a, Ioana Otilia Ghinea b, Isabelle Baussanne a, Jean-Paul Xuereb a
,
,
Rodica Dinica c, Martine Demeunynck a
*
a Department of Pharmacomolecular Chemistry, UMR 5063 & FR 2607, CNRS, Joseph Fourier University, BP 53, 38041 Grenoble cedex 9, France
b Dunarea de Jos University of Galati, Faculty of Food Science and Engineering, Department of Food Science,
Food Engineering and Applied Biotechnology, 111 Domneasca Street, Galati, Romania
c Dunarea de Jos University of Galati, Faculty of Science and Environment, Department of Chemistry, Physics and Environment, 111 Domneasca Street,
800201 Galati, Romania
a r t i c l e i n f o
a b s t r a c t
Article history:
The lipase-catalysed reaction of aliphatic amine with ethyl propiolate was investigated using benzyl-
amine as reference amine. The conditions were optimised to favour the 1,2-addition, i.e., formation of N-
benzylprop-2-ynamide, versus the 1,4-addition. Immobilised Candida antarctica lipase (CALB) was found
to be the most efficient enzyme, and the reactions were performed in solvents, such as tBME, dioxane or
toluene. The methods were used to prepare propiolamides from aliphatic amines in good to excellent
yields. The reactivity of O- and S-nucleophiles was compared in the same conditions.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 19 March 2013
Received in revised form 14 April 2013
Accepted 18 April 2013
Available online 25 April 2013
Keywords:
Activated alkyne
Candida antarctica
Aminolysis
Lipase
1. Introduction
Therefore, propiolamides are generally prepared from propiolic
acid with intermediate formation of activated esters.9 Recently, an
With the development of the biocompatible click reactions,
molecules containing activated electron-deficient or strain alkynes
are becoming very attractive synthons to functionalise bio-
molecules. Propiolic acid derivatives, both esters and amides, have
gained interest because of their good reactivity as dipolarophiles
for metal free click chemistry1e3 and in other cycloaddition re-
actions.4 These electron-deficient alkynes are useful reactants for
the Huisgen cycloaddition with azide, performed at room temper-
ature in water and in absence of metal catalyst.5
efficient synthesis of propiolamides was reported using trime-
thylsilyl propiolic acid as starting material.10,11
In an effort to design alternative methodologies for the prepa-
ration of propiolamides, we turned our attention to the lipase-
catalysed reactions. The biological role of lipases is the hydrolysis
of aliphatic esters in aqueous media, but they may also efficiently
catalyse the reverse reaction of esterification in non-aqueous media
(organic solvents, ionic liquids). Therefore lipases were optimised
to catalyse numerous reactions of industrial interest. Immobilisa-
tion of lipases confers to the enzymes a good stability that enables
their use in organic solvents and temperatures (up to 60 ꢀC for
Candida antarctica lipase B for instance). The separation steps are
also simplified thus allowing the development of continuous pro-
cesses.12,13 The enzyme-catalysed reactions of acrylate esters with
amines and alcohols have been studied in detail,13,14 but so far there
are only a few reports of lipase-catalysed reactions of propiolic
ester. In 1989, Gotor successfully realised the aminolysis of ethyl
propiolate with aniline in the presence of Candida cylindracea lipase
(now known as Candida rugosa lipase or CRL) in CCl4.15,16 However,
the authors reported an important limitation, the aliphatic amines
only giving the 1,4-addition in the same conditions. It was also
mentioned that the aminolysis did not occur with porcine
Preparation of propiolic esters,6,7 thioesters and amides gener-
ally involves the use of coupling reagents (DCC or EDC carbodii-
mides for examples) and the reactions proceed with low to good
yields. There are only few examples of the use of propiolyl chloride,
prepared from propiolic acid and PCl5 and immediately engaged in
the next reaction8 with alcohol or amine. Propiolic anhydride,
generated in situ from propiolic acid and DCC, has been reported for
synthesis of propiolamides, although with moderate yields.
* Corresponding author. Tel.: þ33 (0)476635314; e-mail address: martine.de-
meunynck@ujf-grenoble.fr (M. Demeunynck).
y
ꢀ
Present address: Lycee franco-libanais MLF-AEFE, Verdun Beyrouth, Lebanon.
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.04.093

5496
S. Bonte et al. / Tetrahedron 69 (2013) 5495e5500
pancreatic lipase or papain. Later, the authors published the ami-
nolysis with ammonia in dioxane catalysed by C. antarctica lipase.17
When the current study was on-going, an example of trans-
esterification of alcohols with ethyl acetylene dicarboxylate and
propiolate was published by Deloisy and collaborators.18 These
reactions were catalysed by C. rugosa lipase (CRL) using petroleum
ether as solvent.
trans-Z,E-dienamine 5 was isolated as the sole product.23e25 We
checked the uncatalysed reactivity of benzylamine with ethyl pro-
piolate in the conditions that were later used in our study (stoichi-
ometry, solvent, temperature). As shown in Table 1 (entry 27), we
obtained a mixture of the enaminoesters 3 and 4 in a 67/23 Z/E ratio.
