Lipase-catalyzed kinetic resolution of 1-(1,3-benzothiazol-2-ylsulfanyl) propan-2-ol with antifungal activity: A comparative study of transesterification versus hydrolysis

Article abstract of DOI:10.1016/j.tet.2013.04.014

A study of chemoenzymatic synthesis of both enantiomers of 1-(1,3-benzothiazol-2-ylsulfanyl)propan-2-ol was carried out. Several commercially available lipase preparations were tested as biocatalysts in the kinetic resolution process of target compound by enantioselective transesterification and/or hydrolysis. CAL-B (Novozym 435) was found to be the optimal catalyst. The lipase-mediated hydrolysis approach appeared to be superior to the transesterification reaction. Absolute configuration of the obtained alcohol was postulated, applying modified Mosher's methodology. The inhibitory activity of the synthesized benzothiazole derivatives against pathogenic fungi was checked.

Full text of DOI:10.1016/j.tet.2013.04.014

Tetrahedron xxx (2013) 1e6
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Lipase-catalyzed kinetic resolution of 1-(1,3-benzothiazol-2-
ylsulfanyl)propan-2-ol with antifungal activity: a comparative study
of transesterification versus hydrolysis
*
Pawe1 Borowiecki, Marcin Fabisiak, Zbigniew Ochal
Warsaw University of Technology, Faculty of Chemistry, Noakowskiego St. 3, 00-664 Warsaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
A study of chemoenzymatic synthesis of both enantiomers of 1-(1,3-benzothiazol-2-ylsulfanyl)propan-2-
ol was carried out. Several commercially available lipase preparations were tested as biocatalysts in the
kinetic resolution process of target compound by enantioselective transesterification and/or hydrolysis.
CAL-B (Novozym 435) was found to be the optimal catalyst. The lipase-mediated hydrolysis approach
appeared to be superior to the transesterification reaction. Absolute configuration of the obtained alcohol
was postulated, applying modified Mosher’s methodology. The inhibitory activity of the synthesized
benzothiazole derivatives against pathogenic fungi was checked.
Received 28 December 2012
Received in revised form 19 March 2013
Accepted 4 April 2013
Available online xxx
Keywords:
1,3-Benzothiazoles
Lipase-catalyzed kinetic resolution
Double derivatization
Fungicidal activity
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
To the best of our knowledge, the kinetic enantiomers resolution
(KR) of 1-(1,3-benzothiazol-2-ylsulfanyl)propan-2-ol (ꢀ)-4 is un-
1,3-Benzothiazole derivatives are an extremely important group
of heterocyclic compounds that occupy a prominent place in me-
dicinal chemistry as pharmaceuticals and drug intermediates. They
exhibit a wide range of pharmacological properties, such as antitu-
mor, antiviral, antimicrobial, fungicidal, anthelmintic, antidiabetic,
anticonvulsant, anti-inflammatory, antiallergic, and antioxidant
agents.1 Such various biological applications made the benzothiazole
template a privileged structure fragment in modern medicinal
chemistry stimulating new achievements in searching for novel
derivatives with improved biological activity.
The availability of both enantiomers is essential to the synthesis
of chiral compounds for determination of structureeactivity re-
lationship since it is hard to predict the isomer, which expresses the
biological activity. These facts prompted us to develop a new
method of preparation of optically active 1,3-benzothiazole build-
ing block. Nowadays, biotransformation2 is a very frequently used
method of enantiomers resolution, and if the separated molecules
posses a hydroxyl group connected to chiral carbon the method of
choice is a lipase-catalyzed esterification of the alcohol or hydro-
lysis of the corresponding acetate.3 Their potential stems primarily
from a broad substrate specificity, high regio-, and enantiosele-
lectivity4 as well as very mild reaction condition.5
known so far. Up to now, only the bioreduction approach, leading
solely to one enantiomeric form, was described.6 Moreover, the
absolute configuration of the resultant isomer has not been un-
ambiguously determined. Therefore, to fill this gap we have in-
vestigatedindetailthetransesterificationoftheracemic alcohol(ꢀ)-4
and, forcomparison, thehydrolysisofthecorrespondingacetate(ꢀ)-5
by lipases, in order to obtain both expected enantiomers (Scheme 1).
Moreover, inhibitory activity of the synthesized prochiral ketone 3
and racemic mixtures of the (ꢀ)-4 were tested against a panel of
microorganisms including plant pathogenic fungi. Additionally, the
toxicity measurements of all the obtained enantioenriched products
[(S)-(ꢁ)-4a, (R)-(þ)-5a, (S)-(ꢁ)-5b, (R)-(þ)-4b] werealsoevaluated, in
order to understand their structural implications on the inhibition of
mycelium growth.
2. Results and discussion
2.1. Synthesis of racemic ( )-4 and ( )-5
The aim of this work was to determine optimum conditions for
enzymatic kinetic resolution of 1-(1,3-benzothiazol-2-ylsulfanyl)
propan-2-ol (ꢀ)-4, a potential antifungal agent. Substrates for the
enzymatic study [(ꢀ)-4 and (ꢀ)-5] were synthesized as presented
in Scheme 1. In the first step, according to the methodology
reported by D’Amico et al.,7 ketone (3) was readily obtained in 78%
* Corresponding author. Tel.: þ48 22 234 73 27; fax: þ48 22 628 27 41; e-mail
addresses: zbigniew.ochal.ztibsl@gmail.com, ochal@ch.pw.edu.pl (Z. Ochal).
