Heterocycles 32. Efficient kinetic resolution of 1-(2-arylthiazol-4-yl) ethanols and their acetates using lipase B from Candida antarctica

Article abstract of DOI:10.1016/j.molcatb.2013.05.005

In this paper we describe the chemoenzymatic synthesis of new enantiomerically enriched (R)- and (S)-1-(2-arylthiazol-4-yl)ethanols and their acetates by enzymatic enantioselective acetylation of the racemic alcohols rac-2a-d and by methanolysis of the corresponding racemic esters rac-3a-d mediated by lipase B from Candida antarctica (CaL-B) in non-aqueous media. In terms of stereoselectivity and activity, both procedures, acylation and alcoholysis, gave similar good results (50% conversion, E 〉 200). The absolute configuration of the kinetic resolution products was determined by a detailed ¹H NMR study of the Mosher's derivatives of (S)-2b.

Full text of DOI:10.1016/j.molcatb.2013.05.005

Accepted Manuscript
Title: Heterocycles 32. Efficient kinetic resolution of
1-(2-arylthiazol-4-yl)ethanols and their acetates using lipase B
from Candida antarctica
˘
¨
˘ ˘
˘
Author: Denisa Hapau Jurgen Brem Madalina Moisa
Monica-Ioana Tos¸a Florin Dan Irimie Valentin Zaharia
PII:
S1381-1177(13)00129-X
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2013.05.005
MOLCAB 2679
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
17-1-2013
26-4-2013
11-5-2013
˘
˘
Please cite this article as: D. Hapau, J. Brem, M. Moisa, M.-I. Tos¸a, F.D. Irimie, V.
Zaharia, Heterocycles 32. Efficient kinetic resolution of 1-(2-arylthiazol-4-yl)ethanols
and their acetates using lipase B from Candida antarctica, Journal of Molecular
Catalysis B: Enzymatic (2013), http://dx.doi.org/10.1016/j.molcatb.2013.05.005
This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.

Research highlights
• Kinetic resolution of 1-(2-arylthiazol-4-yl)ethanols and their acetates is described.
• Several lipases were screened as biocatalysts.
• CaL-B efficiently catalyzed both acetylation and methanolysis (ee>90%,
E>>200).
• The absolute configuration of the products was determined by the Mosher’s
method.
Page 1 of 22

Heterocycles 32. Efficient kinetic resolution of 1-(2-arylthiazol-4-yl)ethanols and
their acetates using lipase B from Candida antarctica
Denisa Hapăua, Jürgen Bremb, Mădălina Moisăb, Monica-Ioana Toşab, Florin Dan
Irimieb, Valentin Zahariaa,1
aIuliu Haţieganu University of Medicine and Pharmacy, Department of Organic
Chemistry, Ro-400012 Cluj-Napoca, Victor Babeş 41, Romania
bBabeş-Bolyai University of Cluj-Napoca, Department of Biochemistry and Biochemical
Engineering, Ro-400028 Cluj-Napoca, Arany János 11, Romania
Keywords
Biocatalysis; kinetic resolution; thiazole; chiral secondary alcohols
1Corresponding author. Tel.: +40-264-594-148; fax: +40-264-597-257; e-mail adress: vzaharia@umfcluj.ro
(V. Zaharia)
Page 2 of 22

Abstract
In this paper we describe the chemoenzymatic synthesis of new enantiomerically
enriched (R)- and (S)-1-(2-arylthiazol-4-yl)ethanols and their acetates by enzymatic
enantioselective acetylation of the racemic alcohols rac-2a-d and by methanolysis of the
corresponding racemic esters rac-3a-d mediated by lipase B from Candida antarctica
(CaL-B) in non-aqueous media. In terms of stereoselectivity and activity, both
procedures, acylation and alcoholysis, gave similar good results (50% conversion,
E>>200). The absolute configuration of the kinetic resolution products was determined
by a detailed 1H NMR study of the Mosher’s derivatives of (S)-2b.
1. Introduction
Optically active compounds as single enantiomers have currently a considerable
pharmacological importance because chirality causes a high selectivity in interaction with
protein receptors and provides specific action to the medication. It is well known that the
enantiomers may possess different potency and pharmacoselectivity, due to their different
interaction with pharmacological receptors [1]. Consequently, chiral compounds are
becoming even more important in therapy and in academic research. In particular,
enantiomerically pure secondary alcohols are valuable key synthetic intermediates for
various compounds of biological and medicinal interest [2].
Biocatalytic processes have been intensely studied and have become a useful and
green alternative in stereoselective synthesis due to their multiple advantages [3]. Among
them, lipase mediated kinetic resolutions have found broad applications in the synthesis
of various optically active intermediates such as secondary alcohols, amines, amino
alcohols, epoxides, amino acids and carboxylic acids [4]. Lipases possess the same
enantiomeric preference in both acylation and alcoholysis/hydrolysis process and this
constitutes a viable access to the both enantiomeric forms of the chiral products [5].
Furthermore, lipases maintain their stability and selectivity even in organic solvents,
because of their oil-water interfacial activation [4].
Page 3 of 22

