Promiscuous behavior of Rhizomucor miehei lipase in the synthesis of N-substituted β-amino esters

Article abstract of DOI:10.1002/ejoc.201101624

A mild and efficient procedure for the aza-Michael addition of amines to acrylates by using lipases as catalysts is reported. Various lipases, mono- and bifunctional amines, alkyl acrylates, and reaction parameters were studied. Under the optimal conditio

Full text of DOI:10.1002/ejoc.201101624

FULL PAPER
DOI: 10.1002/ejoc.201101624
Promiscuous Behavior of Rhizomucor miehei Lipase in the Synthesis of
N-Substituted β-Amino Esters
Leandro N. Monsalve,[a] Florencia Gillanders,[a] and Alicia Baldessari*[a]
Keywords: Enzyme catalysis / Amino esters / Michael addition
A mild and efficient procedure for the aza-Michael addition
of amines to acrylates by using lipases as catalysts is re-
ported. Various lipases, mono- and bifunctional amines, alkyl
acrylates, and reaction parameters were studied. Under the
optimal conditions, Rhizomucor miehei lipase showed high
selectivity. It catalyzed the formation of the Michael monoad-
duct as the only product in high yield and purity. Moreover,
when diamines were used as nucleophiles, the lipase cata-
lyzed the addition of only one of the two amino groups, show-
ing in this case high substrate specificity. This promiscuous
and highly selective behavior displayed by Rhizomucor
miehei lipase allowed us to obtain 22 N-substituted β-amino
esters, 15 of them being new products.
tion yields can be obtained when catalysts are employed.
Moreover, they shorten the reaction times, allow a broader
range of solvents, and generally require milder reaction con-
ditions and stoichiometric substrate loadings.
Introduction
β-Amino esters are an important group of compounds,
among which natural products with biological activities can
be found.[1] These compounds are also useful as synthetic
precursors for many bioactive heterocyclic[2–4] and β-pep-
toid[5] compounds. Moreover, β-amino esters are monomers
in the synthesis of poly(β-aminoester)s, linear cationic poly-
mers used as efficient gene delivery vectors.[6,7] These poly-
mers contain both an amino group interacting with poly-
anionic DNA through electrostatic interaction and a de-
gradable region, such as a hydrolyzable linkage.[8] These
characteristics make them useful for targeted delivery of an-
titumor agents, such as cisplatin, minimizing its toxicity to
healthy tissues and increasing its drug efficacy.[9]
The synthesis of β-amino esters can be carried out by
following several strategies.[10,11] The most widely used
methods are the Mannich reaction,[12] the N-alkylation of
amines with β-haloesters.[4] and the Michael addition of
amines to α,β-unsaturated esters.[3,13–15]
In particular, the aza-Michael addition is a powerful tool
for carbon–nitrogen bond formation. The reaction between
an amine and an α,β-unsaturated carbonyl compound can
occur spontaneously in some cases in the absence of sol-
vent[16] or if solvents with high dielectric constants such as
methanol, ethanol, or tetrahydrofuran are employed.[3,14,15]
An excess amount of the amine is often employed in these
cases to maximize product yield. Various catalysts have also
been used for Michael additions, including acids,[17]
bases,[18] Lewis acids,[9,13] and ionic liquids.[19] Higher reac-
It is well known that enzymes are useful as biocatalysts
in a variety of reactions. In particular, enzyme promiscuity
has been thoroughly studied over the last years. For in-
stance, many hydrolases are able to catalyze reactions that
are completely different from those they originally evolved
to perform: Michael addition, Markovnikov addition, and
aldol condensation are a few examples.[20] Regarding the
aza-Michael reaction, it is interesting to mention the aspar-
tase-catalyzed addition of ammonia to fumaric acid was
scaled-up for the commercial production of aspartic
acid.[21] Using secondary amines and fluorine as double-
bond activators, lipases have also been used as catalysts in
Michael-type additions.[22] In the presence of lipases,
Michael addition can compete with aminolysis of the ester
to afford amides, and it is interesting to control the preva-
lence of one of these reactions over the other by applying
several strategies. The optimization of the reaction condi-
tions to minimize Michael-type side products allowed sub-
stituted acrylamides to be obtained in high yield.[23,24] On
the other hand, a solvent engineering strategy was used to
control the lipase selectivity in a Michael addition reac-
tion,[25] and a polyamidoamine oligomer containing a com-
pletely regular structure was synthesized through both ami-
nolysis and Michael addition reactions catalyzed by a li-
pase.[26]
Most of the previous reports on enzyme-catalyzed aza-
Michael reactions show the results obtained on the conju-
gate addition of primary and secondary amines to various
acceptors,[27–29] but the application of bifunctional amines
such as alkanolamines or diamines as nucleophiles remains
unexplored. Considering this fact, in this work we studied
[a] Laboratorio de Biocatálisis,
Departamento de Química Orgánica y UMYMFOR,
Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires, Ciudad Universitaria,
Pabellón 2, Piso 3. C1428EGA. Buenos Aires, Argentina
E-mail: alib@qo.fcen.uba.ar
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Eur. J. Org. Chem. 2012, 1164–1170

Promiscuous Behavior of the Lipase Rhizomucor miehei
the chemoselective behavior of lipases in the synthesis of much faster than Michael additions.[24] PSL also catalyzed
several N-substituted β-amino esters by using various both reactions, and we obtained a mixture of esters and
mono- and bifunctional amines as nucleophiles in aza- amides when this catalyst was used. On the other hand,
Michael addition reactions (Scheme 1).
CRL showed a poor performance. LIP was found to be the
most efficient lipase to catalyze, in a chemoselective way,
the addition reaction with alkylamine 2c, alkanolamine 2d,
and diamine 2h. Only the amino group of alkanolamine 2d
was reactive in the formation of the product, and the hy-
droxy function remained unaltered, as we observed in pre-
vious work with this class of compounds.[24,30]
Moreover, by using 1,3-diaminopropane (2h), LIP
showed a high substrate specificity because the reaction
product, ethyl N-(3-aminopropyl)-β-alaninate (3h), with a
free NH2 group in the molecule was unable to react with
another ethyl acrylate molecule when this catalyst was em-
ployed. N-Substituted β-amino esters containing a free hy-
droxy or amino group are very attractive as monomers in
the synthesis of polyesters with polar pendant groups.
