Study of the chemoselectivity in the aminolysis reaction of methyl acrylate catalysed by lipase B from Candida antarctica

Article abstract of DOI:10.1002/adsc.200505025

The aminolysis reaction of methyl acrylate (MA) with N,N-dimethyl-1,3- propanediamine (DMAPA) in organic solvents has been optimised using lipase B from Candida antarctica (CAL-B) as biocatalyst. A kinetic study about the influence of the reactant concentrations, organic solvent, temperature and enzyme form has been developed focused on minimising the formation of the Michael addition products. The economic efficiency of this process has been finally investigated by reusing the enzyme in the best reaction conditions, thereby observing no significant loss of activity after three reaction cycles.

Full text of DOI:10.1002/adsc.200505025

FULL PAPERS
Study of the Chemoselectivity in the Aminolysis Reaction of
Methyl Acrylate Catalysed by Lipase B from Candida antarctica
a
a
b
´
Oliver Torre, Vicente Gotor-Fernandez, Ignacio Alfonso,
Luis Fernando Garcꢀa-Alles,a Vicente Gotora,*
Departamento de Quꢀmica Organica e Inorganica, Facultad de Quꢀmica, Universidad de Oviedo, 33006 Oviedo, Spain
a
´
´
Fax: (þ34)-98-510-3448, e-mail: vgs@fq.uniovi.es
b
´
´
´
Departamento de Quꢀmica Inorganica y Organica, Universidad Jaume I, Campus del Riu Sec, 12071 Castellon, Spain
Fax: (þ34)-964-728-214, e-mail: alfonso@qui.uji.es
Received: January 19, 2005; Accepted: March 18, 2005
Abstract: The aminolysis reaction of methyl acrylate products. The economic efficiency of this process
(MA)
with
N,N-dimethyl-1,3-propanediamine has been finally investigated by reusing the enzyme
(DMAPA) in organic solvents has been optimised us- in the best reaction conditions, thereby observing no
ing lipase B from Candida antarctica (CAL-B) as bio- significant loss of activity after three reaction cycles.
catalyst. A kinetic study about the influence of the re-
actant concentrations, organic solvent, temperature
and enzyme form has been developed focused on Keywords: amides; aminolysis; Candida antarctica li-
minimising the formation of the Michael addition pase B; green chemistry; Michael addition
Introduction
building block in the preparation of polymers
(Scheme 1). Currently, the chemical synthesis of 3 re-
Compounds containing amides frequently show impor- quires the use of copper catalysts and high temperatures
tant biological activities[1] and industrial applications[2] (over 2108C) so the application of an enzyme could dra-
and, consequently, their syntheses have been extensive- matically decrease both the contamination factor and
ly studied over the years.[3] Although the conversion of the waste of energy.[9] Consequently, biocatalysis ap-
esters to amides is a useful synthetic operation, it usually plied to this process could provide a sustainable alterna-
presents some disadvantages in terms of drastic reaction tive to a chemical reaction of industrial interest.[10]
conditions, long reaction times or strong alkali metal as
catalyst, which usually are not compatible with sensitive
functionalities. Biocatalysis offers a clean and ecological Results and Discussion
way to perform chemical processes under mild reaction
conditions and with high degrees of selectivity and thus, In the reaction between 1 and 2, four reaction products
it could be an interesting alternative to provide this type can be observed depending on the reaction conditions,
of substrates.
The use of lipases to generate amide bonds in organic
solvents has been extensively studied over the last dec-
ades since Klibanov[4] and Wong[5] applied the aminoly-
sis reaction in peptide synthesis.[6] However, the first ex-
ample of a lipase-catalysed aminolysis reaction was re-
ported by our group in the reaction of racemic ethyl 2-
chloropropionate with different aliphatic and aromatic
amines.[7] From that moment, the aminolysis reaction
has been recognised as a useful tool for the synthesis
of interesting nitrogenated compounds.[8]
We report here a comprehensive study of all possible
parameters affecting the reaction between methyl acryl-
ate (MA, 1) and N,N-dimethyl-1,3-propanediamine
(DMAPA, 2) focused on the enzymatic synthesis of N-
(4-methyl-4-azapentyl)acrylamide (3), an important (3).
Scheme 1. Synthesis of N-(4-methyl-4-azapentyl)acrylamide
Adv. Synth. Catal. 2005, 347, 1007–1014
DOI: 10.1002/adsc.200505025
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Oliver Torre et al.
