A practical high-throughput screening system for enantioselectivity by using FTIR spectroscopy

Article abstract of DOI:10.1002/chem.200304885

For the first time FTIR spectroscopy has been applied to the measurement of enantiomeric purity. The underlying concept is based on the use of pseudo-enantiomers that are ¹³C-labeled at appropriate positions. Upon applying Lambert-Beer's law in

Full text of DOI:10.1002/chem.200304885

FULL PAPER
A Practical High-Throughput Screening System for Enantioselectivity by
Using FTIR Spectroscopy
Patrick Tielmann,[a] Matthias Boese,[b] Martin Luft,[b] and Manfred T. Reetz*[a]
Dedicated to Professor Reinhard W. Hoffmann on the occasion of his 70th birthday
Abstract: For the first time FTIR
spectroscopy has been applied to the
measurement of enantiomeric purity.
The underlying concept is based on the
use of pseudo-enantiomers that are 13C-
labeled at appropriate positions. Upon
applying Lambert Beer×s law in the
determination of the concentrations of
both enantiomers, the ee values are
accessible, accuracy to within Æ5% of
the true values being possible. The
application of a commercially available
high-throughput FTIR system results in
a slightly decreased accuracy (Æ7% for
the ee values), but this allows a through-
put of up to 10000 samples per day. The
method is of interest in the area of
combinatorial asymmetric catalysis and
directed evolution of enantioselective
enzymes.
Keywords: asymmetric catalysis
combinatorial chemistry ¥ directed
evolution high-throughput
screening ¥ IR spectroscopy
¥
¥
using high-throughput ee-screening systems. This fundamen-
tally new approach to the creation of asymmetric catalysts is
based on the combination of appropriate molecular biological
methods for random gene mutagenesis and protein expression
coupled with screening systems for the determination of
thousands of ee values in a short period of time. Last but not
least the opportunities that metagenome DNA panning[7]
offers to organic chemists interested in asymmetric catalysis
also depend on fast and precise ee-screening systems. In this
regard it has been estimated that more than 99% of all
enzymes occurring in nature are still unexplored.[7] It can be
estimated that an enormous biodiversity can be investigated
by collecting genes in the environment and expressing the
corresponding enzymes in recombinant microorganisms. Fig-
ure 1 summarizes these three main approaches to the creation
of large libraries of enantioselective catalysts.
We and others have previously developed various high-
throughput screening systems for enantioselectivity which are
in most cases complementary. For example, ee assays based on
UV/Vis spectroscopy,[8] IR thermography,[9] capillary array
electrophoresis,[10] and even special forms of GC[11] were
devised in our laboratory, allowing between 700 and 20000 ee
determinations per day. The accuracy in the ee value ranges
between Æ2% and Æ5%. Further ee-screening systems were
developed by other groups by applying pH indicators or
fluorescence,[12] circular dichroism,[13] enzymatic methods,[14]
DNA arrays,[15] immunoassays,[16] or mass spectrometry of
mass-tagged diastereomeric substrates.[17] In most cases a
precision of Æ10% was reported, which suffices in some, but
not all, cases.
Introduction
The catalytic synthesis of enantiomerically pure or enriched
organic compounds is of great interest to industry and
academia. Two major options for reaching this goal are
available, namely synthetic catalysts such as transition-metal
complexes[1] or biocatalysts such as enzymes[2] or catalytic
antibodies.[3] In the former case a lot of trial-and-error is still
needed despite rational approaches such as ligand design by
molecular modeling. Recently, an alternative approach has
been developed which makes use of combinatorial methods in
the parallel synthesis of large numbers of chiral homogeneous
catalysts.[4, 5] Nevertheless, the actual size of the libraries has
been limited so far to less then 50 100 catalysts. Intriguing
opportunities appear possible if larger libraries could be
generated and screened. The challenges in this area of
research include the development of new strategies for the
synthesis of modular ligands and the development of high-
throughput screening systems for the rapid determination of
enantioselectivity.[5] In addition to initial progress in this field,
which has been loosely called ™combinatorial asymmetric
catalysis∫, recent research in the area of directed evolution
already has provided large libraries of potentially enantiose-
lective biocatalysts[6] which likewise need to be evaluated by
[a] Prof. Dr. M. T. Reetz, Dr. P. Tielmann
Max-Planck-Institut f¸r Kohlenforschung
Kaiser-Wilhelm-Platz 1, 45470 M¸lheim/Ruhr (Germany)
Fax : (‡49)-208-306 2985
E-mail: reetz@mpi-muelheim.mpg.de
[b] Dr. M. Boese, M. Luft
In addition to these methods we have also developed a new
and convenient approach for measuring ee values based on
Bruker Optik GmbH
Rudolf-Plank-Strasse 23, 76275 Ettlingen (Germany)
3882
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/chem.200304885
Chem. Eur. J. 2003, 9, 3882 3887

3882 3887
precision (Æ2 3%).[19] For this
purpose flow-through NMR
systems need to be used, these
being commercially available.
