Biocatalytic synthesis of chiral N-(2-hydroxyalkyl)-acrylamides

Article abstract of DOI:10.3109/10242422.2011.578212

The preparation of a series of novel chiral N-(2-hydroxylalkyl)-acrylamides through a lipase-catalyzed resolution of racemic alkanolamines is described. The absolute stereochemistry and enantiomeric excess of the products were determined by a modified Mosher's method. The method was validated for this particular case by the synthesis of an enantiomerically pure product. Moreover, the stereoselective behavior of the lipase in this reaction is discussed.

Full text of DOI:10.3109/10242422.2011.578212

Biocatalysis and Biotransformation, March–June 2011; 29(2–3): 87–95
ORIGINAL ARTICLE
Biocatalytic synthesis of chiral N-(2-hydroxyalkyl)-acrylamides
LEANDRO N. MONSALVE, EDUARDO M. RUSTOY & ALICIA BALDESSARI
Laboratorio de Biocatálisis, Departamento de Química Orgánica y UMYMFOR, Facultad de Ciencias Exactas y
Naturales, Universidad de Buenos Aires, Pabellón 2, Piso 3, Ciudad Universitaria, C1428EGA, Buenos Aires,
Argentina
Abstract
The preparation of a series of novel chiral N-(2-hydroxylalkyl)-acrylamides through a lipase-catalyzed resolution of racemic
alkanolamines is described. The absolute stereochemistry and enantiomeric excess of the products were determined by a
modified Mosher’s method.The method was validated for this particular case by the synthesis of an enantiomerically pure
product. Moreover, the stereoselective behavior of the lipase in this reaction is discussed.
Keywords: Candida antarctica lipase B, N-(hydroxyalkyl)-acrylamides, stereoselectivity, Mosher’s method, primary alcohol
Introduction
2006). Similarly, Gotor’s group reported the enzy-
Lipases, working in non-aqueous media, have been
widely used for several synthetic reactions (Bom-
marius & Riebel 2004; Carrea & Riva 2008). They
are well known for their high enantioselectivity
(Faber 2000; Buchholz et al. 2005), a property that
has found them widespread application in the syn-
thesis of enantiomerically pure compounds (Gotor
et al. 2007). They have been employed for resolving
racemic mixtures of several highly functionalized
chiral molecules such as amino acids (Forró & Fülöp
2010) and amino alcohols (Turcu et al. 2010).
We have applied lipases as catalysts in a variety
of reactions such as esterification, transesterifica-
tion, aminolysis and even polymerization, which pro-
duced various steroid and terpenoid derivatives,
substituted amides, polyacrylamides and polyamido-
amines with pharmacological properties and biomed-
ical applications (Rustoy & Baldessari 2005; Rustoy
et al. 2007; Monsalve et al. 2008, 2009, 2010; Quin-
tana & Baldessari 2009). Among them, lipase-
catalyzed aminolysis of ethyl acrylate under controlled
reaction conditions allowed us to obtain a series of
N-hydroxyalkylacrylamides, which are useful starting
materials for the production of polymer matrices
employed in electrophoresis (Rustoy & Baldessari
matic synthesis of N-(3-dimethylaminopropyl)-
acrylamide (Torre et al. 2005).
Chiral acrylamides are useful compounds in
organic synthesis.They can act as chiral inductors in
controlling the stereochemistry of cycloadditions
(Nyerges et al. 2005) and the tacticity of chiral poly-
meric matrices applied in stereoselective separations
(Tian et al. 2010), as well as monomers in the syn-
thesis of chiral thermoresponsive amphiphilic co-
networks (Tobis et al. 2010).
Considering these potential applications and
extending our work on this subject, in the present
paper we describe the enzymatic synthesis and ste-
reochemical characterization of a series of chiral
N-(2-hydroxyalkyl)-acrylamides (2a–2d). For this
purpose, we performed a lipase-catalyzed aminolysis
of ethyl acrylate with the corresponding racemic
2-hydroxyalkyamines (1) (Scheme 1).
Previous work in this area includes the studies of
Puertas et al. (1993) describing the enantioselective
conversion of racemic amines to (R)-acrylamides
using Candida antarctica lipase B.
Among the methods available to determine the
absolute stereochemistry of chiral compounds, NMR
spectroscopy using chiral derivatizing agents such
Correspondence: A. Baldessari, Laboratorio de Biocatálisis, Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de
Buenos Aires, Pabellón 2 – Piso 3, Ciudad Universitaria C1428EGA, Buenos Aires, Argentina. Tel: ϩ54-11-4576-3300 ext. 262. Fax: ϩ54-11-4576-3385.
E-mail: alib@qo.fcen.uba.ar; alibhome@yahoo.com.ar
(Received 3 January 2011; revised 1 April 2011; accepted 22 March 2011)
ISSN 1024-2422 print/ISSN 1029-2446 online © 2011 Informa UK, Ltd.
DOI: 10.3109/10242422.2011.578212

88 L. N. Monsalve et al.
R
HN
O
OH
using a Bruker (Billerica, MA, USA) AC-200 spec-
trometer operating at 200.13 MHz and 50.32 MHz
NH2
CAL B
+
1
for H and 13C, respectively, and a Bruker AM-500
HO
O
O
Et
R
NMR instrument operating at 500.14 MHz and
125.76 MHz for 1H and 13C, respectively. Chemical
shifts are reported in δ units (ppm) relative to TMS
set at 0 ppm, and coupling constants are given in
Hertz. High-resolution mass spectra (HRMS) were
recorded on a Bruker microTOF-Q II operating in
electrospray ionization mode.