Having established the optimal catalyst, screening of the
solvent was performed to determine the optimal solvent for
this biocatalytic aza-Michael reaction. With the belief that
this reaction was only slightly affected by non-enzymatic
Scheme 1.
Results and Discussion
Reaction Conditions
In a previous report, by using ethyl acrylate and alkanol- acid/base catalysis and by considering previous re-
amines as reactants, we obtained N-hydroxyalkylacryl- ports,[25,28,29] three low-polarity solvents, hexane, toluene,
amides through an aminolysis reaction on the ester, cata- and diisopropyl ether (DIPE), were tested.[31]
lyzed by Candida antarctica lipase (CAL B).[24] The enzy-
The performance of the solvent was not the same for
matic reaction of ethyl acrylate and the alkanolamines was every case and depended on the nature of the nucleophile.
carried out at 30 °C by using an alkanolamine/ester ratio The experiments (Table 1, Entries 1–9) clearly identified
equal to 1, 0.08 m ester concentration, a ratio enzyme/sub- hexane as the most efficient solvent for the reaction with
strate (E/S) equal to 2, diisopropyl ether as solvent, and ethyl acrylate and alkylamine 2c (Table 1, Entry 1) and di-
p-benzoquinone as radical inhibitor. Under these reaction amine 2h (Table 1, Entry 7), whereas DIPE seemed to be
conditions CAL B worked in a highly chemoselective way the best choice when using alkanolamine 2d as the nucleo-
to afford the amide as the only product in high yield. It was phile (Table 1, Entry 6).
possible to avoid the production of addition products and
a polymeric material that were detected as secondary prod- of ethanolamine (2d) to ethyl acrylate was attempted with
ucts working under less-controlled conditions. the use of hexane as the solvent, we obtained double adduct
Interestingly, when the lipase-catalyzed Michael addition
With the aim to favor the synthesis of addition products, 3d* instead of 3d (Table 1, Entry 4). This difference could
in the present work we studied the enzymatic aza-Michael be attributed to the scarce solubility of ethanolamine in
reaction. In this case, we used three types of nucleophiles hexane. A neat phase separation was observed even at a
as Michael donors: alkylamines 2a–c, alkanolamines 2d–g, very low concentrations (0.012 m). This phenomenon
and diamines 2h–k. To begin, it was necessary to determine should create a large excess of ethyl acrylate in the hexane
the conditions under which lipase-catalyzed Michael ad- phase and thus drive the reaction to the formation of
dition prevailed over amide or polymer formation with the double Michael adducts.
different nucleophiles. Therefore, the reactions of ethyl acry-
Regarding the optimum temperature (Table 1, Entries 10
late (1a) as the Michael acceptor and phenethylamine (2c), and 11), we also performed the addition reaction with 2c
ethanolamine (2d), or 1,3-propanediamine (2h) as the nu- and 2d as Michael donors at 55 °C. The results showed that
cleophile were chosen as model reactions. The influence of an increase in temperature had no significant effect on the
various reaction parameters such as lipase source, solvent, conversion to the desired products. Therefore, 30 °C was
E/S ratio, substrate concentration, and temperature was chosen as the reaction temperature in every experiment.
studied.
To determine the best enzyme/substrate (E/S) ratio, reac-
Four commercially available lipases from various sources tions with various biocatalyst concentrations were carried
were screened for their catalytic activity towards the aza- out (Table 1, Entries 12–16). An increase in conversion with
Michael addition reaction: CAL B, Rhizomucor miehei li- an increase in E/S from 0.1 to 5 was observed, whereas
pase (LIP), Pseudomonas cepacia lipase (PSL), and Candida E/S = 1 was enough to catalyze the reaction under the pres-
rugosa lipase (CRL). We observed that the most important ent parameters (Table 1, Entry 14).
differences in their performance were due to the chemo-
Then, we decided to study the effect of substrate concen-
selectivity achieved in the reaction. In accordance with pre- tration in the spontaneous and enzymatic aza-Michael reac-
vious reports, CAL B-catalyzed aminolysis reactions were tion. A high substrate concentration can have some conse-
Eur. J. Org. Chem. 2012, 1164–1170
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1165

L. N. Monsalve, F. Gillanders, A. Baldessari
FULL PAPER
Table 1. Optimization of reaction parameters for the lipase-cata-
lyzed aza-Michael addition of ethyl acrylate (1a) to amines 2c,d,h.[a]
Entry
R
Solvent
T (°C)
E/S
Product (% conv.)
Solvent
1
2
3
4
5
6
7
8
9
(CH2)2Ph
(CH2)2Ph
(CH2)2Ph
(CH2)2OH
(CH2)2OH
(CH2)2OH
(CH2)3NH2
(CH2)3NH2
(CH2)3NH2
hexane
toluene
DIPE
hexane
toluene
DIPE
hexane
toluene
DIPE
30
30
30
30
30
30
30
30
30
1
1
1
1
1
1
1
1
1
3c (92)
3c (75)
3c (66)
3d* (80)[b]
3d (71)
3d (83)
3h (100)
3h (86)
3h (65)
Figure 1. Conversion of 3d with enzyme (solid lines) and without
enzyme (dashed lines). Inactivated lipase was also used (dotted
line). Reaction conditions: Enzyme: LIP, solvent = DIPE, E/S = 1,
T = 30 °C. With enzyme: c = 0.12 m (᭣), c = 0.012 m (᭢). Without
enzyme: c = 0.12 m (᭹), c = 0.012 m (᭿). Inactivated enzyme: c =
0.12 m (♦).
Temperature
10
11
(CH2)2Ph
(CH2)2OH
hexane
DIPE
55
55
1
1
3c (91)
3d (86)
E/S
12
13
14
15
16
(CH2)2OH
(CH2)2OH
(CH2)2OH
(CH2)2OH
(CH2)2OH
DIPE
DIPE
DIPE
DIPE
DIPE
30
30
30
30
30
0.1 3d (12)
0.5 3d (40)
1
3d (84)
2.5 3d (91)
3d (90)
only 36%. From these experiments we concluded that the
most adequate concentration for the enzyme-catalyzed reac-
tion is 0.12 m.