We also developed a second
NMR-based ee-screening sys-
tem that also makes use of
flow-through NMR systems,
derivatization by a chiral re-
agent being necessary in this
case. Precision amounts to
Æ5% of the true ee value.[19]
We now wish to present yet
Figure 1. Sources of large libraries of potentially enantioselective catalysts.
another high-throughput ee-
screening system based on the
same general principle, but in
this case using FTIR spectroscopy. Appropriate isotopic
labeling of one enantiomer of a given (R)/(S) pair leads to
pseudo-enantiomers which in a mixture can be distinguished
by FTIR spectroscopy. As will be seen, this allows the
determination of as many as 10000 ee values per day with a
good degree of accuracy (Æ7%). Since FTIR spectroscopy is
a cheap and readily available analytical technique that is
available in almost all laboratories, we expect this method to
become frequently applied. It is applicable to the evaluation
of large libraries of synthetic chiral catalysts. Moreover, it is of
particular interest in the analysis of biocatalytic reactions
because the ee values can be measured directly in culture
supernatants, making a time-consuming and expensive work-
up unnecessary.
isotopic labeling which leads to so called pseudo-enantiomers
and pseudo-meso-compounds (Scheme 1). The underlying
principle is restricted to the desymmetrization of prochiral
compounds bearing reactive enantiotopic groups and to the
kinetic resolution of chiral coumpounds.
Results and Discussion
To evaluate the applicability of FTIR spectroscopy for the
determination of ee values for a given substrate, especially
with regard to accuracy, the ™best∫ position at which isotopes
are introduced needs to be determined. To illustrate the
method, we considered the kinetic resolution of esters, which
is often accomplished by the catalytic action of lipases.[21] In
doing so 13C labeling of carbonyl groups was chosen for
several reasons:
a) Carbonyl groups provide intensive vibrational bands in an
IR spectrum, allowing for easy and precise determination
of the concentration of the compounds by applying
Lambert Beer×s law.
b) In the spectral region between 1600 and 1800 cmÀ1, which
is typical for carbonyl stretching vibrations, almost no
absorption of other functional groups appear, eliminating
interferences with other vibrational bands.
c) The 13C-labeled compounds can be easily prepared be-
cause reactive reagents with 13C-labeled carbonyl groups
such as 1-13C-acetyl chloride are commercially available.
d) The absorption maxima of the carbonyl stretching vibra-
tion is shifted by 40 to 50 cmÀ1 to lower wavenumbers by
introducing a 13C-label, which prevents the overlap of the
two carbonyl bands.[22]
Scheme 1. a) Asymmetric transformation of a mixture of pseudo-enan-
tiomers involving cleavage of the functional groups FG and isotopically
labeled FG*. b) Asymmetric transformation of a mixture of pseudo-
enantiomers involving either cleavage or bond formation at the functional
group FG; isotopic labeling at R2 is indicated by the asterisk. c) Asym-
metric transformation of a pseudo-meso-substrate involving cleavage of the
functional group FG and labeled FG*. d) Asymmetric transformation of a
pseudo-prochiral substrate involving cleavage of the functional group FG
and labeled FG*.
To determine the ee values of such isotopically labeled
compounds in a fast and precise manner, two different
approaches have been put into practice. The first one makes
use of deuterated substrates that can be easily analyzed by
standard MS techniques like electrospray ionization (ESI) or
matrix-assisted laser-desorption ionization (MALDI).[18]
A
throughput of up to 10000 samples per day can be reached
with an accuracy of better than Æ5%.
