Lipase-catalyzed aza-michael reaction on acrylate derivatives

Article abstract of DOI:10.1021/jo400268u

A methodology has been developed for an efficient and selective lipase-catalyzed aza-Michael reaction of various amines (primary and secondary) with a series of acrylates and alkylacrylates. Reaction parameters were tuned, and under the optimal conditions it was found that Pseudomonas stutzeri lipase and Chromobacterium viscosum lipase showed the highest selectivity for the aza-Michael addition to substituted alkyl acrylates. For the first time also, some CLEAs were examined that showed a comparable or higher selectivity and yield than the free enzymes and other formulations.

Full text of DOI:10.1021/jo400268u

Article
pubs.acs.org/joc
Lipase-Catalyzed Aza-Michael Reaction on Acrylate Derivatives
Peter Steunenberg,† Maarten Sijm,† Han Zuilhof,†,§ Johan P. M. Sanders,‡ Elinor L. Scott,‡
and Maurice C. R. Franssen*,†
†Laboratory of Organic Chemistry, Wageningen University, Dreijenplein 8, 6703 HB Wageningen, The Netherlands
‡Biobased Commodity Chemicals, Wageningen University, Bornse Weilanden 9, 6708 WG Wageningen, The Netherlands
§Department of Chemical and Materials Engineering, King Abdulaziz University, Jeddah, Saudi Arabia
S
* Supporting Information
ABSTRACT: A methodology has been developed for an
efficient and selective lipase-catalyzed aza-Michael reaction of
various amines (primary and secondary) with a series of
acrylates and alkylacrylates. Reaction parameters were tuned,
and under the optimal conditions it was found that
Pseudomonas stutzeri lipase and Chromobacterium viscosum lipase showed the highest selectivity for the aza-Michael addition
to substituted alkyl acrylates. For the first time also, some CLEAs were examined that showed a comparable or higher selectivity
and yield than the free enzymes and other formulations.
the Cal B-catalyzed aza-Michael addition of imidazoles to
acrylic monomers, the most efficient hydrolase was found to be
lipase M.17 The influence of solvents was examined, and it was
found that solvents with a higher log P value led to higher
conversions.
Since lipases are hydrolytic enzymes, Michael addition can
compete with aminolysis of the ester. The chemoselectivity of
these types of enzyme-catalyzed reactions was studied in more
detail by Gotor et al.,18 focusing on minimizing the amount of
formation of the Michael addition products. Conversely, other
studies tried to optimize the formation of the 1,4-addition
product.19−21 It was demonstrated that the polarity of the
reaction media has an influence on the chemoselectivity
between Michael addition and aminolysis.19
INTRODUCTION
■
Since the pioneering work of Klibanov in the 1980s, enzymatic
reactions in organic media have attracted a lot of attention
because of their enhanced space−time yields, lack of side
reactions, and enhanced enantioselectivities.1−5 Lipase B from
Candida antarctica (Cal B) is usually taken as the enzyme for
use in organic media. This is due to its high stability, activity,
and wide range of substrates and media that can be applied.6−8
This high tolerance for organic media is not uncommon, as
some enzymes, like native lipases, are active at aqueous−
organic interfaces to hydrolyze fats.9,10 Several reactions have
been reported to be catalyzed by Cal B,11−13 including the
Michael addition, an unexpected reaction for hydrolytic
enzymes. The Michael reaction or Michael addition (1,4-
addition) is a nucleophilic addition of a nucleophile to an α,β-
unsaturated carbonyl compound. In the case of a nitrogen
nucleophile, the reaction is called an aza-Michael addition (see
Scheme 1).
Recently, Baldessari et al.22 have shown that Lipozyme RM
IM catalyzes the formation of the Michael adduct of
benzylamine to ethyl and butyl acrylate. However, lipases like
Cal B and the ones from Pseudomonas cepacia and Candida
rugosa formed considerable amounts of the aminolysis
product.23 Lipozyme RM IM was found to catalyze the
addition of alkylamines and alkanolamines to unsubstituted
acrylates chemoselectively. These N-substituted β-amino acid
esters containing a free hydroxyl or amine group are potential
monomers in the synthesis of polyesters.
Scheme 1. General Reaction Scheme for Lipase-Catalyzed
Aza-Michael Reactions
a
It has been shown previously that depending on the substrate
concentration, solvent, and choice of enzyme applied, either an
aminolysis reaction or an aza-Michael addition between ethyl
acrylate or N-methyl-1,3-diaminopropane occurs.24 These
acrylamides or aminoesters were later converted by enzymatic
polymerization into polyamidoamines (PAMAMS), an interest-
ing class of polymers for biomedical applications.24 The
enzymes have a dual behavior, because the enzymatic reaction
a
R1: Me, Et, Bu, t-Bu; R2: H, Me; R3: H, Me, Ph; R4/R5: aromatic,
aliphatic, etc.
The first example of a Michael addition in organic media
catalyzed by enzymes was reported in 1988 by Kitazume et al.14
Later, lipase-catalyzed Michael-type addition of various primary
and secondary amines to acrylonitrile were reported.15,16
Different preparations of CalB led to the Michael adduct,
with Chirazyme L-2 showing the highest reaction rate,15 while
Novozyme 435 gave the best results for primary amines.16 For
Received: February 5, 2013
Published: March 27, 2013
© 2013 American Chemical Society
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Table 1. Screening of Lipases for the Chemoselective Addition of Benzylamine 1 to Acrylate 3
conversion
conversion vs blank
activity
(kU/g)
1,4-addition 3
aminolysis 4
a
b
,
c
b,d
entry
enzyme
(%)
(%)
(%)
(%)
1
2
3
4
5
Chirazyme L-1 carrier-fixed lyophilized
Chirazyme L-5 carrier-fixed lyophilized
Chirazyme L-9 c.f. C2 lyophilized
77
35
235
13
80
66
14
80
60
50
30
12
4
96
97
4
3
63
62
83
>99
61
61
52
68
54
58
48
76
87
72
62
53
78
81
99
85
77
68
95
58
41
21
21
42
59
20
20
11
27
13
17
7
98
2
Chirazyme L-9 c.f. lyophilized
91
9
Candida antarctica lipase on acrylic resin/Novozyme 435
87
13
2
6, 7 Lipase N
98
8
Lipase AP6
96
4
9
Lipase G
100
97
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
Lipase PS
3
Lipase AH
100
100
100
96
0
Lipase R-10
0
Lipase PS-800
840
20
22
3
0
Lipase AK
35
46
31
21
13
37
40
58
44
36
27
54
17
4
Lipozyme TL IM
90
10
3
Lipozyme RM IM
97
Lipoprotein Lipase Amano 100s
Aspergillus niger lipase
30
3
100
100
96
0
0
Chromobacterium viscosum lipase (CvL)
Pseudomonas stutzeri Lipase (PSL)
CLEA Candida antarctica lipaseB
CLEA Pseudomonas stutzeri lipase (PSL)
CLEA Thermomyces lanuginosa lipase (TL) ST
CLEA Thermomyces lanuginosa lipase (TL) OM1
CLEA Thermomyces lanuginosa lipase (TL) OM2
CLEA Rhizomucor miehei lipase (RM) ST
blank
42
17
17
17
510
580
530
67
4
97
3
90
10
0
100
97
3
96
4
97
3
e
99
1
100
0
a
b
c
Names as bought from supplier, literature name can vary. Based on GC−MS analysis. Reaction conditions: 0.1 mmol of benzylamine, 0.1 mmol
d
of methyl acrylate, 5 mg of enzyme, and 0.3 mL of hexane were added to a sealed vessel and stirred at 60 °C for 4 h. Blank: conversion under same
conditions without enzyme, conversion vs blank shows the conversion realized by the addition of the enzyme. Conversion after 4 h.
e
of ethyl acrylate and several alkanolamines gives solely the
product of aminolysis.25
reacted with a bifunctional agent (such as glutaraldehyde) to
form a cross-linked gel.33 The only aza-Michael additions
known to us, which were catalyzed by a CLEA, were described
by Gotor-Fernandez et al.36 They have shown that the Alcalase
CLEA from Bacillus licheniformis can be used for an enzymatic
aza-Michael addition of secondary amines to acrylonitrile.
Up to now, the best results for the aza-Michael addition of
various amines to acrylates were obtained with Novozyme 435
or Lipozyme RM IM. The main drawback of these biocatalysts
is that for the addition of the amines to substituted acrylates
(such as crotonate or methacrylate) low chemoselectivity was
observed and the product of aminolysis was obtained in
considerable amounts. In this paper, we have screened a broad
range of lipases for the aza-Michael addition to various
acrylates. We were able to select an enzyme that even for the
substituted acrylates shows high conversion and selectivity.
Using a large array of primary and secondary amines we have
produced the corresponding Michael adducts in good yields.
A series of interesting β-amino acids can be produced by the
enzymatic aza-Michael addition of various aliphatic and
aromatic amines to acrylates and methyl-substituted derivatives.
β-Amino acids are a useful class of compounds that can serve as
synthetic precursors for many bioactive compounds. β-Amino
acid esters are used as versatile building blocks in the
production of polyamides and for the synthesis of linear
cationic polymers used as gene delivery vectors. In addition, an
enzymatic process has been used for the synthesis of
enantiomerically pure (S)-3-aminobutanoic acid.26,27 The
reaction steps in this process comprise an initial enzyme
(Novozyme 435) catalyzed aza-Michael addition, followed by
enzymatic resolution via aminolysis. After hydrolysis and
removal of the N-benzyl moiety, the desired (S)-3-amino-
butanoic acid was obtained.
The class of enzymes used for the catalyzed aza-Michael
addition is lipase. Lipases are among the most important
enzymes in terms of applications in current industry/
biotechnology.28−31 Lipases can be applied in reactions in
different formulations, like free enzymes or immobilized on
beads, e.g., Novozyme 435, i.e., CalB absorbed to acrylic beads.
