Candida antarctica lipases acting as versatile catalysts for the synthesis of enantiopure (R)- and (S)-1-(2-phenylthiazol-4-yl)ethanamines This paper is dedicated to the anniversary of 95th birthday of Professor Valer Fǎrcǎ?anu.

Article abstract of DOI:10.1016/j.molcatb.2014.05.007

The synthesis of both enantiomers of four new phenylthiazole-based amines by enantiomer-selective acylation of racemic amines and by hydrolysis of the corresponding racemic amides using lipase B from Candida antarctica (Novozyme 435) as chiral catalyst was performed with good yields and excellent enantioselectivities. In order to prevent the frequently occurring partial racemization of enantiopure amides during chemical hydrolysis to the corresponding (R)-amines, the deprotection of the N-acylated (R)-enantiomers by mild enzymatic hydrolysis with lipase A from C. antarctica immobilized on Celite was also developed.

Full text of DOI:10.1016/j.molcatb.2014.05.007

Accepted Manuscript
Title: Candida antarctica lipases acting as versatile catalysts
for the synthesis of enantiopure (R)- and
(S)-1-(2-phenylthiazol-4-yl)ethanamines
˘ ˘
˘
Author: Alexandra Radu Madalina Elena Moisa Monica
Ioana Tos¸a Norbert Dima Valentin Zaharia Florin Dan Irimie
PII:
S1381-1177(14)00157-X
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2014.05.007
MOLCAB 2955
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
4-3-2014
30-4-2014
15-5-2014
˘
Please cite this article as: A. Radu, M.E. Moisa, M.I. Tos¸a, N. Dima, V. Zaharia,
F.D. Irimie, Candida antarctica lipases acting as versatile catalysts for the synthesis of
enantiopure (R)- and (S)-1-(2-phenylthiazol-4-yl)ethanamines, Journal of Molecular
Catalysis B: Enzymatic (2014), http://dx.doi.org/10.1016/j.molcatb.2014.05.007
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1
Graphical Abstract
Candida antarctica lipases acting as versatile catalysts for the synthesis of enantiopure (R)‐ and
(S)‐1‐(2‐phenylthiazol‐4‐yl)ethanamines
Alexandra Radua, Mădălina Elena Moisăa, Monica Ioana Toşaa, Norbert Artur Dimaa,
Valentin Zahariab, Florin Dan Irimiea
aBabeş-Bolyai University of Cluj-Napoca, Department of Biochemistry and Biochemical
Engineering, Ro-400028 Cluj-Napoca, Arany János 11, Romania
bIuliu Haţieganu University of Medicine and Pharmacy, Department of Organic Chemistry,
Ro-400012 Cluj-Napoca, Victor Babeş 41, Romania
O
R
CaL-B
n-Pr-COOEt
H2N
HN
H2N
+
N
N
N
S
S
S
R
R
( )
R
R
acetonitrile
(S)
CaL-A
water
O
O
N
CaL-B
water
HN
H2N
HN
+
S
R
R
R
Cl
(S)
(R)
Page 1 of 19

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Candida antarctica lipases acting as versatile catalysts for the synthesis of enantiopure
(R)- and (S)-1-(2-phenylthiazol-4-yl)ethanamines
Alexandra Radua, Mădălina Elena Moisăa, Monica Ioana Toşaa, Norbert Dimaa, Valentin
Zahariab, Florin Dan Irimiea
aBabeş-Bolyai University of Cluj-Napoca, Department of Biochemistry and Biochemical
Engineering, Ro-400028 Cluj-Napoca, Arany János 11, Romania
bIuliu Haţieganu University of Medicine and Pharmacy, Department of Organic Chemistry,
Ro-400012 Cluj-Napoca, Victor Babeş 41, Romania
This paper is dedicated to the anniversary of 95th birthday of Professor Valer Fărcășanu
Keywords
phenylthiazole, chiral ethanamines, enzymatic kinetic resolution, lipases, amide hydrolysis
Abstract
The synthesis of both enantiomers of four new phenylthiazole-based amines by enantiomer-
selective acylation of racemic amines and by hydrolysis of the corresponding racemic amides
using lipase B from Candida antarctica (Novozyme 435) as chiral catalyst was performed
with good yields and excellent enantioselectivities. In order to prevent the frequently
occurring partial racemization of enantiopure amides during chemical hydrolysis to the
corresponding (R)-amines, the deprotection of the N-acylated (R)-enantiomers by mild
enzymatic hydrolysis with lipase A from Candida antarctica immobilised on Celite was also
developed.
1. Introduction
Enantiopure amines and their derivatives are important chiral building blocks and
final products especially in the agrochemical, food, pharmaceutical and fine chemical
industries. In particular, arylethanamines present valuable industrial applications in the
synthesis of pesticides [1].
Heterocyclic ring systems, including thiazole as a well known integral part of all
available penicillins, are found as key active component of many drugs, as recently pointed
[2]. For instance, 2-phenyl-thiazole derivatives were obtained and a detailed study of their
selective antiflaviviral activity was realized [3].
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With increasing demand for this type of compounds and interest in environmental
aspects, research in this field has led to efforts to establish viable synthethic methods that
meet the principles of green chemistry. In this regard, biocatalytic approaches using enzymes
as chiral catalysts, especially in organic solvents, are extensively applied.
Several chemocatalytic methods for the preparation of various enantiomerically
enriched arylethanamines are described in literature. One of the most common methods for
the enantioselective synthesis of amines is the Ru- or Rh-catalysed reductive amination of
carbonyl compounds [4]. Another procedure uses a microwave technique to produce the (S)-
enantiomer of some heteroaryl-ethanamines from the corresponding N-protected α-L-amino
acids [5]. Rhodium-catalyzed hydrogenation of N-sulfonyl ketimine [6] is also described for
the asymmetric synthesis of ethanamines. In addition to these, Sigamide-catalyzed
enantioselective reduction of ketimines with trichlorosilane also produced selectively the (S)-
enantiomer of various ethanamines [7].