FULL PAPERS
acrylamide 3 for the aminolysis reaction between 1 and
2, Michael adduct 4 due to Michael addition of 2 to 1, Mi-
chael-amide compound 5 for firstly Michael reaction of
1 and 2 and subsequent aminolysis reaction of 2 and 4,
and di-Michael product 6 by a double Michael reaction,
between 1 and 2 to form 4 and a subsequent Michael ad-
dition of 2 to 4 (Scheme 2). The formation of Michael
adducts in enzymatic processes catalysed by Candida
antarctica lipase B has been previously reported by our
group using secondary amines and acrylonitrile[11] and
other authors using thiols and a,b-unsaturated carbonyl
compounds.[12] Different parameters have been studied
in order to optimise the formation of 3, such as enzyme,
organic solvent, ester and amine concentrations, amine
addition form, temperature, amount of biocatalyst and
enzyme recycling.
Scheme 2. Study of the enzymatic process between MA (1)
and DMAPA (2): considered parameters and possible prod-
ucts of the reaction.
Influence of the Enzyme and its Preparation
In our experience, lipase B from Candida antarctica
(CAL-B) is by far the best biocatalyst for the prepara- favoured the Michael addition, forming preferably 4
tion of acrylamides,[13] so an inspection of the effect of (entries 3–9); in fact, 3 was not observed in some cases.
polymer support was studied using 1,4-dioxane as sol- The use of other forms of the biocatalyst such as CAL-B
vent. An immobilised catalyst (Novozyme 435), sup- (Chirazyme) did not result in a better selectivity (entry
ported (Chirazyme), lyophilised, denatured from Novo- 11) and even other biocatalysts like PSL also favoured
zyme species, and Pseudomonas cepacia lipase (PSL) the formation of 4 (entries 12 and 13).
were considered in this study (Table 1). CAL-B Novo-
Lyophilised CAL-B (entry 3) catalysed 7 times faster
zyme (entry 2) presented the highest reaction rate in the Michael addition than in the absence of catalyst (en-
the formation of 3 while other enzymatic preparations try 1). Moreover, the support seemed not to have any in-
Table 1. Kinetics of 3 and 4 after 1 h of reaction between 10% (v/v) of 1 and 2.5% (v/v) of 2 using 50 mg of enzyme in 1,4-
dioxane.
Entry
Enzyme
T [8C]
mmol of 3
mmol of 4
Ratio of 3/4
V0 (3)[a]
V0 (4)[a]
1
2
3
4
5
6
7
8
9
10
11
12
13
–
30
30
30
30
30
30
30
30
30
40
40
40
40
0.000
0.229
0.002
0.000
0.009
0.000
0.053
0.040
0.000
0.000
0.200
0.001
0.034
0.012
0.043
0.089
0.028
0.160
0.048
0.080
0.139
0.020
0.009
0.059
0.038
0.109
0.000
5.381
0.020
0.000
0.058
0.000
0.658
0.289
0.000
0.032
3.416
0.015
0.309
0.0
5.3
0.0
0.0
0.2
0.0
1.5
0.6
0.0
0.0
4.3
0.0
0.6
0.2
0.7
1.4
0.5
4.9
0.6
2.8
4.0
0.2
0.2
0.9
0.6
1.8
CAL-B (Novozyme)
CAL-B (lyophilised)
CAL-B (denatured)[b]
CAL-B 1[c]
CAL-B 2[d]
CAL-B 3[e]
CAL-B 4[f]
BSA[g]
–
CAL-B (Chirazyme)
PSL[h]
PSL-C[i]
[a]
Initial rate; units: mM minꢀ1
CAL-B denatured in refluxing water during 10 h.
CAL-B supported over glyoxyl-Agarose polymer.
CAL-B supported over glyoxyl-Sepabeads polymer.
CAL-B supported over C18-Sepabeads polymer.
CAL-B supported over glutaraldehyde-Sepabeads polymer.
Bovine serum albumin.
.
[b]
[c]
[d]
[e]
[f]
[g]
[h]
[i]
Pseudomonas cepacia lipase (lyophilised).
Pseudomonas cepacia lipase (supported over polymer).
1008
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Aminolysis Reaction of Methyl Acrylate Catalysed by Lipase B
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fluence on the formation of 4 as a hydrophobic support
such as C18-Sepabeads (entry 7) does not diminish its
rate of formation, so the main influence on the reaction
rate was the presence of enzyme in the reaction media.
To clearly show if catalysis of the Michael addition oc-
curs in the lipase active site or on the enzyme surface,
CAL-B was substituted by bovine serum albumin
(BSA), a protein which lacks active sites but has a simi-
lar structure to the lipase. Assays with this protein (entry
9) showed similar results to that obtained without bioca-
talyst. Moreover, performing the reaction with lyophi-
lised CAL-B also supported our proposal, as the Mi-
chael addition is faster than without any catalyst (com-
pare entries 1 and 3) or in the presence of BSA (compare
entries 3 and 9).