In this paper, we report the results of our study of the lipase-
catalysed reactivity of ethyl propiolate with aliphatic amines.
Benzylamine was used as a typical amine. We first screened the
ability of a panel of lipases to catalyse the chemoselective 1,2-
addition. The reactions were performed in various organic sol-
vents in order to select the best conditions (enzyme/solvent couple)
that were then applied to the preparation in larger scale of pro-
piolamides that will be further used as dipolarophiles in coupling
reactions in our group. The chemoselectivity was also checked by
comparing the reactivity with O- and S-nucleophiles.
Table 1
Reaction of benzylamine with ethyl propiolate. Influence of the nature of the en-
zyme and of the solvent on the chemoselectivity
Entry
Lipase
Solvent
2
3
4
5
1
2
3
4
5
6
7
8
CRL
CRL
CRL
CRL
CRL
CRL
CALB
CALB
CALB
CALB
CALB
CALB
CALB
CALB
PPL
PPL
PPL
PPL
PPL
PPL
PPL
PPL
P. cepacia
P. cepacia
LIP
LIP
No enzyme
EOP
0
0
0
0
20
7
0
0
0
93
78
39
93
98
2
0
0
6
0
63
59
37
55
50
54
25
67
18
2
37
41
nd
38
30
37
nd
33
0
0
0
48
6
0
1
59
0
63
4
13
4
Traces
0
0
0
1
0
3
23
0
2
Dioxane
Isooctane
CH3CN
Toluene
tBME
EOP
MOI
MOP
Dioxane
Isooctane
CH3CNa
Toluene
tBME
EOP
MOI
MOP
Dioxane
Isooctane
CH3CN
Toluene
tBME
Dioxane
tBME
Dioxane
tBME
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
0
2. Results and discussion
4
35
7
Traces
21
0
Using enzymes in organic solvent is a large field of research to
develop alternative procedures to known organic reactions. The
difficulty is the numbers of parameters that influence the chemo-,
regio- and stereoselectivities, such as enzyme nature, temperature,
dilution, solvent or stoichiometry. We took as starting conditions,
the data reported by Gotor’s13,14 and Castillo’s19 groups for the li-
pase-catalysed reaction of nucleophiles (alcohols or amines) with
acrylic esters. We focused our study on the influence of the nature
of the solvent and of the enzyme, which clearly appeared as key
factors for the selectivity.
As shown in Scheme 1, controlling the chemoselectivity of the
addition of amine to ethyl propiolate is challenging as this reaction
may lead to four main compounds: propiolamide 2 resulting from
the 1,2-addition, Z- or E-aminoacrylates 3 and 4 resulting from the
1,4-addition, and s-trans-Z,E-dienamino ester 5 issued from two
successive 1,4-additions.
2
0
61
59
62
58
57
41
59
50
67
56
51
19
63
36
41
37
35
39
29
41
35
33
35
30
11
27
0
0
8
0
5
15
63
0
0
4
3
5
Dioxane
0
Benzylamine (0.2 mmol) was added to a suspension of the chosen enzyme (80 mg)
in 1 ml of the chosen solvent. The mixture was shaken for 1 min before adding ethyl
propiolate (0.6 mmol). The reactions were performed in a flask gently stirred at
40 ꢀC for 15 h otherwise mentioned. The ratios in the different compounds were
determined from the 1H NMR integrations.
a
Presence of by-products suggested by a series of multiplets between 6 and
8 ppm in the 1H NMR spectrum. Enzymes: Candida rugosa lipase (CRL), porcine
pancreatic type II lipase (PPL), Candida antarctica lipase B (CALB), lipozyme RM (LIP)
and Pseudomonas cepacia lipase. tBME¼tert-butyl methyl ether, EOP¼EOPipNTf2,
MOP¼MOPyrroNTf2 and MOI¼MOImNTf2.
We then performed the reaction following the conditions re-
ported by Gotor15,16 for the lipase-catalysed amidation of aniline
with ethyl propiolate, using C. cylindracea lipase (now known as C.
rugosa (CRL)) in CCl4. We found out that CRL catalysed the amida-
tion of benzylamine in CCl4, but with a low conversion rate (30%
after 5 h of stirring at 60 ꢀC).