Heterocyclic ring systems, presenting a diverse array of pharmacophores, can be
commonly found in the active components of many medicines. The need for highly
enantiopure heterocyclic building blocks and intermediates containing reactive functional
groups is a challenge for the modern research.
As a part of our interest in development stereoselective methods for preparation of
optically active ethanols bearing a heteroaromatic moiety [6], encouraged by the results
concerning the enzymatic kinetic resolution of racemic 1-(2-aryl-4-methyl-thiazol-5-
yl)ethanols [7], we turned our attention to the lipase mediated synthesis of enantiopure
(R)- and (S)-1-(2-arylthiazol-4-yl)ethanols (R)- and (S)--2a-d and their (R)- and (S)-
acetates (R)- and (S)--3a-d (Scheme 1).
The absolute configuration of the novel enantiopure compounds was determined by a
detailed 1H NMR study of the Mosher’s derivatives of (S)-2b.
2. Experimental
2.1. Analytical methods
1
The H-NMR and 13C-NMR spectra were recorded in CDCl3 solution on a Brucker
Avance DPX-300 spectrometer operating at 300 and 75 MHz, respectively. Chemical
shifts on the  scale are expressed in ppm values from TMS as internal standard. Mass
spectra were recorded on GC-MS Shimadzu QP 2010 Plus spectrometer using direct
injection and EI or NCI ionization at 30-70 eV.
High performance liquid chromatography analyses were conducted with an Agilent
1200 instrument using a Chiralpak IB column (4.6 x 250 mm). In all cases, a gradient
separation procedure was first established, using a mixture of n-hexane and 2-propanol as
eluent, all at 1 mL/min flow rate. The gradient separation procedure and the retention
times of the enantiomers are showed in Table 1. Thin layer chromatography (TLC) was
carried out using Merck Kieselgel 60 F254 sheets. Spots were visualized by treatment
with 5% ethanolic phosphomolybdic acid solution and heating of the dried plates.
Preparative chromatographic separations were performed using column chromatography
on Merck Kieselgel 60Å (63-200 m). Optical rotations were determined on a
Bellingham-Stanley ADP 220 polarimeter using chloroform as solvent. Melting points
were determined on open glass capillaries using an Electrothermal IA 9000 instrument.
Page 4 of 22

2.2. Reagents and solvents
The commercial chemicals and solvents were products of Aldrich or Fluka. All
solvents were purified and dried by standard methods as required.
Lipase B from Candida antarctica (CaL-B, Novozym 435) was purchased from
Novozymes, Denmark. Lipases from Candida rugosa (CrL), Mucor miehei (MML) and
hog pancreas (PPL) were purchased from Fluka. Lipases from Pseudomonas fluorescens
(AK free), Burkholderia cepacia (BcL) were from Amano, Europe, England. Lipase A
from Candida antarctica covalently immobilized (IMMCal-A T2–150) was purchased
from Chiralvision.
2.3. Chemical synthesis of racemic alcohols and their acetates (rac-2,3a-d)
The synthesis of racemic secondary alcohols rac-2a-d and their acetates rac-3a-d is
described in Scheme 1. The 2-arylthiazole-4-carbaldehydes 1a-d were synthesized as
previously described in the literature, using the Hantzsch condensation of various
thioamides with 1,3-dichloroacetone, followed by Sommelet reaction [8]. Further, using
methyl magnesium iodide as Grignard reagent, the racemic alcohols rac-2a-d were
obtained. Racemic acetates rac-3a-d were prepared by chemical acetylation of the
corresponding alcohols with acetic anhydride.
2.3.1. Synthesis of racemic 1-(2-arylthiazol-4-yl)ethanols (rac-2a-d)
To a stirred solution of methyl magnesium iodide, prepared from magnesium (5.6
mmol, 0.134 mg) and iodomethane (5.6 mmol, 0.79 g, 0.348 mL) in diethyl ether (5 mL),
under argon, a solution of 2-arylthiazol-4-carbaldehyde (1a-d) (3.73 mmol) dissolved in
diethyl ether (10 mL) was added slowly. The reaction mixture was stirred at 50°C for 2
hours. After quenching the reaction mixture by the addition of saturated ammonium
chloride solution (12 mL) at 0°C, the organic layer was separated and the aqueous layer
was extracted with diethyl ether (2  10 mL). The combined organic layer was dried over
anhydrous Na2SO4 and the solvent was removed by rotatory evaporation. The crude
product was purified by column chromatography using CH2Cl2/acetone (25:1 v/v) as
eluent.
Page 5 of 22

1
2.3.1.1. 1-(2-phenylthiazol-4-yl)ethanol (rac-2a): Yield: 80%; clear oil; H NMR  300
MHz, CDCl3   1.60d, J = 6.1 Hz, 3H 5.04q, J = 6.1 Hz, 1H 7.11(s, 1H); 7.32-
7.50m, 3H 7.81-8.00m, 2H 13C NMR  75 MHz, CDCl3   22.9; 66.6; 112.9;
126.6; 128.9; 130.1; 133.4; 161.7; 168.5; HRMS: M+ found (M+ calculated for
C11H11NOS): 205.0565 (205.0561); ESI+-MS: m/z (%): 206 (M+1, 16); 205 (M, 31); 190
(100); 162 (63); 121 (15); 104 (15); 77 (20); 59 (19); 45 (12); 43 (24).
2.3.1.2. 1-(2-m-tolylthiazol-4-yl)ethanol (rac-2b): Yield: 82%; clear oil; 1H NMR  300
MHz, CDCl3):   1.62 (d, J = 6.4 Hz, 3H); 2.41 (s, 3H); 5.04 (q, J = 5.0 Hz, 1H); 7.10
(s, 1H); 7.23 (d, J = 7.5 Hz, 1H); 7.32 (t, J = 7.6 Hz, 1H); 7.72 (d, J = 7.6 Hz, 1H); 7.77
(s, 1H); 13C NMR  75 MHz, CDCl3   21.3; 22.9; 66.7; 112.4; 123.7; 127.0; 128.8;
130.9; 133.4; 138.7; 161.5; 168.6; HRMS: M+ found (M+ calculated for C12H13NOS):
219.0722 (219.0718); ESI+-MS: m/z (%): 220 (M+1, 29); 219 (M, 45); 204 (100); 176
(59); 135 (10); 118 (16); 91 (14); 59 (13); 45 (10); 43 (12).
1
2.3.1.3. 1-(2-p-tolylthiazol-4-yl)ethanol (rac-2c): Yield: 78%; clear oil; H NMR  300
MHz, CDCl3):   1.60 (d, J = 6.5 Hz, 3H); 2.38 (s, 3H); 5.03 (q, J = 6.5 Hz, 1H); 7.1 (s,
1H); 7.22 (d, J = 8.0 Hz, 2H); 7.81 (d, J = 8.1 Hz, 2H) 13C NMR  75 MHz, CDCl3  
21.4; 22.8; 66.5; 112.1; 126.4; 129.5; 130.8; 140.2; 161.4; 168.5; HRMS: M+ found (M+
calculated for C12H13NOS): 219.0721 (219.0718); ESI+-MS: m/z (%): 220 (M+1, 20);
219 (M, 46); 204 (100); 176 (61); 135 (10); 118 (20); 91 (15); 59 (16); 45 (12); 43 (16).
1
2.3.1.4. 1-(2-p-bromophenylthiazol-4-yl)ethanol (rac-2d): Yield: 80%; clear oil; H
NMR  300 MHz, CDCl3):   1.59 (d, J = 6.5 Hz, 3H); 5.00 (q, J = 6.3 Hz, 1H); 7.13 (s,
1H); 7.53 (d, J = 8.6 Hz, 2H) 7.77 (d, J = 8.6 Hz, 2H); 13C NMR  75 MHz, CDCl3  
22.9; 66.6; 113.0; 124.2; 127.9; 132.0; 132.3; 161.9; 167.0; HRMS: M+ found (M+
calculated for C11H10BrNOS): 282.9662 (282.9666); ESI+-MS: m/z (%) = 286 (M+1,
81Br, 17); 285 (M, 81Br, 39); 284 (M+1, 79Br, 21); 283 (M, 79Br, 37); 270 (87); 268 (100);
242 (43); 240 (44); 201 (6); 199 (6); 184 (5); 182 (5); 161 (30); 134 (9); 59 (21); 45 (20);
43 (18).
Page 6 of 22

