Biocatalytic Reduction of 2-Monosubstituted 3-Thiazolines Using Imine Reductases

Article abstract of DOI:10.1002/jhet.3437

The application of a straightforward biocatalytic technology for the reduction of racemic 2-monosubstituted 3-thiazolines, which are easily prepared via Asinger-multicomponent reaction, is reported. The biocatalytic reduction yields racemic 2-monosubstituted 3-thiazolidines, which are difficult to be prepared by means of classic chemical routes, in moderate to high yields. Moreover, our study clarifies the stereochemical reaction course of the biocatalytic reduction. Furthermore, the efficiency of this biocatalytic technology is demonstrated in an experiment at an elevated substrate concentration of 60?mM leading to 96% conversion.

Full text of DOI:10.1002/jhet.3437

Month 2019
Biocatalytic Reduction of 2-Monosubstituted 3-Thiazolines Using
Imine Reductases
*
Karla Wagner, Nils Weißing, and Harald Gröger
Nadine Zumbrägel,
Chair of Organic Chemistry I, Faculty of Chemistry, Bielefeld University, Universitätsstrasse 25, 33615 Bielefeld,
Germany
*E-mail: harald.groeger@uni-bielefeld.de
Received August 16, 2018
DOI 10.1002/jhet.3437
Published online 00 Month 2019 in Wiley Online Library (wileyonlinelibrary.com).
The application of a straightforward biocatalytic technology for the reduction of racemic 2-
monosubstituted 3-thiazolines, which are easily prepared via Asinger-multicomponent reaction, is reported.
The biocatalytic reduction yields racemic 2-monosubstituted 3-thiazolidines, which are difficult to be pre-
pared by means of classic chemical routes, in moderate to high yields. Moreover, our study clarifies the ste-
reochemical reaction course of the biocatalytic reduction. Furthermore, the efficiency of this biocatalytic
technology is demonstrated in an experiment at an elevated substrate concentration of 60 mM leading to
96% conversion.
J. Heterocyclic Chem., 00, 00 (2019).
INTRODUCTION
and non-selective conversions are conceivable options.
Notably, both options can be synthetically useful for
various purposes. In the following, we report an approach
towards a straightforward synthesis of 2-monosubstituted
3-thiazolidines. Moreover, our study gives an insight into
the clarification of the stereochemical research issue
towards recognition of the absolute configuration of the
stereogenic center in 2-position.
The 3-thiazolidine scaffold represents an important
heterocyclic structural motif in both medicinal chemistry
and agrochemistry [1–5]. Furthermore, this heterocycle
is widely present in natural products and is a key
structure in penicillins as presumably most prominent
example. For their preparation, a range of methods are
conceivable. As 3-thiazolines are readily accessible via
Asinger-synthesis starting from cheap commodity
chemicals [6–9], their reduction appears as an attractive
retrosynthetic route (Scheme 1). For decades, however,
the realization of such an approach remained a challenge
because numerous established methods for C¼N double
bond reduction failed due to, for example, undesired side-
reactions and catalyst deactivation by sulfur [9–12]. Very
recently, we could demonstrate that these hurdles could be
overcome when applying enzyme catalysis. In detail, 3-
thiazolines have been efficiently converted into the
corresponding 3-thiazolidines when utilizing imine
reductases (IREDs) as biocatalysts and glucose as reducing
agent in combination with a glucose dehydrogenase
(GDH) for in situ regeneration of the cofactor NADPH
[12]. Stereochemical aspects have been studied as well and
when starting from prochiral 3-thiazolines as substrates,
high enantioselectivities are obtained [12].
RESULTS AND DISCUSSION
In the first step, two 2-monosubstituted 3-thiazolines
serving as model substrates for this class of compounds
were prepared through modified Asinger-synthesis
starting from preformed 2-chloroisobutyraldehyde,
sodium
hydrosulfide,
ammonia,
and
either
isobutyraldehyde or pivaldehyde. Following a literature-
known procedure [8,11,13], the 2-monosubstituted 3-
thiazolines rac-1a and rac-1b bearing either an isopropyl
or a tert-butyl substituent were obtained in yields of 63%
and 89%, respectively (Scheme 2). In the next step, the
racemic 3-thiazolidines (rac-2a–b) were prepared by
chemical reduction using catecholborane (HBcat) as
reference compounds for analytical purpose (Scheme 2).