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.04.014
Please cite this article in press as: Borowiecki, P.; et al., Tetrahedron (2013), http://dx.doi.org/10.1016/j.tet.2013.04.014

2
P. Borowiecki et al. / Tetrahedron xxx (2013) 1e6
Scheme 1. Preparation and lipase-catalyzed kinetic resolution of (ꢀ)-1-(1,3-benzothiazol-2-ylsulfanyl)propan-2-ol (ꢀ)-4 and its acetate (ꢀ)-5. Reagents and conditions: (i) KOH
(1 equiv), ethanol, 15 min at 80 ꢂC, than chloroacetone (1 equiv), 20 h at rt (ii) NaBH4 (4.85 equiv), methanol, 0e5 ꢂC, 20 h; (iii) CH3COCl (1 equiv), Et3N (1 equiv),
D, toluene, 4 h; (iv)
vinyl acetate (1.3 equiv), enzyme, solvent, 32 ꢂC, 250 rpm; (v) Novozym SP 435, solvent/H2O, 32 ꢂC, 250 rpm.
isolated yield by reacting chloroacetone (2) with potassium hy-
droxide activated 1,3-benzothiazol-2-thiolate (1). In our hands, the
reduction of the carbonyl group in ketone (3) with sodium tetra-
hydroborate resulted in 1-(1,3-benzothiazol-2-ylsulfanyl)propan-
2-ol (ꢀ)-4 preparation in excellent yield (95%). In order to prepare
an analytical standard of racemic resolution product as well as the
substrate (ꢀ)-5 for an enzyme-catalyzed hydrolysis studies, race-
mic alcohol (ꢀ)-4 was esterified with an equimolar amount of
acetyl chloride in toluene in the presence of triethylamine. This
procedure provided racemic acetyl ester (ꢀ)-5 with quite satisfying
yield reaching up to 80%.
It is well known that in enzymatic resolutions enantioselectivity
depends strongly on enzyme specificity and solvent used.9 Taking
this into account we decided to determine the most convenient
medium for the reaction. Our preliminary studies have shown that
toluene, and particularly, ether solvents (DIPE, TBME) in the KR of
alcohol (ꢀ)-4 were superior to other media. Generally, the reactions
were conducted with 1.3-molar excess of vinyl acetate as an acyl
donor in the above mentioned organic solvents at 32 ꢂC. It should
be emphasized that in our kinetic resolution experiments of 1-(1,3-
benzothiazol-2-ylsulfanyl)propan-2-ol (ꢀ)-4, a very small amount
of enzyme was used as biocatalyst (5e10-% mass equivalent with
respect to the substrate). The progress of the enzymatic processes
was monitored by GC. Reactions were stopped at conversion close
to 50%. The products obtained [(ꢁ)-4a and (þ)-4b] were separated
by chromatography and their enantiomeric excesses were de-
termined using chiral HPLC.
2.2. Enzymatic KR of ( )-4 via transesterification reaction
Herein the results of racemic benzothiazolic alcohol (ꢀ)-4 enan-
tiomers separation in chemoenzymatic lipase-mediated trans-
esterification are reported. For the preliminary screening of the
enzyme-mediated acetylation of (ꢀ)-4 we have chosen various li-
pases preparations, including: Amano A (Aspergillus niger), CCL
(Candida cylindracea), Amano AK (Pseudomonas fluorescens), Amano
PS (Burkholderia cepacia), Amano M (Mucor javanicus), and immobi-
lized enzymatic preparations: Thermomyces lanuginosusdImmobead
150, Chirazyme L-9 (Mucor miehei), Chirazyme L-2 (Candida antarc-
tica), Novozym SP 435 (C. antarctica). Out of the nine lipases that were
tested, those which required long reaction times (>13 days) for ca.
50% substrate conversion or gave very low enantioselectivity were
not studied further. Those which performed adequately are shown in
Table 1.
The best results from the point of view of enantioselectivity and
enantiopurity of formed ester (þ)-4b (Table 1, entries 3e5,
E¼48e88, ee¼93e96%) were achieved by acylation of racemic al-
cohol (ꢀ)-4 with vinyl acetate in the presence of Amano PS. Un-
fortunately, these reactions progressed rather slowly compared to
those catalyzed by immobilized enzymes (Chirazyme L-2 or
Novozym SP) and hardly reached conversions close to 37e42% even
after 6 days. In turn, the stereochemical outcome concerning
enantiopurity of remaining alcohol (ꢁ)-4a displayed moderate
values (ee¼55e73%). Despite this, the screening experiments re-
veal useful information: (i) the reactions carried in ethers run sig-
nificantly faster than those in toluene, (ii) DIPE was found to be the
most advantageous solvent considering the enzyme activity and
finally, (iii) Novozym 435 was slightly more effective catalyst than
Chirazyme L-2 (Table 1: entries 1 vs 8 and 2 vs 10).