Another formulation that shows interesting properties are
cross-linked enzyme aggregates (CLEAs).32−35 CLEAs are
prepared by precipitation of the enzymes, which are then
RESULTS AND DISCUSSION
■
An initial screening of various enzymes for the addition of
various amines to acrylates was performed. The standard
reaction that was used is the aza-Michael addition of
benzylamine 1 to methyl acrylate 2a. The results of this
screening are reported in Table 1.
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Table 2. Screening of Lipases for Chemoselective Addition of Benzylamine (1) to Methyl Crotonate (5a)
conversion
conversion vs blank
activity
(kU/g)
1,4-addition 6
aminolysis 7
a
,
b
a,c
entry
enzyme
(%)
(%)
(%)
(%)
1
2
3
Chirazyme L-1 carrier-fixed lyophilized
Chirazyme L-9 carrier-fixed lyophilized
69
35
28
45
235
80
95
100
90
5
0
62
79
Candida antarctica lipase B on acrylic resin (Novozyme
435)
14
10
4
Lipase PS
48
83
68
66
50
69
74
84
76
68
36
34
14
49
32
32
14
35
40
50
42
34
0
30
840
20
22
3
96
97
4
3
5
Lipase PS-800
6
Lipase AK
97
3
7
Lipozyme TL IM
76
24
5
8
Lipozyme RM IM
95
9
Chromobacterium viscosum lipase (CvL)
Pseudomonas stutzeri lipase (PSL)
CLEA Candida antarctica lipase B
CLEA Pseudomonas stutzeri lipase (PSL)
CLEA Thermomyces lanuginosa lipase (TL) OM2
CLEA Rhizomucor miehei lipase (RM)
42
17
17
17
530
67
99
1
10
11
12
13
14
15
95
5
85
15
2
98
97
3
100
100
0
blank
0
a
b
Based on GC−MS analysis. Reaction conditions: 0.1 mmol of benzylamine, 0.1 mmol of methyl crotonate, 5 mg of enzyme, and 0.3 mL of hexane
c
were added to a sealed vessel and stirred at 60 °C for 3 days. Blank: conversion under same conditions without enzyme, conversion vs blank shows
the conversion realized by the addition of the enzyme.
Table 3. Screening of Lipases for Chemoselective Addition of Benzylamine 1 to Methyl Methacrylate 8
conversion
conversion vs blank
activity
(kU/g)
1,4-addition 9
aminolysis 10
a
,
b
a,c
entry
enzymes
(%)
(%)
(%)
(%)
1
2
3
Chirazyme L-1 carrier fixed lyophilized
47
39
40
71
235
13
100
100
82
0
0
Chirazyme L-9 c.f. lyophilized
49
80
Candida antarctica lipase on acrylic resin/Novozyme
435
14
18
4
Lipase PS-800
69
46
26
32
61
80
81
84
81
10
9
61
37
17
23
0
840
20
22
3
100
94
0
6
5
Lipase AK
6
Lipozyme TL IM
100
100
100
100
80
0
7
Lipozyme RM IM
0
8
Chromobacterium viscosum lipase (CvL)
Pseudomonas stutzeri lipase (PSL)
CLEA Candida antarctica lipase B
CLEA Pseudomonas stutzeri lipase (PSL)
CLEA Thermomyces lanuginosa lipase (TL) OM2
CLEA Rhizomucor miehei lipase (RM)
42
17
17
17
530
67
0
9
71
72
75
72
0
0
10
11
12
13
14
20
0
100
100
100
100
0
0
blank
0
a
b
Based on GC−MS analysis. Reaction conditions: 0.1 mmol of benzylamine, 0.1 mmol of methyl methacrylate, 5 mg of enzyme, and 0.3 mL of
c
hexane were added to a sealed vessel and stirred at 60 °C for 4 days. Blank: conversion under same conditions without enzyme, conversion vs blank
shows the conversion realized by the addition of the enzyme.
It was observed that indeed all lipases do catalyze this
reaction, although their catalytic effect can vary. The chemo-
selectivity of most enzymes is strongly toward the 1,4-addition
product, indicating that a fully chemoselective reaction might
be feasible. As can be seen from Table 1, Candida antarctica
lipase B (Cal B), formulated as Novozyme 435, shows the
highest conversion after 4 h (>99%). However, a drawback of
Novozyme 435 as a catalyst for these aza-Michael reactions is
that the selectivity is relatively low, 87:13 for 1,4-addition/
aminolysis (entry 5). A higher selectivity was obtained for
lipases from Chromobacterium viscosum (CvL) (96:4; entry 18)
and Pseudomonas stutzeri (PSL) (97:3; entry 19), but the
conversion is lower, 85% and 77%, respectively. It can also be
concluded from Table 1 that the enzymes with the highest
activity do not give the highest conversion or selectivity.
Lipozyme TL IM (Thermomyces lanuginosa, TL) shows a high
conversion with low selectivity (entry 14), and Lipozyme RM
IM, like all other Rhizomucor miehei formulations, shows a
higher selectivity for the 1,4-addition, but lower conversion
(entry 15). This is in accordance with the recent report of
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Baldessari et al.,22 although they did not report on aminolysis.
For the first time we here also show that a CLEA of various
lipases can be used for the aza-Michael addition of benzylamine
and methyl acrylate. From Table 1, it can be concluded that a
CLEA gives a higher conversion than other formulations while
retaining a high selectivity. This makes CLEAs comparable with
Novozyme 435. CLEAs of Cal B, PSL, RM, and TL were tested
on their conversion and selectivity for the aza-Michael addition
reaction. Especially CLEA TL OM2, a CLEA of Thermomyces
lanuginosa developed for organic media, showed a higher
selectivity for the Michael addition than the lipozyme RM IM
and CLEA ST (standard) or CLEA OM1 (organic media/old
version). Remarkably, Pseudomonas stutzeri lipase gave a very
good conversion and selectivity, although the enzyme has a
much lower activity per gram than other enzymes (based on
triglyceride conversion). The enzymes and formulations that
gave the best results with the unsubstituted acrylates were also
tested for crotonate and methacrylate (Table 2).
Scheme 2. Aza-Michael Reaction of Diamines and
Ethanolamine to Butyl Acrylate Catalyzed by PSL and CvL
methacrylate or ethyl crononate was carried out (Scheme 3),
and now the addition reaction was completely selective and
Scheme 3. Aza-Michael Reaction of Diamines and
Ethanolamine to Substituted Acrylates Catalyzed by PSL and
CvL
The data obtained for the aza-Michael addition of benzyl-
amine 1 with methyl crotonate 5a were compared to the results
of Priego et al.,19 who optimized this reaction to give high
conversion and selectivity for the product of 1,4-addition. Their
best results were obtained for lipase-PS 800, but this
formulation is no longer commercially available. Here it is
also shown that CvL and PSL give a higher selectivity for the
desired aza-Michael adduct (Table 2, entries 9 and 10), though
in lower yield than Novozyme 435 (entry 3). This can be
circumvented by the use of CLEA PSL, which improved the
conversion (76%) and retained the high selectivity (entry 12).
Finally, the aza-Michael addition of benzylamine 1 and
methyl methacrylate 8 was studied. The most promising lipases
and formulations were screened under conditions that were
found to be optimal for the previous described additions.
Selectivity and conversion were compared to Novozyme 435.
Here again, CvL and PSL showed higher selectivity (Table 3,
entries 8 and 9), this time for PSL even conversion is higher.
This is also the case for Lipase PS-800 (entry 4). Additionally
some CLEAs were screened and here CLEA PSL gave higher
yield and higher selectivity for the 1,4-addition of benzylamine
to methyl methacrylate (entry 11).
Baldessari et al. showed recently that N-substituted β-amino
acids could be formed catalyzed by Lipozyme RM IM starting
from various unsubstituted acrylates and ethanolamine and 1,3-
propanediamine.22 However, no reaction was observed when
substituted acrylates were used. In the current work, PSL and
CvL were found to catalyze the aza-Michael addition of
ethanolamine and various diamines to substituted acrylates.
These enzymes were less selective for the addition of the
amines to unsubstituted acrylates because both amine
functionalities of the diamines underwent an aza-Michael
addition leading to diester 15 (see Scheme 2). When 3 equiv
of the acrylate 2b was added, full conversion was observed and
the diester 15 was obtained in a 4:1 ratio. Diester 15 can be
seen as a promising building block for application in polymer
chemistry. The 1,4-addition of ethanolamine to butyl acrylate
selectively gave the mono adduct, with the amine as the
nucleophile, at a conversion of 78% for PSL and 69% for CvL.
When diamines such as 1,3-propanediamine were added to
ethyl acrylate, again a double addition was observed. It appears
that chain length or type of ester did not have any effect on the
outcome of the reaction in terms of chemoselectivity. Finally,
instead of unsubstituted acrylates, a lipase-catalyzed (PSL or
CvL) aza-Michael addition of 1,3-propanediamine and methyl
methyl substituted N-substituted β-amino acids with a free
amine function could be prepared (see Table 4). Afterward, the
addition reaction was also carried out with ethanolamine and
1,4-diaminobutane. In all examples, only monoaddition took
place without transesterification or aminolysis. All products
were isolated and purified, except for compound 23, which
polymerized during bulb-to-bulb distillation.