The second method makes use of 1H NMR spectroscopy of
13C-labeled compounds, which allows the analysis of up to
1400 samples per day with an exceptionally high degree of
Keeping these advantages in mind we synthesized (R)-1-
phenylethyl acetate ((R)-1), (S)-(1-phenylethyl)-1-13C-acetate
Chem. Eur. J. 2003, 9, 3882 3887
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M. T. Reetz et al.
FULL PAPER
((S)-13C-1), (R)-N-1-phenylethylacetamide ((R)-4) and (S)-N-
(1-phenylethyl)-1-13C-acetamide ((S)-13C-4), which are possi-
ble pseudo-enantiomeric substrates for enzyme-catalyzed
hydrolytic kinetic resolutions (Schemes 2 and 3). Figure 2
shows part of the FTIR spectrum of a 1:1 mixture of (R)-1 and
(S)-13C-1, illustrating the anticipated shift of the respective
carbonyl stretching vibration which allows quantification of
the pseudo-enantiomers.
the molar coefficients of absorbance by applying Lambert
Beer×s law E ˆ e ¥ c ¥ d (Figure 3, Table 1).
With these coefficients in hand, the exploitation of the
FTIR spectra of different synthetic mixtures of the labeled
and nonlabeled enantiomeric compounds was possible. After
applying an automated baseline correction to the spectra and
correcting the absorbance of one enantiomer in the synthetic
mixtures by the absorbance of the other enantiomer at this
position, the accuracy of the
pseudo-enantiomeric
system
based on 1-phenylethyl acetate
turned out to be excellent, spe-
cifically within Æ3% in compar-
ison to the ee values determined
by chiral GC (Table 2).
In the case of the phenylethyl
acetamides (R)-4 and (S)-13C-4,
the precision decreased slightly
(Æ5% of the true ee values), but
this is still quite acceptable (Ta-
ble 3). It is currently not clear
why the small deviations are all
positive, that is, whether a sys-
tematic source of error is in-
volved.
Scheme 2.
Encouraged by these results,
we decided to carry out high-
throughput measurements with
commercially available HTS-
Scheme 3.
Figure 2. Part of an FTIR spectrum of a 1:1 mixture of (R)-1 and (S)-13C-1.
Figure 3. Diagram for the determination of the molar coefficients of
À1
13
~
,
*
absorbance of (R)-1 ( , absorption maximum: 1751 cm ) and (S)- C-1 (
absorption maximum: 1699 cmÀ1) by linear regression.
These compounds were used to evaluate the accuracy of a
possible high-throughput screening system by measuring
different synthetic mixtures of the pseudo-enantiomers with
a standard FTIR spectrometer and checking the ee values by
chiral GC analysis. To apply Lambert Beer×s law in the
calculation of the concentrations of the pseudo-enantiomeric
substances, the molar coefficients of absorbance had to be
determined. For this reason we prepared solutions of (R)-1
and (S)-13C-1 in cyclohexane and (R)-4 and (S)-13C-4 in 1,2-
dichloroethane with different concentrations. After measur-
ing the corresponding absorbances at the absorption maxima
of the carbonyl stretching vibration, we were able to calculate
FTIR systems. The analysis was performed on a Tensor 27
FTIR spectrometer coupled to a HTS-XT system (Bruker
Optik GmbH) which is able to analyze the samples on 96 or
384 well microtiter plates. The plates are equipped with a
silicon plate for IR transmittance. Moreover, we attempted to
measure the ee values in culture supernatants, which would
avoid a time-consuming and expensive workup of biocatalytic
reactions in water. In the case of the MS- or NMR-based
assays this additional step is indispensable. Such a simplifica-
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Chem. Eur. J. 2003, 9, 3882 3887

High-Throughput Screening for Enantioselectivity
3882 3887
Table 1. Molar coefficients of absorbance (e) of the pseudo-enantiomers
calculated by linear regression.
surprisingly well. The accuracy of the ee values of the
unknown samples, which were automatically calculated by
the software Opus,[23] decreased slightly (Æ7% of the true ee
value determined by chiral GC) (Table 4).
Compound
e
Correlation
coefficient
[LcmÀ1 molÀ1
]
1
2
3
4
(R)-1
550.6
374.9
477.1
403.2
0.999
0.999
0.995
0.996
(S)-13C-1
(R)-4
Table 4. Comparison of the ee values of synthetic mixtures of (R)-4 and
(S)-13C-4 determined by chiral GC and FTIR using a high-throughput
FTIR spectrometer.