2.3.2. Synthesis of racemic 1-(2-arylthiazol-4-yl)ethyl acetates (rac-3a-d) by
chemical acetylation of the corresponding racemic 1-(2-arylthiazol-4-yl)ethanols
(rac-2a-d)
To a solution of racemic 1-(2-arylthiazol-5-yl)ethanol (rac-2a-d, 0.6 mmol) in dry
dichloromethane (3 mL), triethylamine (0.66 mmol, 66.8 mg, 92.1 μL), acetic anhydride
(0.66 mmol, 67.32 mg, 62.2 μL) and 4-dimethylaminopyridine (0.016 mmol, 2 mg) were
added. The mixture was stirred at room temperature overnight and then quenched with
water (10 mL). The isolated organic layer was drie0d over anhydrous Na2SO4 and the
solvent was distilled off by rotatory evaporation. The crude product was purified by
column chromatography on silica gel (eluent: dichloromethane: acetone = 25:1 (v/v)) to
afford rac-3a-d.
2.3.2.1. 1-(2-phenylthiazol-4-yl)ethyl acetate (rac-3a): Yield: 91%; clear oil; 1H NMR 
300 MHz, CDCl3   1.68 d, J = 6.6 Hz, 3H 2.11 s, 3H 6.09 q, J = 6.6 Hz, 1H
13
7.18 s, 1H 7.40-7.42 m, 3H 7.93-7.96 m, 2H C NMR  75 MHz, CDCl3  
20.1; 21.2; 68.7; 114.9; 126.5; 128.7; 130.0; 133.4; 157.0; 168.2; 170.1; HRMS: M+
found (M+ calculated for C13H13NO2S): 247.0670 (247.0667); ESI+-MS: m/z (%) = 248
(M+1, 2); 247 (M, 8); 204 (100); 188 (21); 162 (10); 104 (11); 77 (7); 45 (11); 43 (19).
2.3.2.2. 1-(2-m-tolylthiazol-4-yl)ethyl acetate (rac-3b): Yield: 90%; clear oil; 1H NMR 
300 MHz, CDCl3   1.67 (d, J = 6.6 Hz, 3H); 2.11 (s, 3H); 2.40 (s, 3H); 6.08 (q, J =
6.6 Hz, 1H); 7.17 (s, 1H); 7.22 (d, J = 7.6 Hz, 1H); 7.30 (t, J = 7.6 Hz, 1H); 7.71 (d, J =
7.6; 1H); 7.77 (s, 1H); 13C NMR  75 MHz, CDCl3   20.3; 21.3; 68.8; 114.8; 123.8;
127.0; 128.7; 130.8; 133.4; 138.6; 157.0; 168.6; 170.2; HRMS: M+ found (M+ calculated
for C14H15 NO2S): 261.0820 (261.0823); ESI+-MS: m/z (%) = 262 (M+1, 2); 261 (M, 9);
218 (100); 202 (16); 200 (9); 176 (9); 118 (12); 43 (15).
1
2.3.2.3. 1-(2-p-tolylthiazol-4-yl)ethyl acetate (rac-3c): Yield: 92%; clear oil; H NMR 
300 MHz, CDCl3   1.67 (d, J = 6.6 Hz, 3H); 2.11 (s, 3H); 2.38 (s, 3H); 6.07 (q, J =
6.6 Hz, 1H); 7.15 (s, 1H); 7.22 (d, J = 8.0 Hz, 2H); 7.83 (d, J = 8.2 Hz, 2H); 13C NMR 
75 MHz, CDCl3   20.3; 21.3; 21.4; 68.8; 114.5; 126.5; 129.5; 130.9; 140.3; 157.0;
168.6; 170.3; HRMS: M+ found (M+ calculated for C14H15NO2S): 261.0821 (261.0823);
Page 7 of 22