2
1
a: R: -CH3; b: R: -CH2CH3; c: R: -(CH2)2CH3; d: R: -(CH2)3CH3
Scheme 1. Lipase-catalyzed synthesis of 2a–2d.
as 2-methoxy-2-(trifluoromethyl)-phenylacetic acid
(MTPA) has been widely used in the case of stereo-
genic centers bearing either hydroxyl or amine
groups (Pehk et al. 1993). More recently, this meth-
odology has been applied for the derivatization of
chiral primary alcohols, thus determining the abso-
lute configuration of stereogenic centers at a two-
bond distance from the hydroxyl group that reacts
with MTPA (Akiyama et al. 2003; Galman & Hailes
2009). Moreover, the modified Mosher’s method has
been used to determine the absolute configuration
of primary alcohols with chiral methyl groups at C-2
(Czuba et al. 2003).
In this work we used the modified Mosher’s
method for determining the absolute configuration
and enantiomeric ratio of the enzyme-catalyzed reac-
tion products.We also synthesized a Mosher’s ester of
an enantiomerically pure N-(2-hydroxyalkyl)-acryl-
amide in order to test the reliability of the method.
Enzymatic synthesis of N-(2-hydroxyalkyl)-
acrylamides 2a to 2d under standard conditions
Hydroxyalkylacrylamides were prepared as follows:
0.9 mmol of the corresponding rac-2-amino alcohol
was dissolved together with 0.9 mmol of ethyl acrylate
in 7.5 mL of diisopropylether. CALB (100 mg) was
added to the solution and then the reaction was allowed
to proceed in an orbital shaker (33°C, 200 rpm) and
monitored by TLC. Once the reaction reached its
maximum conversion, the enzyme was filtered off and
the solvent evaporated. The resulting hydroxyalky-
lacrylamide was purified by flash chromatography
(acetone).
Synthesis of 2a under microwave irradiation
Substrate 1a was dissolved in diisopropylether and
CALB was added as described for the enzymatic
synthesis of 2a under standard conditions.The reac-
tion was allowed to proceed under microwave irra-
diation with magnetic stirring in a closed vessel.The
temperature was fixed to 30°C by automated control
of irradiation power. The vessel was continuously
refrigerated with an air current at 0°C during the
reaction. The product 2a was isolated as described
(83% yield, racemic).
Materials and methods
General remarks
Candida antarctica lipase B (CALB) (Novozym® 435;
7400 PLU/g) was purchased from Codexis, Inc.
(Pasadena, CA, USA).The enzyme was used ‘straight
from the bottle’. Chemical reagents and solvents were
sourced from Aldrich (Buenos Aires, Argentina),
Sigma (Buenos Aires, Argentina), Fluka (Buenos
Aires, Argentina), or J.T. Baker (Tampa, FL, USA).
Enzymatic reactions were carried out in an Innova
4000 digital incubator shaker (New Brunswick Sci-
entific Co.) (Edison, NJ, USA) at 200 rpm. Reactions
under microwave irradiation were performed using a
Discovery monomode microwave reactor (CEM
Corporation) (Matthews, NC, USA). Reactions were
followed by TLC on Merck (Buenos Aires, Argen-
tina) silica gel 60F-254 aluminum sheets (0.2 mm
thickness). For flash chromatography Merck silica gel
60 (60–230 mesh) was used. Optical rotations were
measured with a Perkin Elmer (Waltham, MA, USA)
343 polarimeter (solvents are indicated). FTIR spec-
tra were obtained on a Shimadzu (Columbia, MD,
USA) FTIR-8300 spectrophotometer. 1H NMR and
13C NMR spectra were recorded in CDCl3 as solvent
(S)-N-(2-hydroxy-1-methylethyl)-acrylamide, (S)-2a
Yellowish oil, 42% yield, 32% ee (determined by
integration of H-1′a and H-1′b proton signals of its
(R)-MTPA derivative, (S)-3a). [α]2D5: –22.0° (c ϭ 1,
CHCl3). IR (film)/(cm–1): 3292 br (OH), 2971,
1650 (C ϭ O). 1H NMR (200.13 MHz, CDCl3), δ
1.19 (dd, 3H, J ϭ 6.2 Hz, –CH3), 3.17 (m, 1H,
–CH2–OH), 3.50 (m, 1H, –CH2–OH), 3.93 (m, 1H,
–CH(NH)–), 5.60 (dd, 1H, J ϭ 9.4, 1.7 Hz, ϭ CH2),
6.15 (dd, 1H, J ϭ 16.9, 9.4 Hz, ϭ CH(CO)), 6.29
(m, 1H, J ϭ 16.9, 1.7 Hz, ϭ CH), 13C NMR (50.32
MHz; CDCl3), δ 20.83 (CH3), 47.07 (CH), 67.16
(CH2OH), 126.87 (ϭ CH2), 130.56 (ϭ CH–),
169.47 (C ϭ O); m/z (HRMS) ([MϩH]ϩ) calcd. for
C6H12NO2, 130.0868; found 130.0864.

Biocatalytic synthesis of chiral N-(2hydroxyalkyl)-acrylamides 89
(S)-N-[1-(hydroxymethyl)-propyl]-acrylamide, (S)-2b
Synthesis of MTPA derivatives
Yellowish oil, 60% yield, 35% ee (determined by
integration of H1′a and H1′b proton signals of its
(R)-MTPA derivative, (S)-3b). [α]25D: –23.3° (c ϭ 1,
CHCl3). IR (film), ν (cm–1): 3300 br (OH), 2970,
1650 (C ϭ O). 1H NMR (200.13 MHz, CDCl3), δ
(ppm): 0.90 (3H, t, J ϭ 7.4 Hz, –CH3); 1.50 (2H,
m, CH3–CH2–); 3.52 (2H, m, –CH2–OH); 3.87
(1H, m, –CH(NH)–); 5.60 (dd, J ϭ 8.4 and 1.8
Hz, ϭ CH2); 6.15 (dd, J ϭ 16.9 and 8.4
Hz, ϭ CH(CO)–); 6.25 (m, J ϭ 16.9 and 1.8
Hz, ϭ CH). 13C NMR (50.32 MHz, CDCl3), δ
(ppm): 10.55 (CH3), 24.10 (CH2), 53.26 (CHNH),
64.42 (CH2OH), 126.55 (ϭ CH2), 130.90 (ϭ CH),
166.47 (C ϭ O). HRMS, m/z: 144.1019. ([MϩH]ϩ).