Scheme 1. Reactivity of benzylamine 1 with ethyl propiolate 2: structures of major
In the search of more efficient catalysts, a selection of enzymes
was evaluated in different solvents. The reactions were performed
at 40 ꢀC overnight (15 h) in open round-bottom flasks, and
quenched by filtration of the enzyme. The reaction mixtures were
then quantified by 1H NMR. The results are collected in Table 1. In all
the tested conditions, the conversion was quantitative. The course
of the reaction was strongly dependent of both the nature of the
enzyme and of the solvent. The 1,2-addition was favoured by the
use of the less polar solvents (entries 10, 13, 14 or 26), the best
selectivity being obtained in toluene, dioxane or tert-butyl methyl
ether (tBME). Ionic liquids only favoured 1,4-addition (entries 1,
7e9 or 15e17). From the five enzymes tested in this study, CALB
emerged as the enzyme of choice with near chemoselective 1,2-
addition in dioxane and tBME (entries 10, 13 and 14). As found
earlier in CCl4, CRL gave a low ratio of 1,2-addition but only in
toluene (entry 5). Lipozyme (LIP) was also able to catalyse the
reaction products.
The reactions were monitored by TLC and the product mixtures
were identified by 1H NMR by comparison with literature data.
Relative yields in the different products were determined using the
following typical 1H NMR signals in CDCl3 of each product: CH
singlet at 2.85 ppm (4.17 ppm in DMSO-d6) and the CH2 doublet at
4.25 ppm for 2; CH multiplet at 6.92e6.97 ppm, the NH multiplet at
8.13 ppm and CH2 doublet at 4.25 ppm for 3; CH multiplet at
7.61e7.66 ppm for 4 and CH doublet at 6.00 ppm (1H, d) and broad
NH multiplet at 9.3 ppm for 5.
In the absence of catalyst, amines are known to quickly react with
ethyl propiolate in a 1,4-Michael addition.20e22 The enaminoesters
3/4 have thus been prepared in excellent yields in polar solvents
(H2O, CH3CN, MeOH, DMF) in less than 1 h at room temperature,
with a Z/E ratio¼2/1. At higher temperatures (over 60 ꢀC), the s-

S. Bonte et al. / Tetrahedron 69 (2013) 5495e5500
5497
amide formation albeit with lower selectivity compared to CALB
(63% in tBME, see entry 26). Two enzymes, Pseudomonas cepacia
and porcine pancreatic lipase (PPL), did not catalyse the 1,2-
addition, as 2 was only obtained as traces in dioxane and tBME
with PPL (entries 18 and 22), or in tBME with P. cepacia (entry 24).
In order to optimise the procedure, CALB was therefore selected
for further reactions. As shown in Table 2, the reactions were per-
formed in various conditions in the solvents selected above (tBME,
dioxane or toluene). Rising temperature up to 60 ꢀC had no sig-
nificant effect on the chemoselectivity (compare entries 1 and 4). In
an effort to rapidly test a variety of conditions the reactions were
performed either in 1.5 ml microtubes and stirred in an orbital
shaker (Thermoshaker) settled at the desired temperature, or
magnetically stirred in an oil bath. Surprisingly, we observed that
the stirring process had an effect on both conversion ratio and
chemoselectivity. Gentle magnetic stirring (250 rpm) was the most
efficient process with complete conversion after 15 h (overnight)
reaction at 50 ꢀC (entries 5, 8 and 10). With the orbital shaker, the
reaction kinetics appeared slower, and did not always go to com-
pletion, even when reaction time was extended to 48 h (see, e.g.,
the entries 1e3). This result emphasises the need of efficient stir-
ring process allowing contact of the reactants with the immobilised
enzyme, taking into account that the uncatalysed reaction proceeds
quickly in solution. The reactions worked well in the three solvents.
Using dry dioxane only slightly influenced the course of the re-
action in favour of the 1,2-addition (compare entries 9 and 11).
Increasing the reaction time had a low effect on the amide forma-
tion, and confirmed the stability of the amide thus formed in these
conditions, however, with time the enamides 3 and 4 slowly dis-
appeared in favour of the amino-diene 5 (compare entries 6 and 7).
As the uncatalysed 1,4-addition is a fast process, we thought that
the order in which the reactants were added to the enzyme sus-
pension might influence the product mixture. In our procedure the
enzyme was suspended in the solvent and the first reactant, ben-
zylamine, was added. The mixture was shaken for 1 min, before
adding ethyl propiolate. In one experiment (entry 12), the ethyl
propiolate was added at first to the lipase suspension and, indeed,
slightly lower yield of amide 2 (compare entries 1 and 12) was
obtained. At the opposite, increasing the time during which ben-
zylamine was left in contact with the enzyme (5 min instead of
1 min) was in favour of amide formation (compare entries 1 and
13). From these experiments, we can hypothesise that the
interaction of benzylamine with the enzyme decreases the amount
of free amine, and therefore limits the non-catalysed 1,4-addition
that quickly occurs in solution in the presence of ethyl propiolate.