In accordance with previous work [11,12], low yields were
obtained for rac-2a–b (43% and 31%), thus underlining
the difficulty for the selective reduction of 3-thiazolines 1
with established reducing agents such as metal hydrides.
With the starting materials and reference compounds in
hand, analytical methods for determining the conversion
A remaining open question, however, is related to the
reduction of 2-monosubstituted 3-thiazolines of type 1
(Scheme 1). As such molecules are racemic when being
prepared via Asinger-synthesis, in the presence of
enzymes as chiral catalysts both enantiomeric recognition
© 2019 Wiley Periodicals, Inc.

N. Zumbrägel, K. Wagner, N. Weißing, and H. Gröger
Vol 000
Scheme 1. Conceptual approaches towards 3-thiazolidines 2 by combination of Asinger-synthesis and enzymatic C¼N reduction using imine reductases.
[Color figure can be viewed at wileyonlinelibrary.com]
Scheme 2. Synthesis of 2-monosubstituted 3-thiazolines 1 and their corresponding 3-thiazolidine derivatives 2 via Asinger-synthesis and subsequent
reduction. [Color figure can be viewed at wileyonlinelibrary.com]
(by achiral GC) and enantioselectivity (by chiral SFC-
HPLC) were established (see Experimental section).
Attracted by the recent successful utilization of the IRED
from Mycobacterium smegmatis [14] for the reduction of
a range of prochiral 3-thiazolines [12], we next conducted
synthetic experiments at substrate concentrations of
20 mM rac-1a and rac-1b in order to get an insight into
the suitability of this IRED to form the desired products.
Towards this end, we used a tailor-made recombinant
whole-cell biocatalyst [12], which contains the IRED
from M. smegmatis and the GDH from Bacillus subtilis
for in situ recycling of the cofactor NADPH (utilized in
catalytic amount) using D-glucose as a co-substrate
(Table 1). We were pleased to find that the racemic 3-
thiazolines rac-1a and rac-1b were converted to the
corresponding 3-thiazolidines 2 in the presence of the
whole-cell catalyst (7.5 mg/mL lyophylized whole-cells)
after 24 h of reaction time at 30°C. Even under non-
optimized reaction conditions rac-1a was converted with
a good conversion of 77%, whereas conversion of rac-1b
was lower with 21% under the same reaction
conditions (Table 1). We assume that the lower
conversion towards reduction of rac-1b is due to the
more sterically demanding tert-butyl substituent at R1
compared with the isopropyl substituent.
enantioselective reaction course are of preparative interest
because in the first case, a racemic resolution is
conceivable (albeit at a maximum yield of 50% for each
enantiomer), whereas the latter case enables an elegant
route with (theoretically) full conversion to racemic 3-
thiazolidines 2 (which are difficult to form by means of
other routes). Utilizing chiral supercritical fluid
chromatography-high performance liquid chromatography
(SFC-HPLC), we analyzed products 2a and 2b after
derivatization and found that both enantiomers are present
to the same extent and the enantiomeric excess is therefore
0% ee (Table 1). However, it should be added that
obtaining a racemate does not necessarily mean a
non-enantioselective reduction as the stereogenic center is
an acetal-type carbon which might be prone to
racemization in particular in case of the saturated 3-
thiazolidines (e.g., due to reversible ring cleavage and
formation). Although taking into account the neutral
reaction conditions such an effect is unlikely, we wanted
to clarify this issue. Therefore, the enantiomeric excess
of the remaining starting material had to be determined.
Because it was not possible to separate the enantiomers of
rac-1a and rac-1b by means of chiral GC, we performed a
biotransformation of rac-1b at an elevated substrate
concentration of 60 mM, using 5 mg/mL of IRED
cell crude extract from M. smegmatis and stopped the
biotransformation at a low conversion of 6%. After
separation of the remaining starting material and product
by flash column chromatography, the remaining starting
material was used as starting material in a non-
After this proof of concept for the usefulness of IREDs to
synthesize 2-substituted 3-thiazolidines 2 through reduction
of the corresponding 3-thiazolines, we wanted to get an
insight into the stereoselectivity of this biocatalytic
reduction. In principle, both an enantioselective and a non-
Journal of Heterocyclic Chemistry
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Biocatalytic Reduction of 2-Monosubstituted 3-Thiazolines
Table 1
Initial biotransformation of 2-monosubstituted 3-thiazolines rac-1a–b.