5
[a] Reaction conditions: LIP; substrate concentration = 0.12 m;
ethyl acrylate/amine
HOCH2CH2N(CH2CH2COOEt)2.
Finally, with the aim of discarding the occurrence of un-
specific catalysis by the protein or the enzyme support, we
performed the addition of ethanolamine to ethyl acrylate
by using a thermally inactivated LIP at a substrate concen-
tration of 0.12 m. The results are also depicted in Figure 1
(dotted line). Comparison of these results with those ob-
tained in the absence of the enzyme (Figure 1, dashed line)
allowed us to conclude that neither denatured protein nor
the support served as a catalyst for this reaction. Therefore,
the active site of LIP is involved in the biocatalytic reaction.
=
1. [b] 3d*: double Michael adduct,
quences: a spontaneous Michael reaction is more likely to
occur due to an increase in the dielectric constant of the
reaction medium, the lipase-catalyzed reaction rate may be
limited due to diffusion processes to the heterogeneous cat-
alyst, and loss of selectivity. We prepared solutions of dif-
ferent concentrations (0.012–1.2 m) of ethyl acrylate and
ethanolamine (1:1) in DIPE. The solutions were incubated
at 30 °C and the reaction was allowed to proceed in both
the presence and absence of LIP. The conversion to product
3d was monitored by GC, and the results are shown in Fig-
ure 1.
Preparation of N-Substituted β-Amino Esters
Once the experimental conditions were optimized, we de-
At the highest concentration (1.2 m), a spontaneous reac- cided to apply the enzymatic aza-Michael addition to other
tion was very fast and no difference was observed between donors and acceptors. The results, expressed as yield of iso-
the presence and absence of the enzyme in terms of a de- lated product, for ethyl and butyl acrylate as Michael ac-
crease in the ethyl acrylate and ethanolamine concentra- ceptors and various donors belonging to the above-men-
tions, but in both cases the reaction was not selective and tioned three types, alkylamines, alkanolamines, and di-
a complex mixture of products including some polymeric amines, are summarized in Table 2.
material was obtained. At a very low concentration of the
A remarkable difference in product yield was observed
substrate (0.012 m), the enzymatic reaction was selective for different alkylamines, and phenethylamine (2c) was
and only Michael adduct 3d was obtained, but the conver- found to be the best substrate for the reaction. Thus, prod-
sion was low (30%, Figure 1). In the absence of enzyme, uct 3c was obtained in higher yield (85%) than 3a and 3b
the amount of 3d in the reaction was insignificant.
by using n-propylamine and isopropylamine, respectively
Through the results obtained working at a concentration (Table 2, Entries 1–3).
of 0.12 m, we observed that the enzymatic reaction turned
It can be seen that the best yields were obtained with
remarkably faster than the spontaneous one as the substrate alkanolamines and diamines as donors. These nucleophiles
concentration was increased. In this case, the conversion to have another polar group besides the nucleophilic amino
3d reached 100% in 12 h, whereas in the absence of LIP, group. With the exception of 3g with 64% yield, which
under the same reaction conditions, the conversion was could be attributed to some steric hindrance in alkanol-
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Eur. J. Org. Chem. 2012, 1164–1170

Promiscuous Behavior of the Lipase Rhizomucor miehei
Table 2. Rhizomucor miehei lipase catalyzed aza-Michael addition to the classification of lipases on the basis of their binding
reaction of amines 2a–k to alkyl acrylates 1a and 1b.[a]
sites to ester substrates, LIP belongs to the group of lipases
that have a large alcohol binding cleft but a narrow acyl
binding cleft.[32] As it has been proposed, the lipase-cata-
lyzed aza-Michael reaction would start with the accommo-
dation of the Michael acceptor.[28] This could explain why
ethyl and n-butyl acrylate, with the same small acyl group
are good substrates and the bulky alkyl crotonate or meth-
acrylate are not. Regarding the alcohol moiety, the struc-
tural geometry of LIP allowed both alkyl groups to be ac-
cepted.
Entry
Amine
Alkyl acrylate
Solvent
Product
(% yield)
1
2
3
4
5
6
7
8
2a
2b
2c
2d
2d
2e
2f
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
1b
1b
1b
1b
1b
1b
1b
1b
1b
1b
1b
1b
hexane
hexane
hexane
DIPE
hexane
DIPE
3a (55)
3b (45)
3c (85)
3d (76)
3d* (76)[b]
3e (71)
3f (83)
LIP is applied in a variety of chemical processes due to
its high activity and stability.[33] Under the above-men-
tioned conditions, its substrate specificity was also remark-
able. Therefore, a single Michael addition product could be
obtained in very good yield in every case without any sec-
ondary product and none of these products were able to
react as Michael donors. In the case of diamines as donors,
this effect highlights even more the substrate specificity, as
the NH2 group in reaction products 3h–k remained unreac-
tive.
DIPE
DIPE
2g
2h
2i
3g (64)
3h (100)
3i (89)
9
hexane
hexane
hexane
hexane
hexane
hexane
hexane
DIPE
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
2j
3j (91)
2k
2a
2b
2c
2d
2d
2e
2f
3k (99)[c]
4a (66)
4b (60)
4c (87)
4d (75)
4d* (81)[d]
4e (92)
4f (80)
hexane
DIPE
DIPE
Conclusions
2g
2h
2i
2j
2k
DIPE
4g (86)
4h (100)
4i (87)
In this work we describe for the first time the synthesis
of N-substituted-β-amino esters by application of lipases in
the aza-Michael addition of mono- and bifunctional amines
to α,β-unsaturated esters. Twenty-two N-substituted-β-
amino esters and two N,N-disubstituted double Michael ad-
ducts were obtained. Fifteen of them, including the double
Michael adducts, are new products.