Table 4. Aza-Michael Addition of Substituted Acrylates with
Diamines and Ethanolamine Catalyzed by CvL and PSL
Amine
Michael
conversion
conversion
a,b
a
,
b
entry X(CH2)nCH2NH2 acceptor product (%) PSL
(%) CvL
1
2
3
4
5
6
7
8
n = 1, X = NH2
n = 1, X = NH2
n = 2, X = NH2
n = 2, X = NH2
n = 3, X = NH2
n = 3, X = NH2
n = 1, X = OH
n = 1, X = OH
5b
8
16
17
18
19
20
21
22
23
79
69
81
68
75
64
82
78
72
63
75
61
70
54
74
72
5b
8
5b
8
5b
8
a
b
Based on GC−MS analysis. Reaction conditions: 0.1 mmol of
benzylamine, 0.1 mmol of methyl methacrylate, 5 mg of enzyme, and
0.3 mL of hexane were added to a sealed vessel and stirred at 60 °C for
4 days.
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Table 5. Solvent Engineering for the PSL- and CvL-Catalyzed Addition of Benzylamine 1 to Methyl Methacrylate 8
a
b
a
b
a
b
entry
solvent system
ET(30)
conversion Cal B (%)
result Cal B
conversion PSL (%)
result PSL
conversion CvL (%)
result CvL
1
n-hexane
toluene
iPr−O-iPr
MTBE
31.0
33.9
34.1
34.7
35.3
36.0
36.5
37.4
38.3
39.2
40.1
80
76
87
80
81
77
74
84
93
93
93
65
71/29/0
34/66/0
18/47/35
17/83/0
20/67/13
17/83/0
29/67/4
13/76/11
17/47/36
14/43/43
19/36/45
67/33/0
80
71
60
76
70
62
4
100/0/0
63/37/0
62
69
54
69
56
51
56
61
83
83
87
45
100/0/0
86/14/0
85/15/0
71/29/0
89/11/0
84/16/0
87/13/0
70/30/0
82/10/8
83/11/7
81/8/11
98/2/0
2
3
50/39/11
72/28/0
50/40/10
78/22/0
100/0/0
73/27/0
74/18/8
72/21/6
65/19/16
85/15/0
4
5
Ph-O-Ph
dioxane
Me-THF
THF
6
7
8
25
88
84
90
54
c
9
3:1
c
10
11
12
1:1
c
1:3
DCM
40.7
a
b
c
Based on GC−MS analysis. Ratio 1,4-addition/aminolysis/1,4-addition and successive aminolysis in percent. Ratio of hexane/2-methyl-2-butanol.
Reaction conditions: 0.1 mmol of benzylamine, 0.1 mmol of methyl methacrylate, 5 mg of enzyme, and 0.3 mL of solvent were added to a sealed
vessel and stirred at 60 °C for 4 days.
As described previously, enzyme performance can be
influenced by changing the solvent system. The complexity of
these solvent−protein interactions makes it hard to quantify
this effect. Polarity, hydrophobicity and other solvent
parameters did not show much correlation in their effects on
the enzymatic activity. Laane et al.37 showed that highly apolar
solvents (log P > 4) retained the highest enzymatic activity in
most cases. On the basis of this report, the choice was made to
try a broad spectrum of solvents, as functional group interaction
of the solvent most likely plays a role along with various other
interactions. However, from the screening of enzymes no clear
trend could be observed based on log P.
In order to determine optimum catalyst loading, a series of
experiments were carried out with different loadings of the
enzyme (see Figure S1, Supporting Information). The amount
of catalyst is dependent on the activity; however, these activities
are measured in aqueous solutions. These values of course can
give an indication for amount of enzyme to be used, but in
order to be efficient and generate less waste, optimal loading
was determined. All samples contained an identical concen-
tration of substrate, only the amount of enzyme was varied. It
turned out that PSL is a very active enzyme, even at low
loadings. It reaches maximum conversion rate with 13 U/mL (3
mg/mL) even though the enzyme has only 17 U/mg activity in
aqueous systems. For CvL a higher loading of the enzyme is
necessary to achieve optimum conversion rates, stabilization
occurs at 65 U/mL (17 mg/mL). From these experiments it
can also be seen that for reactions catalyzed by CvL hardly any
product of aminolysis is observed. For PSL the product of
aminolysis is observed, as well as the product of subsequently
1,4-addition and aminolysis, that are formed at higher enzyme
loadings.
From Figure 1 it can be observed that for reactions catalyzed
by Novozyme 435, the benchmark, the result does not vary
considerably by varying the concentration of the substrate. The
overall conversion stays between 70 and 80%, and with increase
of the substrate concentration, the chemoselectivity decreased.
For PSL we see a similar pattern, at lower concentrations
chemoselectivity is high and mainly the product of 1,4-addition
is formed. At higher concentrations a substantial amount of the
product of both 1,4-addition and aminolysis is formed. For CvL
another pattern can be observed, at higher concentrations the
conversion increases, but the chemoselectivity remains.
To verify if the reaction occurs in the enzyme active site or
that the enzyme catalyzes the reaction as a general acid/base
catalyst, the enzyme was inactivated by heating at 120 °C for 5
days. Afterward the reaction between morpholine and methyl
acrylate was studied using inactivated enzyme. As can be seen
from Figure S2 (see the Supporting Information) the enzymatic
activity of the inactivated enzymes of PSL and CvL is
comparable to the noncatalyzed reaction (when one takes
into account the final conversion), whereas the enzymes as
received showed an increased conversion.
An analogous comparison was made by Priego et al.19 for the
addition of benzylamine to crotonate based on Reichardt’s
ET(30) scale.38,39 These authors used n-hexane, toluene,
diisopropyl ether, THF, and 2-methyl-2-butanol (2M2B) as
solvents. If we use the same solvent series for the aza-Michael
addition of benzylamine 1 to methacrylate 8 catalyzed by PSL
and CvL, a comparable order of chemoselectivity is observed19
(see Table 5). n-Hexane gives for all enzymes the best results
(entry 1), and for PSL conversion is 80% with a selectivity of
100% for the product of 1,4-addition. Although the same
solvent (hexane) shows only 58% conversion in combination
with CvL, it does show full selectivity toward 1,4-addition.
Compared to Cal B this can be seen as an improvement: with
CalB the conversion is high (80%), but the chemoselectivity is
clearly lower (71:29). The 1,4-addition product and the
aminolysis product are somewhat difficult to separate, which
might induce that this selectivity might become the
determining factor for the choice of enzyme.
Looking to some solvents specifically, it becomes visible that
2M2B indeed enhances enzyme activity. This was already
proposed by Klibanov in 200140 and is explained by formation
of hydrogen bonds with the protein. The enzyme is “loosened
up” and gets activated by the organic molecules, mimicking
water molecules. At the same time, either the previous
mentioned activation or the possibly polarity of 2M2B
decreases selectivity toward the 1,4-addition product. At the
same time an increasing amount of product is seen that is
formed by successive 1,4-addition and aminolysis. The
enhancing effect of 2M2B on the rate of reaction, like other
polar solvents, should be applicable to a lot of enzyme-catalyzed
reactions, but the decrease in selectivity prevents use of this
solvents.
With the selected lipases from Pseudomonas stutzeri and
Chromobacterium viscosum the aza-Michael addition of various
amines and acrylates was catalyzed (Tables 6−8). The results
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of the ester. We also tested other esters like tert-butyl and
benzyl. They showed high conversion for the aza-Michael
addition, but do not undergo aminolysis. Methyl substitution
on the acrylate influences the conversion clearly, giving lower
yield for the products starting from crotonate or methacrylate
compared to acrylate.
CONCLUSIONS
■
Lipases from Pseudomonas stutzeri (PSL) and Chromobacterium
viscosum (CvL) are excellent catalysts for the aza-Michael
addition of amines to substituted or unsubstituted acrylates
(high product selectivity, good yields). In an extensive
comparative study other lipases, including Novozyme 435,
were shown to be less effective. The selective Michael addition
of diamines to these substituted acrylates was also achieved.
The use of CLEAs of various lipases for this aza-Michael
addition was shown, and especially the lipase CLEAs of
Thermomyces lanuginosa OM2 and PSL gave the 1,4-adduct of
the acrylates and benzylamine with high selectivity and in good
yields.
EXPERIMENTAL SECTION
Materials and Methods. H NMR spectra were recorded at 400
■
1
MHz and 13C NMR at 100 MHz. All spectra are referenced to residual
proton solvent signal. Abbreviations used include singlet (s), doublet
(d), doublet of doublets (dd), quartet (q), triplet (t) doublet of triplets
(dt), symmetric multiplet (sm), and unresolved multiplet (m). HR-MS
analyses were obtained by an electrospray ionization (ESI) apparatus
using time-of-flight (TOF). IR spectra were recorded using a
spectrometer mounted with a ATR platinum diamond. GC-MS
analysis was done using a DB5MS column, length 30 m, internal size
0.25 mm, film thickness 0.25 μm, split ratio 30:1, flow 0.831 mL/min,
injector temperature: 250 °C, oven temperature: 6 min on 40 °C,
raised by 20 °C per minute up to 250 °C, hold time 6 min. MS scan
range: 50−550 amu. For enzymatic activity, data were used as obtained
from supplier. Alternatively, the activity was determined using a pH-
stat, 1 U catalyzes the release of 1 μmol of butyric acid per minute
from tributyrin ((10% v/v) in 25 mM phosphate buffer, pH 7.5 and 40
°C), titrated with 0.1 M NaOH. Methyl acrylate, tert-butyl acrylate,
methyl methacrylate, methyl crotonate, ethyl crotonate, hexane,
toluene, diisopropyl ether, and MBTE (all anhydrous) were purchased
from Sigma Aldrich. The enzymes used are lipases (EC 3.1.1.3).
Novozyme 435 (14000 U/g), Lipozyme RM IM (3000 U/g) and
Lipozyme TL IM (22000 U/g) were generous gifts from Novozymes,
Pseudomonas stutzeri lipase FE 117 (17500 U/g), CLEA Pseudomonas
stutzeri lipase 117 OM (17600 U/g), CLEA Candida antarctica lipase B
102-ST (17000 U/g), CLEA Rhizomucor miehei lipase 105 ST (67000
U/g) and CLEA Thermomyces lanuginosa lipase 104 ST (510000 U/g),
104 OM1 (580000 U/g) and 104 OM2 (530000 U/g) were purchased
or a generous gift from CLEA technologies (Delft, The Netherlands).