Entry
Substrate
Conversion [%]
77
ee of product [%]
1
0
2
21
0
Scheme 3. Biotransformations and subsequent chemical reductions of remaining starting material 1b for clarification of the stereochemical reaction
course. [Color figure can be viewed at wileyonlinelibrary.com]
Journal of Heterocyclic Chemistry
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N. Zumbrägel, K. Wagner, N. Weißing, and H. Gröger
Vol 000
Scheme 4. Process development and optimized biotransformation for the racemic 3-thiazoline rac-1a. [Color figure can be viewed at wileyonlinelibrary.
com]
enantioselective chemical reduction using catecholborane.
The corresponding 3-thiazolidine 2b was then analyzed by
means of chiral SFC-HPLC, after derivatization with
phenylisocyanate (Scheme 3). As a result, we found the
same ratio of both enantiomers of 2b, corresponding to an
enantiomeric excess of 0% ee. This result indicates that
the IRED from M. smegmatis catalyzes the reduction of
rac-2b in a non-enantioselective fashion regarding the
chiral center at C2, allowing a (theoretically) full
conversion towards the racemic 3-thiazolidine due to the
recognition of both enantiomers of the substrate rac-1b.
Thus, we assume that this stereochemical behavior is
general also when using other 2-substituted 3-thiazolines
in the presence of the IRED from M. smegmatis.
After having characterized the enzymatic reaction in terms
of activity and conversion as well as enantioselectivity, we
were further interested to extend this reaction towards an
efficient synthetic transformation. Thus, an initial
biotransformation experiment at an elevated substrate
concentration of 60 mM rac-1a was carried out (Scheme 4).
As a biocatalyst, again the tailor-made recombinant whole
cell biocatalyst [12] containing both required enzymes
(IRED and GDH) in overexpressed form was utilized. After
65 h of reaction time at 30°C, we were pleased to find that
the reduction of rac-1a proceeds smoothly, leading to the
formation of the desired 3-thiazolidine rac-2a with an
excellent conversion of 96% (Scheme 4).
and formation of the desired 3-thiazolidine products as a
racemate. In addition, the efficiency of this type of
biotransformation has been demonstrated by means of an
experiment at a 10 mL scale and at a substrate
concentration of 60 mM, leading to the formation of the
desired product with 96% conversion.
EXPERIMENTAL
General information.
The commercially available
reagents (Acros, Alfa Aesar, Merck, VWR, Acros, Fisher
Scientific, TCI) were used without further purification.
Solvents were used in high-grade purity or purified prior
to use. NMR spectra were recorded on Bruker Advance
III 500 or Bruker Advance III 500HD at a frequency of
500 MHz (1H) or 126 MHz (13C). The chemical shift δ is
given in ppm and referenced to the corresponding solvent
signal (CDCl3). Coupling constants (J) are given in Hz.
Nano-ESI mass spectra were recorded using an Esquire
3000 ion trap mass spectrometer (Bruker Daltonik
GmbH, Bremen, Germany) equipped with a standard
nano-ESI source. Samples were introduced by static
nano-ESI using in-house pulled glass emitters. Nitrogen
served both as the nebulizer gas and the dry gas. Nitrogen
was generated by a Bruker nitrogen generator NGM 11.