The influence of the enzyme source, the substrate struc-
ture, and various reaction parameters on the results was
analyzed. After an enzyme screening it was concluded that
hexane
hexane
hexane
hexane
4j (94)
4k (93)[c]
[a] Reaction conditions: Enzyme = LIP, substrate concentration =
0.12 m, E/S = 1, T = 30 °C. reaction time = 16 h, 200 rpm. [b] 3d*:
double Michael adduct, HOCH2CH2N(CH2CH2COOEt)2. [c] T =
55 °C. [d] 4d*: double Michael adduct, HOCH2CH2N(CH2CH2-
COOnBu)2.
amine 2g, the rest of the products were obtained in yields LIP was the best biocatalyst in terms of yield and selectiv-
ranging from 71 to 100% (Table 2, Entries 4–12). ity. All reactions, except that with 1,12-diaminododecane
Regarding the reactivity of the diamines, only 2k, con- (55 °C), were performed at room temperature. A strong in-
taining the longest alkyl chain, required a higher reaction fluence of the solvent on the nature of the donors was ob-
temperature to reach the maximum product yield (Table 2, served. Hexane was the solvent of choice for amines and
Entries 12 and 24). From the results it seems that the chain diamines and DIPE for alkanolamines. With alkanolamines
length between both amino groups did not influence mainly in hexane, the enzyme was not selective and double Michael
the reaction yields. The best performance was achieved with adducts were obtained. The substrate concentration played
the shorter chain of the three methylene groups, as with an important role in enhancing the enzyme catalysis over
both acceptors 1a and 1b, their respective products 3h and the spontaneous reaction. Biocatalyst efficiency was limited
4h were obtained in quantitative yield.
by high substrate concentration in which a mixture of prod-
Concerning the structure of the Michael acceptor on the ucts was obtained.
reaction performance, two tendencies were observed. First,
By comparing the performances of mono- and bifunc-
increasing the O-alkyl chain length (Table 2, Entries 13–24) tional amines as Michael donors, the highest yields were
did not affect the reaction and the same type of product achieved when the nucleophile had a polar group besides
(i.e., 4a–k) in similar yields to those obtained for 3a–k were the nucleophilic amine. Regarding the structure of the es-
obtained. Second, if methyl substituents were attached to ters, the chain length of the O-alkyl substituent did not in-
the double bond no reaction products were obtained. Nei- fluence the reaction performance. However, the presence of
ther methyl methacrylate (with a methyl substituent at C3) methyl groups on the double bond did not allow the reac-
nor methyl crotonate (with a methyl substituent at C4) gave tion to proceed.
any product with ethanolamine (2d) or 1,3-diaminopropane
In summary, due to their high selectivity and promiscu-
(2h), which are nucleophiles that served as the best sub- ous behavior, the Rhizomucor miehei lipase proved to be an
strates for the reaction with acrylates 1a and 1b. According efficient biocatalyst to synthesize a variety of N-substituted-
Eur. J. Org. Chem. 2012, 1164–1170
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1167

L. N. Monsalve, F. Gillanders, A. Baldessari
FULL PAPER
1 H), 2.82 (t, J = 6.6 Hz, 2 H), 3.19 (br. s), 4.04 (q, J = 6.9 Hz, 2
H) ppm. 13C NMR (50.2 MHz): δ = 14.0, 22.2, 34.2, 42.0, 48.5,
β-amino esters with potential application as starting materi-
als in the synthesis of biomedical polymers.
60.3, 172.3 ppm. FTIR (film): ν = 3397, 1735, 1632 cm–1. HRMS:
˜
calcd. for C8H18NO2 [M + H] 160.13321; found 160.13368.
Ethyl N-(2-Phenylethyl)-β-alaninate (3c): Colorless liquid. Yield:
Experimental Section
188.8 mg (85%). Physical properties as described previously.[33] 1
H
Enzymes and Materials: Hexane, ethyl acetate, and toluene were
purchased from Merck Argentina and diisopropyl ether (DIPE)
from J. T. Baker. Chemical reagents were purchased from Sigma–
Aldrich de Argentina. Ethyl acrylate, n-butyl acrylate, and methyl
methacrylate were a generous gift from Clariant Argentina. Can-
dida rugosa lipase (CRL, type VII, 706 U/mg solid) was purchased
from Sigma–Aldrich de Argentina, Pseudomonas lipase: Lipase PS
Amano (PSL, 33,200 U/g) was purchased from Amano Enzyme
USA Co., and Candida antarctica B lipase (CAL B, Novozym 435,
7400 PLU/g) and Rhizomucor miehei lipase (Lipozyme RM IM,
7800 U/g) were a generous gift from Novozymes Spain. Enzymes
were used “straight from the bottle”. Inactivated enzyme was pre-
pared by heating the biocatalyst at 105 °C in an oven for 2 h.
NMR (200.1 MHz): δ = 1.23 (t, J = 6.9 Hz, 3 H), 2.51 (t, J =
6.6 Hz, 2 H), 2.72 (t, J = 5.1 Hz, 2 H), 2.82 (t, J = 6.6 Hz, 2 H),
2.91 (t, J = 5.1 Hz, 2 H), 4.11 (q, J = 6.9 Hz, 2 H), 7.22 (dd, J =
6.9, 2.9 Hz,1 H), 7.27 (ddd, J = 6.9, 2.2, 2.9 Hz, 2 H), 7.30 (ddd, J
= 6.9, 2.2, 2.9 Hz, 2 H) ppm. 13C NMR (50.2 MHz): δ = 14.1, 34.3,
35.9, 44.7, 50.7, 60.4, 126.1, 126.3, 128.4, 128.6, 172.5 ppm. FTIR
(film):
ν =
˜
3313, 1733, 1650, 843 cm–1. HRMS: calcd. for
C13H20NO2 [M + H] 222.14886; found 222.14874.
Ethyl N-(2-Hydroxyethyl)-β-alaninate (3d): Colorless liquid. Yield:
110.3 mg (76%). Physical properties and 1H NMR and FTIR spec-
troscopic data as described previously.[14] 13C NMR (50.2 MHz): δ
= 14.1, 34.6, 44.4, 50.8, 60.5, 60.6, 172.6 ppm. HRMS: calcd. for
C7H16NO3 [M + H] 162.11247; found 162.11188.