The optimised procedure for propiolamide formation was
therefore the CALB catalysed reaction in tBME or dioxane at 50 ꢀC
overnight (15 h), under magnetic stirring.
The optimised conditions were applied to the scale-up of N-
benzyl propionamide 2 synthesis. The CALB catalysed reaction of
8.16 mmol of benzylamine with 2 equiv of ethyl propiolate in tBME
gave the desired propionamide 2 in 67% yield as a pale yellow oil
and the s-trans-Z,E adduct 5 in 15% yield as a solid. To evaluate the
scope and limits of this process, the reaction was also performed at
1.5 mmolar scale with various amines (Table 3). Dioxane was cho-
sen as solvent for its higher solubility properties.
Table 3
Scope of the process
Entry
Starting amines
Main reaction products (isolated yields)
1
2
3
4
5
H2NCH2CO2H 14
No reaction
6
Table 2
CALB catalysed reaction of benzylamine with ethyl propiolate: process optimisation
Entry
Solvent
T (ꢀC)
Time (conversion)
2
3
4
5
The reactions with CALB were performed at 1.5 mmol scale of amine in dioxane, at
50 ꢀC overnight using 3 equivalents of ethyl propiolate.
1
2
3
4
5
6
7
8
tBME
tBME
tBME
tBME
50 ꢀC
5 h (90%)
74
83
83
75
98
58
62
93
33
93
53
66
90
14
0
0
11
0
17
7
7
22
2
25
20
6
8
0
0
7
0
9
0
0
6
0
3
50 ꢀC
24 h (89%)
48 h (90%)
5 h (100%)
15 h (100%)
24 h (95%)
48 h (94%)
15 h (100%)
24 h ((90%)
15 h (100%)
24 h (100%)
5 h (90%)
16
16
5
50 ꢀC
60 ꢀC
The naphthalimide derivative 7 is of interest for us, and the
propiolamides 9, 11 and 13 have been described as intermediates in
the synthesis of more complex molecules.
tBME
50 ꢀC/MS
50 ꢀC
2
Toluene
Toluene
Toluene
Diox.
nd
30
0
37
4
11
8
0
50 ꢀC
The reaction worked well with primary amines and was com-
patible with the presence of larger aromatic substituent (entries 1
and 2). In the case of tryptamine (entry 2), the lower ratio of con-
version was probably due to limited solubility of the starting amine
that was recovered in 25% yield after treatment. Compound 11
(entry 3) that was formed in excellent yield, is of major interest as
bifunctional reagent for orthogonal click chemistry. Surprisingly, no
reaction occurred with the amino acid 14 (entry 5). The starting
amine was recovered after washing of the supported enzyme with
methanol. As exemplified by entry 6, the reaction of secondary
amine only gave the 1,4-addition with formation of the E isomer as
the major product.26
50 ꢀC/MS
50 ꢀC
9
10
11
12
13
Diox.
50 ꢀC/MS
50 ꢀC
Dry diox.
tBMEa
tBMEb
11
12
3
50 ꢀC
50 ꢀC
5 h (90%)
Unless otherwise mentioned the reactions were performed by adding benzylamine
(0.2 mmol) to the suspension of CALB (80 mg) in the solvent, the mixture was
shaken for 1 min before adding ethyl propiolate (0.6 mmol). The mixtures were then
kept at the chosen temperature in a thermoshaker unless otherwise mentioned (MS
for magnetic stirring). The ratios in the different compounds were determined from
the 1H NMR integrations.
a
Ethyl propiolate added at first to the enzyme suspension, the mixture was
shaken for 1 min and then benzylamine was added.
b
The chemoselectivity of CALB catalysed reactions with ethyl
propiolate was further investigated by comparing the reactivity with
The mixture of benzylamine and CALB was shaken 5 min before adding ethyl
propiolate. tBME¼tert-butyl methyl ether.

5498
S. Bonte et al. / Tetrahedron 69 (2013) 5495e5500
O- and S-nucleophiles, using benzyl alcohol and benzyl mercaptan,
under similar conditions. A first example of enzyme-catalysed
transesterification with ethyl propiolate has been recently pub-
lished18 using C. rugosa lipase (CRL) in petroleum ether. The present
study showed that CALB also efficiently catalysed the reaction. The
reaction of benzyl alcohol with ethyl propiolate was performed at
50 ꢀC for 6 h in various solvents. As shown in Table 4, gentle mag-
netic stirring clearly emerged as the most effective process, with
toluene as organic solvent. The alcohol-ester equilibrium may be
displaced using 5 A molecular sieves to trap ethanol, but we ob-
served that using open glassware (round-bottom flask or test tube),
allowed evaporation of the ethanol released during the reaction and
displaced the equilibrium in favour of ester 18.