ESI+-MS: m/z (%) = 262 (M+1, 2); 261 (M, 10); 218 (100); 202 (17); 200 (9); 176 (10);
118 (13); 43 (14).
2.3.2.4. 1-(2-p-bromophenylthiazol-4-yl)ethyl acetate (rac-3d): Yield: 91%; white
solid; m.p.: 54-55ºC; 1H NMR  300 MHz, CDCl3   1.66 (d, J = 6.6 Hz, 3H); 2.11 (s,
3H); 6.06 (q, J = 6.6 Hz, 1H); 7.20 (s, 1H); 7.54 (d, J = 8.6 Hz, 2H); 7.81 (d, J = 8.6 Hz,
2H); 13C NMR 75 MHz, CDCl3   20.2; 21.3; 68.6; 115.3; 124.2; 128.0; 132.0;
132.4; 157.3; 167.0; 170.2; HRMS: M+ found (M+ calculated for C13H12BrNO2S):
81
81
324.9769 (324.9772); ESI+-MS: m/z (%) = 328 (M+1, Br, 7); 327 (M, Br, 11); 326
79
79
(M+1, Br, 6); 325 (M, Br, 10); 284 (100); 282 (98); 268 (29); 266 (30); 242 (6); 240
(6); 187 (10); 102 (8); 84 (10); 58 (15); 45 (26); 43 (49).
2.4. Analytical scale enzyme mediated biotransformation of 1-(2-arylthiazol-4-
yl)ethanols (rac-2a-d) and their esters (rac-3a-d)
2.4.1. Enzymatic kinetic resolution of racemic 1-(2-phenylthiazol-4-yl)ethanol (rac-
2a) with vinyl acetate and different lipases
To a solution of racemic 1-(2-phenylthiazol-4-yl)ethanol rac-2a (20 mol, 4.1 mg) in
vinyl acetate (200 μL) lipase (10 mg) was added. The reaction mixture was shaken at
1300 rpm at room temperature, for 5 hours. For HPLC analysis, samples were taken from
the reaction mixture (20 μL), diluted to 2000 μL with a mixture of n-hexane and 2-
propanol (39:1 v/v) and filtered before injection. Data on conversion and enantiomeric
composition of the products for the tested lipases are presented in Table 2.
2.4.2. Enzymatic kinetic resolution of racemic 1-(2-phenylthiazol-4-yl)ethanol (rac-
2a) with vinyl acetate in different solvents with CaL-B
To a solution of racemic 1-(2-phenylthiazol-4-yl)ethanol rac-2a (50 mol, 10.25 mg)
in different solvents (500 μL) mentioned in Table 3, vinyl acetate (200 mol, 18.45 μL)
and lipase CaL-B (2.5 mg) were added. The reaction mixtures were shaken at 1300 rpm
at room temperature. Samples were analyzed by HPLC similarly as described in Section
2.4.1. Data on conversion and enantiomeric composition of the products for the tested
solvents are presented in Table 3. The acylation of racemic alcohols rac-2b-d was tested
Page 8 of 22

by a similar procedure using the optimal conditions previously found for rac-1a (Table
4).
2.4.3. Enzymatic alcoholysis of racemic 1-(2-arylthiazol-4-yl)ethyl acetates (rac-3a-d)
To a solution of rac-3a (25 mol, 6.175 mg) in different solvents (250 μL) mentioned
in Table 5, CaL-B (2.5 mg) and methanol (250 mol, 8 mg, 10 μL) were added. The
reaction mixture was shaken at 1300 rpm at room temperature. Samples were analyzed by
HPLC similarly as described in Section 2.4.1. Further enzymatic alcoholysis of rac-3b-d
were performed similarly, using the optimal conditions previously obtained for rac-3a.
Data on conversion and enantiomeric composition of the products are presented in Table
5.
2.5. Preparative scale enzyme mediated biotransformation of 1-(2-arylthiazol-4-
yl)ethanols (rac-2a-d) and their acetates (rac-3a-d)
2.5.1. Preparative scale enzymatic acetylation of racemic alcohols rac-2a-d
Into the mixture of racemic 1-(2-arylthiazol-4-yl)ethanol (rac-2a-d, 1 mmol) in toluene
(10 mL), vinyl acetate (4 mmol, 344 mg, 369 L) and lipase CaL-B (50 mg) were added.
The reaction mixture was shaken at 1300 rpm at room temperature. The reactions were
monitored by HPLC and were stopped at an approx. 50% conversion. The enzyme was
removed by filtration. Solvents were removed by rotatory evaporation and the crude
product was purified by column chromatography on silicagel using a mixture of
dichloromethane: acetone 25:1 v/v as eluent, resulting both optically pure (S)-1-(2-
arylthiazol-4-yl)ethanols (S)-2a-d and (R)-1-(2-arylthiazol-4-yl)ethyl acetates (R)-3a-d.
2.5.2. Preparative scale enzymatic methanolysis of rac-3a-d
To a solution of rac-3a-d (0.5 mmol) in methyl tert-butyl ether (MTBE, 5 mL), CaL-B
(50 mg) and methanol (5 mmol) were added. The reaction mixture was shaken at 1300
rpm at room temperature. The reactions were monitored by HPLC and were stopped at an
approx. 50% conversion. The enzyme was removed by filtration. Solvents were removed
by rotatory evaporation and the crude product was purified by column chromatography
Page 9 of 22