Helium served as cooling gas for the ion trap and collision
gas for MSn experiments. HRMS-ESI mass spectra are
recorded using an Agilent 6220 time-of-flight mass
spectrometer (Agilent Technologies, Santa Clara, CA,
USA) in extended dynamic range mode equipped with a
Dual-ESI source, operating with a spray voltage of
2.5 kV. Nitrogen served both as the nebulizer gas and the
dry gas. Nitrogen was generated by a nitrogen generator
NGM 11. Samples are introduced with a 1200 HPLC sys-
tem consisting of an autosampler, degasser, binary pump,
column oven, and diode array detector (Agilent Technolo-
gies, Santa Clara, CA, USA) using a C18 Hypersil Gold
column (length: 50 mm, diameter: 2.1 mm, particle size:
CONCLUSION
The successful application of a biocatalytic reduction
technology using IREDs towards the reduction of
racemic 2-monosubstituted 3-thiazolines is reported,
yielding the corresponding 3-thiazolidines in moderate to
high conversions. Furthermore, an insight into the
stereochemical reaction course is provided revealing the
absence of an enantiopreference of the biocatalyst for one
of the enantiomers of the 3-thiazoline substrates of type 1
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Month 2019
Biocatalytic Reduction of 2-Monosubstituted 3-Thiazolines
[ppm] 195.32 (C1), 69.64 (C2(CH3)2), 26.20
1.9 μm) with a short gradient (in 4 min from 0% B to 98%
B, back to 0% B in 0.2 min, total run time 7.5 min) at a
flow rate of 250 μL/min and column oven temperature of
40°C. HPLC solvent A consists of 94.9% water, 5% aceto-
nitrile and 0.1% formic acid, solvent B of 5% water, 94.9%
acetonitrile and 0.1% formic acid. The mass axis was
externally calibrated with ESI-L Tuning Mix (Agilent
Technologies, Santa Clara, CA, USA) as calibration
standard. EI mass spectra were recorded using an Autospec
X magnetic sector mass spectrometer with EBE geometry
(Vacuum Generators, Manchester, UK) equipped with a
standard EI source. Samples were introduced by push rod
in aluminum crucibles if not otherwise noted. Ions were
accelerated by 8 kV in EI mode. Flash column chromatog-
raphy was performed using Biotage “Isolera One” flash
chromatography system with cyclohexane/ethyl acetate
mixtures.
=
(C2(CH3)2). MS (EI) m/z calculated for C4H7ClO [M]+:
106.02, found: 106.0. The analytical data correspond with
literature data [12].
General procedure 1 for the synthesis of 3-thiazolines
(according to references [11,13]).
Sodium hydrosulfide
monohydrate (0.10 mol), ammonia-solution (13.3 M,
22.5 mL), and aldehyde (0.10 mol) are mixed and cooled
to 0°C. α-Chlorinated aldehyde (0.10 mol) is dissolved in
dichloromethane (100 mL) and added to the yellow
mixture, keeping the temperature under 10°C. The
reaction mixture is stirred overnight at room temperature,
phases are separated, and the aqueous phase is extracted
with dichloromethane (3 × 30 mL). The combined
organic phases are dried over magnesium sulfate; the
solvent is evaporated in vacuo; and the crude product is
purified by flash column chromatography.
rac-2-Isopropyl-5,5-dimethyl-3-thiazoline (rac-1a).
Recombinant expression of imine reductases.
The
rac-1a is prepared according to general procedure 1 using
sodium hydrosulfide monohydrate (14.8 g, 200 mmol),
ammonia-solution (13.3 M, 30 mL), and isobutanal
(18.2 mL, 200 mmol), yielding rac-1a (19.8 g,
126 mmol, 63%) as colorless oil after flash column
chromatography (5–28% ethyl acetate in cyclohexane).
1H NMR (500 MHz, CDCl3): δ [ppm] = 0.93 (d,
J = 6.7 Hz, 3H, C2-CH(CH3)2), 1.02 (d, J = 6.8 Hz, 3H,
C2-CH(CH3)2), 1.52 (s, 3H, C5-(CH3)2), 1.53 (s, 3H,
C5-(CH3)2), 2.13–2.09 (m, 1H, C2-CH(CH3)2), 5.62–5.61
(m, 1H, C2-H), 7.03 (d, J = 2.6 Hz 1H, C4-H). 13C
NMR (126 MHz, CDCl3): δ [ppm] = 18.3 (C2-CH
(CH3)2), 19.9 (C2-CH(CH3)2), 29.3 (C5-(CH3)2), 29.7
(C5-(CH3)2), 34.8 (C2-CH), 63.5 (C5), 90.8 (C2), 168.4
(C4). HRMS (ESI) m/z calculated for C8H16NS
[M + H]+: 158.0998, found: 158.0994. IR (neat) [cmꢀ1]:
2958, 2924, 2870, 1650, 1362.