High rates and unsatisfactory enantioselectivities of the re-
actions catalyzed by Chirazyme L-2 and Novozym 435 prompted us
to cut by half the amount of the used enzyme catalyst. This move
has slowed down the reactions, although none significant influence
on the enantioselectivity was observed. The obtained results are
similar to those of previous attempts (Table 1: entries 6 and 8 vs 7
and 9), but in the case of reaction performed in TBME a subtle
positive improvement was observed (Table 1: entries 10 vs 11).
Due to the unsatisfactory E and ee values in the lipase-catalyzed
transesterification, the reverse process, namely the lipase-catalyzed
hydrolysis of the corresponding acetate (ꢀ)-5, was performed.
Table 1
Results of enzymatic transesterification of 1-(1,3-benzothiazol-2-ylsulfanyl)propan-
2-ol (ꢀ)-4
Entry Enzyme
Solvent t [h]
Chirazyme DIPEa
1.5 47
L-2
TBMEa
Conv.c [%] eeOH d [%] eeOAcd [%] Ee
1
2
71
65
80
74
19
13
3
47
a
3
4
5
Amano PS PhCH3
DIPEa
306
165
158
38
42
37
58
67
55
96
94
93
88
65
48
TBMEa
a
6
7
8
9
10
11
Novozym
SP 435
PhCH3
PhCH3
DIPEa
DIPEb
3
7
47
45
67
63
73
69
69
71
76
77
82
84
77
80
15
15
22
21
16
19
b
1.5 47
4.5 46
2.5 47
TBMEa
TBMEb
4
47
2.3. Enzymatic KR of ( )-5 via hydrolysis reaction
a
Conditions: (ꢀ)-4 100 mg, lipase 10 mg, solvent 3 mL, vinyl acetate 50 mg
(1.3 equiv), 32 ꢂC, 250 rpm.
b
As it was stated in the previous section, Novozym 435 was su-
perior to other enzymes studied, and so it was chosen as the bio-
catalyst for further investigations. Since the enzymatic reactions
are highly solvent-dependent, we turned our attention to the se-
lection of the appropriate medium. Initially we compared the ef-
fectiveness of three co-solvents systems (Table 1: entries 1e3)
Conditions: (ꢀ)-4 100 mg, lipase 5 mg, solvent 3 mL, vinyl acetate 50 mg
(1.3 equiv), 32 ꢂC, 250 rpm.
c
The conversion (%) was calculated from the ee of the unreacted alcohol (ees) and
the product (eep) according to the formula conv.¼eeOH/(eeOHþeeOAc).
d
Determined by HPLC analysis using Chiralcel OD-H chiral column.
e
Calculated according to Chen et al.,8 using the equation: E¼{ln
[(1ꢁconv.)(1ꢁeeOH)]}/{ln[(1ꢁconv.)(1þeeOH)]}.
Please cite this article in press as: Borowiecki, P.; et al., Tetrahedron (2013), http://dx.doi.org/10.1016/j.tet.2013.04.014

P. Borowiecki et al. / Tetrahedron xxx (2013) 1e6
3
composed of wateresaturated toluene and DIPE or TBME, re-
spectively. In these lipase-catalyzed hydrolysis reactions, the
enantioselectivities (E values) were as low as for asymmetric
transesterification, except for the reaction carried out in TBME/H2O
mixture (Table 2, entry 3, E¼97). The experiments showed that
when the reaction reached high conversion level, the ee value of
the remaining ester (ꢁ)-5b was fairly good but yet not fully satis-
fying (Table 2, entry 2, ee¼92%).
sign of this parameter (þ or -) bears the information about the
relative position of L1/L2 substituents, with respect to the aniso-
tropic group (phenyl group of MPA). The negative value of DdRS
,
which corresponds to the signal of the protons of L1, and the plus
sign for the protons belonging to L2 indicate an (R)-configuration of
the investigated alcohol (þ)-5a, as depicted in Fig. 1.
Table 2
Results of enzymatic hydrolysis of 1-(1,3-benzothiazol-2-ylsulfanyl)propan-2-ol
acetate (ꢀ)-5
Entry Enzyme
Solvent
t [h] Conv.c [%] eeOHd [%] eeOAcs [%] Ee
1
2
3
4
Novozym PhCH3/H2Oa 507 43
12
62
94
9
92
89
92
1
13
97
SP 435
DIPE/H2Oa
TBME/H2Oa
TBME/H2Ob
54 60
78 49
16 48
Fig. 1. Description of substituents for determination of the absolute configuration
of (þ)-5a.
>99
659
a
Conditions: (ꢀ)-5 100 mg, lipase 10 mg, organic solvent saturated with distilled
water 3 mL, 32 ꢂC, 250 rpm.
b
Conditions: (ꢀ)-5 200 mg, lipase 20 mg, organic solvent saturated with distilled
The above results of the examination of the proton NMR data of
the (R)- and (S)-MPA ester derivatives are consistent with the three-
dimensional structure proposed (see Fig. 2), which is based on the
empirical assumption that in MPA esters of monofunctional sec-
ondary alcohols, in the most populated conformer the methoxy
group of MPA, the carbonyl group, and the proton attached to the
stereogenic center of the alcohol are in the same plane.
water 6 mL, 32 ꢂC, 250 rpm.
c
The conversion was calculated from the ee of the unreacted ester (ees) and the
product (eep) according to the formula conv.¼eeOAc/(eeOAcþeeOH).
d
Determined by HPLC analysis using Chiralcel OD-H chiral column.