Calcd for C7H14NO2: 144.1025.
The reaction was carried out under anhydrous con-
ditions.To a stirred solution of the acrylamide (0.40
mmol) in CH2Cl2 (5 mL), triethylamine (54 μL,
0.40 mmol) and MTPA chloride (100 mg, 0.40
mmol) in CH2Cl2 (2 mL) were added and the reac-
tion mixture was stirred for 12 h at room tempera-
ture. The solvent was evaporated and the product
was purified using flash chromatography (hexane–
EtOAc, 1:2, v/v).
(R)-N-[1-(hydroxymethyl)-propyl]-acrylamide,
(R)-2b and (2R,2′S)-2′-(acrylamido)-propyl-3,3,3-
trifluoro-2-methoxy-2-phenylpropanoate (2R,2′S)-3a
Colorless oil, 60% yield, 32% ed (from integrations
of H1′a and H1′b 1H NMR signals from (S)-3a and
(S)-4a), [α]2D5: –24.4 (C ϭ 1, CHCl3); IR (KBr)
(cm–1) 3290br, 2938s, 1749s, 1663s; 1H NMR
(500.14 MHz; CDCl3), δ 1.40 (3H, dd, J ϭ 6.3, 6.4
Hz, –CH3), 3.49 (3H, ds, OCH3) 4.30 (1H, m,
CHNH) 4.33 (0.36H, dd, J ϭ 4.0, 11.2 Hz, CHO
(2R,2′R)), 4.37 (1.28H, d, J ϭ 4.0, 4.1 Hz (2R,2′S)),
4.46 (0.36H, dd, J ϭ 4.1, 11.2 Hz, CHO (2R,2′R)),
5.63 (1H, ddd, J ϭ 1.1, 1.2, 10.3, 10.4 Hz, ϭ CH),
5.98 (1H, ddd, J ϭ 10.3, 10.4, 16.9, 17.0 Hz, ϭ CH),
(S)-N-[1-(hydroxymethyl)-butyl]-acrylamide, (S)-2c
Yellowish oil, 65% yield, 51% ee (determined by
integration of H1′a and H1′b proton signals of its
(R)-MTPA derivative, (S)-3c). [α]25D: –29.2°
(c ϭ 1, CHCl3). IR (film), ν (cm–1): 3302 br (OH),
1
2969, 1650 (C ϭ O). H NMR (200.13 MHz,
CDCl3), δ (ppm): 0.88 (3H, t, J ϭ 6.9 Hz, –CH3);
1.35 (2H, m, CH3–CH2–); 1.48 (2H, m, –CH2–
CH(NH)–); 3.58 (2H, m, –CH2–OH); 3.99 (1H,
m, –CH(NH)–); 5.60 (dd, J ϭ 9.2 and 2.2 Hz,
(CO)–CH2 ϭ); 6.14 (dd, J ϭ 16.9 and 9.2
Hz, ϭ CH2); 6.22 (m, J ϭ 16.9 and 2.2
Hz, ϭ CH(CO)–). 13C NMR (50.32 MHz, CDCl3),
δ (ppm): 13.94 (CH3), 19.30 (CH3CH2), 33.28
(CH2), 51.58 (CHNH), 64.95 (CH2OH), 126.55
(ϭ CH2), 130.88 (ϭ CH), 166.36 (C ϭ O). HRMS,
m/z: 158.1176 ([MϩH]ϩ). Calcd for C8H16NO2:
158.1181.
6.22 (1H, ddd, J
ϭ 1.1, 1.2, 16.9, 17.0
Hz, ϭ CH(CO)), 7.41 (3H, m, Ph); 7.50 (2H, m,
Ph);13C NMR (125.76 MHz;CDCl3),δ 16.3 (CH3),
44.1 and 44.4 (CHNH), 54.5 and 54.7 (CH3O),
68.0 and 68.2 (CH2O), 84.4 and 84.6 (CPh), 123.4
(q, JCF ϭ 287.9 Hz), 126.8 (CH2ϭ), 127.3 (Ph),
128.6 (Ph), 129.7 (Ph), 130.4 (CHϭ), 132.0 (Ph),
165.3 (C ϭ O, amide), 166.5 and 166.6 (C ϭ O,
ester);m/zHRMS:[MϩH]ϩ calcd.forC16H19F3NO4,
346.1266; found 346.1260.