All these chemical methods require expensive reagents, hard work-up and are time
consuming. In contrast, the enzymatic approaches are easier to handle, don’t need special
reaction conditions and most importantly, they are “eco-friendly”. Biocatalytic approaches
for obtaining enantiomerically enriched ethanamines commonly use hydrolytic enzymes or
transaminases. Lipases were succesfully used in the kinetic resolution of amines through N-
acylation reactions [1,8]. Hoff and co. reported [9] the synthesis of some chiral N-benzyl-N-
methyl-1-(naphthalen-1-yl)ethanamines and their in vitro antifungal activity using in the key
step, the enzymatic kinetic resolution of the racemic 1-(naphthalen-1-yl)ethanamine,
immobilized lipase B from Candida antarctica (commercialized as Novozyme 435) as
catalyst, ethyl 2-methoxyacetate as acyl donor and hexane as solvent at 60 °C with excellent
yield and enantioselectivity (E > 200). A detailed study [10] regarding the kinetic resolution
of three racemic ethanamines by acetylation using variously immobilized Candida antarctica
lipase B indicates that the temperature effect on the selectivity and activity strongly depends
on the substrate structure and on the used immobilization method. The CaL-B-catalyzed
acylation of various amines and amino-alcohols with myristic acid as acyl donor was also
investigated with the aim to explain and control the chemoselectivity of this enzyme, in the
specific acylation of bifunctional substrates [11]. The same enzyme was used in the resolution
of racemic 1-aryl-allylamines with moderate to excellent enantioselectivity (ee 63.5-99.9%)
[12a]. Caffeic acid amides in high enantiopuritiy (ee 98.5%) were obtained by Novozyme
435-catalyzed aminolysis of racemic ester in isooctane at 70 °C [12b]. Recently, a continuous
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flow process for the kinetic resolution of rac-1-phenylethylamine using ethyl acetate as acyl
donor and Novozyme 435 as catalyst with high enantiomeric ratios (E > 200) and short
residence time (40 minutes) at 70 °C was reported [13].
A series of primary amine enantiomers were obtained using ω-transaminase from
Vibrio fluvialis [14], Arthrobacter sp. sol-gel immobilized [15a] or with E. coli whole cell
with ω-transaminase activity [15b].
One of the most frequently used biocatalytic methods is the enzymatic kinetic
resolution of a racemate with an adequate hydrolytic enzyme. Enantioselectivity, high
stability and commercial availability in both free and immobilized forms have made lipases
(EC 3.1.1.3) attractive kinetic resolution catalysts either through the acylation of a
nucleophylic functionality (amine or an alcohol) or through the deacylation (hydrolysis,
alcoholysis or interesterification) of an amidic or esteric group of the target molecule.
Our aim has been to prepare both enantiomers of four phenylthiazole-based
ethanamines in a highly enantiomerically enriched form by a chemoenzymatic pathway
(Scheme 1). Using the immobilized lipase B from Candida antarctica (Novozyme 435) in
the enzymatic kinetic resolution of racemic ethanamines rac-4a-d through N-acylation and of
racemic ethanamides rac-5a-d through hydrolysis, a new enzymatic procedure was
developed.
2. Experimental Section
2.1. Materials and Methods
All reagents were purchased from Aldrich or Fluka and used as received. Solvents and
acyl donors for enzymatic reactions were stored over molecular sieves unless otherwise
stated. Lipase from Pseudomonas fluorescens (AK) was obtained from Amano, England,
while lipase from Candida rugosa (CrL) was purchased from Fluka. Lipase A from Candida
antarctica free or immobilized by adsorption on Celite (CaL-A) was a gift from Professor
Liisa T. Kanerva, University of Turku. Immobilized lipase B from Candida antarctica (CaL-
B, Novozyme 435) was purchased from Novozyme, Denmark and free CaL-B from Chiral
Vision, Netherlands. Lipase PS from Burkholderia cepacia (Pseudomonas cepacia) was
purchased from Amano, Aldrich. The enzymatic reactions were performed at 23 °C (room
temperature) or at 45 °C.
The 1H and 13C NMR spectra were recorded on Bruker spectrometers operating at 300
or 400 MHz and 75 or 100 MHz, respectively, at 25 ºC. Data for mass spectra analysis (ESI+-
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MS) were taken on a VG 7070E mass spectrometer operating at 70 eV. HRMS data were
measured in ESI+ mode with Bruker Avance microOTOF-Qquadrupole-TOF spectrometer.
Thin layer chromatography (TLC) was performed on Merck Kieselgel 60F254 sheets.
Spots were visualized by treatment with 5% ethanolic phosphomolybdic acid solution and
heating. Melting points were determined by hot plate method and are uncorrected. The
optical rotations were determined with Bellingham-Stanley ADP 220 polarimeter, using
chloroform for 5a-d and methanol for 4a-d (c = 1mg/mL) as solvents; [α]D25 values are given
in units of 10-1deg×cm2×g-1.