E-values were calculated using the equation: E¼{ln[(1ꢁconv.)(1ꢁeeOAc)]}/{ln
e
[(1ꢁconv.)(1þeeOAc)]}.
In turn, for the alcohol (þ)-5a, even at conversion below 49%, ee
values up to 94% were observed (Table 2, entry 3). Next, we decided
to scale-up selected enzymatic hydrolysis of racemic ester (ꢀ)-5
(Table 2, entry 4). Unexpectedly, the reaction in larger scale under the
same conditions as before proceeded with higher rate than in small
scale (Table 2, entry 3). Shortening of the reaction time provided
enzymatic assay with the superbly high enantioselectivity (E¼659)
of the almost enantiomerically pure alcohol (þ)-5a (>99% ee).
2.4. Determination of stereochemistry of alcohol (D)-5a
The stereochemical preference of the Novozym 435 lipase in the
hydrolysis reaction of (ꢀ)-5 was determined by assignment of the
absolute configuration of the newly created chiral center in the
faster reacting enantiomer (þ)-5a. This was assayed by the modi-
fied Mosher’s methodology as described by Riguera et al.10 in which
the investigated alcohol enantiomer (þ)-5a was transformed into
two diastereomeric esters with the use of chiral auxiliary reagents
as depicted in Scheme 2. In our study (R)- and (S)-enantiomers of
methoxyphenylacetic acid (MPA) have been used in a double de-
rivatization procedure.
Fig. 2. The 1H NMR spectra of the (R)- and (S)-MPA derivatives of chiral alcohol (þ)-5a.
With red color are marked protons shielded by phenyl ring of chiral auxiliary, blue
labels stand for unaffected protons.
To substantiate the usefulness of this method in case of the
secondary alcohols with benzothiazole moiety and the appropri-
ateness of the outlined above assumptions, a few points should be
stressed: (i) chiral environment provided by the derivatizing agent
(MPA) covalently associated with the considered alcohol (þ)-5a
leads to significant differences in chemical shifts especially of the
protons attached to the carbon atoms bound to the chiral center, an
area where the maximum magnetic shielding occur, (ii) if the ab-
solute configuration of the faster reacting enantiomer of (þ)-5a is
(R) on C(12) carbon then the phenyl ring of chiral auxiliary reagent
projects strongly its magnetic anisotropy effect toward H(110)
proton in (R)-MPA ester (6), while the same proton in the (S)-MPA
ester (7) remains unaffected. The opposite phenomenon is ob-
served for methyl group protons H(140), which are placed in space
Scheme 2. Assignment of the absolute configuration of alcohol (þ)-5a.
Next, the 1H NMR spectra of the resulting diastereoisomers were
recorded and compared. The differences in chemical shifts of the
protons located in the neighborhood of the stereogenic carbon
were measured to give a DdRS value (see equation below).
(
DdRSL1
DdRSL2
¼
¼
d
L1ðRÞ ꢁ
L2ðRÞ ꢁ
d
d
L1ðSÞ ¼ 3:48 ꢁ 3:59 ¼ ꢁ0:11 < 0
L2ðSÞ ¼ 1:41 ꢁ 1:26 ¼ ꢁ0:15 > 0
ð þ Þ ꢁ 5a
d
The calculation of the corresponding differences expressed as
DdRS are prerequisite for the configurational assignment, since the
Please cite this article in press as: Borowiecki, P.; et al., Tetrahedron (2013), http://dx.doi.org/10.1016/j.tet.2013.04.014

4
P. Borowiecki et al. / Tetrahedron xxx (2013) 1e6
so that they become shielded in (S)-MPA derivatives (7) while in
(R)-MPA ester (6) they are unaffected. Assignation of the absolute
configuration confirmed that the lipase-catalyzed hydrolysis of
(ꢀ)-5 by Novozym 435 preparation proceeded with the usual
‘Kazlauskas-type’ enantiomer selectivity.11
separated by an efficient and straightforward lipase-catalyzed ki-
netic resolution. The chemoenzymatic route presented herein have
been used for the first time for 1-(1,3-benzothiazol-2-ylsulfanyl)
propan-2-ol. This method compares advantageously with the
Baker’s yeast-induced asymmetric reduction in term of simplicity
and possibility of simultaneous obtaining both enantiomers with
a very high enantiopurity. We have shown that transesterification
and hydrolysis processes are complementary reactions of value for
obtaining both isomeric forms of the title compound with a very
high enantioselectivity. Our results clearly point that Novozym
435-catalyzed enzymatic hydrolysis is highly efficient method and
can be readily extended to other substrates of this type. Assignment
of the absolute configuration revealed that Novozym 435 behaved
2.5. Antifungal activity assay
The in vitro biological evaluation of the prepared compounds
was conducted with six strains of pathogenic fungi viz., Alternaria
alternata, Botrytis cinerea, Fusarium culmorum, Phytophthora
cactorum, Rhizoctonia solani, and Blumeria graminis. The results of
the assessment are listed in Table 3. Initially, only compounds
Table 3
Fungicidal efficiency of benzothiazole derivatives (%)
Comp. concn
A. alternata
B. cinerea
200 g/ml
g/ml
F. culmorum
200 g/ml
g/ml
P. cactorum
200 g/ml
g/ml
R. solani
200 g/ml
g/ml
B. graminis
100
m
g/ml
m
m
m
m
1000 mg/ml
20
m
20
m
20
m
20 m
3
0
30
30
30
45
50
50
0
40
0
60
0
50
0
60
20
60
20
70
20
0
0
40
0
0
50
45
60
55
70
(ꢀ)-4
60
20
50
10
60
20
60
20
70
30
60
20
60
20
70
20
70
30
80
30
60
30
50
20
70
30
60
30
70
30
(S)-(ꢁ)-4aa
(R)-(þ)-5ab
(S)-(ꢁ)-5bc
(R)-(þ)-4bd
a
ee¼71%.