Analytical Methods: Reactions were carried out in a Sontec incu-
bator shaker (Scientifica Argentina) at 200 rpm at the indicated
temperature. All reactions were monitored by TLC with Merck
TLC sheets (Silica gel 60 F254, aluminum support). Flash column
chromatography was carried out by using silica gel 60 (0,040–
0.063 mm) purchased from Merck. The percentage of conversion
was determined by GC analysis with a Finnigan Focus GC,
Thermo Electron Co. instrument, the capillary column being HP-
ULTRA-1, 25 mϫ 0.2 mm, film thickness 0.11 μm, (5 min at 50 °C,
10 °C/min, 280 °C, inlet 180 °C, detector 300 °C). tR: ethyl acrylate:
5.18 min, ethyl N-(2-hydroxyethyl)-β-alaninate (3d): 15.92 min,
ethanolamine: 24.21 min. BHT (in DIPE as stabilizer) was used as
Ethyl
N-(3-Ethoxy-3-oxopropyl)-N-(2-hydroxyethyl)-β-alaninate
(3d*): Colorless liquid. Yield: 89.7 mg (76%). 1H NMR
(200.1 MHz): δ = 1.27 (t, J = 6.9 Hz, 6 H), 2.47 (t, J = 6.6 Hz, 4
H), 2.61 (t, J = 6.6 Hz, 2 H), 2.82 (t, J = 6.6 Hz, 4 H), 3.60 (t, J =
6.6 Hz, 2 H), 4.15 (q, J = 6.9 Hz, 4 H) ppm. 13C NMR (50.2 MHz):
δ = 14.1, 32.6, 49.1, 55.9, 58.9, 60.5, 172.5 ppm. FTIR (film): ν =
˜
3467, 1732 cm–1. HRMS: calcd. for C12H24NO5 [M
262.16545; found 262.16517.
+ H]
Ethyl N-(3-Hydroxypropyl)-β-alaninate (3e): Colorless liquid. Yield:
112.0 mg (71%). H NMR (200.1 MHz): δ = 1.25 (t, J = 6.9 Hz, 3
H), 1.68 (m, J = 6.6 Hz, 2 H), 2.48 (t, J = 6.6 Hz, 2 H), 2.77 (t, J
= 6.6 Hz, 2 H), 2.87 (t, J = 6.6 Hz, 2 H), 3.78 (t, J = 6.6 Hz, 2 H),
4.13 (q, J = 6.9 Hz, 2 H) ppm. 13C NMR (50.2 MHz): δ = 14.2,
1
1
internal standard (tR: 19.59 min). H NMR and 13C NMR spectra
were recorded with a Bruker AC 200 NMR instrument by using
CDCl3 as solvent. Chemical shifts were reported in δ units relative
to TMS set at 0 ppm. High-resolution mass spectra were recorded
with a Bruker microTOF-Q II mass spectrometer (ionization mode:
ESI). FTIR measurements were performed with a Shimadzu FTIR-
8300 spectrophotometer in film with KBr windows. Melting points
were determined with a Fisher-Jones melting point apparatus.
30.6, 34.4, 44.9, 49.6, 60.5, 64.2, 172.6 ppm. FTIR (film): ν = 3409,
˜
1724, 1643 cm–1. HRMS: calcd. for C8H18NO3 [M + H] 176.12818;
found 176.12833.
Ethyl N-(4-Hydroxybutyl)-β-alaninate (3f): Colorless liquid. Yield:
141.4 mg (83%). H NMR (200.1 MHz): δ = 1.21 (t, J = 6.9 Hz, 3
1
H), 1.60 (m, J = 6.6 Hz, 4 H), 2.51 (t, J = 6.6 Hz, 2 H), 2.63 (t, J
= 6.6 Hz, 2 H), 2.85 (t, J = 6.6 Hz, 2 H), 3.52 (t, J = 6.6 Hz, 2 H),
4.10 (q, J = 6.9 Hz, 2 H) ppm. 13C NMR (50.2 MHz): δ = 14.1,
Preparation of N-Substituted-β-amino Esters: The typical procedure
consisted in the preparation of a solution containing the α,β-unsat-
urated ester (0.9 mmol) and the corresponding amine (0.9 mmol) in
the indicated solvent (7.5 mL). Lipozyme (100 mg) (or equivalent
amount of lipases from various sources) was added to the solution,
and the resulting suspension was shaken at 30 °C and 200 rpm. The
course of the reaction was followed by TLC and GC. Once the
reaction reached its maximum conversion, the enzyme was filtered
and washed with the solvent (3ϫ). The filtered solvents were evapo-
rated under reduced pressure, and the residue was purified by flash
chromatography and characterized by 1H NMR, 13C NMR, and
FTIR spectroscopy and HRMS.
27.8, 31.9, 33.6, 44.3, 49.2, 60.5, 62.2, 172.8 ppm. FTIR (film): ν
˜
= 3417, 1716, 1638 cm–1. HRMS: calcd. for C9H20NO3 [M + H]
190.14377; found 190.14383.
Ethyl N-(2-Hydroxy-1,1-dimethylethyl)-β-alaninate (3g): Colorless
1
liquid. Yield: 109.0 mg (64%). H NMR (200.1 MHz): δ = 1.06 (s,
6 H), 1.26 (t, J = 6.9 Hz, 3 H), 2.17 (br. s), 2.47 (t, J = 6.6 Hz, 2
H), 2.77 (t, J = 6.6 Hz, 2 H), 3.30 (s, 2 H), 4.14 (q, J = 6.9 Hz, 2
H) ppm. 13C NMR (50.2 MHz): δ = 14.2, 27.1, 35.2, 37.1, 60.6,
67.7, 53.6, 171.0 ppm. FTIR (film): ν = 3403, 1732, 1652 cm–1.
˜
HRMS: calcd. for C9H20NO3 [M + H] 190.14377; found 190.14414.