Taking all this into account, we concluded that the li-
pase is able to catalyse the Michael addition reaction
and we suggest that the polymer support can affect the
enzymatic activity, as very usually observed in biotrans-
formations. Besides, it is clear that the best preparation
of CAL-B for aminolysis reaction is Novozyme 435.
Figure 1. Reaction rates in the Michael addition catalysed by
CAL-B at different concentrations of MA in different organ-
ic solvents at 308C.
done to assure that no products remained trapped on
the catalyst surface. Best ratios corresponded to the
process occurring in 1,4-dioxane which completely dis-
Influence of the Organic Solvent
Even in the absence of biocatalyst, polar solvents fav- solved the reactants and the products of the reaction,
oured the formation of the Michael adduct as observed as none of them were observed after an additional
in the comparative study between hexane, tert-butyl wash with MeOH (entry 1). Non-polar solvents as
methyl ether (TBME) and acetonitrile (MeCN) repre- Et2O (entry 5) or hexane (entry 8) presented problems
sented in the Figure 1. Small differences were observed due to the low solubility of the reactants, so high concen-
in the reactions between hexane and TBME; however, a trations of products were obtained after washing with
more polar solvent like MeCN increased the reaction MeOH.
rate in the addition of DMAPA to methyl acrylate.[14]
To extend the analysis to a wide range of solvents in
the presence of enzyme, the amounts of 3 and 4 formed Influence of the Ester and Amine Concentrations
were quantified in the reaction using 1 and 2, both at a
2.5% (v/v) concentration (Table 2). After usual work- As mentioned before, CAL-B is able to promote both
up, an extra wash of the solid support with MeOH was Michael addition and aminolysis reaction, with the li-
Table 2. Influence of the organic solvent in the addition of MA to DMAPA (2.5% v/v) at 308C using 50 mg of CAL-B and
1 mL of solvent.
Entry Solvent
Initial rate[a]
Conversion [%] after 1 h[b]
After wash with MeOH[c]
V0 3 V0 4 V0 3/V0 4
2
3
4
3/4
3 [%]
4 [%]
1
2
3
4
5
6
7
8
9
MeCN
1,4-Dioxane
THF
CH2Cl2
Et2O
TBME
Toluene
Hexane
0.9
2.2
1.4
0.5
2.0
1.5
1.7
1.2
1.0
0.3
0.4
0.4
0.4
0.5
0.6
0.5
0.5
0.9
7.3
3.5
1.3
5.0
3.0
2.8
2.4
4.8
51.24
38.95
41.84
68.93
2.54
47.94
39.88
15.13
36.88
25.26
53.57
36.57
15.65
74.85
36.60
44.92
29.86
51.45
21.50
6.20
1.17
8.64
3.16
1.09
5.50
3.50
3.40
3.72
5.93
1
–
8
0.6
8
5
2
47
3
1
0.8
2
0.4
1
–
–
–
–
11.59
14.42
13.61
10.46
13.20
8.03
1,4-Dioxane-toluene (1: 1) 2.4
8.67
[a]
[b]
[c]
Units: mM minꢀ1
.
Measured by GC and referred to initial amine concentration (0.2 M).
Measured by GC considering that all amine reacted as 100%.
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Oliver Torre et al.
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pase being more efficient for the aminolysis process. Ad-
Using 1,4-dioxane, the most suitable solvent for the
ditionally, Michael addition also occurs without the par- enzymatic reaction, the kinetics in the formation of all
ticipation of the biocatalyst. Each process has a different possible products were studied modifying the amount
rate constant, depending on many parameters. In a first of CAL-B (Table 3). Reaction with 10% of MA led to
approach, we decided to minimise the non-catalysed the best results as the amide was obtained as the major
chemical reaction in order to obtain higher yields of 3. product (entries 1, 5, and 7) and high concentrations of
With this in mind, the effect on the reaction rate was MA led to the increase of the Michael adduct obtaining
studied at different concentrations of ester and amine.
high proportions of 4 when 100% of MAwas used (entry
4). Low concentrations of amine led to a complete reac-
tion with MA, which made it impossible to calculate the
kinetics at short reaction times. This high reactivity also
meant that the formation of the Michael amide product
Ester Concentration
Firstly, the kinetics of the formation of 4 was studied in 5 was appreciable. An increase in the amount of CAL-B
the chemical reaction at different concentrations of 1 us- allowed the formation of slightly higher concentrations
ing limonene as internal standard (Figure 2). As expect- of the desired product (entries 5 and 7).
ed, reactions at high concentrations favoured the forma-
tion of the Michael adduct in the absence of enzyme.