b
ee>99%.
c
ee¼92%.
d
ee¼96%.
3 and (ꢀ)-4 were assessed for their fungicidal efficiency. The
strongest activity was observed in the case of (ꢀ)-4 with the
considerably high toxicity toward both R. solani and B. graminis
microbes, this giving a promising look for determination of bi-
ological behavior of its single enantiomers. Thus, additional study
of the inhibition growth activity in the presence of optically active
derivatives [(S)-(ꢁ)-4a, (R)-(þ)-5a, (S)-(ꢁ)-5b, (R)-(þ)-4b] were
performed and the results obtained enabled the identification of
some correlations between the configuration at the chiral center
and the toxicity properties of the separated enantiomers. To our
surprise, only a little difference was observed for opposite en-
antiomers, nevertheless antifungal activity data clearly indicate
that the (R)-isomers are more benign derivatives, both the alcohol
[(R)-(þ)-5a] as well as the ester [(R)-(þ)-4b]. Comparison of
(S)-alcohol and (S)-ester or (R)-alcohol and (R)-ester activities
indicated that in all cases the acetyl derivatives are more active
against investigated fungal strains suggesting that the lip-
ophilicity factor is the one, which exerts better inhibitory activity.
All of the tested compounds exhibited quite strong activity and
wide antifungal action. It is worth to note that (R)-(þ)-4 ester
in kinetic enantiomers resolution of (ꢀ)-4 as selective Kazlauskas
catalyst, forming (R)-alcohol after hydrolysis. Biological evaluation
confirmed our assumptions that compounds based on the 1-(1,3-
benzothiazol-2-ylsulfanyl)propan-2-ol core-structure can be ap-
plied in the synthesis of potentially valuable agro-chemical chiral
synthons.
4. Experimental section
4.1. General details
All commercially available reagents (Aldrich, Fluka, and POCH)
were used without further purification. Novozym SP 435 (lipase
from C. antarctica B immobilized on a macroporous acrylic resin,
>10,000 U/g) was purchased from Novozymes A/S, Amano PS
(native lipase from B. cepacia earlier Pseudomonas cepacia,
>23.000 U/g) was purchased from Amano Pharmaceutical Co., Ltd.,
and Chirazyme L-2, c.-f., C2, lyo. (lipase from C. antarctica, type B)
was purchased from Roche Molecular Biochemicals and were used
without any pretreatment. Melting points were obtained with an
MPA100 Optimelt SRS apparatus. Thin-layer chromatography was
carried on TLC aluminum plates with silica gel Kieselgel 60 F254
(Merck) (0.2 mm thickness film) using UV light as visualizing agent.
Preparative plate chromatography was performed with PSC-
Fertigplatten Kieselgel 60 F254 (20ꢃ20 cm with 2 mm thickness
layer). The chromatographic analyses (GLC) were performed with
an HP Series II 5890 instrument equipped with a flame ionization
detector (FID) and fitted with HP-50þ (30 m) semipolar column.
Helium (2 mL/min) was used as carrier gas; Tinjector¼280 ꢂC,
Tcolumn¼100 ꢂC (3 min) and 100e260 ꢂC (10 ꢂC/min). Column
posses excellent activity at 200 mg/ml concentration showing 80%
inhibition of P. cactorum colony growth. In conclusion, an appre-
ciable difference was observed when comparing the racemic and
optically active compounds, however a clear tendency could not
be identified.
3. Conclusion
Biologically and pharmaceutically attractive enantiomers of
racemic 1,3-benzothiazole-based secondary alcohol (ꢀ)-4 were
Please cite this article in press as: Borowiecki, P.; et al., Tetrahedron (2013), http://dx.doi.org/10.1016/j.tet.2013.04.014

P. Borowiecki et al. / Tetrahedron xxx (2013) 1e6
5
chromatography was performed using Silica gel 60 (Merck) of
40e63 m. A mixture of 20% ethyl acetate/hexane was used as el-
461, 425 cmꢁ1; NMR dH (400 MHz, CDCl3) 1.35 (3H, d, J¼6.32 Hz,
CH3), 3.34 (1H, dd, J¼14.34, 7.11 Hz, CH2), 3.51 (1H, dd, J¼14.23,
3.16 Hz, CH2) 3.88 (1H, br s, OH), 4.21e4.30 (1H, m, CH), 7.27e7.45
(2H, m, Ph), 7.70e7.87 (2H, m, Ph); NMR dC (100 MHz, CDCl3) 22.5,
42.1, 67.4, 121.0, 121.2, 124.6, 126.2, 135.3, 152.3, 167.7; HRMS (ESIþ,
m/z): [MþH]þ, found 225.9984. C10H11NOS2 requires 226.0355;
HPLC [hexane/i-PrOH (95:5); f¼0.4 mL/min]: tR¼41.4 min,
45.2 min.