on silicagel using a mixture of dichloromethane: acetone 25:1 v/v as eluent, resulting
both optically pure (R)-1-(2-arylthiazol-4-yl)ethanols (R)-2a-d and (S)-1-(2-arylthiazol-4-
yl)ethyl acetates (S)-3a-d.
MS and NMR spectra of the optically active compounds were indistinguishable from
those of their racemates.
2.6. Preparation of the Mosher’s esters
A solution of (S)-α-methoxy-α-trifluoromethylphenylacetic acid ((S)-MTPA, 0.064
mmol, 15 mg) and N,N'-dicyclohexylcarbodiimide (0.077 mmol, 15.8 mg) in anhydrous
dichloromethane (2 mL) was stirred for 30 min followed by addition of (S)-2b (0.128
mmol, 28.032 mg) and 4-dimethylaminopyridine (1.5 mg). The reaction mixture was
stirred overnight at room temperature. After removing the solvent by rotatory
evaporation, the crude product was purified by column chromatography on silicagel,
using a mixture of dichloromethane: acetone 25:1 (v/v) as eluent. The (R)-MTPA-(S)-2b
diastereomer was obtained using a similar procedure.
3. Results and discussion
3.1. Chemical synthesis
As illustrated in Scheme 1, the 2-arylthiazole-4-carbaldehydes 1a-d were transformed
onto the corresponding 1-(2-arylthiazol-4-yl)ethanols (rac-2a-d) by a Grignard reaction
using methyl magnesium iodide as nucleophile. The corresponding acetates rac-3a-d
were obtained by the chemical acetylation of rac-2a-d with acetic anhydride.
3.2. Enzymatic synthesis
To investigate the stereoselectivity of the kinetic resolution process and the enzymatic
activity, first the chromatographic enantiomeric separation of rac-2,3a-d was established,
using various HPLC chiral columns. The retention times of the enantiomers are shown in
Table 1.
3.2.1. Analytical scale biotransformation
3.2.1.1. Analytical scale enzymatic acylation of rac-2a-d
Page 10 of 22

In order to obtain the highest enantiopurities for the kinetic resolution products, various
conditions were screened for the enantiomer selective acylation process. Racemic alcohol
rac-2a was used as the model substrate for the optimization of the enzymatic procedure.
First the acetylation of rac-2a in the presence of various lipases in pure vinyl acetate was
tested (Scheme 1, Table 2).
According one reported theory [9], lipase B from Candida antarctica (CaL-B) is a
useful biocatalyst for the kinetic resolution of secondary alcohols with an substituent
smaller than n-propyl and the other one much larger. This is due to the steric
requirements of the stereoselectivity pocket, a small cavity of the active site involved in
the chiral recognition of the substrate [10]. As a confirmation of this theory, in our work
CaL-B proved to be the most active and selective enzyme for the kinetic resolution of
rac-2a, a secondary alcohol which possesses the same structural particularities enounced
above.
Lipases MML and AK free showed also high enantioselectivity but moderate activity
for the acetylation of rac-2a (Table 2, entries 2, 4).
Several studies have already reported that the nature of the solvent could significantly
influence the activity and selectivity of the lipase [11]. The acetylation of rac-2a
mediated by CaL-B with vinyl acetate in several solvents was tested. While the
enantioselectivity of the enzyme was found to be high in most of the cases, a strong
solvent influence upon the enzyme activity was observed (Table 3). Toluene proved to be
the most appropriate solvent for the efficient CaL-B mediated acetylation (E>>200, c
~50% after 5.5 hours) of rac-2a (Table 3, entry 5). Further a kinetic study was performed
for determining the specific activity of CaL-B in the acetylation of rac-2a at 25 C.
Samples were taken periodically (10 min) and subjected to chiral HPLC separation.
Based on the areas under the HPLC traces corrected with the specific molar absorbance
of substrate 2a (ε2a = 7548.9 M-1×cm-1) and product 3a (ε3a = 6943.4 M-1×cm-1), the
amounts of the substrate (50 μmol), vinyl acetate (100, 150, or 200 μmol) and enzyme
(2.5 mg) used in the reaction, the specific enzyme activity was estimated to be 0.1046
-1
mol×min-1×mgCaL-B for the enzymatic acylation of rac-2a. It was found that the
concentration of vinyl acetate had no influence upon the enzyme activity demonstrating
that the rate-limiting stage of the process is the acyl-enzyme substrate reaction. The same
Page 11 of 22

protocol was successfully used also for the kinetic resolution of alcohols rac-2b-d
(E>>200, c ~50% after 6-9 hours), as shown in Table 4.
3.2.1.2. Analytical scale enzymatic alcoholysis of rac-3a-d
In order to obtain the opposite enantiomers of the resolution products, (R)-2a-d
and (S)-3a-d, the analytical scale enzymatic alcoholysis of racemic 1-(2-arylthiazol-4-
yl)ethyl acetate rac-3a was further investigated. In accordance with the values shown in
Table 5, entries 1-3, the best results were achieved in MTBE using CaL-B and methanol
(10 equiv) as nucleophile (E >>200). To verify the optimal conditions found for the rac-
3a enzymatic methanolysis, the same procedure was further extended to the other
racemic acetates rac-3b-d. Similar good results were obtained in most cases (Table 5,
entries 4-6). Compared to the CaL-B mediated acetylation of rac-2a-d, the enzyme
activity for the methanolysis of rac-3a was lower (0.018 mol×min-1×mgCaL-B-1). In both
cases, when brominated rac-2,3d were used as substrate (Table 4, entry 4; Table 5, entry
6), a decreased CaL-B activity was observed.
3.2.2. Preparative scale synthesis of both (R)- and (S)-2,3a-d
Using the optimal conditions found for the analytical scale reactions, first the semi-
preparative scale enzymatic synthesis of both (S)- and (R)-2,3a-d was performed.
Whereas the enzymatic acetylation afforded the (R) enantiomers of acetates (R)-3a-d and
(S) enantiomers of the alcohols (S)-2a-d, alcoholysis of the racemic acetates rac-3a-d,
yielded the opposite enantiomeric forms (R)-2a-d and (S)-3a-d. No significant
differences were found for the reaction rates and for the enantiopurities of the resolution
products, compared with those found for the analytical scale reactions (Table 6). Higher
amounts of both racemic ethanol rac-2a and acetate rac-3a (5 mmols each) were used as
starting materials in reliable preparative scale reactions in order to demonstrate the
usefulness of the developed procedure for multi-gram scale enzymatic kinetic resolutions.
Compared with those found for the analytical or semipreparative scale reactions, no
significant differences were observed (ee  99% for both products, in both enzymatic
kinetic resolution processes, see supplementary data).
Page 12 of 22