The enantiomeric separation of rac-4a-d and rac-5a-d was performed by High
Performance Liquid Chromatography (HPLC) conducted with Agilent 1200 and 1260
systems. Therefore the separation of the racemic ethanamides was accomplished on Chiralcel
OJ-H column (4.6 × 250 mm) using n-hexane: 2-propanol in different proportions at a flow
rate of 1.0 mL/min as mobile phase. The enantiomeric separation of the racemic ethanamines
rac-4a,b was performed on a Chiralpak ZWIX(+) column (4.0 × 250 mm) using acetonitrile:
methanol (50 mM formic acid, 25 mM DEA): H2O (78:28:2, v/v/v) at 1.0 mL/min flow rate
as mobile phase, while the separation of rac-4c,d on Chiralcel OJ-H column was obtained
with n-hexane: 2-propanol (0.02% DEA) as eluent at 0.9 mL/min flow rate. Table 1 contains
data for the HPLC chromatographic separation of the racemic derivatives.
The determination of the E value was based on the equation E = ln [(1-c)(1-eeS)]
/ln[(1-c)(1+eeS)], with c = eeS/(eeS+eeP) using linear regression (E as the slope of the line
ln[(1-c)(1-eeS)] versus ln[(1-c)(1+eeS)]) [16].
2.2. Chemical synthesis
2.2.1. Synthesis of rac-2a-d.
One of the aldehydes 1a-d (1 mmol) dissolved in diethyl ether (5 mL) was added into
the ice-cooled solution of the Grignard reagent prepared by treating CH3I (1.2 equiv.) with
magnesium (1.2 equiv.) and I2 (a crystal) in dry diethyl ether at reflux under argon
atmosphere until the magnesium was consumed. The mixture was refluxed overnight. After
cooling, saturated ammonium chloride solution was added dropwise under vigorous stirring
on an ice bath. The separated organic layer was next extracted with CH2Cl2 (3 × 5 mL), dried
on anhydrous Na2SO4 and evaporated in vacuum. The crude product was purified by column
chromatography using CH2Cl2: CH3OH (9:1, v/v) as eluent, affording the pure racemic
alcohols rac-2a-d as yellow semi-solids with 57-70% yields.
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2.2.2. Synthesis of racemic azides (rac-3a-d)
Diphenyl phosphonic azide (1.5 equiv.) was added to a solution of rac-2a-d (1 mmol)
dissolved in dry degassed toluene (2 mL). The mixture was cooled at 0 °C and neat DBU (1.5
equiv.) was added under argon atmosphere. The reaction was stirred for 3 h at 0 °C, then
slowly warmed to room temperature and the reaction mixture was stirred for an additional 16
h. The resulting two-phase mixture was washed with water (2 × 20 mL) and 5% HCl solution.
(20 mL). The organic layer, dried over anhydrous Na2SO4, was further concentrated in
vacuum and purified on silica gel with CH2Cl2 as eluent providing the desired azides rac-3a-
d as yellow liquids with 65-95% yield.
2.2.3. Synthesis of racemic amines (rac-4a-d)
The reduction of racemic azides rac-3a-d to amines rac-4a-d was performed
according to a procedure described in literature [17]. To the solution of azides (1 mmol) and
ammonium chloride (2.6 equiv.) in ethyl alcohol (27 mL) and water (9 mL), activated zinc
powder (1.3 equiv.) was added. The mixture was stirred vigorously at reflux and after
approximately 4 h all the reactions were completed (monitored by TLC). Finally, ethyl
acetate and aqueous ammonia (25%) were added and the mixture was filtered to remove the
unreacted zinc. The isolated organic layer was dried over anhydrous Na2SO4, the solvent was
removed under reduced pressure and the residue was purified on silica gel using sequentially
two eluents: firstly, CH2Cl2: CH3OH (9:1, v/v) was used to recover the unreacted azide and
then CH2Cl2: CH3OH (8:2, v/v) was used to elute the racemic amines. The final products rac-
4a-d were obtained as yellow semi-solids with 90-97% yields.
2.2.3.1. 1-(2-phenylthiazol-4-yl)ethanamine (rac-4a). Yield 97%; yellow semi-solid;
1H NMR (300 MHz, CDCl3, 25 °C): δ = 1.51 (d, J = 7 Hz, 3H); 1.91 (broad signal); 4.27 (q, J
= 7 Hz, 1H); 7.04 (s, 1H); 7.43 (m, 3H); 7.93-7.96 (m, 2H) ppm. 13C NMR (75 MHz, CDCl3,
25 °C): δ = 23.52; 48.15; 111.90; 125.68; 129.02; 130.05; 133.89; 163.31; 168.31 ppm.
HRMS: M+ found (M+ calculated for C11H12N2S): 204.0729 (204.0721); ESI+-MS: m/z (%):
204 (M, 12); 203 (M-1, 7); 189 (100); 85 (15); 77 (19); 58 (19); 43 (49); 27 (12).
2.2.3.2. 1-(2-m-tolylthiazol-4-yl)ethanamine (rac-4b). Yield 94%; yellow semi-solid;
1H NMR (300 MHz, DMSO-d6, 25 °C): δ = 1.49 (d, J = 7 Hz, 3H); 1.84 (broad signal); 2.38
(s, 3H); 4.37 (q, J = 7 Hz, 1H); 7.29-7.42 (m, 2H); 7.62 (s, 1H); 7.73-7.78 (m, 2H) ppm. 13C
NMR (75 MHz, DMSO-d6, 25 °C): δ = 21.26; 22.41; 47.33; 113.9; 123.40; 126.51; 129.19;
130.94; 133.14; 138.64; 159.02; 166.98 ppm. HRMS: M+ found (M+ calculated for
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C12H14N2S): 218.0893 (218.0878); ESI+-MS: m/z (%): 219 (M+1, 4); 218 (M, 6); 203 (100);
86 (9); 57 (12); 43 (17); 28 (20).