(2S,2′S)-2′-(acrylamido)-propyl-3,3,3-trifluoro-
2-methoxy-2-phenylpropanoate, (2S,2′S)-4a
(S)-N-[1-(hydroxymethyl)-pentyl]-acrylamide, (S)-2d
Yellowish oil, 72% yield, 67% ee (determined by
integration of H1′a and H1′b proton signals of its
(R)-MTPA derivative, (S)-3d). [α]2D5: –45.9° (c ϭ 1,
CHCl3); IR (film), ν (cm–1): 3308 br (OH), 2974,
1650 (C ϭ O). 1H NMR (200.13 MHz; CDCl3), δ
0.89 (t, 3H, J ϭ 6.6 Hz, CH3–); 1.32 (m, 4H, CH3–
(CH2)2–), 1.54 (m, 2H, –CH2–CH(NH)–), 3.62
(m, 2H, CH2–OH), 3.99 (m, 1H, –CH(NH)–), 5.65
(dd, 1H, J ϭ 9.5, 2.2 Hz, (CO)–CH2ϭ), 6.14 (dd,
1H, J ϭ 17.2, 9.5 Hz, CH2ϭ), 6.22 (m, 1H,
J ϭ 17.2, 2.2 Hz, ϭCH(CO)); 13C NMR (50.32
MHz, CDCl3), δ 13.94 (CH3), 19.30 (CH3CH2),
33.28 (CH2CHNH), 51.58 (CHNH), 64.95
(CH2OH), 126.55 (CH2ϭ), 130.88 (ϭCH), 166.36
(C ϭ O); m/z (HRMS) [MϩH]ϩ calcd. for
C9H18NO2, 172.1338; found 172.1339.
Colorless oil, 58% yield 32% ed (from integrations
of H1′a and H1′b 1H NMR signals from (S)-3a and
1
(S)-4a); [α]2D5: ϩ32.1° (c ϭ 1, CHCl3). H NMR
(500.14 MHz, CDCl3), δ 1.40 (3H, dd, J ϭ 6.3, 6.4
CH3), 3.49 (3H, ds, OCH3), 4.30 (1H, m, CHNH),
4.33 (0.68H, dd, J ϭ 4.0, 11.2 Hz, CHO (2S,2′S)),
4.37 (0.32H, d, J ϭ 4.0, 4.1 Hz (2S,2′R)), 4.46
(0.68H, dd, J ϭ 4.1 and 11.2 Hz, CHO (2S,2′S)), 5.63
(1H, ddd, J ϭ 1.1, 1.2, 10.3, 10.4 Hz, CH ϭ), 5.98
(1H, ddd, J ϭ 10.3, 10.4, 16.9, 17.0 Hz, ϭ CH),
6.22 (1H, J ϭ 1.1, 1.2, 16.9, 17.0 Hz, ϭ CH(CO)),
7.41 (3H, m, Ph); 7.50 (2H, m, Ph). 13C NMR
(125.76 MHz; CDCl3): δ 16.3 (CH3), 44.1 and 44.4
(CHNH), 54.5 and 54.7 (CH3O), 68.0 and 68.2
(CH2O), 84.4 and 84.6 (CPh), 123.4 (q, JCF ϭ 287.9

90 L. N. Monsalve et al.
Hz), 126.8 (CH2ϭ), 127.3 (Ph), 128.6 (Ph), 129.7
(Ph), 130.4 (CHϭ), 132.0 (Ph), 165.3 (C ϭ O,
amide), 166.5 and 166.6 (C ϭ O, ester).
(2R,2′S)-2′-(acrylamido)-pentyl-3,3,3-trifluoro-
2-methoxy-2-phenylpropanoate, (2R,2′S)-3c
Colorless oil, 72% yield, 45% ed (from integrations
of H1′a and H1′b 1H NMR signals from (S)-3c and
(S)-4c). [α]2D5: –31.2° (c ϭ 1, CHCl3). IR (KBr), ν
(2R,2′S)-2′-(acrylamido)-butyl-3,3,3-trifluoro-
2-methoxy-2-phenylpropanoate, (2R,2′S)-3b
1
(cm–1): 3310 br, 2913 s, 1764 s, 1655 s. H NMR
(500.14 MHz, CDCl3), δ (ppm): 0.90 (3H, dt,
J ϭ 7.3 and 7.5 Hz, CH3); 1.45–1.62 (4H, m,
(CH2)2); 3.54 (3H, ds, OCH3); 4.31 (1H, m,
–CH(NH)–); 4.35 (0.30H, dd, J ϭ 4.4 and 11.8 Hz,
CHO (2R,2′R)); 4.37 (1.40H, d, J ϭ 4.2 and 4.4
Hz (2R,2′S)); 4.46 (0.30H, dd, J ϭ 11.8 and 4.2 Hz,
CHO (2R,2′R)); 5.60 (1H, ddd, J ϭ 1.1, 10.3 and
10.4 Hz, ϭ CH); 6.01 (1H, ddd, J ϭ 10.3. 10.4,
16.7 and 17.0 Hz, ϭ CH); 6.27 (1H, ddd, J ϭ 1.1,
16.7 and 17.0 Hz, ϭ CH(CO)); 7.39 (3H, m, Ph);
7.55 (2H, m, Ph). 13C NMR (125.76 MHz, CDCl3),
δ (ppm): 11.8 (CH3), 17.3 (CH2), 30.5 and 30.7
(CH2), 50.2 and 50.3 (CHNH), 54.9 and 55.1
(CH3O), 67.8 and 68.0 (CH2O), 84.8 and 84.9
(CPh), 124.7 (q, JCF ϭ 287.0 Hz), 127.7 (ϭ CH2),
128.0 (Ph), 128.9 (Ph), 129.7 (Ph), 130.8 (ϭ CH),
133.4 (Ph), 165.1 (C ϭ O, amide), 166.8 (C ϭ O,
ester). HRMS m/z: 374.1601 ([MϩH]ϩ). Calcd for
C18H23F3NO4: 374.1579.