Chromobacterium viscosum lipase (42000 U/g) and Aspergillus niger
lipase (3000 U/g) were generous gifts from Biocatalysts. Chirazyme L-
1 carrier-fixed lyophilized (235000 U/g, Burkholderia cepacia),
Chirazyme L-5 carrier-fixed lyophilized (Candida antarctica B, 13000
U/g) Chirazyme L-9 c.f. C2 lyophilized, (Rhizomucor miehei, 80000
U/g), Chirazyme L-9 c.f. lyophilized (Rhizomucor miehei, 66000 U/g)
were obtained from Boehringer Mannheim, Lipase AK (Pseudomonas
fluorescens, 20000 U/g), Lipase G (Penicilium cyclopium, 50000 U/g),
Lipase N (Rhizopus sp., 80000 U/g) Lipase AP6 (Aspergillus sp., 60000
U/g), Lipase PS-800 (Pseudomonas cepacia, 840000 U/g) Lipase R-10
(Humicola lanuginosa, 4000 U/g), Lipase AH (Pseudomonas cepacia,
12000 U/g), and Lipoproteine Lipase Amano 100s (Burkholderia sp.,
30000 U/g) were generous gifts from Amano Enzymes, Inc.
Figure 1. Chemoselectivity of Novozyme 435 (a), PSL (b) and CvL
(c) at various substrate concentrations. Reaction conditions: benzyl-
amine: methyl methacrylate 1: 1, 5 mg enzyme and 0.3 mL of hexane
⧫
▲
in a sealed vessel stirred at 60 °C for 4 days: , overall conversion;
Michael adduct; , product of aminolysis; ×, product of aminolysis
and successive Michael addition.
,
■
show that the lipase-mediated aza-Michael addition has a very
broad applicability including linear and cyclic amines, primary
and secondary amines, and substituted acrylate esters with
various alcohol chain lengths. Only allylamine gave a low
product yield. Additionally, it can be seen from Table 6−8 that
cyclic secondary amines show higher conversion than primary
aromatic amines. From the results of the lipase-catalyzed aza-
Michael addition of unsubstituted benzylamines to various
acrylates, it can also be concluded that in accordance with the
results obtained by Lin et al.,17 with increasing the length of the
aliphatic chain of the ester, the conversion decreases. However,
this analogy is not confirmed for the methoxy-substituted
benzylamines 3-methoxybenzylamine and 3,4-methoxybenzyl-
amine. In this case, conversion was around 70 and 80% for the
respective amines, regardless of the length of the aliphatic chain
General Methods. General Method A: Synthesis of Ethyl 3-((2-
Aminoethyl)amino)butanoate (16). This Method Is Representative
for Compounds 16−23. Ethylenediamine (0.6 g, 10 mmol) and
methyl methacrylate (1.0 g, 10 mmol) or ethyl crotonate (1.15 g, 10
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Table 6. Yields for PSL-Catalyzed Aza-Michael Additions of Various Amines to Methyl and Ethyl Acrylate
a
b
Based on GC−MS analysis. Reaction conditions: 0.1 mmol of amine, 0.1 mmol of MMA, 5 mg of enzyme, and 0.3 mL of hexane were added to a
sealed vessel and stirred at 60 °C for 4 h.
mmol) were dissolved in 6 mL of hexane, and 50 mg enzyme was
added. The reaction mixture was stirred at 60 °C, 250 rpm in a sealed
vial for 4 days. Enzymes were filtered off and washed with EtOAc (3 ×
5 mL). Evaporation of solvents gave a yellow to light-brown oil. The
crude product was purified by bulb-to-bulb distillation.
General Method B: Synthesis of Methyl 3-(Benzylamino)-
Propanoate (3). This Method Is Representative for Compounds
24−45 and 3. Benzylamine (1.07 g, 10 mmol) and methyl acrylate
(0.86 g, 10 mmol) were dissolved in 6 mL of hexane, and 50 mg
enzyme was added. The reaction mixture was stirred at 60 °C, 250 rpm
in a sealed vial for 4 h. Enzymes were filtered off and washed with
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Table 7. Yields for PSL-Catalyzed Aza-Michael Additions of Various Amines to Butyl Acrylate
a
b
Based on GC−MS analysis. Reaction conditions: 0.1 mmol of amine, 0.1 mmol of MMA, 5 mg of enzyme, and 0.3 mL of hexane were added to a
sealed vessel and stirred at 60 °C for 4 h.
EtOAc (3 × 5 mL). Evaporation of solvents gave a yellow, transparent
oil. The crude product was purified over SiO2 using DCM/MeOH
95:5 as mobile phase.
used as catalyst. Yield: 69% (liquid, 1.11 g, 7.1 mmol). 1H NMR
(CDCl3) δ (ppm): 3.59 (s, 3H), 2.79 (m, 1H), 2.67 (t, 2H, J = 6.0
Hz), 2.5−2.4 (m, 4H), 1.28 (bs, NH), 1.07 (d, 2H, J = 6.8 Hz). 13C
NMR (CDCl3) δ (ppm): 176.0, 52.4, 52.0, 51.3, 41.3, 39.9, 15.0, IR
(neat) ν: 3005, 2938, 2830, 1727, 1456, 1256, 1197, 1169, 832 cm−1.
HR-MS: mass calcd for C7H17N2O2 [M + H]+ 161.1290, mass
measured [M + H]+ 161.1286.
General Method C: Synthesis of Methyl 3-(Benzylamino)-2-
methylpropanoate (9). This Method Is Representative for
Compounds 46−51 and 9. Benzylamine (1.07 g, 10 mmol) and
methyl methacrylate (1.0 g, 10 mmol) were dissolved in 6 mL of
hexane, and 50 mg of enzyme was added. The reaction mixture was
stirred at 60 °C, 250 rpm in a sealed vial for 4 days. Enzymes were
filtered off and washed with EtOAc (3 × 5 mL). Evaporation of
solvents gave a yellow to light-brown oil. The crude product was
purified over SiO2, using EtOAc/hexane 1:1 as mobile phase. Other
compositions of mobile phases used are DCM/MeOH 95:5 and
MTBE/MeOH 98:2, which yielded comparable results.
Ethyl 3-((3-Aminopropyl)amino)butanoate (18). Prepared accord-
ing to general method A from 1,3-propanediamine (0.75 g, 10 mmol)
and ethyl crotonate (1.15 g, 10 mmol). PSL (50 mg) was used as
1
catalyst. Yield: 81% (liquid, 8.1 mmol, 1.53 g). H NMR (CDCl3) δ
(ppm): 4.14 (q, 2H, J = 7.0 Hz), 3.09 (m, 1H), 2.8−2.6 (m, 4H), 2.42
(dd, 1H, J = 6.8, J = 14.5 Hz), 2.34 (dd, 1H, J = 6.8, J = 14.5 Hz) 1,60
(sm, 2H), 1.26 (brt, 4H, J = 7.0 Hz) 1.12 (d, J = 6.8 Hz, 3H). 13C
NMR (CDCl3) δ (ppm): 172.2, 60.2, 50.2, 44.7, 41.4, 40.3, 33.8, 20.4,
14.1 IR (neat) ν: 3097, 2964, 2929, 2851, 1752, 1446, 1296, 1178, 845,
721 cm−1. HR-MS: mass calcd for C9H21N2O2 [M + H]+ 189.1603,
mass measured [M + H]+ 189.1599.
Methyl 3-((3-Aminopropyl)amino)-2-methylpropanoate (19).
Prepared according to general method A from 1,3-propanediamine
(0.75 g, 10 mmol) and methyl methacrylate (1.0 g, 10 mmol). PSL (50
mg) or CvL was used as catalyst. Yield: 68% (liquid, 1.20 g, 7.0 mmol).
1H NMR (CDCl3) δ (ppm): 3.69 (s, 3H), 2.90 (m, 1H), 2.74 (t, 2H, J
= 7.0 Hz), 2.6−2.5 (m, 4H), 1,61 (sm, 2H), 1.21 (d, 2H, J = 6.8 Hz)
1.1 (bs, (NH)). 13C NMR (CDCl3) δ (ppm): 176.1, 52.7, 51.4, 47.5,
40.3, 39.8, 33.4, 15.2 IR (neat) ν: 3099, 2934, 2844, 1726, 1458, 1258,
1197, 832, 757 cm−1. HR-MS: mass calcd for C8H19N2O2 [M + H]+
175.1446, mass measured [M + H]+ 175.1441.
Compounds 25,20 3,20 36,22 37,22 44,22 and 4522 were described
previously, and their NMR spectra are in accordance with literature.
Ethyl 3-((2-Aminoethyl)amino)butanoate (16). Prepared accord-
ing to general method A from ethylenediamine (0.7 g, 10 mmol) and
ethyl crotonate (1.15 g, 10 mmol). PSL (50 mg) was used as catalyst.
1
Yield: 79% (liquid, 1.39 g, 8.1 mmol). H NMR (CDCl3) δ (ppm):
4.14 (q, 2H, J = 7.0 Hz), 3.09 (sm, 1H), 2.8−2.6 (m, 4H), 2.42 (dd,
1H, J = 7.5, J = 15.0 Hz), 2.35 (dd, 1H, J = 6.5, J = 15.0 Hz), 1.40 (bs,
3H, (NH)), 1.26 (t, J = 7.0 Hz, 3H) 1.11 (d, 2H, J = 6.5 Hz). 13C
NMR (CDCl3) δ (ppm): 171.9, 59.7, 49.6, 49.2, 41.6, 41.4, 20.1, 13.8
IR (neat) ν: 2928, 2974, 2853, 1727, 1447, 1295, 1179, 1028, 845, 801
cm−1. HR-MS: mass calcd for C8H19N2O2 [M + H]+ 175.1446, mass
measured [M + H]+ 175.1442.