Ethyl N-Propyl-β-alaninate (3a): Colorless liquid. Yield: 87.5 mg
(55%). H NMR (200.1 MHz): δ = 0.81 (t, J = 7.3 Hz, 3 H), 1.15
(t, J = 6.9 Hz, 3 H), 1.42 (m, J = 7.3 Hz, 2 H), 2.46 (t, J = 6.6 Hz,
2 H), 2.51 (t, J = 7.3 Hz, 2 H), 2.81 (t, J = 6.6 Hz, 2 H), 3.49 (br.
s), 4.03 (q, J = 6.9 Hz, 2 H) ppm. 13C NMR (50.2 MHz): δ = 11.4,
1
Ethyl N-(3-Aminopropyl)-β-alaninate (3h): Colorless liquid. Yield:
1
156.8 mg (100%). H NMR (200.1 MHz): δ = 1.15 (t, J = 6.9 Hz,
3 H), 1.53 (m, J = 7.0 Hz, 2 H), 2.39 (t, J = 6.6 Hz, 2 H), 2.57 (t,
J = 7.0 Hz, 2 H), 2.65 (t, J = 7.0 Hz, 2 H), 2.77 (t, J = 6.6 Hz, 2
H), 4.02 (q, J = 6.9 Hz, 2 H) ppm. 13C NMR (50.2 MHz): δ = 14.0,
14.0, 22.4, 34.0, 44.5, 51.1, 60.2, 172.3 ppm. FTIR (film): ν = 3403,
˜
1732, 1647 cm–1. HRMS: calcd. for C8H18NO2 [M + H] 160.13321;
found 160.13379.
33.5, 34.5, 40.3, 44.9, 47.4, 60.1, 172.6 ppm. FTIR (film): ν = 3363,
˜
1733, 1643, 1561 cm–1. HRMS: calcd. for C8H19N2O2 [M + H]
175.14410; found 175.14397.
Ethyl N-Isopropyl-β-alaninate (3b): Colorless liquid. Yield: 71.7 mg
1
(45%). H NMR (200.1 MHz): δ = 1.01 (d, J = 6.2 Hz, 6 H), 1.16
Ethyl N-(6-Aminohexyl)-β-alaninate (3i): Colorless liquid. Yield:
(t, J = 6.9 Hz, 3 H), 2.48 (t, J = 6.6 Hz, 2 H), 2.76 (m, J = 6.2 Hz, 173.2 mg (89%). 1H NMR (200.1 MHz): δ = 1.20–1.70 (m, 8 H),
1168
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© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2012, 1164–1170

Promiscuous Behavior of the Lipase Rhizomucor miehei
1.25 (t, J = 6.9 Hz, 3 H), 2.50 (t, J = 6.6 Hz, 2 H), 2.59 (t, J =
= 0.93 (t, J = 6.9 Hz, 6 H), 1.36 (m, J = 6.9 Hz, 4 H), 1.61 (m, J
6.9 Hz, 2 H), 2.67 (t, J = 6.9 Hz, 2 H), 2.86 (t, J = 6.6 Hz, 2 H), = 6.9 Hz, 4 H), 2.47 (t, J = 6.6 Hz, 4 H), 2.61 (t, J = 6.6 Hz, 2 H),
4.13 (q, J = 6.9 Hz, 2 H) ppm. 13C NMR (50.2 MHz): δ = 14.1,
2.82 (t, J = 6.6 Hz, 4 H), 3.60 (t, J = 6.6 Hz, 2 H), 4.07 (t, J =
26.8, 27.2, 30.1, 33.6, 34.9, 41.9, 45.1, 49.5, 60.4, 172.7 ppm. FTIR
6.9 Hz, 4 H) ppm. 13C NMR (50.2 MHz): δ = 13.7, 19.1, 30.6, 32.5,
(film): ν = 3360, 1735, 1653, 1560 cm–1. HRMS: calcd. for
49.1, 56.0, 58.9, 64.5, 172.6 ppm. FTIR (film): ν = 3446, 1735 cm–1.
˜
˜
C11H25N2O2 [M + H] 217.19105; found 218.19133.
HRMS: calcd. for C16H32NO5 [M
318.22839.
+ H] 318.22750; found
Ethyl N-(8-Aminooctyl)-β-alaninate (3j): Colorless liquid. Yield:
1
200.1 mg (91%). H NMR (200.1 MHz): δ = 1.26 (t, J = 6.9 Hz, 3
Butyl N-(3-Hydroxypropyl)-β-alaninate (4e): Colorless liquid. Yield:
1
H), 1.30–1.43 (m, 12 H), 2.51 (t, J = 6.6 Hz, 2 H), 2.60 (t, J =
6.9 Hz, 2 H), 2.67 (t, J = 6.9 Hz, 2 H), 2.87 (t, J = 6.6 Hz, 2 H),
4.14 (q, J = 6.9 Hz, 2 H) ppm. 13C NMR (50.2 MHz): δ = 14.1,
26.8, 27.2, 29.3, 29.4, 30.0, 33.8, 34.7, 42.2, 45.0, 49.7, 60.3,
168.3 mg (92%). H NMR (200.1 MHz): δ = 0.87 (t, J = 6.9 Hz, 3
H), 1.31 (m, J = 6.9 Hz, 2 H), 1.48–1.70 (m, 4 H), 2.45 (t, J =
6.6 Hz, 2 H), 2.80 (t, J = 6.6 Hz, 2 H), 2.82 (t, J = 6.6 Hz, 2 H),
3.09 (br. s), 3.70 (t, J = 6.6 Hz, 2 H), 4.02 (t, J = 6.9 Hz, 2 H) ppm.
13C NMR (50.2 MHz): δ = 14.0, 19.0, 27.8, 30.7, 34.2, 44.8, 49.2,
172.8 ppm. FTIR (film): ν = 3324, 1733, 1639, 1560 cm–1. HRMS:
˜
60.5, 62.2, 172.5 ppm. FTIR (film): ν = 3314, 1732, 1646 cm–1.
calcd. for C13H29N2O2 [M + H] 245.22290; found 245.22235.
˜
HRMS: calcd. for C10H22NO3 [M
204.15960.