2.2.3.3. 1-(2-p-tolylthiazol-4-yl)ethanamine (rac-4c). Yield 93%; yellow semi-solid;
1H NMR (300 MHz, DMSO-d6, 25 °C): δ = 1.42 (d, J = 7 Hz, 3H); 1.89 (broad signal); 2.35
(s, 3H); 4.23 (q, J = 7 Hz, 1H); 7.31 (d, J = 9, 2H); 7.48 (s, 1H); 7.83 (d, J = 9, 2H) ppm. 13C
NMR (75 MHz, DMSO-d6, 25 °C): δ = 21.17; 22.37; 54.96; 113.73; 126.06; 130.17; 130.55;
140.06; 161.37; 167.00 ppm. HRMS: M+ found (M+ calculated for C12H14N2S): 218.0908
(218.0878); ESI+-MS: m/z (%): 218 (M, 19); 203 (100); 176 (17); 159 (17); 118 (21); 101
(16); 85 (24); 57 (20); 43 (47); 27 (7).
2.2.3.4. 1-(2-(4-chlorophenyl)thiazol-4-yl)ethanamine (rac-4d). Yield 90%; yellow semi-
1
solid; H NMR (400 MHz, CD3OD, 25 °C): δ = 1.49 (d, J = 7 Hz, 3H); 4.22 (q, J = 7 Hz,
1H); 7.32 (s, 1H); 7.41 (d, J = 6 Hz, 2H); 7.88 (d, J = 9 Hz, 2H) ppm. 13C NMR (100 MHz,
CD3OD, 25 °C): δ = 22.76; 48.69; 114.71; 128.86; 130.24; 133.47; 137.04; 163.17; 168.27
ppm. HRMS: M+ found (M+ calculated for C11H11ClN2S): 238.0356 (238.0331); ESI+-MS:
m/z (%): 239 (M,7); 226 (37Cl, 13); 225 (35Cl, 100); 224 (37Cl, 35); 222 (35Cl, 8); 196 (34);
179 (38); 111 (32); 85 (12); 45 (54); 28 (28).
2.2.4. Synthesis of racemic amides (rac-5a-d)
DMAP (1%) in pyridine (4 equiv.) and the acylating agent (4 equiv.) were added into
the solution of rac-4a-d (1 mmol) in dry CH2Cl2 (15 mL). The mixture was stirred at room
temperature for 3 h and then quenched with water (15 mL). The isolated organic layer was
dried over anhydrous Na2SO4, the solvent evaporated in vacuum and the crude product was
purified by column chromatography on silica gel with CH2Cl2: CH3OH (9.5:0.5, v/v) as
eluent giving rac-5a-d as yellow semi-solids with 82-90% yields.
2.2.4.1. N-(1-(2-phenylthiazol-4-yl)ethyl)butyramide (rac-5a). Yield 90%; yellow semi-solid;
1H NMR (300 MHz, CDCl3, 25 °C): δ = 0.95 (t, J = 7.5 Hz, 3H); 1.55 (d, J = 6 Hz, 3H);
1.62-1.75 (m, 2H); 2.19 (t, J = 7.5 Hz, 2H); 5.30 (dq, app. quin, J = 6 Hz, 6 Hz, 1H); 6.23
(bd, J = 6 Hz, 1H); 7.07 (s, 1H); 7.43-7.48 (m, 3H); 7.92-7.95 (d, J = 9 Hz, 2H) ppm. 13C
NMR (75 MHz, CDCl3, 25 °C): δ = 13.89; 19.29; 22.20; 38.95; 45.78; 113.86; 126.66;
129.08; 130.23; 158.56; 163.44; 169.60; 171.63 ppm. HRMS: M+ found (M+ calculated for
C15H18N2OS): 274.1136 (274.1140); ESI+-MS: m/z (%): 274 (M, 9); 203 (100); 186 (13); 104
(9); 85 (8); 43 (18); 27 (9).
2.2.4.2. N-(1-(2-m-tolylthiazol-4-yl)ethyl)butyramide (rac-5b). Yield 87%; yellow semi-solid;
1H NMR (300 MHz, CDCl3, 25 °C): δ = 0.94 (t, J = 7.5 Hz, 3H); 1.55 (d, J = 6 Hz, 3H);
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1.62-1.74 (m, 2H); 2.19 (t, J = 7.5 Hz, 2H); 2.42 (s, 3H); 5.27 (dq. app. quin, J = 9 Hz, 6 Hz,
1H); 6.28 (bd, J = 9 Hz, 1H); 7.06 (s, 1H); 7.22-7.32 (m, 2H); 7.70-7.75 (m, 2H) ppm. 13C
NMR (75 MHz, CDCl3, 25 °C): δ = 13.87; 19.26; 21.50; 22.14; 38.90; 45.78; 113.88; 123.88;
127.17; 128.96; 131.03; 133.61; 138.83; 158.57; 168.94; 172.22 ppm. HRMS: M+ found (M+
calculated for C16H20N2OS): 288.1322 (288.1296); ESI+-MS: m/z (%): 288 (M, 12); 218 (16);
217 (100); 200 (14); 118 (8); 85 (13); 43 (26); 41 (14); 27 (8).