3.3. The absolute configuration of the kinetic resolution products
The absolute configuration of the novel enantiopure compounds was determined by a
1
detailed H NMR study of the Mosher’s derivatives of (-)-2b. According to one
procedure already described in the literature [12], the enantiomerically pure alcohol (-)-
2b remained untransformed during the CaL-B-mediated acetylation was esterified with
both (R)-MTPA and (S)-MTPA. The resulting diastereomers were differentiated by their
1H NMR spectra (Fig.1-3).
As shown in Figure 1 and 2, in the case of (S)-MTPA-(-)-2b the strong diamagnetic
effect of the phenyl group caused the proximal CH3 protons to be more shielded,   1.73
ppm, when compared with their analogues in (R)-MTPA-(-)-2b (  1.79 ppm) where the
distance between the phenyl and the methyl group is higher. The methoxy protons of the
(S)-MTPA-(-)-2b were also found to be more shielded,  3.56 ppm, in comparison with
the methoxy protons of the (R)-MTPA-(-)-2b ( 3.61 ppm), due to the diamagnetic
effect of the proximal 2-phenylthiazole moiety.
The proton of (R)-MTPA-(-)-2b located at the 5-position of the thiazole ring was
found to be more shielded ( 6.99 ppm) when compared with the (S)-MTPA-(-)-2b (
7.19 ppm).
Based on these data, the (S)-(-) absolute configuration was assigned for (-)-2b
remained untransformed during the CaL-B mediated acylation.
Configurations of all the enantiomers obtained by the CaL-B catalyzed acetylations
were assigned as (S) on the basis of the same sign of their specific rotation.
4. Conclusions
An efficient enzymatic kinetic resolution of 1-(2-arylthiazol-4-yl)ethanol
derivatives and their acetates has been achieved. For the enantioselective acylation
process, the best results were obtained using CaL-B (5 mg/mL), vinyl acetate (4 equiv)
and toluene as solvent. CaL-B proved to be an efficient biocatalyst also for the
methanolysis (10 equiv) of the corresponding acetates rac-3a-d, using methyl tert-butyl
ether as solvent. The rate of the enzymatic reaction was higher for the acylation process,
compared to methanolysis.
Page 13 of 22

These results were a valuable prerequisite for the preparative scale production of
(R)- and (S)-2,3a-d.
The absolute configuration of (-)-2b isolated as unreacted enantiomer during the
1
CaL-B mediated acetylation of rac-2b was assigned to be (S) using a H NMR study of
its Mosher’s derivatives. These results confirmed that in this case the stereochemical
outcome of the enzymatic kinetic resolution respect the Kazlauskas empirical rule [13].
Acknowledgements
This work was supported by a grant of the Romanian National Authority for Scientific
Research, CNDI– UEFISCDI, project number PN-II-PT-PCCA-2011-3.1-1268.
References
[1] E. Francotte, W. Lindner, Chirality in Drug Research, Wiley-WCH: Weinheim,
2006, pp.18-24.
[2] V. Farina, J.T. Reeves, C.H. Senanayake, J.J. Song, Chem. Rev. 106 (2006) 2734-
2793.
[3] (a) S. Caner, E. Groner, L. Levy, Drug Discov. Today 9 (2004) 105-110; (b) M.
Breuer, K. Ditrich, T. Habicher, B. Hauer, M. Keßeler, R. Stürmer, T. Zelinski,
Angew. Chem., Int. Ed. 43 (2004) 788 – 824; (c) A. Schmid, J.S. Dordick, B. Hauer,
A. Kiener, M. Wubbolts, B. Witholt, Nature 409 (2001) 258-268.
[4] A. Zaks, A.M. Klibanov, Science 224 (1984) 1249-1251; U.T. Bornscheuer, R.J.
Kazlauskas, Hydrolases in Organic Synthesis: Regio- and Stereoselective
Biotransformations, Wiley-VCH: Weinheim-New York, 2006; A. Kamal, A.M.
Azhar, T. Krishajni, M.S. Malik, S. Azeeza, Coordin. Chem. Rev. 252 (2008) 569-
592.
[5] A. Ghanem, H.Y. Aboul-Enein, Tetrahedron: Asymmetry 15 (2004) 3331-3351.
[6] L.C. Bencze, C. Paizs, M.I. Toşa, F.D. Irimie, Tetrahedron: Asymmetry 22 (2011)
675-683; J. Brem, A. Liljeblad, C. Paizs, M.I. Toşa, F.D. Irimie, L.T. Kanerva,
Tetrahedron: Asymmetry 22 (2011) 315-322; M. Toşa, S. Pilbák, P. Moldovan, C.
Paizs, G. Szatzker, G. Szakács, L. Novák, F.D. Irimie, L. Poppe, Tetrahedron:
Asymmetry 19 (2008) 1844-1852; C. Paizs, M.I. Toşa, V. Bódai, G. Szakács, I.
Page 14 of 22