Methyl 3-((2-Aminoethyl)amino)-2-methylpropanoate (17). Pre-
pared according to general method A from ethylenediamine (0.7 g, 10
mmol) and methyl methacrylate (1.0 g, 10 mmol). PSL (50 mg) was
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Table 8. Yields for PSL-Catalyzed Aza-Michael Additions of Various Amines to Methacrylates
a
b
Based on GC−MS analysis. Reaction conditions: 0.1 mmol of amine, 0.1 mmol of MMA, 5 mg of enzyme, and 0.3 mL of hexane were added to a
sealed vessel and stirred at 60 °C for 4 days.
Ethyl 3-((4-Aminobutyl)amino)butanoate (20). Prepared accord-
ing to general method A from 1,4-butanediamine (0.88 g, 10 mmol)
and ethyl crotonate (10 mmol, 1.15 g). PSL (50 mg) or CvL was used
as catalyst. Yield: 75% (liquid, 7.5 mmol, 1.52 g). 1H NMR (CDCl3) δ
(ppm): 4.11 (q, 2H, J = 7.5 Hz), 3.08 (sm, 1H), 2.6−2.4 (m, 4H), 2.42
(dd, 1H, J = 7.5, J = 15.0 Hz), 2.34 (dd, 1H, J = 6.5, J = 15.0 Hz), 1.41
(m, 4H), 1.14 (t, 3H, J = 7.5 Hz), 1.14 (bs, 3H, (NH)), 1.09 (d, 3H, J
= 6.5 Hz). 13C NMR (CDCl3) δ (ppm): 172.3, 60.2, 50.6, 44.7, 41.5,
40.3, 33.6, 20.4, 14.1. IR (neat) ν: 2958, 2930, 2871, 1728, 1462, 1308,
1175, 1120, 1064, 738 cm−1. HR-MS: mass calcd for C10H23N2O2 [M
+ H]+ 203.1759, mass measured [M + H]+ 203.1753
(m, 2H). 13C NMR (CDCl3) δ (ppm): 173.2, 54.2, 51.5, 32.1, 26.1,
24.3. IR (neat) ν: 2934, 2853, 2776, 1738, 1437, 1198, 1169, 1113
cm−1. HR-MS: mass calcd for C9H17NO2 [M + H]+ 172.1332, mass
measured [M + H]+ 172.1326.
Methyl 3-Morpholinopropanoate (25). Prepared according to
general method B from morpholine (0.87 g, 10 mmol) and methyl
acrylate (0.86 g, 10 mmol). PSL (50 mg) was used as catalyst. Yield:
1
95% (liquid, 1.64 g, 9.5 mmol). H NMR (CDCl3) δ (ppm): 3.63 (s,
3H), 3.62 (m, 4H), 2.62 (t, 2H, J = 6.5 Hz), 2.44 (t, 2H, J = 6.5 Hz),
2.38 (m, 4H). 13C NMR (CDCl3) δ (ppm): 172.8, 67.2, 54.1, 52.9,
51.8, 31.9. IR (neat) ν: 3342, 2974, 2950, 2828, 1731, 1454, 1193,
1171, 734, 697 cm−1. HR-MS: mass calcd for C8H16NO3 [M + H]+
174.1125, mass measured [M + H]+ 174.1119.
Methyl 3-((4-Aminobutyl)amino)-2-methylpropanoate (21). Pre-
pared according to general method A from 1,4-butanediamine (0.9 g,
10 mmol) and methyl methacrylate (1.0 g, 10 mmol). PSL (50 mg) or
Methyl 3-(4-Methylpiperazin-1-yl)propanoate (26). Prepared
according to general method B from 1-methylpiperazine (1.0 g, 10
mmol) and methacrylate (0.86 g, 10 mmol). PSL (50 mg) was used as
1
CvL was used as catalyst. Yield: 64% (liquid, 1.21 g, 6.5 mmol). H
NMR (CDCl3) δ (ppm): 3.59 (s, 3H), 2.82 (m, 1H), 2.67 (t, 2H, J =
7.0 Hz), 2.4−2.3 (m, 4H), 1,41 (m, 4H), 1.21 (bs, (NH)) 1.06 (d, 3H,
J = 6.8 Hz). 13C NMR (CDCl3) δ (ppm): 176.3, 52.8, 52.0, 49.6, 42.1,
39.9, 31.5, 27.4, 15.3, IR (neat) ν: 2929, 2849, 1728, 1457, 1361, 1495,
1196, 1167, 1125, 833 cm−1 HR-MS: mass calcd for C9H21N2O2 [M +
H]+ 189.1603, mass measured [M + H]+ 189.1600.
1
catalyst. Yield: 95% (liquid, 1.76 g, 9.5 mmol). H NMR (CDCl3) δ
(ppm): 3.61 (s, 3H), 2.65 (t, 2H, J = 7.4 Hz), 2.44 (t, 2H, J = 8.0 Hz),
2.37 (bs, 8H), 2.22 (s, 3H). 13C NMR (CDCl3) δ (ppm): 172.5, 55.0,
53.5, 52.9, 51.6, 46.0, 32.1. IR (neat) ν: 2939, 2879, 2794, 2690, 1736,
1458, 1283, 1161, 1085 cm−1. HR-MS: mass calcd for C9H18N2O2 [M
+ H]+ 187.1441, mass measured [M + H]+ 187.1434.
Ethyl 3-((2-Hydroxyethyl)amino)butanoate (22). Prepared accord-
ing to general method A from ethanolamine (0.8 g, 10 mmol) and
ethyl crotonate (1.15 g, 10 mmol). PSL (50 mg) or CvL was used as
Methyl 3-(Allylamino)propanoate (27). Prepared according to
general method B from allylamine (0.57 g, 10 mmol) and methacrylate
1
1
catalyst. Yield: 82% (liquid, 1.44 g, 8.1 mmol). H NMR (CDCl3) δ
(0.86 g, 10 mmol). PSL (50 mg) was used as catalyst. Yield: n.d. H
(ppm): 4.14 (q, 2H, J = 7.0 Hz), 3.63 (m, 2H), 3.15 (m, 1H), 2.82 (m,
1H), 2.72 (m, 1H), 2.39 (m, 2H), 1.59 (m, 1H, (NH)), 1.28 (t, 3H, J
= 7.0 Hz) 1.14 (d, 3H, J = 6.5 Hz). 13C NMR (CDCl3) δ (ppm):
172.4, 60.9, 60.4, 49.9, 48.3, 41.5, 20.3, 14.1 IR (neat) ν: 3295, 2933,
2873, 1725, 1637, 1560, 1459, 1182, 1054, 1027 cm−1. HR-MS: mass
calcd for C8H18NO3 [M + H]+ 176.1286, mass measured [M + H]+
176.1282.
NMR (CDCl3) δ (ppm): 5.88 (sm, 1H), 5.15 (m, 2H), 3.67 (s, 3H),
3.25 (dt, 2H, J = 6.0, J = 1.5 Hz), 2.87 (t, 2H, J = 6.5 Hz), 2.51 (t, 2H,
J = 6.5 Hz), 1.65 (bs, (NH)). 13C NMR (CDCl3) δ (ppm): 173.2,
136.6, 116.0, 52.2, 51.6, 44.3, 34.5. IR (neat) ν: 3322, 3077, 2952,
1733, 1643, 1437, 1194, 995 cm−1. HR-MS: mass calcd for C7H14NO2
[M + H]+ 144.1014, mass measured [M + H]+ 144.1014.
Methyl 3-((4-Methoxybenzyl)amino)propanoate (28). Prepared
according to general method B from 4-methoxybenzylamine (1.37 g,
10 mmol) and methacrylate (0.86 g, 10 mmol). PSL (50 mg) was used
as catalyst. Yield: 74% (liquid, 1.65 g, 7.4 mmol). 1H NMR (CDCl3) δ
(ppm): 7.19 (m, 2H), 6.78 (m, 2H), 3.72 (s, 3H), 3.66 (s, 2H), 3.60
(s, 3H), 2.81 (t, 2H, J = 6.5 Hz), 2.46 (t, 2H, J = 6.5 Hz), 1.52 (bs,
Methyl 3-(Piperidin-1-yl)propanoate (24). Prepared according to
general method B from piperidine (0.85 g, 10 mmol) and methyl
acrylate (0.86 g, 10 mmol). PSL (50 mg) was used as catalyst. Yield:
1
95% (liquid, 1.62 g, 9.5 mmol). H NMR (CDCl3) δ (ppm): 3.60 (s,
3H), 2.62 (m, 2H), 2.44 (m, 2H), 2.33 (m, 4H), 1.50 (m, 4H), 1.40
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(NH)). 13C NMR (CDCl3) δ (ppm): 173.2, 158.6, 132.3, 129.2, 113.8,
53.5, 53.1, 51.5, 44.4, 30.9. IR (neat) ν: 2997, 2952, 2835, 1731, 1611,
1511, 1457, 1437, 1243, 1169, 1032, 812 cm−1. HR-MS: mass calcd for
C12H18NO3 [M + H]+ 224.1268, mass measured [M + H]+ 224.1274.