(S)-13C-4
ee by FTIR
[%]
ee by GC
[%]
Deviation
[%]
Table 2. Comparison of the ee values of synthetic mixtures of (R)-1 and
(S)-13C-1 determined by chiral GC and FTIR using a standard FTIR
spectrometer.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
87.1 (S)
85.3 (S)
87.0 (S)
57.4 (S)
56.9 (S)
52.3 (S)
97.4 (R)
97.5 (R)
94.5 (R)
11.5 (R)
10.4 (R)
11.1 (R)
33.5 (S)
30.5 (S)
33.4 (S)
90.8 (S)
52.0 (S)
90.2 (R)
12.4 (R)
29.8 (S)
3.7
5.5
3.8
5.4
4.9
0.3
7.2
7.3
4.3
0.9
2.0
1.3
3.7
0.7
3.6
ee by GC
[%]
ee by FTIR
[%]
Deviation
[%]
1
2
3
4
5
6
7
8
9
100 (S)
94.4 (S)
89.7 (S)
71.0 (S)
40.2 (S)
2.6 (S)
39.8 (R)
71.0 (R)
89.7 (R)
94.0 (R)
99.7 (R)
100 (S)
93.9 (S)
87.5 (S)
70.1 (S)
37.8 (S)
0.2 (R)
42.7 (R)
73.9 (R)
93.1 (R)
95.7 (R)
100 (R)
0
0.5
2.2
0.9
2.4
2.8
2.9
2.9
3.4
1.7
0.3
10
11
Although the present method has not yet been applied in a
specific project concerning combinatorial asymmetric transi-
tion metal catalysis or directed evolution of enantioselective
enzymes, high-throughput in the range of 10000 samples per
day is technically possible under these conditions. For this
purpose Bruker has already coupled the microplate stacking
device TWISTER1 (Zymark) to its microplate reader. In this
combination which is controlled by OPUS software (Bruker),
40 IR microplates can be measured automatically. To perform
the sample loading at high throughput, the autosampler
microlab 4000 SP (Hamilton) was tested successfully. Both
formats (96 and 384) of the Bruker silicon microplates were
suitable to be loaded automatically with different types of
samples (proteins, cells, culture media).
The surprisingly good accuracy connected with the high
sample throughput and the opportunity to measure the ee
values directly in culture supernatants makes this approach
very valuable for the analysis of biocatalytic reactions in
water. Furthermore, the selectivity factors E in kinetic
resolutions can also be obtained because this system provides
the concentration of each enantiomer which allows the
conversion of the reaction to be calculated.[24]
Table 3. Comparison of the ee values of synthetic mixtures of (R)-4 and
(S)-13C-4 determined by chiral GC and FTIR using a standard FTIR
spectrometer.
ee by GC
[%]
ee by FTIR
[%]
Deviation
[%]
1
2
3
4
5
6
7
8
9
100 (S)
93.4 (S)
89.3 (S)
69.8 (S)
40.0 (S)
0.4 (S)
39.6 (R)
71.4 (R)
88.6 (R)
93.4 (R)
100 (R)
100 (S)
95.1 (S)
90.2 (S)
70.3 (S)
37.1 (S)
4.7 (R)
45.1 (R)
75.4 (R)
93.3 (R)
96.5 (R)
100 (R)
0
1.7
0.9
1.5
2.9
5.1
5.5
4.0
4.7
3.1
0
10
11
tion would be of great value in projects concerning the
directed evolution of enantioselective enzymes.
For this reason 19 synthetic mixtures of (R)-4 and (S)-13C-4
in DMSO (0.20m) were prepared, of which 14 were used to
calibrate the system, while the remaining samples were used
as unknown mixtures. We diluted 20 mL of each mixture with
180 mL of culture supernatant (E. coli in LB-medium)
containing extracellular proteins, nutrients like glucose and
other components. After placing 3 mL of each mixture three
times on a 384 microtiterplate and drying in an desiccator for
30 min at 220 mbar and ambient temperature, the microtiter
plate was transferred into the HTS-XT system. The measure-
ments were performed with a resolution of 8 cmÀ1 and 10
scans per sample resulting in a total time for each sample of
8.9 s. We were pleased to discover that the system works
Conclusion
We have devised a high-throughput FTIR system for the fast
and precise determination of the enantiomeric purity of chiral
acetates or acetamides. In the case of enzyme catalysis,
supernatants can be used without any workup. A throughput
of up to 10000 samples per day is possible by directly
measuring the ee values in culture supernatants, the accuracy
amounting to Æ7% of the true value as checked by GC. This
avoids the time-consuming and expensive workup of the
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M. T. Reetz et al.