Amine Concentration
At this point we considered it important to increase si-
multaneously the amount of amine and ester (Table 4)
and, as expected, high concentrations of reactants pro-
moted a drop in the selectivity of the process. Best con-
ditions for the formation of the amide were obtained at
low concentrations of amine and ester which minimise
the formation of Michael adducts (entry 1). A lack of se-
lectivity was observed at 10% of MA and DMAPA, es-
pecially when small amounts of enzyme were used (en-
tries 3, 5, and 7).
Form of Addition of Amine
To improve the ratio between 3 and 4 in the formation of
the amide, the rate of the addition of DMAPA was con-
sidered (Table 5). Good selectivity was achieved when
the addition was done by continuous flow addition com-
pared with the reaction with the amine present since the
start of the reaction (entries 1 and 2); in fact, better re-
sults were obtained if the addition was done during lon-
ger times (entry 3).
Figure 2. Michael adduct formation versus time course at dif-
&
~
ferent concentrations of MA in TBME at 308C ( , 100%;
,
^
*
60%; , 30%; , 10%).
Table 3. Reactions with varying amounts of CAL-B and concentrations of 1 in 1,4-dioxane at 308C.
Entry
CAL-B [mg]
[MA] [%]
Conversion [%][a]
Rate [mM·minꢀ1
]
3
4
5
6
3
4þ5þ6
1
2
3
4
5
6
7
8
50
50
50
50
80
80
100
100
10
30
60
100
10
30
67.62
62.61
50.38
40.78
69.78
58.43
71.64
66.12
25.44
34.45
45.55
44.38
23.29
39.33
22.35
32.14
6.52
1.33
0.60
0.25
6.52
1.36
5.60
0.95
0.42
1.61
3.46
14.59
0.41
0.88
0.41
0.79
6.55
11.6
n.m.[b]
n.m.[b]
5
4.7
n.m.[b]
n.m.[b]
6.9
11.5
n.m.[b]
n.m.[b]
n.m.[b]
n.m.[b]
n.m.[b]
n.m.[b]
10
30
[a]
Conversion once the amine had completely disappeared, measured by GC.
n.m.: not measured.
[b]
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Aminolysis Reaction of Methyl Acrylate Catalysed by Lipase B
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Table 4. Reactions with varying amounts of CAL-B, 1, and 2 in 1,4-dioxane at 308C.
Entry
CAL-B [mg]
[1] and [2] [%]
Conversion [%][a]
Rate [mM·minꢀ1
]
3
4
5
6
3
4þ5þ6
1
2
3
4
5
6
7
50
50
50
100
100
150
150
2.5
5
10
5
10
5
10
90.40
72.01
40.75
70.03
45.94
80.92
50.48
1.92
9.18
42.26
10.05
37.06
5.47
7.68
0.00
0.38
1.65
0.29
1.17
0.15
1.07
2.0
4.2
8.6
4.4
14.5
10.3
24.0
0.3
2.3
18.3
1.9
19.6
3.5
31.5
18.42
15.35
18.80
15.33
13.47
16.83
31.62
[a]
Conversion once that amine had completely disappeared, measured by GC.
Table 5. Study of the addition of 2 in portions in the reaction with 1 (10% v/v) using 150 mg of CAL-B in 1,4-dioxane at 308C.
Entry
Addition of 2 [mL/min]
Addition time [h]
Conversion [%][a]
3
4
5
6
1
2
3
–
–
5
9
50.48
65.16
72.68
31.62
14.21
17.53
16.83
6.90
2.96
1.07
5.02
3.88
0.0004
0.0002
[a]
Conversion once the amine had completely disappeared, measured by GC.
Influence of the Temperature
Temperature is a key factor in all biocatalytic processes
as high temperatures usually allow high conversions,
generally in spite ofa loss in the selectivity. The reactions
were studied in the presence of CAL-B over a range of
temperatures between 10 and 608C, and the courses of
the reactions are represented against the ratio of prod-
ucts 3/4 (Figure 3). Formation of 3 was slightly favoured
at higher temperatures up to 458C, while the reaction
conducted at 608C showed a decrease in the selectivity
of the process, probably due to thermal denaturation
of the enzyme.
Amount of Biocatalyst
Figure 3. Ratio of acrylamide 3 and Michael product 4 during
the time course of the reaction between 1 (10% v/v) and 2
(2.5% v/v) in 1,4-dioxane at different temperatures.