Colorless oil, 70% yield, 35% ed (from integrations
of H1′a and H1′b 1H NMR signals from (S)-3b and
(S)-4b). [α]2D5: –27.3° (c ϭ 1, CHCl3). IR (KBr), ν
(cm–1): 3300 br, 2933 s, 1752 s, 1659 s. 1H NMR
(500.14 MHz, CDCl3), δ (ppm): 0.93 (3H, dt,
J ϭ 7.3 and 7.5 Hz, –CH3); 1.54 (2H, m, CH2);
3.52 (3H, ds, OCH3); 4.30 (1H, m, –CH(NH)–);
4.33 (0.34H, dd, J ϭ 3.9 and 11.2 Hz, CHO
(2R,2′R)); 4.37 (1.34H, dd, J ϭ 3.9 and 4.8 Hz
(2R,2′S)); 4.46 (0.34H, dd, J ϭ 4.8 and 11.2 Hz,
CHO (2R,2′R)); 5.63 (1H, J ϭ 1.1, 1.2, 10.3 and
10.4 Hz, ϭ CH); 5.98 (1H, J ϭ 10.3. 10.4, 16.9
and 17.0 Hz, ϭ CH); 6.23 (1H, J ϭ 1.1, 1.2, 16.9
and 17.0 Hz, ϭ CH(CO)); 7.41 (3H, m, Ph); 7.50
(2H, m, Ph). 13C NMR (125.76 MHz, CDCl3), δ
(ppm): 10.2 (CH3), 24.5 and 24.6 (CH2), 49.5 and
49.6 (CHNH), 54.4 and 54.5 (CH3O), 67.0 and
67.1 (CH2O), 84.6 and 84.8 (CPh), 124.0 (q,
J
CF ϭ 288.0 Hz), 127.0 (ϭ CH2), 127.2 (Ph), 128.9
(Ph), 129.9 (Ph), 131.0 (ϭ CH), 133.0 (Ph), 165.5
(C ϭ O, amide), 166.5 (C ϭ O, ester). HRMS, m/z:
360.1417 ([MϩH]ϩ). Calcd for C17H21F3NO4:
360.1423.
(2S,2′S)-2′-(acrylamido)-pentyl-3,3,3-trifluoro-
2-methoxy-2-phenylpropanoate, (2S,2′S)-4c
Colorless oil, 70% yield, 45% ed (from integrations
of H1′a and H1′b 1H NMR signals from (S)-3c and
1
(S)-4c); [α]2D5: ϩ37.7 (c ϭ 1, CHCl ). H NMR
(500.14 MHz, CDCl3), δ (ppm): 03.89 (3H, dt,
J ϭ 7.3 and 7.5 Hz, –CH3); 1.44–1.61 (4H, m,
(CH2)2); 3.53 (3H, ds, OCH3); 4.30 (1H, m,
–CH(NH)–); 4.34 (0.76H, dd, J ϭ 4.4 and 11.8 Hz,
CHO (2S,2′S)); 4.36 (0.48H, d, J ϭ 4.2 and 4.2
Hz (2R,2′S)); 4.45 (0.76H, dd, J ϭ 4.2 and 11.8 Hz,
CHO (2S,2′S)); 5.59 (1H, J ϭ 1.1, 10.3 and 10.4
Hz, ϭ CH); 6.00 (1H, J ϭ 10.3, 10.4, 16.7 and 17.0
Hz, ϭ CH); 6.26 (1H, J ϭ 1.1, 16.7 and 17.0 Hz, ϭ
(2S,2′S)-2′-(acrylamido)-butyl-3,3,3-trifluoro-
2-methoxy-2-phenylpropanoate, (2S,2′S)-4b
Colorless oil, 65% yield, 35% ed (determined by
1
integration of H1′a and H1′b H NMR signals
from (S)-3b and (S)-4b). [α]2D5: ϩ35.7° (c ϭ 1,
CHCl3). 1H NMR (500.14 MHz, CDCl3), δ
(ppm): 0.93 (3H, dt, J ϭ 7.3 and 7.5 Hz, –CH3);
1.54 (2H, m, CH2); 3.52 (3H, ds, OCH3); 4.30
(1H, m, –CH(NH)–); 4.33 (0.52H, dd, J ϭ 3.9 and
11.2 Hz, CHO (2S,2′S)); 4.37 (0.52H, d, J ϭ
3.9 and 4.8 Hz (2S,2′R)); 4.46 (0.74H, dd,
J ϭ 4.8 and 11.2 Hz, CHO (2S,2′S)); 5.63
(1H, J ϭ 1.1, 1.2, 10.3 and 10.4 Hz, ϭ CH);
5.98 (1H, J ϭ 10.3. 10.4, 16.9 and 17.0 Hz, ϭ CH);
6.23 (1H, J ϭ 1.1, 1.2, 16.9 and 17.0 Hz, ϭ
CH(CO)); 7.41 (3H, m, Ph); 7.50 (2H, m, Ph).
13C NMR (125.76 MHz, CDCl3), δ (ppm): 10.2
(CH3), 24.5 and 24.6 (CH2), 49.5 and 49.6
(CHNH), 54.4 and 54.5 (CH3O), 67.0 and 67.1
CH(CO)); 7.38 (3H, m, Ph); 7.54 (2H, m, Ph). 13
C
NMR (125.76 MHz, CDCl3), δ (ppm): 11.8 (CH3),
17.3 (CH2), 30.5 and 30.7 (CH2), 50.2 and 50.3
(CHNH), 54.9 and 55.1 (CH3O), 67.8 and 68.0
(CH2O), 84.8 and 84.9 (CPh), 124.7 (q, JCF ϭ 287.0
Hz), 127.7 (ϭ CH2), 128.0 (Ph), 128.9 (Ph), 129.7
(Ph), 130.8 (ϭ CH), 133.4 (Ph), 165.1 (C ϭ O,
amide), 166.8 (C ϭ O, ester).