2.2.4.3. N-(1-(2-p-tolylthiazol-4-yl)ethyl)butyramide (rac-5c). Yield 84%; yellow semi-solid;
1H NMR (300 MHz, CDCl3, 25 °C): δ = 0.94 (t, J = 7.5Hz, 3H); 1.54 (d, J = 6 Hz, 3H); 1.62-
1.74 (m, 2H); 2.19 (t, J = 7.5 Hz, 2H); 2.39 (s, 3H); 5.26 (dq, app. quin, J = 6 Hz, 6 Hz, 1H);
6.25 (bd, J = 6 Hz, 1H); 7.03 (s, 1H); 7.24 (d, J = 6 Hz, 2H); 7.82 (d, J = 6 Hz, 2H) ppm. 13C
NMR (75 MHz, CDCl3, 25 °C): δ = 13.88; 19.28; 21.57; 22.19; 38.94; 45.80; 113.34; 126.57;
129.73; 131.10; 140.47; 158.47; 168.31; 172.2 ppm. HRMS: M+ found (M+ calculated for
C16H20N2OS): 288.1314 (288.1296); ESI+-MS: m/z (%): 289 (M+1, 2); 288 (M, 12); 218 (16);
217 (100); 200 (12); 118 (8); 85 (7); 43 (26); 41 (14); 27 (8).
2.2.4.4. N-(1-(2-(4-chlorophenyl)thiazol-4-yl)ethyl)butyramide (rac-5d). Yield 82%; yellow
1
semi-solid; H NMR (300 MHz, CDCl3, 25 °C): δ = 0.95 (t, J = 7.5 Hz, 3H); 1.55 (d, J =
9Hz, 3H); 1.75-1.65 (m, 2H); 2.19 (t, J = 9 Hz, 2H); 5.27 (dq, app. quin, J = 9 Hz, 6 Hz, 1H);
6.16 (bd, J = 9 Hz, 1H); 7.09 (s, 1H); 7.41 (d, J = 6 Hz, 2H); 7.88 (d, J = 6 Hz, 2H) ppm. 13C
NMR (75 MHz, CDCl3, 25 °C): δ = 13.89; 19.28; 22.13; 38.94; 45.73; 114.19; 127.86;
129.30; 132.2; 158.34; 169.98; 177.2 ppm. HRMS: M+ found (M+ calculated C15H17ClN2OS),
308.0764 (308.0750); ESI+-MS: m/z (%): 308 (M, 9); 239 (37Cl, 40); 237 (35Cl, 100); 222
(37Cl, 13); 220 (35Cl, 12); 85 (37Cl, 7); 83 (35Cl, 5); 43 (37Cl, 15); 41 (35Cl 7); 27 (3).
2.3. Enzymatic synthesis
2.3.1. Analytical scale N-acylation
Novozyme 435 (12.5 mg) and 3-4 pieces of molecular sieves (4 Å) were weighed in a
reaction vial before rac-4a-d (12.5 μmol) in dry organic solvent (0.5 mL) and the acylation
agent (50 μmol) were added. Reactions proceeded under shaking (1350 rpm) at 23 °C and
were monitored by TLC and HPLC analysis. After approximately 16 h the reactions were
considered to be completed.
2.3.2. Analytical scale hydrolysis
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10 mg of a CaL-B or CaL-A preparation were weighed in a reaction vial before rac-
5a-d or (R)-5a-d (7.3 μmol) were added in water (1 mL). The mixtures were shaken at r. t. or
at 45 °C. The progress of the reactions was monitored by TLC and HPLC analysis.
2.3.3. Preparative scale N-acylation
The N-acylation of racemic amines at preparative scale was performed using
substrate:biocatalyst ratio and reaction conditions as for the analytical scale.
Therefore, Novozyme 435 (1500 mg) and molecular sieves were weighed in a
reaction vial before rac-4a-d (1.5 mmol) and ethyl n-butyrate (6 mmol) were added in ACN
(20 mL). The mixtures were shaken at 1350 rpm at 23 °C for approximately 16 h to reach
50% conversion. The enzyme was filtered off, washed with ACN and the combined organic
phases were concentrated in vacuum. The crude product was purified by column
chromatography on silica gel with CH2Cl2: CH3OH (9.5:0.5, v/v) and CH2Cl2: CH3OH (8:2,
v/v) as eluent. The properties of obtained chiral derivatives are presented in Table 2, Part A.
2.3.4. Preparative scale hydrolysis
Novozyme 435 or CaL-A on Celite (1000 mg) were weighed in a reaction vial before
the aqueous solution of rac-5a-d or (R)-5a-d (20 mL, 0.73 mmol) was added. The mixtures
were shaken at 45 °C. The progress of the reactions was monitored by TLC and HPLC
analysis. When the reactions were completed, the reaction mixtures were freezed and the
water was removed by lyophilization. The products were dissolved in CH2Cl2 and the enzyme
was filtered off. Next, the solution was concentrated in vacuum and the crude product was
purified by column chromatography with CH2Cl2: CH3OH as eluent, with a gradient from
initial 5% (v/v) to 20% (v/v) to yield (R)-amines and (S)-amides with excellent enantiomeric
excesses at approximately 50% conversion or optically pure (R)-4a-d as yellow semi-solids
data presented in Table 2, Part B.
2.4. Absolute configuration
To a solution of rac-2a (1 mmol) in toluene (10 mL), vinyl acetate (4 mmol),
Novozyme 435 (40 mg/mL) and molecular sieves were added. The mixture was shaken at
1350 rpm at room temperature. The process was monitored by TLC and HPLC analysis. The
reaction was completed in approximately 6 hours. The enzyme was filtered off, the solvent
was removed in vacuum and the crude product was purified by column chromatography with
CH2Cl2: acetone (25:1, v/v) as eluent. The isolated (S)-2a was further transformed into the
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azide (R)-3a and next reduced to give (R)-4a following the procedures described above.
Yield: 87%.
3. Results and Discussion
3.1. Chemical synthesis
Based on the methods described in literature, the preparation of amines rac-4a-d from
the corresponding aldehydes 1a-d was first studied. The racemic ethanols rac-2a-d obtained
by Grignard reaction [18] were transfomed into racemic amines rac-4a-d via azide
derivatives rac-3a-d as shown in Scheme 1 (route a, steps I-III).