+ H] 204.15942; found
Ethyl N-(12-Aminododecyl)-β-alaninate (3k): White solid. Yield:
270.4 mg (99%). M.p. 93–95 °C. Physical properties as described
previously.[35] 1H NMR (200.1 MHz): δ = 1.21 (m, 19 H), 1.41 (m,
J = 6.9 Hz, 4 H), 2.47 (t, J = 6.6 Hz, 2 H), 2.54 (t, J = 6.9 Hz, 2
H), 2.63 (t, J = 6.9 Hz, 2 H), 2.81 (t, J = 6.6 Hz, 2 H), 4.14 (q, J
= 6.9 Hz, 2 H) ppm. 13C NMR (50.2 MHz): δ = 14.1, 26.7, 27.2,
29.3, 29.4, 29.6, 32.8, 33.0, 34.2, 41.7, 44.7, 49.6, 60.4, 172.7 ppm.
Butyl N-(4-Hydroxybutyl)-β-alaninate (4f): Colorless liquid. Yield:
1
156.5 mg (80%). H NMR (200.1 MHz): δ = 0.86 (t, J = 6.9 Hz, 3
H), 1.28 (m, J = 6.9 Hz, 2 H), 1.47–1.61 (m, 6 H), 2.47 (t, J =
6.6 Hz, 2 H), 2.60 (t, J = 6.6 Hz, 2 H), 2.81 (t, J = 6.6 Hz, 2 H),
3.50 (t, J = 6.6 Hz, 2 H), 3.65 (br. s), 4.02 (t, J = 6.9 Hz, 2 H) ppm.
13C NMR (50.2 MHz): δ = 13.5, 19.0, 28.0, 30.4, 32.0, 33.8, 44.4,
FTIR (film): ν = 3340, 1732, 1652, 1568 cm–1. HRMS: calcd. for
˜
C17H37N2O2 [M + H] 301.28495; found 301.28507.
49.2, 62.2, 64.3, 172.5 ppm. FTIR (film):
ν = 3312, 1733,
˜
1646 cm–1. HRMS: calcd. for C11H24NO3 [M + H] 218.17507;
found 218.17578.
Butyl N-Propyl-β-alaninate (4a): Colorless liquid. Yield: 11.2 mg
1
(66%). H NMR (200.1 MHz): δ = 0.93 (t, J = 7.3 Hz, 3 H), 0.94
(t, J = 6.9 Hz, 3 H), 1.38 (m, J = 6.9 Hz, 2 H), 1.57 (m, J = 7.3 Hz,
2 H), 1.58 (m, J = 6.9 Hz, 2 H), 2.58 (t, J = 6.6 Hz, 2 H), 2.62 (t,
J = 7.3 Hz, 2 H), 2.92 (t, J = 6.6 Hz, 2 H), 4.09 (t, J = 6.9 Hz, 2
H) ppm. 13C NMR (50.2 MHz): δ = 11.4, 13.5, 18.9, 22.4, 30.4,
Butyl N-(2-Hydroxy-1,1-dimethylethyl)-β-alaninate (4g): Colorless
liquid. Yield: 169.0 mg (86%). H NMR (200.1 MHz): δ = 0.92 (t,
1
J = 6.9 Hz, 3 H), 1.06 (s, 6 H), 1.35 (m, J = 6.9 Hz, 2 H), 1.60 (m,
J = 6.9 Hz, 2 H), 2.00 (br. s), 2.47 (t, J = 6.6 Hz, 2 H), 2.77 (t, J
= 6.6 Hz, 2 H), 3.30 (s, 2 H), 4.09 (t, J = 6.9 Hz, 2 H) ppm. 13C
NMR (50.2 MHz): δ = 13.6, 19.1, 24.0, 30.6, 34.9, 37.1, 51.7, 53.7,
34.0, 44.6, 51.1, 64.2, 172.4 ppm. FTIR (film): ν = 3405, 1736,
˜
1640 cm–1. HRMS: calcd. for C10H22NO2 [M + H] 188.16451;
found 188.16408.
64.5, 67.7, 173.4 ppm. FTIR (film): ν = 3363, 1735, 1653 cm–1.
˜
Butyl N-Isopropyl-β-alaninate (4b): Colorless liquid. Yield:
101.1 mg (60%). Physical properties and FTIR as described pre-
viously.[4] 1H NMR (200.1 MHz): δ = 0.85 (t, J = 6.9 Hz, 3 H),
1.02 (d, J = 6.2 Hz, 6 H), 1.30 (m, J = 6.9 Hz, 2 H), 1.53 (m, J =
6.9 Hz, 2 H), 2.48 (t, J = 6.6 Hz, 2 H), 2.64 (t, J = 6.6 Hz, 2 H),
2.80 (m, J = 6.6 Hz, 1 H), 4.03 (t, J = 6.9 Hz, 2 H) ppm. 13C NMR
(50.2 MHz): δ = 13.5, 18.1, 22.3, 30.5, 34.3, 42.2, 48.5, 64.2,
172.5 ppm. HRMS: calcd. for C10H22NO2 [M + H] 188.16451;
found 188.16496.
HRMS: calcd. for C11H24NO3 [M
218.17590.
+ H] 218.17507; found
Butyl N-(3-Aminopropyl)-β-alaninate (4h): Colorless liquid. Yield:
1
182.1 mg (100%). H NMR (200.1 MHz): δ = 0.92 (t, J = 6.9 Hz,
3 H), 1.37 (m, J = 6.9 Hz, 2 H), 1.56 (m, J = 6.9 Hz, 4 H), 2.38 (t,
J = 7.0 Hz, 2 H), 2.57 (t, J = 6.6 Hz, 2 H), 2.66 (t, J = 7.0 Hz, 2
H), 2.79 (t, J = 6.6 Hz, 2 H), 4.05 (t, J = 6.9 Hz, 2 H) ppm. 13C
NMR (50.2 MHz): δ = 14.0, 19.1, 30.6, 33.5, 34.5, 40.3, 44.9, 47.4,
64.3, 172.3 ppm. FTIR (film): ν = 3381, 1735, 1653, 1558 cm–1.
˜
Butyl N-(2-Phenylethyl)-β-alaninate (4c): Colorless liquid. Yield:
HRMS: calcd. for C10H23N2O2 [M + H] 203.17540; found
203.17522.