m
uent. The enantiomeric excesses of resulting esters and alcohols
were determined by HPLC analysis performed on Shimadzu CTO-
10ASV chromatograph equipped with STD-20A UV detector and
Chiralcel OD-H (Diacel) chiral column using mixtures of n-hexane/
iso-propyl alcohol as mobile phase in appropriate ratios given in
experimental section; the HPLC analyses were executed in an iso-
cratic manner; flow (f) is given in mL/min; retention times (tR) are
given in minutes under conditions given; racemic alcohols and
esters were used as standards. Optical rotations were measured
with an AP-300 automatic polarimeter (ATAGO) in a 0.5 dm long
cuvette. UV spectra were measured with Cary 3 spectrometer (data
were collected on diluted solutions in quartz spectroscopy cells). 1H
and 13C NMR spectra were measured with a Varian Mercury 400BB
spectrometer operating at 400 MHz for 1H and 100 MHz for 13C
4.1.3. 1-(1,3-Benzothiazol-2-ylsulfanyl)propan-2-yl acetate ((ꢀ)-5).
To a solution of 1-(1,3-benzothiazol-2-ylsulfanyl)propan-2-ol (ꢀ)-4
(3 g, 13.3 mmol) in toluene (30 mL) acetyl chloride (1.04 g,
13.3 mmol) and subsequently triethylamine (1.35 g, 13.3 mmol)
were added with stirring at room temperature. The reaction mix-
ture was stirred at reflux for 3 h, then cooled to room temperature,
and the precipitated triethylamine hydrochloride salt was filtered
off. Next, the permeate was concentrated in vacuo and crude
product was purified by gel chromatography (20% ethyl acetate/
hexane) yielding the title compound (ꢀ)-5 (2.85 g, 80%) as a light-
green oil; Rf (20% ethyl acetate/hexane) 0.53; FTIR nmax(neat):
2981, 1734, 1456, 1426, 1369, 1309, 1228, 1126, 1036, 991, 955, 753,
725, 673, 606, 429 cmꢁ1; NMR dH (400 MHz, CDCl3) 1.40 (3H, d,
J¼6.32 Hz, CHCH3), 2.02 (3H, s, (C]O)CH3), 3.49 (1H, dd, J¼13.89,
6.89 Hz, CH2), 3.70 (1H, dd, J¼13.78, 4.74 Hz, CH2), 5.27 (1H, td,
J¼6.49, 4.86 Hz, CH), 7.27e7.46 (2H, m, Ph), 7.72e7.90 (2H, m, Ph);
NMR dC (100 MHz, CDCl3) 19.3, 21.2, 38.1, 69.1, 120.9, 121.4, 124.3,
126.0, 135.2, 152.8, 166.0, 170.2; HRMS (ESIþ, m/z): [MþH]þ, found
268.0106. C12H13NO2S2 requires 268.0461; HPLC [hexane/i-PrOH
(95:5); f¼0.4 mL/min]: tR¼18.6 min, 26.6 min.
nuclei, chemical shifts (d) are given in parts per million (ppm) re-
lated to tetramethylsilane (TMS) as internal standard; signal mul-
tiplicity assignment: s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet; coupling constant (J) are given in hertz (Hz). Mass
spectra were recorded with a Micro-mass ESI Q-TOF spectrometer
at the Mass Spectrometry Laboratory, Institute of Biochemistry and
Biophysics (IBB), PAN. IR spectra of neat samples were recorded on
a PerkineElmer System 2000 FTIR Spectrometer equipped with
a Pike Technologies GladiATRÔ attenuated total reflectance (ATR)
accessory with a monolithic diamond crystal stage and a pressure
clamp.
4.1.1. 1-(1,3-Benzothiazol-2-ylsulfanyl)propan-2-one (3). In a three-
necked round bottom flask equipped with a reflux condenser
ethanolic (66 mL) solution of 1,3-benzothiazole-2-thiol 1 (5 g,
29.9 mmol) was placed. Then potassium hydroxide (1.68 g,
29.9 mmol) was added and the mixture was stirred at 80 ꢂC for
15 min in a gentle stream of dry nitrogen. After cooling the con-
tents of the flask to room temperature chloroacetone 2 (2.77 g,
29.9 mmol) was added and stirring was continued for an addi-
tional 20 h. Next, crude reaction mixture was poured into ice-
water (66 mL) and kept at 0e10 ꢂC for 30 min with stirring. A
yellow precipitate was filtered off, quenched with distilled H2O
(15 mL), and dried at ambient temperature to give desired ketone.