(2R,2′S)-2′-(acrylamido)-hexyl-3,3,3-trifluoro-
2-methoxy-2-phenylpropanoate (2R,2′S)-3d
(CH2O), 84.6 and 84.8 (CPh), 124.0 (q, JCF
ϭ
288.0 Hz), 127.0 (ϭ CH2), 127.2 (Ph), 128.9
(Ph), 129.9 (Ph), 131.0 (ϭ CH), 133.0 (Ph),
165.5 (C ϭ O, amide), 166.5 (C ϭ O, ester).
Colorless oil, 75% yield, 67% ed (from integrations
of H1′a and H1′b 1H NMR signals from (S)-3d and

Biocatalytic synthesis of chiral N-(2hydroxyalkyl)-acrylamides 91
(S)-4d). [α]2D5: –30.5° (c ϭ 1, CHCl3). IR (KBr), ν
Colorless oil, 57% yield. >99% ee (determined
1
(cm–1): 3300 br, 2908 s, 1767 s, 1653 s. H NMR
by integration of H1’a and H1’b proton signals).
1
[␣]2D5: ϩ133.1 (C ϭ 1, CHCl3); H NMR (500.14
(500.14 MHz, CDCl3), δ (ppm): 0.89 (3H, dt,
J ϭ 7.3 and 7.5 Hz, CH3); 1.32 (4H, m, (CH2)2);
1.51 (4H, m, CH2); 3.53 (3H, ds, OCH3); 4.33 (1H,
m, –CH(NH)–); 4.35 (0.15H, dd, J ϭ 4.3 and 11.2
Hz, CHO (2R,2′R)); 4.36 (1.70H, d, J ϭ 4.1 and
4.3 Hz (2R,2′S)); 4.45 (0.15H, dd, J ϭ 4.1 and 11.2
Hz, CHO (2R,2′R)); 5.61 (1H, J ϭ 1.2, 10.1 and
10.5 Hz, ϭ CH); 6.08 (1H, J ϭ 10.1. 10.5, 16.9 and
17.3 Hz, ϭ CH); 6.26 (1H, J ϭ 1.2, 16.9 and 17.3
Hz, ϭ CH(CO)); 7.31 (3H, m, Ph); 7.57 (2H, m,
Ph). 13C NMR (125.76 MHz, CDCl3), δ (ppm):
13.8 (CH3), 22.4 (CH2), 27.9 (CH2), 31.2 (CH2),
48.0 and 48.1 (CHNH), 55.4 and 55.5 (CH3O),
67.3 and 67.4 (CH2O), 84.4 and 84.8 (CPh), 124.5
(q, JCF ϭ 288.0), 126.9 (ϭ CH2), 127.8 (Ph), 128.5
(Ph), 129.7 (Ph), 130.6 (ϭ CH), 132.4 (Ph), 165.0
(C ϭ O, amide), 166.5 and 166.7 (C ϭ O, ester).
HRMS m/z: 388.1700 ([MϩH]ϩ). Calcd for
C19H25F3NO4: 388.1736.
MHz; CDCl3) ␦ 0.92 (3H, t, Jϭ 7.5 Hz, CH3), 1.52
(2H, m, CH2) 3.51 (3H, ds, OCH3), 4.27 (1H, m,
CHNH), 4.36 (2H, dd, Jϭ 3.9, 4.8 Hz, CHO), 5.61
(1H, ddd, J ϭ 1.4, 10.3 Hz, CHϭ), 5,99 (1H, dd,
J ϭ 10.3, 17.2 Hz, CHϭ), 6.22 (1H, dd, J ϭ 1.4,
17.2 Hz, ϭCH(CO)), 7.41 (3H, m, Ph), 7.49 (2H,
m, Ph); 13C NMR (125.76 MHz; CDCl3): ␦
10.2(CH3), 24.5 (CH3), 49.6 (CHNH), 54.4
(CH3O), 67.0 (CH2O), 84.8 (CPh), 124.0 (q, JCF ϭ
288.0 Hz, (CF3)), 127.0 (CH3ϭ), 127.2 (Ph), 128.9
(Ph), 129.9 (Ph), 131.0 (ϭCH), 133.0 (Ph), 165.5
(C ϭ O, amide), 166.5(C ϭ O, ester).
Results and discussion
The lipase-catalyzed aminolysis reaction for the pro-
duction of the chiral N-(2-hydroxyalkyl)-acrylamides
(2a–2d) was studied. To start the work, rac-2a was
chosen as a model substrate in order to optimize the
reaction conditions using the lipase from C. antarc-
tica B as biocatalyst, based on the good results previ-
ously reported using this enzyme in aminolysis
reactions (Puertas et al. 1993; Rustoy & Baldessari
2005, 2006; Torre et al. 2005).
We performed several experiments varying reac-
tion parameters such as enzyme/substrate ratio (E/S)
between 0.1 and 5, ethyl acrylate/2a ratio (A/S)
between 0.5 and 1.2, reagent concentrations from
0.012 M to 1.2 M, temperature (33°C and 55°C)
and solvent (diisopropylether, hexane and toluene).
As a result we chose as standard conditions: diiso-
propylether as solvent, 33°C, an E/S ratio and A/S
ratio of 1, and a concentration 0.12 M of both ethyl
acrylate and hydroxyalkanolamine 2a.
Either higher substrate concentration or higher
temperature favored the formation of aza-Michael
products. The same results were observed when
hexane was used as solvent. This side reaction had
previously been observed in other examples of lipase-
catalyzed aminolysis and it was necessary to find
optimal conditions in each particular case to mini-
mize it (Torre et al. 2005; Monsalve et al. 2010).
Finally, the synthesis of the products 2a–2d was
performed under the optimized conditions described.
Table I (column 3) shows that the chiral hydroxyal-
kylacrylamides were obtained in moderate to good
yields. An increase in yield was observed as the chain
length of the alkyl group R in the stereocenter of the
alkanolamine increased.