1
195.8 mg (87%). H NMR (200.1 MHz): δ = 0.90 (t, J = 6.9 Hz, 3
H), 1.31 (m, J = 6.9 Hz, 2 H), 1.52 (m, J = 6.9 Hz, 2 H), 2.51 (t,
J = 6.6 Hz, 2 H), 2.69 (t, J = 5.1 Hz, 2 H), 2.82 (t, J = 6.6 Hz, 2
H), 2.92 (t, J = 5.1 Hz, 2 H), 4.03 (t, J = 6.9 Hz, 2 H), 7.22 (dd, J
= 6.9, 2.9 Hz, 1 H), 7.27 (ddd, J = 6.9, 2.2, 2.9 Hz, 2 H), 7.32 (ddd,
J = 6.9, 2.2, 2.9 Hz, 2 H) ppm. 13C NMR (50.2 MHz): δ = 13.8,
18.4, 30,9, 34.8, 36.2, 46.4, 49.1, 64.5, 126.1, 126.3, 128.5, 128.7,
Butyl N-(6-Aminohexyl)-β-alaninate (4i): Colorless liquid. Yield:
191.0 mg (87%). Physical properties as described previously.[15] 1
H
NMR (200.1 MHz): δ = 0.93 (t, J = 6.9 Hz, 3 H), 1.20–1.65 (m, 12
H), 2.51 (t, J = 6.6 Hz, 2 H), 2.60 (t, J = 7.0 Hz, 2 H), 2.68 (t, J =
7.0 Hz, 2 H), 2.87 (t, J = 6.6 Hz, 2 H), 4.09 (t, J = 6.9 Hz, 2
H) ppm. 13C NMR (50.2 MHz): δ = 13.7, 19.1, 26.8, 27.2, 30.0,
172.3 ppm. FTIR (film): ν = 3315, 1733, 1650, 845 cm–1. HRMS:
˜
calcd. for C15H24NO2 [M + H] 250.18016; found 250.18044.
30.6, 33.6, 34.7, 42.1, 45.1, 49.7, 64.3, 172.9 ppm. FTIR (film): ν =
˜
3314, 1734, 1657, 1559 cm–1. HRMS: calcd. for C13H29N2O2 [M +
H] 245.22235; found 245.22221.
Butyl N-(2-Hidroxyethyl)-β-alaninate (4d): Colorless liquid. Yield:
127.7 mg (75%). Physical properties as described previously.[14] 1
H
NMR (200.1 MHz): δ = 0.91 (t, J = 6.9 Hz, 3 H), 1.38 (m, J =
6.9 Hz, 2 H), 1.59 (m, J = 6.9 Hz, 2 H), 2.51 (t, J = 6.6 Hz, 2 H),
2.77 (t, J = 6.6 Hz, 2 H), 2.90 (t, J = 6.6 Hz, 2 H), 3.63 (t, J =
6.6 Hz, 2 H), 4.07 (t, J = 6.9 Hz, 2 H) ppm. 13C NMR (50.2 MHz):
δ = 13.7, 19.1, 30.6, 34.6, 44.5, 50.8, 60.6, 64.5, 172.8 ppm. FTIR
Butyl N-(8-Aminooctyl)-β-alaninate (4j): Colorless liquid. Yield:
228.1 mg (94%). H NMR (200.1 MHz): δ = 0.91 (t, J = 6.9 Hz, 3
H), 1.30–1.41 (m, 14 H), 1.54 (m, J = 6.9 Hz, 2 H), 2.53 (t, J =
6.6 Hz, 2 H), 2.62 (t, J = 6.9 Hz, 2 H), 2.66 (t, J = 6.9 Hz, 2 H),
2.88 (t, J = 6.6 Hz, 2 H), 4.05 (t, J = 6.9 Hz, 2 H) ppm. 13C NMR
(50.2 MHz): δ = 13.7, 19.2, 26.8, 27.2, 28.8, 29.2, 29.4, 30.1, 30.6,
1
(film): ν = 3326 1732, 1638 cm–1. HRMS: calcd. for C H NO [M
˜
9
20
3
+ H] 190.14377; found 190.14441.
33.9, 34.7, 42.3, 45.2, 49.9, 64.1, 172.7 ppm. FTIR (film): ν = 3333,
˜
N-(3-Ethoxy-3-oxopropyl)-N-(2-hydroxyethyl)-β-alaninate
Colorless liquid. Yield: 115.7 mg (81%). H NMR (200.1 MHz): δ
(4d*):
1733, 1630, 1560 cm–1. HRMS: calcd. for C15H33N2O2 [M + H]
273.25365; found 273.25394.
1
Eur. J. Org. Chem. 2012, 1164–1170
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1169

L. N. Monsalve, F. Gillanders, A. Baldessari
FULL PAPER
[13] M. Vijender, P. Kishore, B. Satyanarayana, Synth. Commun.
2007, 37, 591–594.
Butyl N-(12-Aminododecyl)-β-alaninate (4k): White solid. Yield:
274.8 mg (93%). M.p. 97–98 °C. H NMR (200.1 MHz): δ = 0.92
1
[14] N. Ogata, T. Asahara, Bull. Chem. Soc. Jpn. 1966, 39, 1486–
(t, J = 6.9 Hz, 3 H), 1.29 (m, 18 H), 1.41 (m, 6 H), 2.44 (t, J =
6.9 Hz, 2 H), 2.53 (t, J = 6.6 Hz, 2 H), 2.66 (t, J = 6.9 Hz, 2 H),
2.87 (t, J = 6.6 Hz, 2 H), 4.06 (t, J = 6.9 Hz, 2 H) ppm. 13C NMR
(50.2 MHz): δ = 13.7, 19.3, 27.2, 27.4, 28.7, 29.3, 29.5, 29.6, 29.8,
29.9, 30.4, 32.9, 34.4, 41.9, 44.8, 49.5, 64.4, 172.7 ppm. FTIR
1490.
[15] K. Sanui, M. Ishida, N. Ogata, Bull. Chem. Soc. Jpn. 1968, 41,
256–259.
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(film): ν = 3340, 1732, 1642, 1570 cm–1. HRMS: calcd. for
˜
C19H41N2O2 [M + H] 329.31625; found 329.31579.
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Published Online: December 27, 2011
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