Methyl 3-((3,4-Dimethoxybenzyl)amino)propanoate (29). Pre-
pared according to general method B from 3,4-dimethoxybenzylamine
(1.67 g, 10 mmol) and methyl acrylate (0.86 g, 10 mmol). PSL (50
1.60 (bs(NH)), 1.26 (t, 3H, J = 7.0 Hz). 13C NMR (CDCl3) δ (ppm):
172.8, 158.6, 132.3, 129.2, 113.8, 60.4, 55.3, 53.2, 44.4, 34.8, 14.2. IR
(neat) ν: 2980, 2906, 2835, 1727, 1611, 1511, 1243, 1172, 811 cm−1.
HR-MS: mass calcd for C13H20NO3 [M + H]+ 238.1424, mass
measured [M + H]+ 238.1430.
Ethyl 3-((3,4-Dimethoxybenzyl)amino)propanoate (35). Prepared
according to general method B from 3,4-dimethoxybenzylamine (1.67
g, 10 mmol) and ethyl acrylate (1.0 g, 10 mmol). PSL (50 mg) was
used as catalyst. Yield: 89% (liquid, 2.37 g, 8.9 mmol). 1H NMR
(CDCl3) δ (ppm): 6.84 (m, 3H), 4.14 (q, 2H, J = 7.2 Hz), 3.89 (s,
3H), 3.88 (s, 3H), 3.73 (s, 2H), 2.88 (t, 2H, J = 6.4 Hz), 2.50 (t, 2H, J
= 6.4 Hz), 1.66 (bs(NH)), 1.23 (t, 3H, J = 7.2 Hz). 13C NMR
(CDCl3) δ (ppm): 172.7, 148.9, 147.9, 132.8, 120.0, 111.2, 111.0, 60.3,
55.8, 53.3, 44.5, 34.7, 14.1. IR (neat) ν: 2937, 2906, 2835, 1727, 1514,
1260, 1233, 1155, 1026, 806 cm−1. HR-MS: mass calcd for C14H22NO4
[M + H]+ 268.1530, mass measured [M + H]+ 268.1535.
1
mg) was used as catalyst. Yield: 71% (liquid, 1.88 g, 0.7 mmol) H
NMR (CDCl3) δ (ppm): 6.84 (m, 3H), 3.87 (s, 3H), 3.85 (s, 3H),
3.72 (s, 2H), 3.67 (s, 3H), 2.88 (t, 2H, J = 6.5 Hz), 2.52 (t, 2H, J = 6.4
Hz), 1.67 (bs, (NH)). 13C NMR (CDCl3) δ (ppm): 173.2, 149.0,
148.1 132.8, 120.1, 111.3, 111.1, 55.9, 55.8, 53.5, 51.5, 44.4, 34.5, IR
(neat) ν: 3326.7, 3026, 2951, 2840, 1731, 1453, 1436, 1169, 734, 697
cm−1. HR-MS: mass calcd for C13H20NO4 [M + H]+ 254.1392, mass
measured [M + H]+ 254.1357.
Methyl 3-(Benzylamino)propanoate (3). Prepared according to
general method B from benzylamine (1.07 g, 10 mmol) and
methacrylate (0.86 g, 10 mmol). PSL (50 mg) was used as catalyst.
Ethyl 3-(Benzylamino)propanoate (36). Prepared according to
general method B from benzylamine (1.07g, 10 mmol) and ethyl
acrylate (1.0 g, 10 mmol). PSL (50 mg) was used as catalyst. Yield:
75% (liquid, 1.56 g, 7.5 mmol) 1H NMR (CDCl3) δ (ppm): 7.20 (m,
4H), 7.15 (m, 1H), 4.08 (q, 2H, J = 7.2 Hz), 3.73 (s, 2H), 2.83 (t, 2H,
J = 6.4 Hz), 2.45 (t, 2H, J = 6.4 Hz), 1.58 (bs (NH)), 1.18 (t, 3H, J =
7.2 Hz). 13C NMR (CDCl3) δ (ppm): 172.8, 140.2, 128.4, 128.1,
126.9, 60.4, 53.8, 44.5, 34.8, 14.2. IR (neat) ν: 3327, 3028, 2981, 2906,
2830, 1728, 1175, 1028, 734, 698 cm−1. HR-MS: mass calcd for
C12H18NO2 [M + H]+ 208.1332, mass measured [M + H]+ 208.1326.
Ethyl 3-(2-Phenylethylamino)propanoate (37). Prepared accord-
ing to general method B from 2-phenylethylamine (1.09 g, 10 mmol)
and ethyl acrylate (1.0 g, 10 mmol). PSL (50 mg) was used as catalyst.
1
Yield: 78% (liquid, 1.51 g, 7.8 mmol). H NMR (CDCl3) δ (ppm):
7.22 (m, 5H), 3.77 (s, 2H), 3.64 (s, 3H), 2.87 (t, 2H, J = 6.5 Hz), 2.50
(t, 2H, J = 6.5 Hz), 1.75 (bs, (NH)). 13C NMR (CDCl3) δ (ppm):
173.1, 140.2, 128.4, 128.0, 126.9, 53.7, 51.5, 44.5, 34.6. IR (neat) ν:
3326.7, 3026, 2951, 2840, 1731, 1453, 1436, 1169.5, 734, 697 cm−1.
HR-MS: mass calcd for C11H16NO2 [M + H]+ 194.1176, mass
measured [M + H]+ 194.1169.
Ethyl 3-(Piperidin-1-yl)propanoate (30). Prepared according to
general method B from piperazine (0.86 g, 10 mmol) and ethyl
acrylate (1.0 g, 10 mmol). PSL (50 mg) was used as catalyst. Yield:
1
95% (liquid, 1.75 g, 9.5 mmol). H NMR (CDCl3) δ (ppm): 4.07 (q,
1
2H, J = 7.5 Hz), 2.59 (t, 2H, J = 7.5 Hz), 2.44 (t, 2H, J = 7.6 Hz), 2.34
(m, 4H), 1.50 (m, 4H), 1.36 (m, 2H), 1.20 (t, 3H, J = 7.5 Hz). 13C
NMR (CDCl3) 172.8, 60.3, 54.3, 54.2, 32.3, 25.9, 24.3, 14.2. IR (neat)
ν: 2980, 2934, 2853, 1734, 1171, 1153, 1075 cm−1. HR-MS: mass
calcd for C10H20NO2 [M + H]+ 186.1489, mass measured [M + H]+
186.1482.
Yield: 70% (liquid, 1.55 g, 7.1 mmol). H NMR (CDCl3) δ (ppm):
7.21 (m, 5H), 4.08 (q, 2H, J = 6.5 Hz), 2.87 (m, 4H), 2.70 (t, 2H, J =
7.5 Hz), 2.44 (t, 2H, J = 7.5 Hz), 1.51 (bs, 1H, (NH)) 1.21 (t, 3H, J =
6.5 Hz). 13C NMR (CDCl3) δ (ppm): 172.4, 139.8, 128.5, 125.9, 60.1,
50.8, 44.1, 36.2, 34.6, 14.0. IR (neat) ν: 3027, 2974, 2950, 2829, 1731,
1495, 1255, 1194, 735, 698 cm−1. HR-MS: mass calcd for C14H22NO2
[M + H]+ 222.1494, mass measured [M + H]+ 222.1498.
Ethyl 3-Morpholinopropanoate (31). Prepared according to
general method B from morpholine (0.87 g, 10 mmol) and ethyl
acrylate (1.0 g, 10 mmol). PSL (50 mg) was used as catalyst. Yield:
Butyl 3-(Piperidin-1-yl)propanoate (38). Prepared according to
general method B from piperidine (0.85 g, 10 mmol) and butyl
acrylate (1.28 g, 10 mmol). PSL (50 mg) was used as a catalyst. Yield:
1
95% (liquid, 9.5 mmol, 1.77 g). H NMR (CDCl3) δ (ppm): 4.07 (q,
1
2H, J = 7.5 Hz), 3.62 (m, 4H), 2.62 (t, 2H, J = 7.5 Hz), 2.5−2.3 (m,
6H), 1.19 (t, 3H, J = 7.5 Hz). 13C NMR (CDCl3) δ (ppm): 174.7,
69.2, 62.7, 56.3, 55.7, 34.5, 16.5. IR (neat) ν: 2958, 2854, 2810, 1731,
1255, 1185, 1115, 1010 cm−1. HR-MS: mass calcd for C9H18NO3 [M
+ H]+ 188.1281, mass measured [M + H]+ 188.1276.
94% (liquid, 2.00 g, 9.4 mmol). H NMR (CDCl3) δ (ppm): 4.01 (t,
2H, J = 6.6 Hz), 2.61 (t, 2H, J = 7.4 Hz), 2.42 (t, 2H, J = 7.4 Hz), 2.32
(m, 4H), 1.54 (m, 6H), 1.35 (m, 4H), 0.86 (t, 3H, J = 8.6 Hz). 13C
NMR (CDCl3) δ (ppm): 172.8, 64.2, 54.3, 54.2, 32.4, 30.7, 25.9, 24.3,
19.1, 13.7. IR (neat) ν: 2934, 2855, 2776, 1735, 1170, 1153, 1114
cm−1. HR-MS: mass calcd for C12H24NO2 [M + H]+ 214.1788, mass
measured [M + H]+ 214.1794.
Ethyl 3-(4-Methylpiperazin-1-yl)propanoate (32). Prepared ac-
cording to general method B from 1-methylpiperazine (1.0 g, 10
mmol) and ethyl acrylate (1.0 g, 10 mmol). PSL (50 mg) was used as
Butyl 3-Morpholinopropanoate (39). Prepared according to
general method B from morpholine (0.87 g, 10 mmol) and butyl
acrylate (1.28 g, 10 mmol). PSL (50 mg) was used as catalyst. Yield:
1
catalyst. Yield: 95% (liquid, 1.90 g, 9.5 mmol). H NMR (CDCl3) δ
(ppm): 4.14 (q, 2H, J = 7.0 Hz), 2.70 (t, 2H, J = 7.0 Hz), 2.5−2.4 (m,
10H), 2.28 (s, 3H), 1.25 (t, 3H, J = 7.0 Hz). 13C NMR (CDCl3) δ
(ppm): 172.5, 60.3, 55.1, 53.5, 52.8, 46.0, 32.3, 14.2. IR (neat) ν: 2938,
2879, 2794, 1733, 1459, 1284, 1180, 1162, 1027 cm−1. HR-MS: mass
calcd for C10H21N2O2 [M + H]+ 201.1598, mass measured [M + H]+
201.1593.