Kmecz, B. Simándi, C. Majdik, L. Novák, F.D. Irimie, L. Poppe, Tetrahedron:
Asymmetry 14 (2003) 1943-1949; J. Brem, C. Paizs, M.I. Toşa, E. Vass, F.D. Irimie,
Tetrahedron: Asymmetry 20 (2009) 489-496; L.C. Bencze, C. Paizs, M.I. Toşa, E.
Vass, F.D. Irimie, Tetrahedron: Asymmetry 21 (2010) 443-450
[7] D. Hapău, J. Brem, V. Zaharia, Tetrahedron: Asymmetry 22 (2011) 2165-2171.
[8] I. Simiti, M. Farkas, Archiv der Pharmazie 307 (1974) 81-88.
[9] D. Rotticci, F. Hæffner, C. Orrenius, T. Norin, K. Hult, J. Mol. Catal. B: Enzym. 5
(1998) 267-272; J.L. Gu, J. Liu, H.W. Yu, J Mol Catal B-Enzym, 72 (2011) 238-247.
[10](a) J. Uppenberg, N. Öehrner, M. Norin, K. Hult, G.J. Kleywegt, S. Patkar, V.
Waagen, T. Anthonsen, T.A. Jones, Biochemistry, 34 (1995) 16838-16851; (b) C.
Orrenius, F. Hæffner, D. Rotticci, N. Öhrner, T. Norin, K. Hult, Biocatal.
Biotransform. 16 (1998) 1-15; P. Braiuca, K. Lorena, V. Ferrario, C. Ebert, L.
Gardossi, Advanced Synthesis & Catalysis, 353 (2011) 1405-1405.
[11]L.C. Bencze, C. Paizs, M.I. Toşa, M. Trif, F.D. Irimie, Tetrahedron: Asymmetry 21
(2010) 1999-2004; J. Brem, M.I. Toşa, C. Paizs, A. Munceanu, D. Matković-
Čalogović, F.D. Irimie, Tetrahedron: Asymmetry 21 (2010) 1993-1998; J. Brem,
M.I. Toşa, C. Paizs, E. Vass, F.D. Irimie, Tetrahedron: Asymmetry 21 (2010) 365-
373; J. Brem, S. Pilbák, C. Paizs, G. Bánóczi, F.D. Irimie, M.I. Toşa, L. Poppe,
Tetrahedron: Asymmetry 22 (2011) 916-923.
[12]I. Ohtani, T. Kusumi, Y. Kashman, H. Kakisawa, J. Am. Chem. Soc. 113 (1991)
4092-4096.
[13]R.J. Kazlauskas, A.N.E. Weissfloch, A.T. Rappaport, L.A. Cuccia, J. Org. Chem. 56
(1991) 2656-2665.
Page 15 of 22

Table legends
Table 1. The retention times of the enantiomers of rac-2,3a-d, using Chiralpak IB column
Table 2. Lipase screening for the enantioselective acetylation of rac-2a in vinyl acetate
Table 3. Solvent screening for the CaL-B mediated acetylation of rac-2a with vinyl acetate
Table 4. CaL-B mediated acetylation of rac-2a-d with vinyl acetate in toluene
Table 5. CaL-B mediated methanolysis of rac-3a-d in various solvents
Table 6. Yields and ee values for the preparative scale synthesis of (R)- and (S)-2,3a-d
Table 1. The retention times of the enantiomers of rac-2,3a-d, using Chiralpak IB column
Gradient
separation
tR
tR
(min)
tR
(min)
tR
(min)
Entry Compound
Compound
Compound
Compound
(min)
Time
%
(min) iPrOH
0-9
1
1
(R)-3a
7.0
(S)-3a
8.4
(S)-2a
14.5
(R)-2a
15.5
9.1-16
20
0-7.5
2
2
3
4
(R)-3b
(R)-3c
(R)-3d
6.0
6.4
6.7
(S)-3b
(S)-3c
(S)-3d
6.8
7.4
7.7
(S)-2b
(S)-2c
(S)-2d
12.6
13.2
13.4
(R)-2b
(R)-2c
(R)-2d
14.0
14.0
14.0
7.6-18
20
0-7.5
2
7.6-18
20
0-7.5
2
7.6-18
15
Table 2. Lipase screening for the enantioselective acetylation of rac-2a in vinyl acetate
Entry
Enzyme
c1 (%)
ee(S)-2a (%)
ee(R)-3a (%)
E
1
2
PPL
5.94
6.29
>99.5
>99.5
»200
»200
MML
21.64
27.48
3
CrL
65.96
30.67
15.82
1.78
4
AK free
CaL-A
BcL PS
CaL-B
27.90
76.42
3.74
38.50
99.50
3.87
>99.5
30.70
>99.5
96.04
»200
8.94
»200
»200
5
6
7
50.99
>99.5
1after 5 hours
Page 16 of 22

Table 3. Solvent screening for the CaL-B mediated acetylation of rac-2a with vinyl acetate
Entry
Solvent
c1 (%)
ee(S)-2a (%)
E
ee(R)-3a (%)
1
2
3
4
5
6
7
8
9
n-Hexane
n-Octane
Cyclohexane
Chloroform
Toluene
37.29
48.80
45.95
1.66
59.16
94.83
84.60
1.68
>99.5
>99.5
>99.5
>99.5
»200
»200
»200
»200
49.75
37.35
29.39
41.08
98.50
59.31
40.15
69.37
>99.5
>99.5
»200
»200
DIPE
THF
96.48
>99.5
82.78
»200
Acetonitrile
MTBE
38.92
63.31
99.35
»200
1after 5 hours 30 min
Table 4. CaL-B mediated acetylation of rac-2a-d with vinyl acetate in toluene
Entry
Compound
Time (h)
c (%)
ee(S)-2a-d (%)
ee(R)-3a-d (%)
E
1
2
3
4
2a
2b
2c
2d
6
6
6
9
50
50
50
50
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
»200
»200
»200
»200
Table 5. CaL-B mediated methanolysis of rac-3a-d in various solvents
Entry
Compound
Solvent
Time (h)
c (%)
ee(R)-2a-d (%) ee(S)-3a-d (%)
E
1
2
3
4
5
6
3a
3a
3a
3b
3c
3d
Acetonitrile
n-Hexane
MTBE
12
12
12
13
13
88
117
30
51.5
51.2
50.3
92.4
83.0
98.0
98.0
87.0
99.3
»200
»200
»200
»200
MTBE
42.0
41.3
>99.5
>99.5
72.3
70
MTBE
MTBE
46.9
98.7
87.3
Page 17 of 22