(2S,2′S)-2′-(acrylamido)-hexyl-3,3,3-trifluoro-
2-methoxy-2-phenylpropanoate (2S,2′S)-4d
Colorless oil, 75% yield, 67% ed (from integrations
of H1′a and H1′b 1H NMR signals from (S)-3d and
(S)-4d). [α]25D: ϩ41.1° (c ϭ 1, CHCl ). 1H NMR
(500.14 MHz, CDCl3), δ (ppm): 0.389 (3H, dt,
J ϭ 7.3 and 7.5 Hz, –CH3); 1.32 (4H, m, (CH2)2);
1.51 (4H, m, CH2); 3.53 (3H, ds, OCH3); 4.33
(1H, m, –CH(NH)–); 4.35 (0.82H, dd, J ϭ 4.3 and
11.2 Hz, CHO (2S,2′S)); 4.36 (0.18H, d, J ϭ 4.1
and 4.3Hz (2S,2′R)); 4.45 (0.82H, dd, J ϭ 4.1 and
11.2 Hz, CHO (2S,2′S)); 5.61 (1H, J ϭ 1.2, 10.1
and 10.5 Hz, ϭ CH); 6.08 (1H, J ϭ 10.1. 10.5,
16.9 and 17.3 Hz, ϭ CH); 6.26 (1H, J ϭ 1.2, 16.9
and 17.3 Hz, ϭ CH(CO)); 7.31 (3H, m, Ph); 7.57
(2H, m, Ph). 13C NMR (125.76 MHz, CDCl3), δ
(ppm): 13.8 (CH3), 22.4 (CH2), 27.9 (CH2), 31.2
(CH2), 48.0 and 48.1 (CHNH), 55.4 and 55.5
(CH3O), 67.3 and 67.4 (CH2O), 84.4 and 84.8
(CPh), 124.5 (q, JCF ϭ 288.0 Hz), 126.9 (ϭ CH2),
127.8 (Ph), 128.5 (Ph), 129.7 (Ph), 130.6 (ϭ CH),
132.4 (Ph), 165.0 (C ϭ O, amide), 166.5 and 166.7
(C ϭ O, ester).
(2S,2′R)-2′-(acrylamido)-butyl-3,3,3-trifluoro-
2-methoxy-2-phenylpropanoate (2S,2′R)-4b
Commercial (R)-2-amino-1-butanol was treated as
described for the synthesis of N-(2-hydroxyalkyl)-
acrylamides 2a to 2d under standard conditions.The
resulting acrylamide (R)-2b was treated as described
for the synthesis of MTPA derivatives.
Optical rotation measurements of 2a–2d showed
that the lipase-catalyzed preparation of N-(2-hydro-
xyalkyl)-acrylamides was stereoselective. We also

92 L. N. Monsalve et al.
Table I. Enzyme-catalyzed synthesis, absolute configuration and
enantiomeric excess of 2a–2d.
has been extensively studied. Kazlauskas’ rule is
intended to predict the behavior of lipases in such
cases and, according to this rule, lipases tend to
hydrolyze esters of chiral secondary alcohols having
absolute configuration (R) faster than their
enantiomer (Kazlauskas et al. 1991). Lipases also
show the same stereochemical preference in trans-
esterification and aminolysis reactions (Puertas
et al. 1993). This model takes into consideration
that the substituent priority can be correlated to
substituent size, although this is not always the case.
The enormous diversity of substrates accepted by
lipases and reaction conditions applied showed that
this rule is not met in some circumstances
(González-Sabín et al. 2005; Xia et al. 2009).
Therefore, we considered it important to investi-
gate the stereoselective behavior of CALB in the
aminolysis reaction carried out in this work and to
determine whether the results could be correlated
with Kazlauskas’ rule.
MTPA
amido ester
% ee
2a–2da
Product
R
% yield
42
2a
CH3
CH3
32% (S)
0
(2R,2′S)-3a
(2S,2′S)-4a
–
2ab
2b
2c
83
60
65
72
–
CH3CH2–
35% (S)
45% (S)
67% (S)
(2R,2′S)-3b
(2S,2′S)-4b
(2R,2′S)-3c
(2S,2′S)-4c
(2R,2′S)-3d
(2S,2′S)-4d
CH3(CH2)2–
CH3(CH2)3–
2d
Reactions were performed under standard conditions, time: 12 h.
aDetermined from the integration of 1H NMR signals for H1′a
and H1′b from both (R)- and (S)-MTPA amido esters.
b
Performed under microwave irradiation, time: 1 h
performed the preparation of 2a under microwave
irradiation, which resulted in a dramatic decrease in
reaction time and an increase in product yield but
the complete loss of stereoselectivity.Without lipase,
under microwave irradiation, the substrate did not
react at all.
With this in mind, we prepared the (R)- and (S)-
MTPA esters of the products 2a–2d in order to
assign their absolute stereochemistry and the degree
of stereoselectivity achieved in each case (Scheme 2).
The derivatives were obtained in good yield (58–75%)
by treatment of each hydroxyalkylacrylamide 2a–2d
with MTPA chloride in triethylamine.
The stereoselective behavior of lipases during
hydrolysis of esters of chiral secondary alcohols
Scheme 2. MTPA esters (2R,2′S)-3a–d and (2S,2′S)-4a–d.

Biocatalytic synthesis of chiral N-(2hydroxyalkyl)-acrylamides 93
(2R,2'S)
their oxymethylene proton signals than those pre-
pared from (S)-primary alcohols.
A
For the (R)-MTPA derivatives of chiral N-(2-
hydroxyalkyl)-acrylamides reported here, we found
Δδ ϭ 0.01 ppm for the major isomer and Δδ ϭ 0.11
ppm for the minor isomer. This indicates that the
absolute stereochemistry of the major isomer is
(2R,2’S) and therefore that (S) seems to be the abso-
lute configuration of the products 2a–2d.