1
90% (liquid, 1.93 g, 9.0 mmol). H NMR (CDCl3) δ (ppm): 4.02 (t,
2H, J = 7.0 Hz), 3.62 (m, 4H), 2.61 (t, 2H, J = 7.5 Hz), 2.46 (t, 2H, J =
7.5 Hz) 2.44 (m, 4H), 1. 57 (m, 2H), 1.34 (m, 2H), 0.87 (t, 3H, J =
7.6 Hz). 13C NMR (CDCl3) 172.4, 66.9, 64.3, 54.0, 53.4, 32.2, 30.7,
19.1, 13.7. IR (neat) ν: 2958, 2855, 2810, 1733, 1458, 1116, 1035, 868,
859 cm−1. HR-MS: mass calcd for C11H22NO3 [M + H]+ 216.1594,
mass measured [M + H]+ 216.1588.
Ethyl 3-(Allylamino)propanoate (33). Prepared according to
general method B from allylamine (0.57 g, 10 mmol) and ethyl
acrylate (1.0 g, 10 mmol). PSL (50 mg) was used as catalyst. Yield:
Butyl 3-(4-Methylpiperazin-1-yl)propanoate (40). Prepared ac-
cording to general method B from 1-methylpiperazine (1.00 g, 10
mmol) and butyl acrylate (1.28 g, 10 mmol). PSL (50 mg) was used as
1
n.d. H NMR (CDCl3) δ (ppm): 5.90 (sm, 1H), 5.16 (m, 2H), 4.15
(q, 2H, J = 7.2 Hz), 3.27 (dt, J = 6.0, J = 1.5 Hz, 2H), 2.88 (t, 2H, J =
6.6 Hz), 2.50 (t, 2H, J = 6.6 Hz), 1.53 (bs, (NH)), 1.27 (t, 3H, J = 7.2
Hz). 13C NMR (CDCl3) δ (ppm): 172.7, 136.7, 115.9, 60.4, 52.2, 44.4,
34.8, 14.2. IR (neat) ν: 3362, 3079, 2981, 2907, 2825, 1729, 1459,
1417, 1179 cm−1. HR-MS: mass calcd for C8H16NO2 [M + H]+
158.1176, mass measured [M + H]+ 158.1170.
Ethyl 3-((4-Methoxybenzyl)amino)propanoate (34). Prepared
according to general method B from 4-methoxybenzylamine (1.37 g,
10 mmol) and ethyl acrylate (1.0 g, 10 mmol). PSL (50 mg) was used
as catalyst. Yield: 71% (liquid, 1.68 g, 7.1 mmol). 1H NMR (CDCl3) δ
(ppm): 7.24 (m, 2H), 6.87 (m, 2H), 4.14 (q, 2H, J = 7.0 Hz), 3.80 (s,
3H), 3.74 (s, 2H), 2.89 (t, 2H, J = 6.4 Hz), 2.52 (t, 2H, J = 6.4 Hz),
1
catalyst. Yield: 95% (liquid, 2.17 g, 9.5 mmol). H NMR (CDCl3) δ
(ppm): 4.09 (t, 2H, J = 6.6 Hz), 2.71 (t, 2H, J = 6.8 Hz), 2.51−2.36
(m, 10H), 2.28 (s, 3H), 1.64−1.57 (m, 2H), 1.43−1.33 (m, 2H), 0.94
(t, 3H, J = 7.2 Hz). 13C NMR (CDCl3) δ (ppm): 172.6, 64.3, 55.1,
53.6, 52.9, 46.0, 32.4, 30.7, 19.1, 13.7. IR (neat) ν: 2936, 2794, 1734,
1458, 1178, 1013 cm−1. HR-MS: mass calcd for C12H25N2O2 [M +
H]+ 229.1916, mass measured [M + H]+ 229.1908.
Butyl 3-(Allylamino)propanoate (41). Prepared according to
general method B from allylamine (0.57 g, 10 mmol) and butyl
acrylate (1.28 g, 10 mmol). PSL (50 mg) was used as a catalyst. Yield:
19% (liquid, 0.35 g, 1.9 mmol). 1H NMR (CDCl3) δ (ppm): 5.83 (sm,
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Article
1H), 5.07 (m, 2H), 4.02 (t, 2H, J = 6.6 Hz), 3.18 (dt, 2H, J = 6.0, J =
1.5 Hz), 2.81 (t, 2H, J = 6.6 Hz), 2.44 (t, 2H, J = 6.6 Hz), 1.57 (m,
2H), 1.46 (bs (NH)), 1.33 (m, 2H), 0.88 (t, 3H, J = 7.4 Hz). 13C
NMR (CDCl3) δ (ppm): 170.4, 134.2, 113.5, 61.9, 49.8, 42.0, 32.4,
28.2, 16.7, 11.2. IR (neat) ν: 3327, 3078, 2960, 2934, 1730, 1461,
1177, 916 cm−1. HR-MS: mass calcd for C10H20NO2 [M + H]+
186.1489, mass measured [M + H]+ 186.1483.
2853, 2809, 1736, 1457, 1116, 1070, 985, 835 cm−1. HR-MS: mass
calcd for C9H18NO3 [M + H]+ 188.1281, mass measured [M + H]+
188.1275.
Methyl 2-Methyl-3-(4-methylpiperazin-1-yl)propanoate (48). Pre-
pared according to general method B from 1-methylpiperazine (1.0 g,
10 mmol) and methyl methacrylate (1.0 g, 10 mmol). PSL (50 mg)
1
was used as catalyst. Yield: 84% (liquid, 1.68 g, 8.4 mmol). H NMR
(CDCl3) δ (ppm): 3.61 (s, 3H), 2.62 (m, 2H), 2.4−2.2 (m, 9H), 2.19
(s, 3H), 1.14 (d, 3H, J = 7.5 Hz). 13C NMR (CDCl3) δ (ppm): 176.5,
61.5, 55.2, 53.2, 51.5, 46.0, 37.8, 15.5. IR (neat) ν: 2938, 2878, 2794,
1737, 1458, 1245, 1168, 1013 cm−1. HR-MS: mass calcd for
C10N21N2O2 [M + H]+ 201.1584, mass measured [M + H]+ 201.1591.
Methyl 3-(Allylamino)-2-methylpropanoate (49). Prepared ac-
cording to general method B from allylamine (0.57 g, 10 mmol) and
methyl methacrylate (1.0 g, 10 mmol). PSL (50 mg) was used as
Butyl 3-((4-Methoxybenzyl)amino)propanoate (42). Prepared
according to general method B from 4-methoxybenzylamine (1.37 g,
10 mmol) and butyl acrylate (1.28 g, 10 mmol). PSL (50 mg) was
1
used as a catalyst. Yield: 86% (liquid, 2.66 g, 8.6 mmol). H NMR
(CDCl3) δ (ppm): 7.24 (m, 2H), 6.87 (m, 2H), 4.08 (q, 2H, J = 7.5
Hz), 3.79 (s, 3H), 3.73 (s, 2H), 2.87 (t, 2H, J = 6.4 Hz), 2.53 (t, 2H, J
= 6.4 Hz), 1.60 (bs, NH), 1.58 (m, 2H), 1.34, (m, 2H), 0.92 (t, 3H, J =
7.0 Hz). 13C NMR (CDCl3) δ (ppm): 172.5, 158.3, 132.0, 129.4,
113.4, 60.5, 55.3, 53.2, 44.4, 34.8, 30.3, 18.8, 13.3. IR (neat) ν: 2957,
2934, 2834, 1728, 1510, 1462, 1243, 1169, 1033, 810 cm−1. HR-MS:
mass calcd for C15H24NO3 [M + H]+ 266.1756, mass measured [M +
H]+ 266.1748.
Butyl 3-((3,4-Dimethoxybenzyl)amino)propanoate (43). Prepared
according to general method B from 3,4-dimethoxybenzylamine (1.67
g, 10 mmol) and butyl acrylate (1.28 g, 10 mmol). PSL (50 mg) was
used as catalyst. Yield: 86% (liquid, 2.53 g, 8.6 mmol). 1H NMR
(CDCl3) δ (ppm): 6.75 (m, 3H), 4.03 (t, 2H, J = 6.6 Hz), 3.86 (s,
3H), 3.80 (s, 3H), 3.67 (s, 2H), 2.83 (t, 2H, J = 6.6 Hz), 2.46 (t, 2H, J
= 6.2 Hz), 1.52 (m, 3H), 1.32 (m, 2H), 0.88 (t, 3H, J = 7.6 Hz). 13C
NMR (CDCl3) δ (ppm): 173.1, 148.2, 147.3, 133.0, 120.1 112.0,
111.1, 64.2, 55.9, 53.6, 44.6, 34.0, 30.8, 19.5, 13.8. IR (neat) ν: 2958,
2873, 2835, 1729, 1514, 1260, 1234, 1028 cm−1. HR-MS: mass calcd
for C16H26NO4 [M + H]+ 296.1843, mass measured [M + H]+
296.1849.