Racemic amides rac-5a-d were obtained by chemical acylation of rac-4a-d with
butyryl chloride in dichloromethane, in the presence of pyridine and a catalytic amount of
DMAP (Scheme 1, route a, step IV).
3.2. Enzymatic synthesis
In order to investigate the stereoselectivity of the reactions involving chiral 1-(2-
phenylthiazol-4-yl)ethanamines rac-4a–d and their amides rac-5a-d, the chromatographic
separation of the enantiomers was first established. The base-line separation of all the
enantiomers of rac-4,5a-d was performed using appropriate HPLC chiral columns. The
chromatographic enantiomer separation of rac-4c,d was successfully performed when small
amounts of diethylamine (DEA) [19] was added to the mobile phase (Table 1).
3.2.3. Analytical scale enzymatic N-acylation of rac-4a-d
Common commercial lipase preparations [25 mg/mL; lipases from Candida rugosa
(CrL), Pseudomonas fluorescens (AK), lipase A from Candida antarctica immobilized by
adsorption on Celite (CaL-A), immobilized lipase B from Candida antarctica (CaL-B,
Novozyme 435) and Amano lipase PS from Burkholderia cepacia (Pseudomonas cepacia)]
were screened for the N-acylation of the model compound, rac-4a, at room temperature.
In order to find the optimal reaction conditions for the N-acylation of rac-4a the
analytical scale enzymatic reactions were conducted in various organic solvents using four
different acyl donors like ethyl acetate, isopropyl n-butyrate, ethyl n-butyrate, ethyl
propionate (4 equiv. each). The tested solvents, methyl tert-butyl ether (MTBE), acetonitrile
(ACN), dichloromethane, toluene and diisopropyl ether (DIPE), were selected as they are
frequently used in organic synthesis and industry and are known for their compatibility with
lipases. The addition of molecular sieve [4 Å; the ratio to the catalyst 1:1 (w/w)] to the
reaction medium was necessary since even small traces of water could promote lipase-
catalysed hydrolytic reactions. In this case, the hydrolysis of the amidic group could lead to
Page 10 of 19

11
the formation of an ammonium salt between the amine and the liberated acid, thus lowering
the enantioselectivity of the kinetic resolution.
Some lipases (CRL, PS-free and AK on Celite) proved to be catalytically inactive
even after 28 hours, while CaL-A showed either no activity or poor selectivity in MTBE with
isopropyl n-butyrate and ethyl n-butyrate.
The highest enantioselectivity (E » 200) and reactivity (c = 50%) for the CaL-B
mediated N-acylation of rac-4a were obtained with ethyl n-butyrate as acyl donor in dry ACN
as solvent after 16 hours (Table 3, entry 3).
3.2.4. Preparative scale enzymatic N-acylation of rac-4a-d
The developed small-scale kinetic resolution method of rac-4a was next used for the
preparative-scale kinetic resolution of all substrates rac-4a-d (1.5 mmol) with ethyl n-
butyrate (6 mmol) and Novozyme 435 (75 mg/mL) in dry ACN (Scheme 1, step V). The
highly enantioselective N-acylations of the reactive (R)-4a-c (E » 200) were all completed (c
~ 50%) in 16 h. Lower selectivity and activity of the enzyme was observed when the
chlorinated rac-4d was subjected to the same biotransformation (E = 125, c ~ 46% after 16
h).
The resolution products [the unreacted (S)-4a-d and the obtained (R)-5a-d] were
isolated at close to 50% theoretical yields (93-97% from the theoretical amounts at 50%
conversion) in highly enantiopure forms (ee 82-99%) (Table 2, Part A).
3.2.5. Kinetic resolution and deprotection of (R)-5a-d by lipase catalyzed hydrolysis
Recently, a very important feature that affords amide hydrolysis by serine proteases
was attributed to the hydrogen bond formed between the substrate’s amide nitrogen and the
enzyme or the substrate itself, thus facilitating the nitrogen inversion during the catalytic
process [20]. Even though lipases have the same catalytic triad, some of them lack this
particular hydrogen bond and, subsequently, the ability to efficiently cleave amides.
However, previous results showing that lipases CaL-A [21-22] and CaL-B [1, 22-23] can
hydrolyze amides in water, encouraged us to study the hydrolysis of the arylethanamides
using lipases as catalysts.
In order to obtain the opposite enantiomeric forms of the enantiomerically enriched 1-
(2-phenylthiazol-4-yl)ethanamines [the (S)-enantiomers], the lipase-mediated kinetic
resolution of rac-5a-d was investigated (Scheme 1, route b). To this extent, several CaL-A
and CaL-B preparations were tested for the selective hydrolysis of the model compound rac-
5a.
Page 11 of 19

12
Analytical scale hydrolytic reactions were performed in water at room temperature
and at 45 °C using four enzyme preparations (Table 4). It is worth mentioning that the
addition of any co-solvent was not necessary. As expected, the results show a greater reaction
rate for the reactions performed at 45 °C. All CaL-B-mediated hydrolysis were highly
enantioselective, the best reactivity was achieved with Novozyme 435 (Table 4, entry 1).
CaL-A (free or immobilized on Celite) proved to be a highly active but non-stereoselective
catalyst for the same transformations. On terms of reactivity, CaL-A on Celite gave the
highest reactivity at 45 °C (Table 4, entry 4).
Novozyme 435-catalysed preparative scale hydrolysis of rac-5a-d proceeded
enantioselectively and afforded the (R)-amines and (S)-amides with excellent enantiomeric
excesses at approximately 50% conversion. The enantioselective hydrolytic reactions were
completed in 80 hours for rac-5a,b and 90 hours for rac-5c,d (Table 2, Part B).