IREDs from M. smegmatis [14] is recombinantly
expressed in Escherichia coli (E. coli) BL21(DE3),
according to a literature-known procedure [12]. A
preculture (10 mL LB medium, containing 100 μg/mL of
carbenicillin) of E. coli BL21(DE3) carrying the
recombinant plasmid is cultivated overnight at 37°C. The
main culture (300 mL TB medium, containing
100 μg/mL of carbenicillin) is inoculated with the
preculture to a final concentration of 1%. The production
of recombinant protein is induced by addition of isopropyl-
β-D-thiogalactopyranoside
(IPTG) (final concentration
0.5 mM) at an OD600 between 0.4 and 0.6. Cultures are
shaken at 25°C for 20 h and 140 rpm and harvested by
centrifugation at 4000 g and 4°C for 30 min. Cell pellets
are stored at ꢀ20°C. Cell disruption is performed after
resuspension of the cells (3 mL 50 mM KPi pH 7 per gram
wet cell weight) by sonification (Bandelin Sonopuls HD
2070) with 3 × 120 s burst (5 cycles, 20% energy) and
120 s intervals and both steps being carried out on ice.
After centrifugation at 21,000 g for 30 min at 4°C, the
crude extract is obtained as supernatant. The protein
concentration is determined via the Bradford assay against
BSA as a concentration standard.
rac-2-(tert-Butyl)-5,5-dimethyl-3-thiazoline (rac-1b).
rac-1b is prepared according to general procedure 1
using sodium hydrosulfide monohydrate (11.1 g,
0.10 mol), ammonia-solution (13.3 M, 22.5 mL), and
2,2-dimethylpropanal (16.3 mL, 100 mmol), yielding
rac-1b (15.3 g, 89.0 mmol, 89%) as colorless oil after
flash column chromatography (5–40% ethyl acetate in
Construction and preparation of whole cell catalyst. The
whole cell catalyst is constructed and prepared according
to reference [12].
cyclohexane). 1H NMR (500 MHz, CDCl3):
δ
[ppm] = 0.99 (s, 9H, C2-C(CH3)3), 1.52 (s, 3H,
C5-(CH3)2), 1.53 (s, 3H, C5-(CH3)2), 5.56 (d, J = 2.7 Hz,
1H, C2-H), 7.03 (d, J = 2.7 Hz, 1H, C4-H). 13C NMR
(126 MHz, CDCl3): δ [ppm] = 26.7 (C2-C (CH3)3), 29.0
(C5-(CH3)2), 29.7 (C5-(CH3)2), 36.1 (C2-C(CH3)3), 63.5
(C5), 95.2 (C2), 168.5 (C4). MS (ESI) m/z calculated for
C9H18NS [M + H]+: 171.11, found: 172.00. The analytical
data correspond with literature data [15].
2-Chloro-2-methylpropanal (according to [12]). Sulfuryl
chloride (41.0 mL, 0.50 mol) is added under cooling at a
vacuum of 900 mbar to isobutanal (46.0 mL, 0.50 mol),
keeping the temperature stable at 40°C. The reaction
mixture is stirred afterwards for 2 h at 45°C and
900 mbar. 2-Chloro-2-methylpropanal (44.4 g, 0.42 mol,
83%) is obtained as colorless liquid by fractional
1
distillation (boiling point 90°C). H NMR (500 MHz,
General procedure 2 for the reduction of 3-thiazolines
CDCl3): δ [ppm] = 9.43 (s, 1H, C1-H), 1.64 (s, 6H,
using catecholborane (according to [11]).