Further purification of the crude product by recrystallization
process from EtOH gave the title compound 3 (5.2 g, 78%) as
a white solid, mp 64e64.5 ꢂC (ethanol) (lit.10 mp 64e65 ꢂC, eth-
anol); Rf (20% ethyl acetate/hexane) 0.38; FTIR nmax(neat): 2967,
1717, 1463, 1427, 1357, 1298, 1232, 1152, 1073, 997, 753, 703, 673,
577, 436, 406 cmꢁ1; NMR dH (400 MHz, CDCl3) 2.40 (3H, s, CH3),
4.26 (2H, s, CH2), 7.28e7.45 (2H, m, Ph), 7.72e7.87 (2H, m, Ph); dC
(100 MHz, CDCl3) 28.8, 43.1, 121.1, 121.5, 124.5, 126.1, 135.4, 152.5,
165.1, 201.7; HRMS (ESIþ, m/z): [MþH]þ, found 223.9879.
4.1.4. Enzymatic transesterification of ((ꢀ)-4). Racemic alcohol
(ꢀ)-4 (100 mg, 0.44 mmol) was dissolved in corresponding organic
solvent (3 mL) as stated in Table 1. Afterward, suspension of lipase
(5e10 mg) and vinyl acetate (50 mg, 0.57 mmol) were added in one
portion. The reaction mixture was shaken (250 rpm) at 32 ꢂC, and
its aliquots were regularly analyzed by gas chromatography. The
reaction was stopped by filtering off the catalyst and the enzyme
was washed using the solvent (3 mL) as applied previously. After
evaporation of the filtrate under reduced pressure, the components
of the mixture were separated by a silica gel column chromatog-
raphy (20% ethyl acetate/hexane) affording enantiomerically
enriched alcohol (S)-(ꢁ)-4a and ester (R)-(þ)-4b, respectively. For
additional data, see Table 1.
4.1.5. Enzymatic hydrolysis of ((ꢀ)-5). Ester (ꢀ)-5 (200 mg,
0.75 mmol) was dissolved in wateresaturated TBME (6 mL).
Novozym SP 435 (20 mg) was added in one portion, and then the
suspension was kept under shaking (250 rpm) with incubation at
32 ꢂC for the amount of time given in Table 2. The degree of hy-
drolysis was followed by GC analysis of the properly treated sample
accordingly to the procedure as given in the text below. The re-
action was terminated by filtering off the enzyme. The phases were
separated and the extraction of the aqueous solution with AcOEt
(3ꢃ5 mL) was performed. The combined organic phases were dried
(MgSO4), filtered, and then concentrated under vacuum. The puri-
fication of the resulting residue by column chromatography [20%
C
10H9NOS2 requires 224.0198.
4.1.2. 1-(1,3-Benzothiazol-2-ylsulfanyl)propan-2-ol
((ꢀ)-4). In
a
two-necked round bottom flask 1-(1,3-benzothiazol-2-
ylsulfanyl)propan-2-one 3 (0.73 g, 3.3 mmol) was dissolved in
methanol (40 mL) and kept under an atmosphere of nitrogen at
ice-water temperature. Subsequently, sodium borohydride (0.6 g,
15.9 mmol) was added portionwise with stirring at a rate sufficient
to maintain the internal temperature at 5 ꢂC. The reaction mixture
was stirred at 0e5 ꢂC for 2 h. Next, the methyl alcohol was
evaporated in vacuo, the residue was suspended in distilled H2O
(30 mL) and extracted with EtOAc (3ꢃ20 mL). The organic layers
were combined and dried (MgSO4). After filtration through short
silica pad and the solvent evaporation under reduced pressure, the
final product (ꢀ)-4 was obtained (0.62 g, 85%) as an orange oil; Rf
(20% ethyl acetate/hexane) 0.22; FTIR nmax(neat): 3346, 2966,
2868, 1452, 1423, 1245, 1127, 1043, 1002, 943, 752, 686, 598, 580,
ethyl acetate/hexane] afforded enantioenriched ester (S)-(ꢁ)-5b
29
[ee¼92%; [
a
]
ꢁ14.86 (c 1.0, CHCl3)] and alcohol (R)-(þ)-5a
þ25.54 (c 1.0, CHCl3)].
D
29
[ee>99%; [
a]
D
4.1.6. Esterification of (R)-(þ)-5a with (R)- or (S)-MPA. A catalytic
amount of DMAP (5 mg) and DCC (44 mg, 0.213 mmol) were added
to the solution of (R)-(þ)-1-(1,3-benzothiazol-2-ylsulfanyl)propan-
2-ol (R)-(þ)-5a (40 mg, 0.178 mmol) and (R)- or (S)-
a-methoxy-a-
phenylacetic acid (29 mg, 0.178 mmol) in dry CH2Cl2 (3 mL), re-
spectively. After 48 h of stirring at room temperature, precipitated
Please cite this article in press as: Borowiecki, P.; et al., Tetrahedron (2013), http://dx.doi.org/10.1016/j.tet.2013.04.014

6
P. Borowiecki et al. / Tetrahedron xxx (2013) 1e6
dicyclohexylurea was removed by filtration and then the urea
cake was rinsed with toluene (3ꢃ2.5 mL). The combined solutions
were evaporated and the product was purified on preparative
chromatographic plate using mixture of 20% ethyl acetate/hexane
as the eluent.
Supplementary data
NMR spectra data for all synthesized compounds. Supplemen-
tary data related to this article can be found at http://dx.doi.org/
10.1016/j.tet.2013.04.014.