24 are characterised by pairs of doublets at 6.68 and 5.79 (J 15 Hz)
for 23 and 7.1 and 5.8 (J 10 Hz) for 24, and the doublet at 3.7 ppm of
the benzylic protons of thiol 21 is replaced by a singlet at 4.02 ppm.
The propargylic proton of the thioester 22 is expected at 3.3 ppm.
The reactions were performed in 1.5 ml microtubes to limit air
oxidation of the thiol into disulphide.
In the chosen conditions (Table 5), no formation of thioester 22
was observed whatever conditions were applied, the starting
mercaptan 21 being mainly recovered at the end of the reaction.
Note that in similar conditions, the uncatalyzed reaction readily
gave a mixture of 23 and 24 (entry 7).
18
ꢁ
Table 5
CALB catalysed reaction of benzyl mercaptan 21 with ethyl propiolate: influence of
the reaction conditions on the reaction mixture composition
Table 4
CALB catalysed reaction of benzyl alcohol 17 with ethyl propiolate: influence of the
reaction conditions on the ester formation
Entry
Solvent
Time
21
23 (E)
24 (Z)
1
2
3
4
5
6
7
Dioxane
Dioxane
CH3CN
CH3CN
tBME
24 h
5 h
24 h
5 h
24 h
5 h
5 h
93
96
94
96
91
>99
90
4
0
3
0
1
0
2
0
6
3
tBME
Traces
5
Traces
2
tBME without enzyme
Entry
Solvent
Stirring process
18
The reactions of benzyl mercaptan (0.2 mmol) with ethyl propiolate (0.6 mmol)
were performed in 1.5 ml capped microtubes and the mixtures were stirred at
50 ꢀC. The ratios in the different compounds were determined from the 1H NMR
integrations, traces of the corresponding disulfide (<4%) may also form.
1
2
3
4
5
6
7
tBME
Toluene
Toluene
Dioxane
Dioxane
Acetonitrile
2-MeeBue2-OH
TS
Magnetic
TS
Magnetic
TS
Magnetic
Magnetic
27
84
66
57
42
54
31
Finally, it was appealing to try the reaction with S,N- or S,O-di-
functional nucleophiles (cysteamine and
respectively).
b-mercaptethanol,
Reaction of benzyl alcohol 17 (0.2 mmol) with ethyl propiolate (0.6 mmol) at 50 ꢀC
for 6 h. The reactions were either performed in open vessels magnetically stirred at
250 rpm or in capped microtubes orbitally shaken at 1000 rpm in a thermoshaker
(TS). The conversion ratio in ester 18 was determined from the 1H NMR integrations.
tBME¼tert-butyl methyl ether.
The reactions were achieved in round-bottom flasks in the
optimised conditions selected for amidation (dioxane, 15 h) and
transesterification (toluene, 6 h). The crude residues obtained
after filtration of the enzyme and evaporation of the solvents,
were analyzed by 1H NMR. As depicted in Scheme 3, the reaction
The reaction was then performed on N-(3-hydroxypropyl)
naphthalimide 19, the hydroxy analogue of 6, to prepare in 79%
yield the ester 20 (Fig. 1).
with
identified as resulting from the selective O-esterification. The
CH2O of -mercaptoethanol is characterised by a triplet at
b-mercaptoethanol 25 gave a major product that was
b
3.75 ppm, had shifted to 4.35 ppm. The CH2SH signals, a quintu-
plet at 2.73 ppm (CH2) and a triplet at 1.45 ppm (SH) in the
starting material, appeared at 2.83 ppm and 1.59 ppm. A singlet at
2.97 ppm, integrating for 1 proton, confirmed the presence of
propiolic CH. Any attempt to purify the mixture only yielded to
the decomposition of 26 with increased formation of by-products.
Nevertheless this method allows the formation of compound
26 that cannot be prepared by usual chemical methodologies
without protection of the thiol function. And indeed, the unca-
talysed reaction gives a 9/1 mixture of Z- and E-ethyl 3-(2-
hydroxyethylthio)acrylates resulting from the 1,4-addition of the
thiol group.22
Fig. 1. Ester resulting from the reaction with N-(3-hydroxypropyl)naphthalimide.
Concerning the reactivity with thiol, the reaction with benzyl
mercaptan 21 may lead 1,2- and 1,4-addition products (formation
of 22 and 23/24, respectively, see Scheme 2). Thioacrylates 23 and
Scheme 3.
Scheme 2. Reaction of benzyl mercaptan with ethyl propiolate: structures of the po-
tential reaction products.