It is known that CAL-B catalyses both aminolysis and
the Michael addition reactions, however, which is the
more favoured enzymatic process? This study was
done in 1,4-dioxane as solvent and using 2.5% (v/v) of
MA and DMAPA with different amounts of enzyme zyme directed the process towards the faster formation
(5, 10, 20, 35, 50, 80, and 150 mg) by measuring the con- of acrylamide 3, the rate of formation of compound 3 in-
version and reaction rate of all possible products at creasing from 0.1 to 2.2 (Table 6), in this manner lower
75 min (Table 6). More catalyst implied a major selectiv- amounts of starting materials are present in the media
ity in the process directed towards the formation of the and the Michael addition is less favoured along the
amide product.
time course. Another key point is that Candida antarcti-
The ratio acrylamide/all possible Michael products ca lipase B is not able to catalyse the formation of com-
was measured along the time course, and it remained pound 5 from 3 through an enzymatic Michael reaction,
constant with respect to the conversion except in the rather compound 4 reacts with 2 to form 5 through an
150 mg reaction (entry 7) where the ratio significantly aminolysis process. This has been checked in independ-
increased (Figure 4). An increase in the amount of en- ent experiments (data not shown).
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Oliver Torre et al.
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Table 6. Influence of the amount of CAL-B used in the reaction between 1 and 2 (2.5% v/v) in 1,4-dioxane at 308C after
75 min.
Entry
CAL-B [mg]
Conversion after 75 min [%][a]
Rate [mM·minꢀ1
]
2
3
4
5
3
4
5
1
2
3
4
5
6
7
5
10
20
35
50
89.54
73.20
50.13
43.77
41.73
21.28
8.74
5.03
5.35
0.08
0.95
4.10
3.08
2.55
3.92
2.85
0.1
0.5
1.0
1.2
1.3
1.7
2.2
0.2
0.3
0.3
0.2
0.1
0.1
0.0
0.0
0.0
0.2
0.1
0.1
0.1
0.1
15.70
33.27
45.83
50.37
70.39
87.01
10.15
12.50
7.32
5.37
4.41
1.40
80
150
[a]
Measured by GC and referred to the initial amount of amine (0.2 M).
Table 7. Study of enzyme recycling in the reaction between 1
and 2 (10%v/v) using 150 mg of CAL-B in 1,4-dioxane at
308C.
Entry
Cycle
t [min]
Conversion of 3 [%][a]
1
2
3
4
1
2
3
4
60
60
80
80
47.56
47.30
46.39
37.42
[a]
Calculated by GC.
Conclusion
The reaction between methyl acrylate and N,N-dimeth-
yl-1,3-propanediamine catalysed by CAL-B has been
studied in order to optimise the formation of the corre-
sponding acrylamide. Non-polar solvents, low concen-
trations of reactants, and temperatures around 458C
favoured the formation of the amide with respect to
the Michael addition product. Also, slow addition of
the amine increased dramatically the formation of the
acrylamide. The potential of CAL-B has been demon-
strated for the synthesis of acrylamides avoiding the
use of extreme temperatures and the copper catalyst re-
quired for the conventional chemical synthesis.
Figure 4. Ratio of acrylamide 3 and Michael products (4 and
5) versus the percent conversion in the reaction between 1
(2.5% v/v) and 2 (2.5% v/v) in 1,4-dioxane at 308C using dif-
ferent amounts of CAL-B.
Study of Enzyme Recycling
Experimental Section
One of the main advantages of supported lipases is their
easy recovery by simple filtration, thus they can be
reused without any significant loss of activity. To take
advantage of this, the reaction of MA and DMAPA cat-
alysed by CAL-B was repeated four times until com-
plete disappearance of the amine, while quantifying
the amount of acrylamide obtained by simple work-up
(Table 7). Under these conditions CAL-B maintained
its activity during three cycles without any significant de-
crease.
General remarks
Candida antarctica lipase B, CAL-B Novozyme 435 (7300 U/g),
was a gift from Novo Nordisk co. CAL-B Chirazyme L-2
(ꢁ200 U/g) is commercially available from Roche Diagnos-
tics, lyophilised CAL-B (590 U/g) was purchased from Fluka.