Nevertheless (R)-4a-d amines could be provided also by the deprotection of (R)-5a-d
amides. In our try to avoid the racemization and due to the instability of the heteroaryl ring
against traditional acid- and base-catalysed hydrolysis we used the CaL-A mediated
hydrolytic deprotection of (R)-5a-d amides. The CaL-A on Celite mediated preparative scale
hydrolytic reactions of (R)-5a-d were performed at 45 °C yielding quantitatively the
corresponding enantiomerically enriched (R)-4a-d amines in 60 hours (Table 2, Part B).
3.3. Determination of the absolute configuration
The absolute configuration of (R)-4a was determined by first preparing the
corresponding alcohol according to the previous method and resolving it with Novozyme 435
in neat vinyl acetate, yielding (S)-2a with 99% ee (Scheme 1, route c) [24]. The subsequent
transformations of (S)-2a to (R)-4a following the procedure described above did not affect
significantly the enantiopurity of the involved compounds. The optical rotation measured for
25
(R)-4a obtained in this way gave [α]D = +6.3 (c = 1.0, CH3OH) at 95% ee, which is in
accordance with the value [α]D25 = +6.4 (c = 1.0, CH3OH) at 99% ee obtained for the CaL-B-
catalyzed hydrolysis of rac-5a in water (Table 2, Part B). It is reasonable to assume that
Novozyme 435 manifests the same enantiopreference with the other substrates too in N-
acylation and hydrolytic reactions. The (R)-enantiopreference of Novozyme 435 shown
herein has proved to be in accordance with the enantiopreference observed before for the
enzyme in N-acylations of arylethanamines [8a, 22].
Page 12 of 19

13
4. Conclusions
An efficient new procedure for the synthesis of enantiomerically enriched (R)- and
(S)-1-(2-phenylthiazol-4-yl)ethanamines has been developed (ee 82-99%, E » 200 except the
chlorinated compound when E = 125). It was found that CaL-B (Novozyme 435) is the
proper catalyst for both enantioselective N-acylation and hydrolysis. The CaL-B-mediated
kinetic resolution of rac-4a-d through N-acylation occured best using ethyl n-butyrate as acyl
donor in acetonitrile as solvent at room temperature. The Novozyme 435-mediated hydrolysis
of rac-5a-d was optimal in water at 45 °C, with no need for additional co-solvents.
Deprotection of (R)-5a-d to the corresponding (R)-4a-d was achieved faster using CaL-A
immobilized on Celite, without affecting the optical purity.
Amide hydrolysis is generally uncommon to lipases which naturally hydrolyze fatty
acid glycerol esters but these studies have proven once more the versatility of this type of
enzymes. The amidase activity of lipases observed here is of great interest for organic
synthesis as it offers an alternative mild method for deprotection of amides or for resolving
racemic amides since chemical decomposition and racemization was avoided.
Acknowledgments
This work was supported by a grant of the Romanian National Authority for Scientific
Research, CNCS – UEFISCDI, project number PN-II-ID-PCE-2011-3-0775.
References
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[7] A.V. Malkov, K. Vranková, S. Stončius, P. Kočovský, J. Org. Chem. 74 (2009) 5839-
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Hietanen, T. Saloranta, R. Leino, L.T. Kanerva, Tetrahedron: Asymmetry 23 (2012)
1629-1632; c) A. Ghanem, Lipase-catalysed kinetic resolution of racemates: a versatile
method for the separation of enantiomers in: F. Toda (Ed.) Enantiomer separation.
Fundamental and Practical Methods, Kluver Academic Publishers, The Netherlands
2004, pp. 193-230; d) J.H. Sun, R.J. Dai, W.W. Meng, Y.L. Deng, Catal. Commun. 11
(2010) 987-991.
[9] T.H.K. Thvedt, K. Kaasa, E. Sundby, C. Charnock, B.H. Hoff, Eur. J. Med. Chem. 68
(2013) 482-496.
[10] Z. Boros, P. Falus, M. Márkus, D. Weiser, M. Oláh, G. Hornyánszky, J. Nagy, L. Poppe,
J. Mol. Catal. B: Enzymatic 85-86 (2013) 119-125.
[11] F. Le Joubioux, Y.B. Henda, N. Bridiau, O. Achour, M. Graber, T. Maugard, J. Mol.
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936-941; b) P. Xiao, S. Zhang, H. Ma, A. Zhang, X. Lv, L. Zheng, J. Biotechnol. 168
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[13] A.S. de Miranda, L.S.M. Miranda, R.O.M.A. de Souza, Org. Biomol. Chem. 11 (2013)
3332-3336.
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Simon, W. Kroutil, L.H. Andrade, Tetrahedron: Asymmetry 24 (2013) 1495–1501.
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[21] A. Liljeblad, P. Kallio, M. Vainio, J. Niemi, L.T. Kanerva, Org. Biomol. Chem. 8 (2010)
886-895.
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J. Org. Chem. 17 (2012) 3288-3294.
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Enzymatic 94 (2013) 88-94.Figure legends
O
HN
Pr
NH2
N3
NH2
OH
OH
II.
III.
V.
+
R
R
R
R
R
R
(S)-4a-d
(S)-2a
(R)-5a-d
rac-2a-d
rac-3a-d
rac-4a-d
II.
IV.
VII.
I.
b
c
a
N3
O
O
R
O
NH2
R
(R)-3a
VI.
HN
Pr
HN
Pr
R
+
R
R
1a-d
III.