S. Bonte et al. / Tetrahedron 69 (2013) 5495e5500
5499
The reaction with cysteamine gave a mixture of two major
4.2. Enzyme screening procedure
products that could not be separated by chromatography. They
were identified as the E and Z isomers of compound 28 resulting
from the reaction of cysteamine with two molecules of ethyl pro-
piolate, i.e., 1,2-addition of the amino group to one ethyl propiolate
and 1,4-addition of the thiol group to a second molecule of ethyl
propiolate. The 1H NMR data were compared to those of similar
compounds.27,28 The cysteamine SH (1.5 ppm) is not present and
the CH2N signal (triplet at 2.69 ppm for the starting cysteamine)
now appeared downfield as a multiplet centred at 3.60 ppm. Two
CH2S triplets can be found at 2.98 and 3.05 ppm (with a 1/0.7 ratio),
the ethyl ester groups are observed as multiplets centred at 1.33
and 4.22 ppm. The presence of characteristic doublets of 1,4-
addition of the thiol to the triple bond are observed at 5.89 and
7.64 ppm (E isomer, J 15 Hz) and 5.95 and 7.07 ppm (Z isomer, J
10 Hz), with a E/Z ratio¼1/0.7. The formation of the propiolamide
bond is indicated by the presence of a large signal at 6.56 ppm and
two singlets at 2.88 and 2.89 ppm with a 1/0.7 ratio for the E and Z
isomers. The difference in reactivity of the thiol group can be at-
tributed to the existence of the cysteamine as internal salt
(ꢁSCH2CH2NHþ3 ) that greatly increases the nucleophilic properties
against triple bond.27
The reactions were performed in triplicate in 1.5 ml microtubes
or in 5-ml test tubes containing a small stirring bar. The nucleo-
phile (benzyl alcohol, thiol or amine, 0.2 mmol) was added to
a suspension of the chosen enzyme (80 mg) in 1 ml of the chosen
solvent. The mixture was shaken for 1 min (ultrasound stirring)
before addition of ethyl propiolate (11 ml, 0.6 mmol). The capped
microtubes were orbitally shaken at 1000 rpm in a thermoshaker
(TS) at the chosen temperature for the desired time. The open test
tubes were magnetically stirred at 250 rpm at the chosen tem-
perature for the desired time. The suspension were then filtered
off to remove the enzyme, the solid was washed twice with EtOH
and Et2O and the solvents were evaporated under reduce pressure.
The residues were then diluted with DMSO-d6 or CDCl3 for NMR
analysis.
4.3. Applications to the preparation of N-substituted
propiolamides
4.3.1. N-Benzylprop-2-ynamide 2. Benzylamine (1 ml, 9.16 mmol)
was added to a suspension of the CALB (2 g) in tBME (10 ml) in
a round-bottom flask. The mixture was mixed for 1 min (ultrasound
stirring) before addition of ethyl propiolate (1.85 ml, 18.3 mmol).
The mixture was gently stirred (250 rpm) overnight at 50 ꢀC. The
suspension was then filtered to remove the enzyme, the solid was
washed twice with CH2Cl2 and the solvents were evaporated under
reduced pressure. Methanol was added to the oily residue. The
white powder was filtered and dried to give s-trans-Z,E compound 5
(422 mg, 1.39 mmol) in 15% yield. The filtrate was evaporated to
dryness to afford an oily residue that was triturated in diethyl ether
to give 2 (980 mg, 6.1 mmol) as a white powder in 67% yield.
The 1H NMR spectra of 29 and 524 were conformed to the lit-
erature data:
3. Conclusion
In the search of efficient route to variously N-substituted pro-
piolamides as activated alkynes, we have optimised the lipase
biocatalysed addition of benzylamine to ethyl propiolate. Immo-
bilised C. antarctica lipase (CALB) emerged as the enzyme of choice
to favour the chemoselective 1,2-addition, using tBME, dioxane or
toluene under overnight (15 h) gentle magnetic stirring at 50 ꢀC. In
these conditions, propiolamide 2 was prepared in gram scale in
good yield, s-trans-Z,E-dienamine 5 being formed as by-products.
The reactions worked well with other primary amines but not
with secondary amines that only gave the corresponding amino-
acrylates.
The chemoselectivity of CALB with N- and S-nucleophiles was
also checked. The transesterification, preparation of propiolic ester,
also worked in good yields in tBME, toluene or dioxane. The best
yields (near quantitative) were observed when the reactions were
carried out in open vessels under gentle magnetic stirring at 50 ꢀC
for 6 h. The transesterification was chemoselective in presence of
thiols, are indicated by the formation of 2-sulfanylethyl prop-2-
Compound 2: 1H NMR (CDCl3, 400 MHz)
5H); 6.33 (br s, 1H); 4.18 (d, 2H, J 8 Hz); 2.85 (s, 1H).