FULL PAPER
drying in vacuum the desired products were obtained as white solids. Yield:
0.73 g (64%).
samples in biocatalytic reactions in water. Extension to other
substrates should also be feasible, provided appropriate 13C
labeling is possible. Thus, we view this new and cheap ee assay
to be a practical screening technique in enantioselective
catalytic and biocatalytic reactions.
(R)-N-1-Phenylethyl acetamide ((R)-4): 1H NMR (CDCl3): d ˆ 1.46 (d, J ˆ
6.9 Hz, 3H; CH3), 1.95 (s, 3H; C(O)CH3), 5.10 (q, J ˆ 7.0 Hz, 1H; CH), 6.03
(s, 1H; NH) 7.24 7.37 ppm (m, 5H; ArH); 13C NMR (CDCl3): d ˆ 21.7,
23.4, 48.8, 126.2, 127.3, 128.6, 143.3, 169.2 ppm; MS: m/z (rel. int.): 163 (54)
‡
[M ], 148 (13), 120 (30), 106 (100), 77 (19), 43 (22); IR (KBr): nÄ ˆ 3265 (N-
À
À
H) 3071, 3022 (C H, ArH), 2980, 2972, 2929 (C H, CH ‡ CH3), 1642
(C O), 1557 (N-H) cmÀ1; elemental analysis (%) calcd: C 73.6, H 8.0, N
ˆ
Experimental Section
8.6; found: C 73.5, H 8.1, N 8.5.
(S)-N-(1-Phenylethyl)-1-13C-acetamide ((S)-4): 1H NMR (CDCl3): d ˆ 1.46
(d, J ˆ 6.9 Hz, 3H; CH3), 1.95 (s, J ˆ 6.0 Hz, 3H; 13C(O)CH3), 5.10 (q,
JC,H ˆ 2.2 Hz, J ˆ 7.0 Hz, 1H; CH), 6.04 (s, 1H; NH) 7.24 7.37 ppm (m, 5H;
ArH); 13C NMR (CDCl3): d ˆ 21.7, 23.4, 48.8, 126.2, 127.3, 128.6, 143.3,
General remarks: The reagents and solvents were obtained from commer-
cial sources and the reagents were generally used without further
purification. All solvents were distilled and stored under argon. The
conditions for gas chromatographic analyses are given in the analytical data
of the relevant compounds. 1H NMR spectra were recorded on a Bruker
AMX 300 (300 MHz) spectrometer. Chemical shifts are reported in ppm
using tetramethylsilane (TMS, d ˆ 0.00 ppm) as internal standard. 13C NMR
spectra were recorded at 75 MHz with CDCl3 as internal reference. Mass
spectra were recorded on a Finnigan MAT8200 and IR data were obtained
using a Perkin Elmer FT1600. High-throughput FTIR measurements
were performed using a Bruker Tensor27 spectrometer in connection with
a High-Throughput-Screening Extension (HTS-XT) system. For data post
processing Opus¹ software (Bruker) was used. Elemental analysis was
carried out in an external laboratory (Mikroanalytisches Labor Kolbe in
M¸lheim/Ruhr). In all cases the amount of 13C labeling was >99%.
‡
169.1 ppm; MS: m/z (rel. int.): 164 (46) [M ], 149 (13), 120 (30), 106 (100),
À
À
77 (22), 44 (40); IR (KBr): nÄ ˆ 3265 (N H) 3049, 3023 (C H, ArH), 2972,
13
2929 (C H, CH ‡ CH3), 1606 ( C O), 1545 (N-H) cmÀ1; elemental
À
ˆ
analysis (%) calcd: C ‡ 13C 73.7, H 8.0, N 8.5; found: C ‡ 13C 73.7, H 8.0, N
8.5.