Pseudomonas cepacia lipase in lyophilised (PSL, 30000 U/g)
and immobilised (PSL-C, 783 U/g) forms are marketed by
Amano Pharmaceuticals. CAL-B preparations 1–4 were a
´
gift from Professor Guisan (CSIC Biocatalysis Institute, Ma-
drid). Chemical reagents obtained from Aldrich, Fluka, Lan-
caster or Prolabo. Solvents were distilled over an appropriate
desiccant under nitrogen. Gas chromatography (GC) was car-
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Aminolysis Reaction of Methyl Acrylate Catalysed by Lipase B
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ried out with flame ionisation detection (FID) and an HP-1 ca-
pillary column (25 mꢂ0.2 mmꢂ0.2 mm) coated with methylsi-
licone gum using nitrogen as carrier gas. In this method the in-
jector and the detector were set at 1508C and 2808C, respec-
tively, initial column temperature was 608C increasing the tem-
perature by 208C/min until 2608C; retention times: 2 at
1.4 min, 3 at 4.4 min, 4 at 4.6 min, 5 at 8.9 min, and 6 at
7.4 min. 1H NMR, 13C NMR and DEPT spectra were recorded
in Bruker AC-200, Bruker AC-300 or Bruker AC-300 DPX
spectrometers using CDCl3 as solvent. The chemical shift val-
ues (d) are given in ppm and the coupling constants (J) are giv-
en in Hz. The positive electrospray ionisation (ESIþ) mode was
used to record mass spectra on a Hewlett-Packard 110 Series
spectrometer. Microanalyses were performed on a Perkin-El-
mer model 2400 instrument.
Influence of the Ester Concentration (Table 3)
Solutions of MA at concentrations 10, 30, 60, and 100% (v/v) in
1,4-dioxane were shaken at 250 rpm and 308C during 5 min in
the presence of CAL-B (Novozyme 435). After that time 50 mL
of an internal standard solution formed from 203.6 mg of limo-
nene and 5 mL of DMAPA were added, and every 10 min ali-
quots were taken and injected in the GC until complete con-
sumption of the amine.
Influence of the Amine Concentration (Table 4)
Mixtures with the same volumes (25, 50 or 100 mL) of MA and
an internal standard solution forming from 203.6 mg of limo-
nene and 5 mL of DMAPA were added to a total volume of
1 mL and different amounts of CAL-B (Novozyme 435). Mix-
tures were shaken at 250 rpm and 308C until complete con-
sumption of the amine, and aliquots were taken every 10 min
and injected in the GC.
Influence of the Enzyme and its Polymer Support
(Table 1)
0.9 mL of 1,4-dioxane and 0.1 mL of MA were mixed in the
presence of 50 mg of enzyme (if necessary) and shaken at
308C and 250 rpm. After 5 min, 25 mL of an internal standard
solution formed by 203.9 mg of limonene and 5 mL of DMAPA
were added. Aliquots were taken every 10 min and injected in
the GC.
Influence of the Form of Addition of Amine (Table 5)
A mixture of 150 mg of CAL-B (Novozyme 435), 0.1 mL of
MA and 0.8 mL of an internal standard solution formed from
19.8 mg of limonene in 10 mL of dioxane was magnetically stir-
red in a round-bottom flask and heated at 308C. After 10 min,
the addition of DMAPA (100 mL at 0.0004 mL/min during 5 h
or 0.0002 mL/min during 9 h) was started. Once the amine was
completely added, aliquots were taken and injected in the GC
until complete consumption of the amine.
Influence of the Organic Solvent (Figure 1)
Solutions of MA (concentrations 10, 30, 60, or 100% v/v) in
1 mL of solvent (hexane, TBME, and MeCN) were shaken at
250 rpm and 308C during 5 min. After that time, 50 mL of an in-
ternal standard solution formed from 202.3 mg of limonene
and 5 mL of DMAPA were added. Aliquots were taken every
5 or 10 min and injected in the GC.
Influence of the Temperature (Figure 3)
A mixture containing 50 mg of CAL-B (Novozyme 435) and
25 mL of MA dissolved in 1,4-dioxane was shaken at 250 rpm
during 5 min. Then, 25 mL of an internal standard solution
formed from 203.9 mg of limonene and 5 mL of DMAPA
were added. Aliquots were taken every 10 min and injected
in the GC.
Influence of the Organic Solvent (Table 2)
To 50 mg of CAL-B (Novozyme 435) 25 mL of MA and 1 mL of
solvent were added. The mixture was shaken at 308C and
250 rpm during 5 min, then 25 mL of an internal standard solu-
tion formed from 203.9 mg of limonene and 5 mL of DMAPA
were added, and every 10 min aliquots were taken and injected
in the GC. After 1 h, the enzyme was filtered off and washed
with 2ꢂ0.5 mL of MeOH. One mL of the filtered solution
was also injected in the GC.
Influence of the Amount of Biocatalyst (Table 6 and
Figure 4)
A mixture of 25 mL of MA, different amounts of CAL-B (No-
vozyme 435) in 1 mL of 1,4-dioxane was shaken at 308C and
250 rpm during 5 min. Then 25 mL of an internal standard sol-
ution formed from 203.6 mg of limonene and 5 mL of DMAPA
were added and aliquots were taken every 10 min and injected
in the GC.