Compound 5: 1H NMR (CDCl3, 400 MHz)
(ppm) 9.25 (br s, 1H);
d (ppm) 7.30e7.44 (m,
d
7.28e7.44 (m, 7H); 6.09 (d, 1H, J 15.7 Hz); 4.41 (d, 2H, J 5.9 Hz), 4.30
(q, 2H, J 7.1 Hz); 4.23 (q, 2H, J 7.1 Hz); 1.40 (t, 3H, J 7.1 Hz); 1.32 (t, 3H,
J 7.1 Hz).
4.3.2. N-[(1,3-Dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-propyl]-
propen-2-ynamide 7. 3-(1,3-Dioxo-1H-benzo[de]isoquinolin-2(3H)-
yl)-propyl amine 629 (400 mg, 1.57 mmol) was dissolved in dioxane
(15 ml) in a round-bottom flask. CALB (1.6 g) was added, and the
ynoate 18 from b-mercaptoethanol.
4. Experimental section
4.1. General
mixture was shaken 1 min before adding ethyl propiolate 2 (443 ml,
4.37 mmol). The mixture was gently stirred (250 rpm) at the 50 ꢀC
overnight. The suspension was then filtered to remove the enzyme,
the solid was washed twice with CH2Cl2 and the solvents were
evaporated under reduced pressure. A methanole1 N HCl mixture
(1/1) was added to the residue. The solid part was filtered, washed
with water and dried. Crystallisation from methanol afforded
compound 7 (611 mg, 1.02 mmol) as a pale yellow solid in 65%
yield.
Melting points were determined using a Reicher Thermovar
apparatus and are uncorrected. NMR spectra were recorded on
Bruker Avance 400 spectrometer using the solvent as the internal
reference (CDCl3 at 7.24 ppm); the chemical shifts are reported in
parts per million, in
d units.
Ethyl propiolate, benzylamine, benzyl alcohol and benzyl mer-
captan purchased from Sigma Aldrich were used without further
purifications. Ionic liquids, EOPipNTf2 (EOP), MOPyrroNTf2 (MOP)
and MOImNTf2 (MOI) were prepared by Nathalie Kardos from
Mp 177e178 ꢀC; 1H NMR (CDCl3, 400 MHz)
d (ppm) 8.65 (dd, 2H,
J 7.4, 1.0 Hz); 8.29 (dd, 2H, J 8.3, 1.0 Hz); 7.82 (dd, 2H, J 8.3, 7.4 Hz);
7.05 (br s, 1H, NH); 4.32 (t, 2H, J 6.1 Hz); 3.36 (m, 2H); 2.04 (m, 2H);
13C NMR (CDCl3, 100 MHz)
d (ppm) 164.7, 152.2, 134.3, 131.6, 131.5,
ꢀ
Universite de Savoie (France).
128.2, 127.1, 122.4, 73.0, 37.3, 36.4, 27.7.
C. rugosa (also called cylindracea) lipase (CRL) (700 U/mg solid)
and type II lipase crude from porcine pancreas (PPL) (190 U/mg
protein) were purchased from Sigma Chemical Co.; C. antarctica
lipase B (CALB) Novozyme 435 (7400 PLU/g) and lipase immobilised
from Rhizomucor miehei (Lipozyme RM IM) (LIP) (7800 U/g) were
generous gifts of Novozymes A/S. Biochemika lipase immobilised in
Sol-Gel-AK from P. cepacia lipase (40 U/g).
4.3.3. N-[2-(1H-Indol-3-yl)ethyl]prop-2-ynamide 9. The reaction
was performed as described for 7, starting from tryptamine 8
(240 mg, 1.5 mmol), ethyl propiolate (443
ml, 4.37 mmol), CALB
(960 mg) in dioxane (9 ml). After filtration and evaporation of the
solvent, CH2Cl2 was added to the residue. The insoluble part was
filtered off and identified as the starting tryptamine (60 mg, 25%).

5500
S. Bonte et al. / Tetrahedron 69 (2013) 5495e5500
The propiolamide 9 was isolated in 33% yield (105 mg, 0.49 mmol)
after column chromatography (elution: CH2Cl2/diethyl ether 5/1).
The 1H NMR was conformed to the literature data:30 1H NMR
mercaptoethanol 25 (210
ml, 3 mmol), CALB (900 mg) (10 ml) and
ethyl propiolate (912
m
l, 9 mmol). The reaction was stirred at 50 ꢀC
for 6 h. After filtration of the enzyme, and evaporation, the crude
(DMSO-d6, 400 MHz)
1H, J 7.7 Hz), 7.34 (d,1H, J 7.7 Hz), 7.16 (s,1H), 7.07 (t,1H, J 7.7 Hz), 6.99
(t, 1H, J 7.7 Hz), 4.10 (s, 1H), 3.31e3.40 (m, 2H), 2.83e2.86 (m, 2H).
d
(ppm) 10.82 (br s, 1H), 8.35 (br s, 1H), 7.52 (d,
residue (239 mg) was analysed by NMR.