Table 6. Yields and ee values for the preparative scale synthesis of (R)- and (S)-2,3a-d
Product1
Yield3 (%) ee (%)
Product2 Yield3 (%) ee (%)
4
4
Entry
[]D
[]D
1
2
3
4
5
6
7
8
(S)-2a
(S)-2b
(S)-2c
(S)-2d
(R)-3a
(R)-3b
(R)-3c
(R)-3d
-8.60
-7.00
(R)-2a
(R)-2b
(R)-2c
(R)-2d
(S)-3a
(S)-3b
(S)-3c
(S)-3d
48
47
48
48
49
47
46
49
98
>99.5
>99.5
98
+8.1
+7.2
48
48
49
48
48
47
48
47
>99.5
98.5
>99.5
>99.5
99
-6.80
+7.0
-10.25
+126.4
+119
+9.8
99
-126.0
-117.5
-111.3
-88.3
98
>99.5
99
+112.8
98
+97
90
99.3
1from the enantioselective acetylation of rac-2a-d mediated by CaL-B
2from the enantioselective methanolysis of rac-3a-d mediated by CaL-B
3isolated yields based on racemic starting material
4c 1.0, CHCl3, t=25ºC
Page 18 of 22

Figure legends
Scheme 1. Synthesis and biotransformation of the 1-(2-phenylthiazol-4-yl)ethanols and their acetates.
Reagents and conditions: I. (a) CH3MgI/diethyl ether; (b) NH4Cl/H2O; II. (CH3CO)2O,
DMAP/Et3N/CH2Cl2; III. CaL-B, vinyl acetate/toluene; IV. CaL-B, MeOH/MTBE.
Figure 1. (S)- and (R)- MTPA derivatives of (S)-2b.
Figure 2. Signals of the CH3 and OCH3 protons from the 1H NMR spectra of (S)-MTPA-(S)-2b and of (R)-
MTPA-(S)-2b.
Figure 3. Signals of the proton located at the 5th-position of the thiazole ring from the the 1H NMR spectra
of (S)-MTPA-(S)-2b and of (R)-MTPA-(S)-2b.
O
R:
R1
OH
O
OH
N
S
I.
III.
+
R
R
R
R
R2
O
1a-d
2a-d
3a-d
rac-
(R)-
2a-d
(S)-
R1
H
R2
H
II.
a
b
c
d
O
O
O
CH3
H
H
O
OH
IV.
+
R
R
CH3
Br
R
rac-3a-d
2a-d
(R)-
3a-d
(S)-
H
Scheme 1. Synthesis and biotransformation of the 1-(2-phenylthiazol-4-yl)ethanols and their acetates.
Reagents and conditions: I. (a) CH3MgI/diethyl ether; (b) NH4Cl/H2O; II. (CH3CO)2O,
DMAP/Et3N/CH2Cl2; III. CaL-B, vinyl acetate/toluene; IV. CaL-B, MeOH/MTBE.
Page 19 of 22

CH3
CH3
S
S
N
N
(S)
(S)
H3C
H3C
H
H
H
H
O
O
Ph
OCH3
H3CO
Ph
(S)
(R)
O
O
CF3
CF3
(R)-MTPA-(S)-2b
(S)-MTPA-(S)-2b
Figure 1. (S)- and (R)- MTPA derivatives of (S)-2b.
(S)-MTPA-(S)-2b
S,S
S,S
-OCH3
-CH3
-CH3
R,S
R,S
3.50
3.00
2.50
2.00
(R)-MTPA-(S)-2b
R,S
R,S
-OCH3
S,S
-CH3
-CH3
S,S
3.50
3.00
2.50
2.00
ppm (t1)
Figure 2. Signals of the CH3 and OCH3 protons from the 1H NMR spectra of (S)-MTPA-(S)-2b
and of (R)-MTPA-(S)-2b.
Page 20 of 22

(S)-MTPA-(S)-2b
CH
7.20
7.10
7.00
6.90
6.80
6.70
6.60
6.50
6.40
6.30
(R)-MTPA-(S)-2b
CH
7.20
7.10
7.00
6.90
6.80
6.70
6.60
6.50
6.40
6.30
ppm (t1)
Figure 3. Signals of the proton located at the 5th-position of the thiazole ring from the the 1H
NMR spectra of (S)-MTPA-(S)-2b and of (R)-MTPA-(S)-2b.
Page 21 of 22

Graphical Abstract
Heterocycles 32. Efficient kinetic resolution of 1-(2-arylthiazol-4-yl)ethanols and
their acetates using lipase B from Candida antarctica
Denisa Hapăua, Jürgen Bremb, Mădălina Moisăb, Monica-Ioana Toşab, Florin Dan
Irimieb, Valentin Zahariaa
aIuliu Haţieganu University of Medicine and Pharmacy, Department of Organic
Chemistry, Ro-400012 Cluj-Napoca, Victor Babeş 41, Romania
bBabeş-Bolyai University of Cluj-Napoca, Department of Biochemistry and Biochemical
Engineering, Ro-400028 Cluj-Napoca, Arany János 11, Romania
O
HO
O
N
O
N
Ar
Ar
CaL-B
methanol
MTBE
S
CaL-B
vinyl acetate
toluene
HO
O
S
(R)-2a-d
(R)-3a-d
N
S
N
S
(CH3CO)2O
Ar
Ar
O
O
Et3N/DMAP
CH2Cl2
HO
N
rac-3a-d
rac-2a-d
N
Ar
H3C
Ar
S
S
Ar =
H3C
Br
(S)-2a-d
(S)-3a-d
a
b
c
d
>90% ee
>98.5% ee
Page 22 of 22