Influence of the Ester Concentration (Figure 2)
Study of Enzyme Recycling (Table 7)
Solutions of MA at concentrations 10, 30, 60, and 100% (v/v) in
1 mL of TBME, were shaken at 250 rpm and 308C during
5 min. After that time, 50 mL of an internal standard solution
formed from 202.3 mg of limonene and 5 mL of DMAPA
were added, and every 5 or 10 min aliquots were taken and in-
jected in the GC.
A mixture of 150 mg of CAL-B, 0.1 mL of MA, 0.8 mL of 1,4-
dioxane and 100 mL of an internal standard solution formed
from 412.9 mg of limonene and 10 mL of DMAPAwas shaken
at 308C and 250 rpm. Consumption of amine was followed by
injecting aliquots in the GC. Once the amine had completely
reacted, the enzyme was filtered and washed with MeOH.
Adv. Synth. Catal. 2005, 347, 1007–1014
asc.wiley-vch.de
ꢁ 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1013

Oliver Torre et al.
FULL PAPERS
This enzyme was recovered for further use under identical re-
action conditions.
er, Bioorg. Med. Chem. 2004, 14, 4919–4923; b) D. X.
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J. Fu, F. Oveisi, C. Blazquez, D. Piomelli, J. Biol. Chem.
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N-(4-Methyl-4-azapentyl)acrylamide (3): Colouꢀrl1ess liquid;
[2] For example: a) O. Marder, F. Albericio, Chim. Oggi,
2003, 21, 35–40; b) K. M. Hosamani, R. S. Pattanashet-
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1
¼
¼
IR (film): n¼1653 (C O), 3076 (C C), 3277 cm (NH); H
NMR (CDCl3, 300 MHz): d¼7.41 (br s, 1H, NH), 6.20 (dd,
1H, H1trans, J¼17.1 Hz, 1.8 Hz), 6.06 (dd, 1H, H2, J¼17.1 Hz,
10.0 Hz), 5.58 (dd, 1H, H1cis, J¼10.0 Hz, 1.8 Hz), 3.41 (m,
2H, H3), 2.39 (t, 2H, H5, J¼6.4 Hz), 2.23 (s, 6H, 2NCH3),
1.68 (m, 2H, H4); 13C NMR (CDCl3, 75.5 MHz): d¼165.4
¼
(C O), 131.4 (C2), 125.2 (C1), 58.7 (C3), 45.3 (2NCH3), 39.5
(C5), 25.7 (C4); MS (ESIþ): m/z¼179 [(MþNa)þ, 100%];
anal. calcd. for C8H16N2O: C 61.51; H 10.32, N 17.93; found:
C 61.4, H 10.4, N 17.9.
8-Methyl-4,8-diazamethyl nonanoate (4): Colourless liquid;
IR (film): n¼1734 (C O), 3318 cmꢀ1 (NH); 1H NMR (CDCl3,
¼
300 MHz): d¼3.58 (s, 3H, OCH3), 2.76 (t, 2H, H4, J¼6.5 Hz),
2.53 (t, 2H, H3, J¼7.1 Hz), 2.40 (t, 2H, H6, J¼6.6 Hz), 2.19 (t,
[7] V. Gotor, R. Brieva, F. Rebolledo, Tetrahedron Lett.
1988, 29, 6973.
2H, H2, J¼7.1 Hz), 2.09 (s, 6H, 2NCH3), 1.53 (m, 2H, H5); 13
C
¼
NMR (CDCl3, 75.5 MHz): d¼172.9 (C O), 57.6 (OCH3), 51.2
(C4), 47.8 (C3), 45.2 (2NCH3), 44.8 (C6), 34.2 (C2), 27.7 (C5);
MS (ESIþ): m/z¼189 [(MþH)þ, 100%], 211 [(MþNa)þ,
30%]; anal. calcd. for C9H20N2O2: C 57.42; H 10.71, N 14.88;
found: C 57.5, H 10.8, N 14.7.
[8] a) V. Gotor, Bioorg. Med. Chem. 1999, 7, 2189–2197;
b) F. van Rantwijk, M. A. P. J. Hacking, R. A. Sheldon,
Monatsh. Chem. 2000, 131, 549–569; c) N. Aoyagi, T.
Izumi, Tetrahedron Lett. 2002, 43, 5529–5531; d) C. Boz-
zo, M. D. Pujol, X. Solans, M. Font-Bardia, Tetrahedron,
2003, 59, 1227–1236; d) M. Fernandez-Perez, C. Otero,
Enz. Microb. Technol. 2003, 33, 650–660; e) F. van Rant-
wijk, R. A. Sheldon, Tetrahedron 2004, 60, 501–509; f) I.
Alfonso, V. Gotor, Chem. Soc. Rev. 2004, 33, 201–209.
[9] a) P. T. Anastas, J. B. Zimmerman, Environ. Sci. Technol.