Butyl 3-(Benzylamino)propanoate (44). Prepared according to
general method B from benzylamine (1.07 g, 10 mmol) and butyl
acrylate (1.28 g, 10 mmol). PSL (50 mg) was used as catalyst. Yield:
65% (liquid, 1.53 g, 0.66 mmol). 1H NMR (CDCl3) δ (ppm): 7.28 (m,
5H), 4.09 (t, 2H, J = 6.8 Hz), 3.80 (s, 2H), 2.89 (t, 2H, J = 6.4 Hz),
2.52 (t, 2H, J = 6.4 Hz), 1.79 (bs, 1H), 1.60 (m, 2H), 1.36 (m,2H),
0.92 (t, 3H, J = 7.2 Hz). 13C NMR (CDCl3) δ (ppm): 172.7, 140.1,
128.3, 128.0, 126.8, 64.2, 53.7, 44.5, 34.7, 30.5, 19.0, 13.6. IR (neat) ν:
3063, 3028, 2959, 2933, 2873, 1729, 1495, 1172, 734, 697 cm−1. HR-
MS: mass calcd for C14H22NO2 [M + H]+ 236.1632, mass measured
[M + H]+ 236.1638.
1
catalyst. Yield: 18% (liquid, 0.28 g, 1.8 mmol). H NMR (CDCl3) δ
(ppm): 5.84 (sm, 1H), 5.05 (m, 2H), 3.63 (s, 3H), 3.19 (dt, 2H, J =
6.0, J = 1.5 Hz), 2.82 (m, 3H), 2.62 (m, 2H), 1.60 (bs (NH)), 1.18 (d,
3H, J = 7.5 Hz). 13C NMR (CDCl3) δ (ppm): 176.3, 136.6, 116.0,
52.2, 52.0, 51.6, 40.0, 15.3. IR (neat) ν: 2945, 2827, 1725, 1520, 1477,
1237, 1190, 833 cm−1 HR-MS: mass calcd for C8H16NO2 [M + H]+
158.1176, mass measured [M + H]+ 158.1175.
Methyl 3-((4-Methoxybenzyl)amino)-2-methylpropanoate (50).
Prepared according to general method B from 4-methoxybenzylamine
(1.37 g, 10 mmol) and methyl methacrylate (1.0 g, 10 mmol). PSL (50
1
mg) was used as catalyst. Yield: 64% (liquid, 1.52 g, 6.4 mmol). H
NMR (CDCl3) δ (ppm): 7.17 (m, 2H), 6.78 (m, 2H), 3.72 (s, 3H),
3.65 (s, 2H), 3.61 (s, 3H), 2.81 (m, 1H), 2.61 (m, 2H), 1.46 (bs
(NH)), 1.10 (d, 3H, J = 7.0 Hz). 13C NMR (CDCl3) δ (ppm): 176.3,
158.6, 132.5, 129.2, 113.8, 55.2, 53.1, 51.6, 40.0, 15.3. IR (neat) ν:
3340, 2950, 2835, 1730, 1511, 1459, 1243, 1171, 1065 cm−1. HR-MS:
mass calcd for C13H20NO3 [M + H]+ 238.1438, mass measured [M +
H]+ 238.1434.
Methyl 3-((3,4-Dimethoxybenzyl)amino)-2-methylpropanoate
(51). Prepared according to general method B from 3,4-dimethox-
ybenzylamine (liquid, 1.67 g, 10 mmol) and methyl methacrylate (1.0
g, 10 mmol). PSL (50 mg) was used as catalyst. Yield: 80% (2.14 g, 8.0
mmol). 1H NMR (CDCl3) δ (ppm): 6.77 (m, 3H), 3.88 (s, 3H), 3.79
(s, 3H), 3.66 (s, 2H), 3.61 (s, 3H), 2.82 (m, 1H), 2.62 (m, 2H), 1.51
(bs, (NH)), 1.17 (d, 2H, J = 7.0 Hz). 13C NMR (CDCl3) δ (ppm):
176.3, 148.9, 148.0, 133.0, 120.1, 111.3, 111.0, 55.9, 55.8, 53.5, 52.1,
51.6, 40.0, 15.3. IR (neat) ν: 2950, 2834, 1730,1514, 1256, 1234, 1027
cm−1. HR-MS: mass calcd for C14H22NO4 [M + H]+ 268.1543, mass
measured [M + H]+ 268.1540.
Butyl 3-(2-Phenylethylamino)propanoate (45). Prepared accord-
ing to general method B from phenylethylamine (1.09 g, 10 mmol)
and butyl acrylate (1.1 g, 10 mmol). PSL (50 mg) was used as catalyst.
Methyl 3-(Benzylamino)-2-methylpropanoate (9). Prepared ac-
cording to general method B from benzylamine (1.07 g, 10 mmol) and
methyl methacrylate (1.0 g, 10 mmol). PSL (50 mg) was used as
1
Yield: 60% (liquid, 1.50 g, 6.1 mmol). H NMR (CDCl3) δ (ppm):
7.21 (m, 5H), 4.07 (t, 2H, J = 7.5 Hz), 2.87 (m, 4H), 2.80 (t, 2H, J =
6.5 Hz) 2.50 (t, 2H, J = 6.5 Hz) 1.58 (m, 2H) 1.52 (brs, (NH)), 1.35
(m, 2H) 0.92 (t, 3H, J = 7.5 Hz). 13C NMR (CDCl3) δ (ppm): 172.7,
140.0, 128.7, 128.4, 126.1, 64.3, 51.0, 45.0, 36.4, 34.0, 30.6, 19.1, 13.7.
IR (neat) ν: 3027, 2957, 2932, 2872, 1729, 1454, 1250, 1166, 1065,
747, 698 cm−1. HR-MS: mass calcd for C15H23NO2 [M + H]+
250.1807, mass measured [M + H]+ 250.1810.
1
catalyst. Yield: 81% (liquid, 1.68 g, 8.1 mmol). H NMR (CDCl3) δ
(ppm): 7.31 (m, 5H), 3.72 (s, 2H), 3.61 (s, 3H), 2.79 (m, 1H), 2.67
(m, 2H), 1.56 (bs (NH)), 1.17 (d, 3H, J = 6.8 Hz). 13C NMR
(CDCl3) δ (ppm): 176.3, 140.3, 128.35, 128.0, 126.9, 53.7, 52.1, 51.6,
40.1, 15.3. IR (neat) ν: 3027, 2974, 2950, 2829, 1731, 1495, 1255,
1194, 735, 698 cm−1. HR-MS: mass calcd for C12H18NO2 [M + H]+
208.1375, mass measured [M + H]+ 208.1370.
Methyl 2-Methyl-3-(piperidin-1-yl)propanoate (46). Prepared
according to general method B from piperidine (0.85 g, 10 mmol)
and methyl methacrylate (1.0 g, 10 mmol). PSL (50 mg) was used as
ASSOCIATED CONTENT
* Supporting Information
Influence of enzyme loading on reaction mixture composition
■
1
catalyst. Yield: 60% (liquid, 1.11 g, 6.0 mmol). H NMR (CDCl3) δ
S
(ppm): 3.69 (s, 3H), 2.65 (m, 1H), 2.53 (dd, 1H, J = 12.5, J = 8.5 Hz),
2.3−2.2 (m, 4H), 2.17 (dd, 1H, J = 12.5, J = 6.0 Hz), 1.45 (m, 4H),
1.32 (m, 2H), 1.06 (d, 3H, J = 7.5 Hz). 13C NMR (CDCl3) δ (ppm):
176.8, 62.4, 54.7, 51.5, 32.9, 25.1, 24.4, 15.7. IR (neat) ν: 2934, 2854,
1739, 1457, 1435, 1352, 1169, 1157 cm−1. HR-MS: mass calcd for
C10H20NO2 [M + H]+ 186.1489, mass measured [M + H]+ 186.1486.
Methyl 2-Methyl-3-morpholinopropanoate (47). Prepared accord-
ing to general method B from morpholine (0.87 g, 10 mmol) and
methyl methacrylate (1.0 g, 10 mmol). PSL (50 mg) was used as
and enzymatic activity of enzyme from supplier vs inactivated
1
enzyme; H and 13C NMR spectra for compounds 3, 9, and
16−51. This material is available free of charge via the Internet,
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: maurice.franssen@wur.nl.
■
1
catalyst. Yield: 68% (liquid, 1.27 g, 6.8 mmol). H NMR (CDCl3) δ
(ppm): 3.69 (s, 3H), 3.72 (m, 4H), 2.70 (m, 2H), 2.47 (m, 2H), 2.40
(m, 2H), 2.33 (m, 1H), 1.26 (d, 3H, J = 6.4 Hz). 13C NMR (CDCl3) δ
(ppm): 176.4, 67.0, 62.0, 53.7, 51.5, 37.6, 15.4. IR (neat) ν: 2953,
Notes
The authors declare no competing financial interest.
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Article
(33) Sheldon, R. A.; Schoevaart, R.; van Langen, L. M. Meth.
Biotechnol. 2006, 22, 31−45.
ACKNOWLEDGMENTS
■
We thank Frank Claassen and Barend van Lagen (Laboratory of
Organic Chemistry, Wageningen University) and Ben van den
Broek (Food and Biobased Research, Wageningen) for their
technical support, Bart Zwijnenburg (Johnson Matthey) for
helpful discussions, and NWO Aspect (Project No. 053.62.031)
for financial support.
(34) Sheldon, R. A. Adv. Synth. Catal. 2007, 349, 1289−1307.
(35) Cao, L.; Langen, L. M.; Janssen, M. H. A.; Sheldon, R. A.
Preparation and properties of crosslinked aggregates of penicillin
acylase and other enzymes. EP 1999-203117, Sep 23, 1999.
(36) Lopez-Iglesias, M.; Busto, E.; Gotor, V.; Gotor-Fernandez, V.
Adv. Synth. Catal. 2011, 353, 2345−2353.
(37) Laane, C.; Boeren, S.; Vos, K.; Veeger, C. Biotechnol. Bioeng.
1987, 30, 81−87.
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