4.1.7. (2R)-1-(1,3-Benzothiazol-2-ylsulfanyl)propan-2-yl
(2R)-me-
References and notes
thoxy(phenyl)ethanoate (6). Yellowish oil; yield 66%; NMR dH
(400 MHz, CDCl3) 1.41 (3H, d, J¼6.32 Hz, CHCH3), 3.36e3.45 (4H, m,
SCH2CH and OCH3), 3.57 (1H, dd, J¼13.89, 4.74 Hz, SCH2CH), 4.75
(1H, s, CHOCH3), 5.30 (1H, td, J¼6.61, 4.86 Hz, CH3CHCH2),
7.26e7.45 (7H, m, Ph), 7.71e7.86 (2H, m, Ph).
1. (a) Sareen, S.; Shinde, D.; Khatri, V.; Sareen, V. Heterocycl. Lett. 2012, 3,
361e377; (b) Facchinetti, V.; Reis, da R.; Raisa, R. B.; Gomes, C.; Vasconcelos, R.
A. T. Mini-Rev. Org. Chem. 2012, 9, 44e53.
€
2. (a) Borgstrom, B. In Lipases, 9th ed.; Brockman, H. L., Ed.; Elsevier: Amsterdam,
1984; (b) Bu’Lock, J.; Kristiansen, B. Basic Biotechnology; Academic: New York,
NY, 1987; (c) Dordick, J. S. Enzyme Microb. Technol. 1989, 11, 194e211; (d)
Ghanem, A. Tetrahedron 2007, 63, 1721e1754; (e) García-Urdiales, E.; Alfonso, I.;
Gotor, V. Chem. Rev. 2011, 111e180.
4.1.8. (2R)-1-(1,3-Benzothiazol-2-ylsulfanyl)propan-2-yl
(2S)-me-
thoxy(phenyl)ethanoate (7). Yellowish oil; yield 26%; NMR dH
(400 MHz, CDCl3) 1.26 (3H, d, J¼6.32 Hz, CHCH3), 3.37 (3H, s, OCH3),
3.49 (1H, dd, J¼14.00, 7.00 Hz, SCH2CH), 3.69 (1H, dd, J¼14.00,
4.74 Hz, SCH2CH), 4.67 (1H, s, CHOCH3), 5.30e5.36 (1H m,
CH3CHCH2), 7.28e7.46 (7H, m, Ph), 7.72e7.89 (2H, m, Ph).
3. (a) Ghandi, N. N. J. Am. Oil Chem. Soc. 1997, 74, 621e634; (b) Itoh, T.; Takagi, Y.;
Tsukube, H. J. Mol. Catal. B: Enzym. 1997, 3, 259e270; (c) Carnell, A. J. J. Mol.
Catal. B: Enzym. 2002, 19e20, 83e92; (d) Kazlauskas, R. J.; Bornscheuer, U. T. In
Biotransformations with Lipases, Biotechnology, 2nd ed.; Kelly, D. R., Ed.; Bio-
transformation 1; Wiley-VCH: Weinheim, 1998; Vol. 8, pp 37e191.
4. Faber, K. Biotransformations in Organic Chemistry; Springer: 19974e5.
5. (a) Klibanov, A. M. Trends Biochem. Sci. 1989, 14, 141e144; (b) Chen, C.-S.; Sih, C.
J. Angew. Chem., Int. Ed. Engl. 1989, 28, 695e707; (c) Halling, P. J. Enzyme Microb.
Technol. 1994, 16, 178e206; (d) Tramper, J. Biotechnol. Bioeng. 1996, 52,
290e295; (e) Lambusta, D.; Nicolosi, G.; Patti, A.; Sanfilippo, C. J. Mol. Catal. B:
Enzym. 2003, 22, 271e277.
Acknowledgements
The project was co-financed by The European Regional De-
velopment Fund under The Innovative Economy Operational Pro-
gramme 2007e2013: ‘Biotransformations for Pharmaceutical and
Cosmetic Industry’ POIG.01.03.01-00-158/09. These studies were
partially supported by Warsaw University of Technology, Faculty of
Chemistry. We express our special gratitude to Professor Jan Plen-
kiewicz and Dr. Sergiusz Dzierzgowski for helpful discussions and
collaborative support while writing this work.
6. Di Nunno, L.; Franchini, C.; Nacci, A.; Scilimati, A.; Sinicropi, M. S. Tetrahedron:
Asymmetry 1999, 10, 1913e1926.
7. D’Amico, J. J.; Campbell, R. H.; Guinn, E. C. J. Org. Chem. 1964, 29, 865e869.
8. Chen, C. S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J. J. Am. Chem. Soc. 1982, 104,
7294e7299.
9. (a) Janssen, A. E. M.; Hailing, P. J. J. Am. Chem. Soc. 1994, 116, 9827e9830; (b)
Carrea, G.; Ottolina, G.; Riva, S. Trends Biotechnol. 1995, 13, 63e70.
10. Seco, J. M.; Quinoa, E.; Riguera, R. Tetrahedron: Asymmetry 2001, 12, 2915e2925.
11. Kazlauskas, R. J.; Weissfloch, A. N. E.; Rappaport, A. T.; Cuccia, L. A. J. Org. Chem.
1991, 56, 2656e2665.
Please cite this article in press as: Borowiecki, P.; et al., Tetrahedron (2013), http://dx.doi.org/10.1016/j.tet.2013.04.014