2003, 37, 95A-101A; b) G. Centi, S. Perathoner, Catal.
Today 2003, 287–297.
[10] a) U. Kragl, M. Eckstein, N. Kaftzik, Curr. Opin. Bio-
technol. 2002, 13, 565–571; b) S. Park, R. J. Kazlauskas,
Curr. Opin. Biotechnol. 2003, 14, 432–437; c) R. A. Shel-
don, F. van Rantwijk, Aust. J. Chem. 2004, 57, 281–289.
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1724–1725.
N-(4-Methyl-4-azapentyl)-8-methyl-4,8-diazanonanamide
´
´
(5): Green oil; IR (film): n¼1653 (C O), 3282 cmꢀ1 (NH); 1H
¼
NMR (CDCl3, 300 MHz): d¼7.90 (br s, 1H, NH), 3.32 (m, 2H,
H6), 2.90 (t, 2H, H4, J¼6.1 Hz), 2.71 (t, 2H, H3, J¼6.9 Hz),
2.32–2.41 (m, 6H, H1þH5þH8), 2.24 (s, 6H, 2NCH3), 2.23
(s, 6H, 2NCH3), 1.62–1.74 (m, 4H, H2þH7); 13C NMR
¼
(CDCl3, 75.5 MHz): d¼172.1 (2C O), 58.1 (C6), 58.0 (C4),
48.1 (C3), 45.6 (C5), 45.5 (2C, 2NCH3), 45.4 (2C, 2NCH3),
38.3 (C8), 35.6 (C1), 27.4 (C2), 26.8 (C7); MS (ESIþ): m/z
¼259 [(MþH)þ, 100%], 281 [(MþNa)þ, 25%]; anal. calcd.
for C13H30N4O: C 60.41; H 11.71, N 21.69; found: C 60.3, H
11.9, N 21.6.
N-4-(4-Methyl-4-azapentyl)iminodimethyl dipropanoate
(6): Orange liquid; IR (film): n¼1734 cmꢀ1 (C O); H NMR
1
¼
(CDCl3, 300 MHz): d¼3.64 (s, 6H, 2OCH3), 2.72 (t, 4H,
H4þH4’, J¼7.0 Hz), 2.43 (m, 8H, H1þH3þH5þH5’), 2.36
(s, 6H, 2NCH3), 1.68 (m, 2H, H2); 13C NMR (CDCl3,
[12] P. Carlqvist, M. Svendehal, C. Branneby, K. Hult, T.
Brinck, P. Berglund, ChemBioChem 2005, 6, 331–336.
´
´
[13] a) M. Quiros, V. M. Sanchez, R. Brieva, F. Rebolledo, V.
Gotor, Tetrahedron: Asymmetry 1993, 4, 1105; b) S. Puer-
tas, R. Brieva, F. Rebolledo, V. Gotor, Tetrahedron 1993,
¼
75.5 MHz): d¼172.9 (2C O), 57.1 (2OCH3), 51.4 (C4þC4’),
49.0 (C1þC3), 44.7 (2C, 2NCH3), 32.3 (C5þC5’), 24.3 (C2);
MS (ESIþ): m/z¼275 [(MþH)þ, 100%], 297 [(MþNa)þ,
90%]; anal. calcd. for C13H26N2O4: C 56.90; H 9.56, N 10.21;
found: C 56.8, H 9.6, N 10.1.
´
49, 4007; c) V. Gotor, E. Menendez, Z. Mouloungui, A.
Gaset, J. Chem. Soc. Perkin Trans. 1 1993, 2453.
[14] For example: a) G. Simonian, N. M. Beylerian, Oxid.
Commun. 2003, 26, 485–491; b) P. Kotrusz, S. Toma,
H.-G. Schmalz, A. Adler, Eur. J. Org. Chem. 2004,
1577–1583; c) Y. Cai, X.-F. Sun, N. Wang, X.-F. Lin, Syn-
thesis 2004, 671–674; d) X. Zheng, Y. Zhang, J. Chem.
Res. (S) 2004, 412–413; e) S. B. Tsogoeva, S. B. Jagtap,
Z. A. Ardemasova, V. N. Kalikhevich, Eur. J. Org.
Chem. 2004, 4014–4019.
Acknowledgements
We thank SNF S. A. (France) for financial support and especial-
´
ly to Cedrick Favero and Marco Wieser for scientific discus-
sions.
References and Notes
[1] For example: a) V. Gududuru, E. Hurh, G. G. Durgam,
S. S. Hong, V. M. Sardar, H. Xu, J. T. Dalton, D. D. Mill-
1014
ꢁ 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
Adv. Synth. Catal. 2005, 347, 1007–1014