1H NMR (CDCl3, 400 MHz)
d (ppm) 4.35 (t. 2H, J 6.7 Hz); 2.97 (s,
1H, CH); 2.83 (m, 2H); 1.59 (t, 1H, J 8.6 Hz).
4.3.4. N-(Prop-2-yn-1-yl)prop-2-ynamide 11. The reaction was
performed as described for 7, starting from propargylamine 10
4.3.10. Ethyl 3-[(2-(prop-2-ynamido)-ethyl)sulfanyl]acrylate. Mix-
ture of E and Z isomers 28. The reaction was performed in dioxane
(5 ml) as described for 7, starting from cysteamine 27 (115 mg,
(32
ml, 1.5 mmol), ethyl propiolate (443 ml, 4.37 mmol), CALB
(130 mg) in dioxane (2 ml). The propiolamide 11 was obtained as an
oil in 95% yield (414 mg, 1.42 mmol).
1.5 mmol), CALB (400 mg) and ethyl propiolate (440 ml, 3.7 mmol).
After filtration of the enzyme, and evaporation, the crude resi-
due was analysed by NMR and correspond to a 1/0.7 mixture of Z/E
isomers.
The 1H NMR was conformed to the literature data:31 1H NMR
(CDCl3, 400 MHz) d (ppm) 6.18 (br s,1H), 4.09 (dd, J 5.2, 2.4 Hz), 2.84
(s, 1H), 2.28 (t, 1H, J 2.4 Hz).
1H NMR (CDCl3, 400 MHz)
d (ppm) 7.64 (d, 1H, J 15.0 Hz), 7.07 (d,
0.7H, J 10.0 Hz), 6.57 (br s,1.5 H), 5.95 (d, 0.7H, J 10.0 Hz), 5.90 (d,1H,
J 15.0 Hz), 4.19e4.22 (m, 3.4H), 3.57e3.54 (m, 3.4H), 3.05 (t, 2H, J
6.8 Hz), 2.98 (t, 1.4H, J 6.8 Hz), 2.89 (s, 1H), 2.88 (s, 0.7H), 1.30e1.34
(m, 5.1H).
4.3.5. N-(2,2-Diethoxyethyl)prop-2-ynamide 13. The reaction was
performed as described for 7, starting from 2,2-diethoxyethylamine
12 (163 ml, 1.5 mmol), ethyl propiolate (443 ml, 4.37 mmol), CALB
(600 mg) in dioxane (6 ml). The propiolamide 13 was isolated in 45%
yield after column chromatography (elution: pentane/diethyl ether).
The 1H NMR was conformed to the literature data:32 1H NMR
Acknowledgements
(CDCl3, 400 MHz)
3.47e3.50 (m, 2H), 3.44 (s, 6H), 2.86 (s, 1H).
d (ppm) 6.20 (br s, 1H), 4.44 (t, 1H, J 5.6 Hz),
This work was supported by a grant of the Romanian National
Authority for Scientific Research, CNCS e UEFISCDI, project number
PN-II-ID-PCE-2011-3-0226 and IOG was financed by the Project
SOP HRD e TOP ACADEMIC 76822. Dr. Luc Choisnard from our
department (DPM) is gratefully acknowledged for his suggestions
and discussions on biocatalysis by lipases.
4.3.6. Ethyl (E)-3-(morpholin-4-yl)acrylate 16. The reaction was
performed as for 7 starting from morpholine 15 (132
ethyl propiolate (443 l, 4.37 mmol), CALB (440 mg) in dioxane
(5 ml). Compound 16 was isolated as an oil (235 mg, 1.32 mmol)
after removal of the enzyme and evaporation of the solvent.
The 1H NMR was conformed to the literature data:26 1H NMR
ml, 1.5 mmol),
m
References and notes
(CDCl3, 400 MHz)
13.0 Hz), 4.16 (q, 2H, J 7.1 Hz), 3.72e3.74 (m, 4H), 3.20e3.25 (m, 4H),
1.28 (t, 2H, J 7.1 Hz).
d (ppm) 7.38 (d, 1H, J 13.0 Hz), 4.72 (d, 1H, J
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19
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d (ppm) 8.63 (dd, 2H,
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4.35e4.38 (m, 4H); 2.83 (s, 1H); 2.21 (quint, 2H, J 7.6 Hz): 13C NMR
(CDCl3, 100 MHz)
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4.3.9. 2-Sulfanylethyl prop-2-ynoate 26. The reaction was per-
formed in toluene as described for 18, starting from
b-