4.48 4.46 4.44 4.42 4.40 4.38 4.36 4.34 4.32
B
(2S,2'S)
(2S,2'S)
However, as this result was difficult to rationalize
in terms of Kazlauskas’ rule, which predicted an (R)-
configuration, we sought additional confirmation.
Therefore the stereochemical outcome of the enzy-
matic aminolysis of ethyl acrylate may need to be
re-examined. Previous studies on the enzyme-
catalyzed hydrolysis and transesterification of esters
(Francalanci et al. 1987) and hydrolysis of amide
(Fadnavis et al. 1999) derivatives of such compounds
showed enantioselectivity towards (R)- or (S)-
enantiomers depending on the catalyst and the reac-
tion conditions. For instance, Carrea and co-workers
(1993) studied the hydrolysis of a series of butyrates
of racemic primary alcohols and found that Pseudo-
monas cepacia lipase showed the same stereochemical
behavior as observed in the present work, producing
the (S)-alcohol.
In order to support our findings, we also prepared
(2S,2′R)-2′-(acrylamido)-butyl-3,3,3-trifluoro-
2-methoxy-2-phenylpropanoate, (2S,2′R)-4b. First,
we used as substrate the pure enantiomer (R)-2-
amino-1-butanol in the enzymatic synthesis of
hydroxyalkylacrylamide (R)-2b and then (R)-2b was
treated with MTPA chloride following the previously
described procedure. 1H NMR analysis of (2S,2′R)-
4b showed the oxymethylene proton signals at
δ ϭ 4.36 and 4.37 ppm, which confirms our stereo-
chemical assignment based on the previous results
employing MTPA esters from β-disubstituted chiral
primary alcohols.
Thus, these results do not accord with Kazlaus-
kas’ rule, which is usually reliable with substrates
having substituents which differ significantly in size.
In the acylation reaction of every chiral alkanolamine
used in this work the enzyme showed an enantiose-
lective behavior opposite to that described by Kazlaus-
kas’ rule, selectively giving the product from reaction
of alkanolamine with (S) configuration instead of
the (R) predicted by the rule. In 1a this could be
explained considering that the hydroxymethyl group
is clearly larger than the methyl group, but in 1b
ethyl and hydroxymethyl substituents are about the
same size and compounds 1c and 1d have larger alkyl
substituent than their hydroxyalkyl substituent.
The opposite configuration observed between
the experimental results and that predicted by the rule
is possibly due to electronic effects (i.e. substituents
4.48 4.46 4.44 4.42 4.40 4.38 4.36 4.34 4.32
Figure 1.1H NMR signals for diastereotopic protons H1′a and
H1′b in the (R)-MTPA amido ester (2R,2′S)-3b (spectrum A)
and the (S)-MTPA amido ester (2S,2′S)-4b (spectrum B).
After purification, the MTPA esters were ana-
lyzed by 1H NMR spectroscopy in CDCl3. The
results observed for compounds (2R,2′S)-3b and
(2S,2′S)-4b were taken as examples and showed sig-
nificant differences in the chemical shift for many
signals. Particularly, signals for the diastereotopic
protons H-1’a and H-1’b of the (R)-MTPA ami-
doester (2R,2′S)-3b for the major isomer were at
δ ϭ 4.36 and 4.37 ppm and for the minor isomer at
δ ϭ 4.33 and 4.44 ppm (Figure 1, spectrum A). For
the (S)-MTPA ester (2S,2′S)-4b these signals were
reversed, so that those corresponding to the major
isomer are observed at δ ϭ 4.33 and 4.44 ppm and
the signals for the minor isomer at δ ϭ 4.36 and 4.37
ppm (Figure 1, spectrum B). The integration of the
above mentioned signals in both cases gave the same
35% ee value for 2b.
The same procedure was applied to study the
enantiomeric purity of the other three products 2a,
2c and 2d. Table 1 (columns 4 and 5) shows these
results.
This pattern for the oxymethylene protons of chi-
ral primary alcohols esterified with MTPA agrees
with previously published results on the determina-
tion of the absolute stereochemistry of a series of
chiral primary alcohols with a methyl group at the
C-2 position (Akiyama et al. 2003) and chiral
1,3-dihydroxyketones (Galman & Hailes 2009). In
these studies the absolute configurations of stereo-
genic centers were assigned by considering the Δδ
between oxymethylene protons. Larger Δδ values of
the (R)-MTPA derivative were diagnostic for R
stereochemistry on the carbon vicinal to oxymethyl-
ene whereas smaller Δδ values of the (R)-MTPA
derivative were diagnostic for the opposite stereo-
chemistry (S). Accordingly, the (S)-MTPA derivatives
of (R)-primary alcohols showed smaller Δδ values for

94 L. N. Monsalve et al.
having a remarkably different polarity) rather than
to a difference in substituent size. Therefore the
chemo- and enantioselectivity of the lipase-catalyzed
N-acylation of chiral alkanolamines may be driven
by the presence of the hydroxyl group, which seems
to accommodate preferably in the less hindered side
of the alcohol-binding site of the lipase. According
to recent research on the enantioselectivity of CALB
(Juhl et al. 2009) the residues that determine the
stereoselectivity of the reaction are larger and
hydrophobic at one side of Ser105 (Trp104, Ile189)
and smaller and more polar at the opposite side
(Gln106, Gly39, Thr40). This would accord with
our results if we assume that the hydroxyl group of
alkanolamines 1a–1d tends to accommodate closer
to the most polar region of the binding site prior to
aminolysis.
support. The authors report no conflicts of interest.
The authors alone are responsible for the content
and writing of the paper.
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