GC-analysis: Hewlett Packard 5890, column: 25 m Ivadex-1/PS086 G/375,
detector: FID, temperature programme: 2208C, 808C 28C per min
2108C, 5 min isotherm, 3208C, gas: 0.8 bar hydrogen, retention time:
26.7 min ((S)-N-(1-phenylethyl)-1-13C-acetamide), 27.9 min ((R)-N-1-phe-
nylethyl acetamide), >99% ee in both cases.
Procedure for the determination of molar coefficients of absorbance of the
labeled and unlabeled 1-phenylethyl acetates: After preparation of a stock
solution (0.200m) of (R)-1-phenylethyl acetate ((R)-1) and (S)-(1-phenyl-
ethyl)-1-13C-acetate ((S)-13C-1) in cyclohexane, the solutions were diluted
with cyclohexane to concentrations of 0.180, 0.160, 0.140, 0.120, 0.100,
0.080, 0.060, 0.040 and 0.020m, respectively (total volume: 1 mL). The
absorbance of the resulting samples was measured with a FTIR spectrom-
eter at the corresponding absorption maxima of the carbonyl stretching
vibration ((R)-1: 1751 cmÀ1, (S)-13C-1: 1699 cmÀ1) with a thickness of the
layers of 25.0 mm. The measurements were carried out with 32 scans and a
resolution of 4 cmÀ1. The molar coefficients of absorbance were determined
by linear regression. The correlation coefficient were in both cases better
than 0.995.
Synthesis of 1-phenylethyl acetates 1: Enantiomerically pure (S)- or (R)-1-
phenylethanol (2) (1.0 g, 8.2 mmol) and pyridine (4 mL) were dissolved in
dichloromethane (30 mL) in a 50 mL N2 flask under argon. After the
solution was cooled with an ice bath, the corresponding 1-13C-labeled or
unlabeled acetyl chloride (0.97 g, 12.3 mmol) was slowly added with a
syringe with appearance of a white solid (pyridine hydrochloride). The
reaction mixture was stirred overnight at ambient temperature and the
resulting red solution was quenched with water while being cooled with an
ice bath. The organic phase was separated and subsequently washed with
1m HCl and brine. After the organic phase was dried with MgSO4, the
solvent was evaporated and the crude products were purified by column
chromatography (SiO2) using dichloromethane as eluent. After removal of
the solvent in vacuum, the desired products were obtained as colorless oils.
Yield: 1.24 g (92%).
The analysis of synthetic mixtures of the pseudo-enantiomers of 1-phenyl-
ethyl acetate was performed under the same conditions at a concentration
of 0.10m.
(R)-1-Phenylethyl acetate ((R)-1): 1H NMR (CDCl3): d ˆ 1.53 (d, J ˆ
6.6 Hz, 3H; CH3), 2.06 (s, 3H; C(O)CH3), 5.88 (q, J ˆ 6.6 Hz, 1H; CH),
7.24 7.37 ppm (m, 5H; ArH); 13C NMR (CDCl3): d ˆ 21.3, 22.2, 72.3, 126.1,
Procedure for the determination of molar coefficients of absorbance of the
labeled and unlabeled N-1-phenylethyl acetamides: After preparation of a
stock solution (0.200m) of (R)-N-1-phenylethyl acetamide ((R)-4) and (S)-
N-(1-phenylethyl)-1-13C-acetamide ((S)-13C-4) in 1,2-dichloroethane, the
solutions were diluted with 1,2-dichloroethane to concentrations of 0.180,
0.160, 0.140, 0.120, 0.100, 0.080, 0.060, 0.040 and 0.020m, respectively (total
volume: 1 mL). The absorbance of the resulting samples was measured
with a FTIR-spectrometer at the corresponding absorption maxima of the
carbonyl stretching vibration ((R)-4: 1676 cmÀ1, (S)-13C-4: 1635 cmÀ1) with
a thickness of the layers of 106.7 mm. The measurements were carried out
with 32 scans and a resolution of 4 cmÀ1. The molar coefficients of
absorbance were determined by linear regression. The correlation coef-
ficient were in both cases better than 0.995.
‡
127.9, 128.5, 141.7, 170.3 ppm; MS: m/z (rel. int.): 164 (25) [M ], 122 (77),
À
À
104 (100), 77 (43); IR (neat): nÄ ˆ 3064, 3034 (C H, ArH), 2982, 2934 (C H,
CH ‡ CH3), 1744 (C O), 1242 (C O)cmÀ1; elemental analysis (%) calcd:
ˆ
À
C 73.3, H 7.3; found: C 72.9, H 7.4.