5a-d
(S)-
-4a-d
(R)
rac-5a-d
NH2
N
S
N
N
R
(R)
N
R=
-4a
S
S
S
Cl
a
b
c
d
Scheme 1. Synthesis and biotransformations of the studied 1‐(2‐phenylthiazol‐4‐yl)ethanamines and
ethanacetamides. Reagents and conditions: I. CH3MgI, diethyl ether; II. (PhO)2PON3/toluene; III. Zn/NH4Cl,
H2O/ethanol; IV. CH3(CH2)2COCl/DMAP/Pyridine/DCM; V. CaL‐B/ ethyl n‐butyrate/ACN; VI. CaL‐B/H2O; VII. CaL‐
A/H2O.
Page 15 of 19

16
Research highlights
•
•
A new procedure for the synthesis of both enantiomerically enriched (R)- and (S)-1-
(2-phenylthiazol-4-yl)ethanamines, based on enzymatic kinetic resolution, was
developed.
The CaL-B-mediated kinetic resolution of racemic amines through N-acylation
occurred optimal using ethyl n-butyrate as acyl donor and acetonitrile as solvent at
room temperature.
•
•
The CaL-B-mediated hydrolysis of racemic amides was optimal in water at 45 °C,
with no need for additional co-solvents.
In order to avoid the chemical decomposition and/or racemization during the
deprotection of (R)-amides to the corresponding (R)-amines, CaL-A on Celite, a
versatile biocatalyst, was used.
Page 16 of 19

17
Table legends
Table 1. HPLC chromatographic separation of the racemic derivatives rac-4,5a-d.
Table 2. Preparative scale enzymatic N-acylation of rac-4a-d (Part A), enzymatic hydrolysis of rac-5a-d (Part
B).
Table 3. N-acylation of rac-4a with CaL-B at 23°C after 16 h.
Table 4. CaL-A and CaL-B-catalyzed analytical scale hydrolysis of rac-5a in water after 30 h.
Table 1. HPLC chromatographic separation of the racemic derivatives rac-4,5a-d.
Retention time (min)
Retention time (min)
Amine
Condition
Amide
Condition
tr (S)
tr (R)
tr (S)
6.1
6.6
5.8
7.5
tr (R)
7.9
7.9
7.8
8.7
4a
4b
4c
4d
70:28:2a
70:28:2a
80:20:2b
90:10:1b
25.3
24.4
9.2
26.7
26.8
10.0
11.2
5a
5b
5c
5d
85:15c
90:10c
85:15c
90:10c
10.7
a ratio of acetonitrile:methanol (FA+DEA):H2O (Chiralpak ZWIX (+), 1.0 mL/min flow rate)
b ratio of n-hexane:2-propanol:DEA (Chiralcel OJ-H, 0.9 mL/min flow rate)
c ratio of n-hexane:2-propanol (Chiralcel OJ-H, 1.0 mL/min flow rate)
Table 2. Preparative scale enzymatic N-acylation of rac-4a-d (Part A) and enzymatic hydrolysis of rac-5a-d
(Part B).
Preparative scale Novozyme 435
Preparative scale Novozyme 435
Part A
Part B
mediated N-acylation of rac-4a-db
mediated hydrolysis of rac-5a-dc
mp
(°C)
-
Yield ee
Yield ee
25
25
Compound
[α]D
E
Compound
[α]D
E
(%)a (%)
(%)a (%)
(S)-4a
(R)-5a
(S)-4b
(R)-5b
(S)-4c
(R)-5c
(S)-4d
(R)-5d
> 99
> 99
> 99
> 99
> 99
> 99
82
(R)-4a
(S)-5a
(R)-4b
(S)-5b
(R)-4c
(S)-5c
(R)-4d
(S)-5d
96
96
93
93
94
96
91
95
> 99
> 99
> 99
98
+6.4
97
97
95
95
93
96
93
93
-6.2
» 200
» 200
110±1
-176.4
+8.5
+175.9
-8.4
-
» 200
» 200
125
» 200
» 200
> 200
113±1
-164.8
+18.8
- 147.4
+10.8
-149.7
+164.2
-18.3
-
> 99
96
135±1
-
+148.8
-11.0
98
96
130±1
94
+151.2
a 50% of the racemates taken as 100% theoretical yields; b after 16h at 23 °; c in water at 45 °C after 80 and 90h
respectively.
Table 3. N-acylation of rac-4a with CaL-B at 23 °C after 16 h.
Entry
Acyl donor
Solvent
c (%)
E
1
isopropyl n-butyrate
toluene
40
» 200
ethyl n-butyrate
ethyl n-butyrate
toluene
ACN
44
50
» 200
» 200
2
3
Page 17 of 19

18
Table 4. CaL-A and CaL-B-catalyzed analytical scale hydrolysis of rac-5a in water after 30 h.
Entry
Enzyme
Temperature (°C) c (%)
ees (%)
eep (%)
E
23
45
23
45
23
45
23
45
28
45
39
44
14
31
55
75
37
80
64
78
3
> 99
> 99
> 99
> 99
18
> 200
> 200
>200
> 200
1
1
CaL-B (Novozyme 435)
2
3
4
CaL-B (Chiral Vision)
CaL-A free
9
20
2
5
4
1
CaL-A on Celite
18
6
1
Page 18 of 19

Research highlights
 A new synthesis of both enantiomers of 1-(2-phenylthiazol-4-yl)ethanamines, by
enzymatic kinetic resolution, was developed
 The CaL-B-mediated kinetic resolution of amines occurred optimal using ethyl n-
butyrate as acyl donor in acetonitrile
 The CaL-B-mediated hydrolysis of racemic amides was optimal in water at 45 °C,
with no need for additional co-solvents.
 To avoid the chemical decomposition and/or racemization during the deprotection
of (R)-amides, CaL-A on Celite was used.
Page 19 of 19