(15.0 mmol) is dissolved in toluene (60 mL), and
3-Thiazoline
C2(CH3)2). 13C NMR (126 MHz, CHCl3):
δ
Journal of Heterocyclic Chemistry
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N. Zumbrägel, K. Wagner, N. Weißing, and H. Gröger
Vol 000
catecholborane (45.0 mmol), dissolved in toluene (15 mL),
is added slowly under argon atmosphere at room
temperature. The reaction mixture is stirred at room
temperature for 48 h; distilled water (dH2O) (60 mL) is
carefully added; the phases are separated; and the organic
phase is extracted with sodium hydroxide solution (2 M,
3×). The combined organic phases are washed with brine,
dried over magnesium sulfate, and the solvent is
evaporated in vacuo. The crude product is purified via
column chromatography.
program: 40–200°C, 10°C/min. tR (rac-1a): 5.5 min; tR
(rac-2a): 6.1 min; tR (rac-1b): 6.0 min; tR (rac-2b):
6.6 min.
General procedure 3 for derivatization of 3-thiazolidines
(according to [11,12]).
3-Thiazolidine (1.25 mmol) is
dissolved in diethylether (10 mL), phenylisocyanate
(1.30 mmol), and cyclohexane (5 mL) are added, and the
reaction mixture is stirred overnight while the solvent is
evaporated in the meantime. The white solid is dried
in vacuo.
rac-2-Isopropyl-5,5-dimethyl-N-phenylthiazolidine-3-
carboxamide (rac-3a). Preparation according to general
rac-2-Isopropyl-5,5-dimethyl-3-thiazolidine (rac-2a).
rac-2a is prepared according to general procedure 2
using rac-1a (2.00 g, 12.7 mmol). rac-2a (0.94 g,
5.43 mmol, 43%) is obtained as colorless oil after
purification via flash column chromatography (5–40%
ethyl acetate in cyclohexane). 1H NMR (500 MHz,
CDCl3): δ [ppm] = 1.00 (d, J = 6.7 Hz, 3H, C2-CH
(CH3)2), 1.05 (d, J = 6.7 Hz, 3H, C2-CH(CH3)2), 1.36
(s, 3H, C5-(CH3)2), 1.48 (s, 3H, C5-(CH3)2), 1.86–1.93
(m, 1H, C2-CH(CH3)2), 2.77 (d, J = 12.3 Hz, 1H,
C4-H2), 3.02 (d, J = 12.3 Hz, 1H, C4-H2), 4.51
(d, J = 7.2 Hz, 1H, C2-H). 13C NMR (126 MHz,
CHCl3): δ [ppm] = 20.8 (C2-CH(CH3)2), 21.2 (C2-CH
(CH3)2), 29.9 (C5-(CH3)2), 32.5 (C5-(CH3)2), 35.1 (C2-
CH (CH3)2), 57.1 (C5), 66.6 (C4), 80.9 (C2). HRMS
(ESI) m/z calculated for C8H18NS [M + H]+: 160.1155,
found: 160.1157. IR (neat) [cmꢀ1]: 2954, 2921, 2863,
1453, 1364.
rac-2-(tert-Butyl)-5,5-dimethyl-3-thiazolidine (rac-2b).
rac-2b is prepared according to general procedure 2
using rac-1b (2.51 g, 15.0 mmol), yielding rac-2b
(0.80 g, 4.62 mmol, 31%) as colorless oil after
purification via flash column chromatography (5–40%
ethyl acetate in cyclohexane). 1H NMR (500 MHz,
CDCl3): δ [ppm] = 1.02 (s, 9H, (C2-C(CH3)3), 1.32 (s,
3H, C5-(CH3)2), 1.48 (s, 3H, C5-(CH3)2), 2.77 (d,
J = 12.2 Hz, 1H, C4-H2), 3.03 (d, J = 12.2 Hz, 1H,
C4-H2), 4.63 (s, 1H, C2-H).13C NMR (126 MHz,
CHCl3): δ [ppm] = 27.2 (C2-C(CH3)3), 29.2 (C5-
(CH3)2), 31.7 (C5-(CH3)2), 34.5 (C2-C(CH3)3), 56.1
(C5), 66.4 (C4), 84.3 (C2). HRMS (ESI) m/z calculated
for C9H20NS [M + H]+: 174.1311, found: 174.1306. IR
(neat) [cmꢀ1]: 2952, 2924, 2863, 1454, 1363.