(S)-(1-Phenylethyl)-1-13C-acetate ((S)-13C-1): 1H NMR (CDCl3): d ˆ 1.53
(d, J ˆ 6.6 Hz, 3H; CH3), 2.06 (d, JC,H ˆ 6.8 Hz, 3H; 13C(O)CH3); 5.88 (q,
JC,H ˆ 3.2 Hz, J ˆ 6.6 Hz, 1H; CH), 7.24 7.37 ppm (m, 5H; ArH); 13C NMR
(CDCl3): d ˆ 21.3, 22.2, 72.3, 126.1, 127.9, 128.5, 141.7, 170.7 ppm; MS: m/z
‡
(rel. int.): 165 (38) [M ], 122 (91), 104 (100), 77 (35), 44 (53); IR (neat): nÄ ˆ
13
À
À
ˆ
3065, 3034 (C H, ArH), 2982, 2934 (C H, CH ‡ CH3), 1691 ( C O), 1206,
1066 (C O)cmÀ1; elemental analysis (%) calcd: C ‡ 13C 73.3, H 7.3; found:
À
C ‡ 13C 73.2, H 7.4.
The analysis of synthetic mixtures of the pseudo-enantiomers of N-1-
phenylethyl acetamide was performed under the same conditions at a
concentration of 0.10m.
GC-analysis: Hewlett Packard 5890, column: 25 m TBCD/OV-1701,
detector: FID, temperature programme: 2308C, 608C 28C per min
1808C, 5 min isotherm, 3508C, gas: 1 bar hydrogen, retention time:
21.7 min ((S)-(1-phenylethyl)-1-13C-acetate), 23.3 min ((R)-1-phenylethyl
acetate), >99% ee in both cases.
High-throughput FTIR measurements: The high-throughput measure-
ments were performed on a Tensor27 spectrometer in connection with a
HTS-XT system provided by Bruker Optik GmbH. For this reason 19
different synthetic mixtures of the pseudo-enantiomers of N-1-phenylethyl
acetamide were dissolved in DMSO (total concentration: 0.20m) and
aliquots of 20 mL were diluted in 180 mL of a culture supernatant (E. coli in
LB-medium). A 3 mL portion of each mixture were transferred onto a 384
microtiterplate equipped with a silicon plate for IR transmittance. This
procedure was repeated three times for every sample. After the micro-
titerplate was dried in a desiccator for 30 min at 220 mbar and ambient
temperature, the plate was placed in the HTS-XT system. Every sample
was measured with a resolution of 8 cmÀ1 and a scan number of 10, so that
the total time for the analysis of each sample amounted to 8.9 s, allowing a
throughput of 9700 samples per day. The resulting spectra were analyzed
Synthesis of N-1-phenylethyl acetamides 4: Enantiomerically pure (S)- or
(R)-1-phenylethylamine (5) (0.90 g, 7.5 mmol) and pyridine (4 mL) were
dissolved in dichloromethane (30 mL) in a 50 mL N2 flask under argon.
After the solution was cooled with an ice bath the corresponding 1-13C-
labeled or unlabeled acetyl chloride (0.59 g, 7.6 mmol) was slowly added
with a syringe. The reaction mixture was stirred overnight at ambient
temperature and the resulting yellow solution was quenched with water
while being cooled with an ice bath. The organic phase was separated and
subsequently washed three times with 1m HCl and once with brine. After
the organic phase was dried with MgSO4, the solvent was evaporated and
the crude products were recrystallized from hexane. After filtration and
3886
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 3882 3887

High-Throughput Screening for Enantioselectivity
3882 3887
with the software Opus and Opus Lab.[23] The first 14 samples were used for
calibration purposes, while the remaining probes were taken as unknown
samples. To evaluate the accuracy of the system, the ee value of each
mixture was independently determined by chiral GC analysis.
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Acknowledgement
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We thank Miss Beata Dittrich for performing the FTIR measurements on a
standard FTIR spectrometer. Parts of this research were supported by a
grant from the EC (QLK3-CT-2001-00519). We also thank the Fonds der
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Received: February 26, 2003 [F4885]
Chem. Eur. J. 2003, 9, 3882 3887
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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