procedure 3 using rac-2a (50.0 mg, 0.29 mmol), yielding
rac-2-isopropyl-5,5-dimethyl-N-phenylthiazolidine-3-
carboxamide (80.0 mg, 0.29 mmol, 99%) as colorless
solid. 1H NMR (500 MHz, CDCl3): δ [ppm] = 0.98
(d, J = 4.4 Hz, 3H, C2-C(CH3)2), 0.99 (d, J = 4.4 Hz,
3H, C2-C(CH3)2), 1.41 (s, 3H, C5-(CH3)2), 1.43 (s, 3H,
C5-(CH3)2), 2.19–2.26 (m, C2-CH(CH3)2), 3.26
(d, J = 11.2 Hz, 1H, C4-H2), 4.04 (d, J = 11.2 Hz, 1H,
C4-H2), 5.30 (d, J = 6.6 Hz, 1H, C2-H), 7.04–7.07
(m, 1H, Ar–H), 7.28–7.30 (m, 2H, Ar–H), 7.35–7.37 (m,
2H, Ar–H). 13C NMR (126 MHz, CHCl3):
δ
[ppm] = 17.6 (C2-CH(CH3)2), 20.1 (C2-CH(CH3)2), 26.5
(C5-(CH3)2), 27.2 (C5-(CH3)2), 34.7 (C2-CH(CH3)2),
52.4 (C5), 62.1 (C4), 70.5 (C2), 120.2 (Ar–C), 123.7
(Ar–C), 129.3 (Ar–C), 138.9 (Ar–C), 154.6 (C¼O).
HRMS (ESI) m/z calculated for C15H23N2OS [M + H]+:
279.1526, found: 279.1521. IR (neat) [cmꢀ1]: 2958,
2917, 1640, 1442, 1376. Tm: 164°C.
rac-2-(tert-Butyl)-5,5-dimethyl-N-phenylthiazolidine-3-
carboxamide (rac-3b). Preparation according to general
procedure 3 using rac-2a (104 mg, 0.58 mmol), yielding
rac-2-(tert-butyl)-5,5-dimethyl-N-phenylthiazolidine-3-
carboxamide (169 mg, 0.578 mmol, 98%) as colorless
1
solid. H NMR (500 MHz, CDCl3): δ [ppm] = 1.02 (s,
9H, C2-C(CH3)3), 1.37 (s, 3H, C5-(CH3)2), 1.47 (s, 3H,
C5-(CH3)2), 3.24 (d, J = 11.7 Hz, 1H, C4-H2), 4.02 (d,
J = 11.7 Hz, 1H, C4-H2), 5.48 (s, 1H, C2-H), 7.05–7.07
(m, 1H, Ar–H), 7.28–7.35 (m, 4H, Ar–H). 13C NMR
(126 MHz, CHCl3): δ [ppm] = 26.1 (C5-(CH3)2), 26.8
(C2-C(CH3)3), 31.1 (C5-(CH3)2), 38.8 (C2-C(CH3)3),
53.2 (C5), 63.5 (C4), 73.7 (C2), 120.3 (Ar–C), 123.8
(Ar–C), 129.4 (Ar–C), 139.0 (Ar–C), 156.4 (C¼O).
HRMS (EI) m/z calculated for C16H25N2OS [M + H]+:
293.1682, found: 293.1676. IR (neat) [cmꢀ1]: 3307,
2968, 2949, 1750, 1632, 1539, 1520. Tm: 162°C.
Achiral
gas
chromatography
analysis.
Gas
chromatography analysis (determination of the conversions
for the biotransformations of 3-thiazolines [rac-1a–b] to
the corresponding 3-thiazolidines [rac-2a–b]) is carried
out using the gas chromatograph system GC-2010 Plus
(Shimadzu, Kyoto, Japan) equipped with ZB-5MSi
column (Phenomenex, 30 m × 0.25 mm × 0.25 μm; N2;
linear velocity 46.9 cm/s split mode 1:10; total flow
28.8 mL/min; purge flow 3.0 mL/min; column flow
2.34 mL/min; pressure 140.4 kPa) and coupled to an
AOC-20i/s auto injector/auto sampler. Temperature
Chiral
supercritical
fluid
chromatography-high
performance liquid chromatography analysis.
Chiral
SFC-HPLC analysis (for determination of the
enantiomeric excess of rac-2a and rac-2b after
derivatization with phenylisocyanate to rac-3a/rac-3b) is
performed using the LC2000 SFC-HPLC system from
Jasco (Easton, USA) with the HPLC column Chiralpak
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2019
Biocatalytic Reduction of 2-Monosubstituted 3-Thiazolines
IC from Daicel (Tokyo, Japan) (supercritical CO2:EtOH
(Et2NH) = 90:10 (0.01), 1 mL/min, 20°C, 10 MPa,
210 nm). Enantiomeric excess is determined based on
area% of the enantiomers. tR (rac-3a): 9.5 min, 12.9 min;
tR (rac-3b): 8.6 min, 10.2 min.
25 mg/mL of lyophilized whole cell catalyst (which is
prepared from a 50 mg/mL of cell suspension in 200 mM
KPi buffer pH 7), 140 mM of D-glucose, 0.1 mM of
NADP+, and 2% of methanol as co-solvent in distilled
water. The reaction mixture is stirred at 30°C for 65 h.
The reaction is stopped by adding 1 mL of 32% NaOH
solution and 20 mL of dichloromethane. Phase separation
is promoted by centrifugation. The organic phase is dried
over magnesium sulfate. The conversions are determined
by analyzing the organic phase by achiral GC, and the
enantiomeric excess is determined by means of chiral
SFC-HPLC, after derivatization of the samples with
phenylisocyanate.
General
procedure
for
biotransformations
of
2-monosubstituted 3-thiazolines.
Biotransformations of
2-monosubstituted 3-thiazolines rac-1a–b are performed
on a 10 mL scale at 30°C and 1000 rpm in 100 mM KPi
buffer pH 7, containing 2% of methanol as co-solvent,
40 mM D-glucose, 20 mM rac-1a–b, 7.5 mg/mL of
lyophilized whole cell catalyst (which is prepared from a
14 mg/mL of cell suspension in 200 mM KPi buffer
pH 7) and 0.1 mM NADP+. After 24 h, the reaction is
stopped by adding 1 mL of 32% NaOH solution and
10 mL of dichloromethane. Phase separation is promoted
by centrifugation. The conversions are determined by
analyzing the organic phase by achiral GC, and the
enantiomeric excess is determined by means of chiral
SFC-HPLC, after derivatization of the samples with
phenylisocyanate according to general procedure 3.
Acknowledgments. We gratefully acknowledge generous
support from the German Federal Ministry of Education and
Research (Bundesministerium für Bildung und Forschung,
BMBF) within the project “Biotechnologie 2020+, Nächste
Generation biotechnologischer Verfahren” (grant number
031A184A). The authors also thank Dr. Hans Iding and Dr.
Dennis Wetzl for providing us with the plasmids encoding for
the imine reductases.
Biotransformation of rac-1b and chemical reduction for
clarification of the stereochemical reaction course.
The
biotransformation for the clarification of the
sterochemical reaction course (10 mL) is performed from
a 60 mM concentration of rac-1b, 5.7 mg/mL of IRED
crude extract from M. smegmatis, 280 U of GDH,
120 mM of D-glucose, 0.1 mM of NADP+, and 2% of
methanol as co-solvent in distilled water. The reaction
mixture is stirred at 30°C for 23.5 h. The reaction is
stopped by adding 1 mL of 32% NaOH solution and
10 mL of dichloromethane. Phase separation is promoted
by centrifugation. The organic phase is dried over
magnesium sulfate. The conversions are determined by
analyzing the organic phase by achiral GC. The solvent is
evaporated in vacuo, product and remaining starting
material rac-1b are separated by flash column
chromatography (5–40% ethyl acetate in cyclohexane).
The isolated, remaining rac-1b is reduced using
catecholborane via general procedure 2 (24 h reaction
time in this case). The corresponding 3-thiazolidine rac-
2b is purified via preparative layer chromatography
and derivatized according to general procedure 3.
The corresponding colorless solid is dissolved in
dichloromethane and analyzed by means of chiral SFC-
HPLC.
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Semi-preparative scale biotransformation of rac-1a. The
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet