A method for high-throughput screening of enantioselective catalysts

Article abstract of DOI:10.1002/(SICI)1521-3773(19990614)38:12<1758::AID-ANIE1758>3.0.CO;2-8

About 1000 catalytic or stoichiometric asymmetric reactions of racemic compounds or prochiral substrates bearing enantiotopic groups can be analyzed per day. In this highly efficient method the enantioselectivity is determined by electrospray ionization mass spectrometry using isotopically labeled substrates. The picture shows the mass spectrum of the mixture obtained upon hydrolysis of 1 to afford the pseudo-enantiomeric products 2 and 3.

Full text of DOI:10.1002/(SICI)1521-3773(19990614)38:12<1758::AID-ANIE1758>3.0.CO;2-8

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[
13] Under DCC-mediated esterification conditions, we observe a small
degree of epimerization of the N-acylprolines, but this does not
interfere with the present application.
possible per day, in this case mutant lipases created by
[4, 5]
directed evolution.
Accordingly, following expression of a
library of mutant genes in E. coli/P. aeruginosa, the bacterial
colonies on agar plates were collected and cultivated individ-
ually in the wells of microtiter plates, each supernatant
containing a mutant lipase suitable for screening. By nature
this particular screening system is restricted to chiral acids and
cannot be used in the evaluation of asymmetric catalytic
reactions involving chiral alcohols, diols, amines, amino
alcohols, alkyl halides, or epoxides. Recently, IR thermog-
raphy was introduced as a means to detect metal- or enzyme-
catalyzed enantioselective reactions, but quantification still
needs to be accomplished.[
[
14] The use of more concentrated samples, containing approximately
25 nmol of desired esters or amides along with the other components
of the acylation reaction mixtures, showed poorer sensitivity and gave
less accurate results (standard deviations for repeat injections of
approximately 5%).
[
[
15] s ˆ [%ee(y ‡ 1) ‡ 100(y � 1)]/[%ee(y ‡ 1) � 100(y � 1)] for 100% ee,
s ˆ y.
16] a) T. F. Spande, H. M. Garraffo, M. W. Edwards, H. J. C. Yeh, L.
Pannell, J. W. Daly, J. Am. Chem. Soc. 1992, 114, 3475 ± 3478; b) J. W.
Daly, J. Nat. Prod. 1998, 61, 162 ± 172; c) Z. M. Chen, M. L. Trudell,
Chem. Rev. 1996, 96, 1179 ± 1193.
[
17] For example, diol 7 was analyzed using the masses corresponding to its
derived diesters containing two units of each mass tag (and ignoring
diesters having one of each of the mass tags). It appears that the first
derivatization of the primary alcohol occurs with little or no
diastereoselectivity, whereas the secondary alcohol is esterified with
s ꢀ 2. Thus, the analysis of chiral compounds such as diols containing
multiple functionalities should also be feasible.
6]
We now describe a method based on electrospray ionization
[7]
mass spectrometry (ESI-MS) which enables the determina-
tion of enantioselectivity in about 1000 catalytic or stoichio-
metric asymmetric reactions per day. Two basically different
stereochemical processes can be monitored by this approach,
namely, kinetic resolution of racemates and asymmetric
transformation of substrates which are prochiral due to the
presence of enantiotopic groups.
[
18] Note, for example, that not all of the compounds analyzed here could
have been accomodated by a single chiral HPLC or GC column. Of
course, the techniques are related in that the chemical interactions
responsible for molecular discrimination in chromatography are likely
to be similar to those giving rise to diastereoselectivity in the
derivatization process that preceeds mass spectrometry readout.
19] Each mass spectrometry injection presently requires approximately
two minutes. The analysis can be made more efficient by combining
compounds of differing molecular weights in each injection.
The underlying principle is based on the use of isotopically
labeled substrates in the form of pseudo-enantiomers or
[
[
[8]
pseudo-prochiral compounds (Scheme 1). The course of the
asymmetric transformationÐthat is, the relative amounts of
reactants and/or productsÐis detected by ESI-MS.[
20] Our present sensitivity limit for quantitative ESI-MS of small
9, 10]
molecules is approximately 500 fmol per mL.[6]
A Method for High-Throughput Screening of
Enantioselective Catalysts**
Manfred T. Reetz,* Michael H. Becker,
Heinz-Werner Klein, and Detlef Stöckigt
Whereas the principles of combinatorial chemistry are well
[
1]
established in pharmaceutical research, extension to the
[
2]
area of catalysis is not as advanced. One reason is that few
general methods for high-throughput screening of heteroge-
neous and homogeneous catalysts have been devised. This
[
3]
applies all the more to enantioselective catalysts. We have
previously developed a screening system for the catalytic
enantioselective hydrolysis of chiral p-nitrophenol esters in
which the course of the reactions of the R- and S-configured
substrates is monitored in a parallel manner by UV/Vis
Scheme 1. a) Asymmetric transformation of a mixture of pseudo-enan-
tiomers involving cleavage of the functional groups FG and labeled
functional groups FG*. b) Asymmetric transformation of a mixture of
pseudo-enantiomers involving either cleavage or bond formation at the
2
functional group FG; isotopic labeling at R is indicated by the asterisk.
c) Asymmetric transformation of
a pseudo-meso substrate involving
[
4]
spectroscopy. With the use of microtiter plates crude
screening of about 800 different enantioselective catalysts is
cleavage of the functional groups FG and labeled functional groups FG*.
d) Asymmetric transformation of a pseudo-prochiral substrate involving
cleavage of the functional group FG and labeled functional group FG*.
[
*] Prof. M. T. Reetz, Dipl.-Chem. M. H. Becker, H.-W. Klein,
Dr. D. Stöckigt
In the case of kinetic resolution, compounds 1 and 2,
differing in absolute configuration and in labeling at the
functional group FG*, are prepared in enantiomerically pure
form and then mixed in a 1:1 manner, simulating a racemate
Max-Planck-Institut für Kohlenforschung
Kaiser-Wilhelm-Platz 1, D-45470 Mülheim an der Ruhr (Germany)
Fax: (‡49)208-306-2985
E-mail: reetz@mpi-muelheim.mpg.de
(Scheme 1a). Following asymmetric functional group trans-
[
**] We thank H. Husmann for performing GC analyses and H. Hinrichs
for performing LC analyses as well as Novo Nordisk (Denmark) for
enzyme samples.
formation (in an ideal kinetic resolution up to 50% con-
version), true enantiomers 3 and 4 are formed, in addition to
1
758
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nonlabeled and labeled achiral products 5 and 6. The ratios of
the total intensities of 1/2 and 5/6 in the mass spectra (m/z
intensities of the quasi-molecular ions) allow for the deter-
mination of enantioselectivity. In some cases it may be
advantageous to use an internal standard in order to
Table 1. Enantiomeric excesses of samples prepared by mixing 15 and 16
as determined by GC and ESI-MS.
Sample
ee [%] (GC)
ee [%] (ESI-MS)
1
2
3
4
5
6
7
8
9
100
91
81
74
60
56
48
28
10
23
40
45
55
65
75
95
100
100
90
79
73
57
54
48
27
10
20
38
43
53
60
74
93
100
[
11]
determine the conversion.
As a variation of this theme, kinetic resolution of the
pseudo-enantiomers 1 and 7, in which labeling occurs at
2
residue R , affords a new pair of pseudo-enantiomers 3 and 8
(Scheme 1b). Based on the m/z intensities of the quasi-
molecular ions of 1/7 and 3/8, the conversion, the enantio-
selectivity, and the selectivity factor (s or E value) can be
obtained. An internal standard is not necessary.
In the case of prochiral substrates having enantiotopic
groups, for example meso compounds (Scheme 1c), the
synthesis of a single pseudo-meso compound suffices, for
example 9, since the stereodifferentiating reaction of interest
delivers a mixture of two pseudo-enantiomers 10 and 11 that
are detectable by MS. The same applies to other pseudo-
prochiral substrates of the type 12 (Scheme 1d).
10
11
12
13
14
15
16
17
Following these encouraging results, the task remained to
Although our method is independent of the type of catalyst
or reagent used, we restrict ourselves in this paper to lipase-
catalyzed reactions. The lipase-catalyzed hydrolytic kinetic
resolution of racemic 1-phenylethyl acetate, corresponding to
Scheme 1a, served as the first example. In exploratory
experiments pseudo-enantiomers 15 and 16 were prepared
in enantiomerically pure form and mixed in various ratios
devise an experimental setup capable of high-throughput
screening of enantioselective reactions. This was achieved by
combining an automated liquid sampler for microtiter plates
with an ESI-MS system, both commercially available
(Scheme 2). With such a configuration about 1000 precise
[
Eq. (1)]. The resulting mixtures were analyzed by gas
Scheme 2. Schematic description of the experimental setup for the
determination of ee values.
determinations of the ee value and the conversion of the
reaction in Equation (1) can be performed in a single day.[ In
other cases the number varies between 700 and 1400, depend-
ing upon the particular substrate. It should be emphasized that
neither chromatographic separation of enantiomers or pseudo-
enantiomers nor of the products is involved. Thus, the method
constitutes a fast and accurate way to determine the enantio-
selectivity and conversion of lipase-catalyzed (or otherwise
induced) hydrolysis reactions leading to chiral alcohols.
Our approach can be used in the kinetic resolution of all
types of chiral compounds, for example in the lipase-catalyzed
stereoselective esterification of 2-phenylpropionic acid using
a 1:1 mixture of 21 and 22 [Eq. (2)],[ which correspond
13]
chromatography in order to ascertain the exact pseudo-ee
values. Thereafter the samples were analyzed employing ESI-
[
12]
MS.
A typical ESI-mass spectrum is shown in Figure 1.
Comparison of the two sets of data shows an agreement of
Æ5% (Table 1). Similar observations were made in control
experiments involving other substrates, thereby demonstrat-
ing the accuracy and precision of such ee determinations.
13]
to Scheme 1b. As before, about 1000 ee determinations can be
performed per day. To exclude possible secondary kinetic
isotope effects, the reaction of the pseudo-enantiomers 21 and
Figure 1. ESI mass spectrum of 15 and 16 (sample 6, Table 1).
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2
2 was compared to that of the true racemate of 21. The data
in Figure 2 demonstrate agreement within the limits of
experimental error.
Figure 2. Comparison of the ee values obtained for the lipase-catalyzed
(
Novo SP 435 lipase, Novo Nordisk) esterification of the racemate of 21 (*)
with that of a 1:1 mixture of 21 and 22 (*).
Finally, the lipase-catalyzed enantioselective hydrolysis of
[
13]
meso-1,4-diacetoxy-2-cyclopentene was investigated, which
is an example of Scheme 1c. Accordingly, the pseudo-meso
compound 25 was first prepared by acylation of (1S,4R)-4-
Figure 3. a) ESI mass spectrum of a sample obtained from the use of a
nonactive mutant lipase in the attempted hydrolysis of 25. b) ESI mass
spectrum of a sample obtained from the use of an active esterase in the
hydrolysis of 25 (ESI-MS: pseudo-ee ˆ 73%; GC: ee ˆ 73%).
acetoxycyclopent-2-ene-1-ol (26) using CD C(O)Cl (>99
3
atom% D). Hydrolysis of 25 afforded the expected mixture
of pseudo-enantiomeric products 26 and 27 [Eq. (3)], which
strates having enantiotopic faces, as in the enantioselective
reduction of ketones or olefins.
Received: March 10, 1999 [Z13137IE]
German version: Angew. Chem. 1999, 111, 1872 ± 1875
were analyzed by MS as described above for the other
substrates. In Figure 3 ESI mass spectra of two representative
samples are depicted, one involving a mutant lipase (P.
Keywords: asymmetric catalysis ´ combinatorial chemistry ´
enzyme catalysis ´ high-throughput screening ´ mass spec-
trometry
[
4]
aeruginosa) having no enzyme activity (no conversion;
Figure 3a) and the other involving an active enzyme (porcine
liver esterase, Sigma) leading to an ee value of 73% (Fig-
ure 3b). In control experiments several samples were also
analyzed by gas chromatography using a chiral stationary
phase. Again, the correspondence between the traditional
chromatographic (slow) and the present method (fast) turned
out to be excellent.
[
1] a) F. Balkenhohl, C. von dem Bussche-Hünnefeld, A. Lansky, C.
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1996, 108, 19 ± 46; Angew. Chem. Int. Ed. Engl. 1996, 35, 17 ± 42;
c) Chem. Rev. 1997, 97, 347 ± 510 (special issue on combinatorial
chemistry); d) S. R. Wilson, A. W. Czarnick, Combinatorial Chem-
istry: Synthesis and Application, Wiley, New York, 1997.
[
2] a) G. Liu, J. A. Ellman, J. Org. Chem. 1995, 60, 7712 ± 7713; b) K.
Burgess, H.-J. Lim, A. M. Porte, G. A. Sulikowski, Angew. Chem.
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H. P. Nestler, U. Piarulli, B. Salom, Liebigs Ann. Recl. 1997, 637 ± 647;
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Sarangapani, E. S. Smotkin, T. E. Mallouk, Science (Washington,
DC) 1998, 280, 1735 ± 1737; f) R. Schlögl, Angew. Chem. 1998, 110,
In summary, we have developed a highly efficient method
to assay the enantioselectivity of asymmetric reactions of
chiral compounds or of prochiral substrates bearing enantio-
[
14]
topic groups. Although isotopically labeled pseudo-enan-
tiomers or pseudo-prochiral compounds need to be prepared,
once this task has been performed a large number of
enantioselective biocatalysts or chemical catalysts can be
screened for the particular reaction in question within a short
time. Since the position of isotopic labeling is chosen so as to
preclude primary isotope effects which might influence the
results, reliable data are obtained. The present method is not
restricted to ESI-MS; preliminary studies show that other
ionization techniques such as matrix-assisted laser desorption/
ionization (MALDI) are also efficient. Other screening
methods are required for systems involving prochiral sub-
2
467 ± 2470; Angew. Chem. Int. Ed. 1998, 37, 2333 ± 2336; g) A.
Holzwarth, H.-W. Schmidt, W. F. Maier, Angew. Chem. 1998, 110,
2788 ± 2792; Angew. Chem. Int. Ed. 1998, 37, 2644 ± 2647; h) J. Klein,
K. W. Lehmann, H.-W. Schmidt, W. F. Maier, Angew. Chem. 1998, 110,
3
557 ± 3561; Angew. Chem. Int. Ed. 1998, 37, 3369 ± 3372; i) X. Gao,
H. B. Kagan, Chirality 1998, 10, 120 ± 124; j) T. Bein, Angew. Chem.
999, 111, 335 ± 338; Angew. Chem. Int. Ed. 1999, 38, 323 ± 326; k) P.
1
Cong, R. D. Doolen, Q. Fan, D. M. Giaquinta, S. Guan, E. W.
McFarland, D. M. Poojary, K. Self, H. W. Turner, W. H. Weinberg,
1
760
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Angew. Chem. Int. Ed. 1999, 38, No. 12

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Angew. Chem. 1999, 111, 508 ± 512; Angew. Chem. Int. Ed. 1999, 38,
84 ± 488.
7
6
[
{[(h -C B H )(h -C B H )U][K (thf) ]} ]:
2
10 12
2
10 12
2
5
2
4
A Metallacarborane Containing the Novel
[
3] a) G. Zandonella, L. Haalck, F. Spener, K. Faber, F. Paltauf, A.
Hermetter, Chirality 1996, 8, 481 ± 489: b) L. E. Janes, R. J. Kazlaus-
kas, J. Org. Chem. 1997, 62, 4560 ± 4561; c) L. E. Janes, A. C.
Löwendahl, R. J. Kazlauskas, Chem. Eur. J. 1998, 4, 2324 ± 2331.
4] M. T. Reetz, A. Zonta, K. Schimossek, K. Liebeton, K.-E. Jaeger,
Angew. Chem. 1997, 109, 2961 ± 2963; Angew. Chem. Int. Ed. Engl.
7
4�
10 12
h -C B H
Ligand**
2
Zuowei Xie,* Chaoguo Yan, Qingchuan Yang, and
Thomas C. W. Mak
[
1
997, 36, 2830 ± 2932.
It has been well-documented that C B H R (R ˆ H, alkyl,
2
10 10
2
[
[
5] M. T. Reetz, K.-E. Jaeger, Top. Curr. Chem. 1999, 200, 31 ± 57.
6] M. T. Reetz, M. H. Becker, K. M. Kühling, A. Holzwarth, Angew. Chem.
aryl) can be reduced by alkali metals to form the nido-
2�
6
C B H R dianion, which can be bound in a h manner to
1
998, 110, 2792 ± 2795; Angew. Chem. Int. Ed. 1998, 37, 2547 ± 2650.
2
10 10
2
[
[
7] J. B. Fenn, M. Mann, C. K. Meng, S. F. Wong, C. M. Whitehouse,
Science (Washington, DC) 1989, 246, 64 ± 71.
8] pseudo-Enantiomers are chiral compounds which differ in two
respects only: opposite absolute configuration and isotopic labeling.
A 1:1 mixture of such compounds has been called a pseudo-racemate
transition metals to afford a series of 13-vertex closo-metal-
[1]
6
5
lacarboranes. Treatment of [(h -C B H )Co(h -C H )] with
2
10 12
5
5
Na/naphthalene followed by reaction with C H Na and CoCl
2
5
5
6
6
gave the 14-vertex closo-metallacarborane [(h :h -C B H )-
2
10 12
5
[2]
(B. Testa, P. Jenner in Drug Fate and Metabolism: Methods and
{Co(h -C H )} ]. The proposed geometry of the cage is the
5 5 2
Techniques, Vol. 2 (Eds.: E. R. Garrett, J. L. Hirtz), Dekker, New
York, 1978, p. 143), but this has been declared a misnomer (E. L. Eliel,
S. H. Wilen, L. N. Mander, Stereochemistry of Organic Compounds,
Wiley, New York, 1994, p. 159).
bicapped hexagonal antiprism; X-ray confirmation of this
species has not been reported. We are interested in this
tetraanion ligand and its bonding mode to transition metals,
and describe herein the isolation and structural character-
[
9] Horeau et al. have described the application of MS to the detection of
isotopically labeled diastereomers, prepared by the reaction of a
compound of unknown configuration with an excess of a 1:1 mixture
of isotopically labeled pseudo-enantiomers: a) A. Horeau, A.
Nouaille, Tetrahedron Lett. 1990, 31, 2707 ± 2710; other use of
pseudo-enantiomers: b) L. R. Sousa, G. D. Y. Sogah, D. H. Hoffman,
D. J. Cram, J. Am. Chem. Soc. 1978, 100, 4569 ± 4576; c) T. Walle, M. J.
Wilson, U. K. Walle, S. A. Bai, Drug Metab. Dispos. 1983, 11, 544 ±
7
4�
ization of the first metallacarborane bearing a h -C B H
2
10 12
ligand.
Interaction between o-C B H and excess K metal in THF
2
10 12
at room temperature followed by treatment with a suspension
of UCl in THF gave, after workup, 1 as deep red crystals in
4
5
8% yield [Eq. (a)]. Compound 1 is extremely air- and
549; d) D. W. Armstrong, Anal. Chem. 1987, 59, 84A ± 91A; e) M. A.
Baldwin, S. A. Howell, K. J. Welham, F. J. Winkler, Biomed. Environ.
Mass Spectrom. 1988, 16, 357 ± 360; f) M. Sawada, H. Yamaoka, Y.
Takai, Y. Kawai, H. Yamada, T. Azuma, T. Fujioka, T. Tanaka, Chem.
Commun. 1998, 1569 ± 1570.
THF
4
o-C
2
B
10
H
12 ‡ 12K ‡ 2UCl
4
�!
(a)
7
6
[
{[(h -C
2
B
10
H
12)(h -C
2
B
10
H
12)U][K
2
(thf)
5
]}
2
] ‡ 8KCl
1
[
9a]
[
10] In principle it should be possible to extend Horeauꢁs method to the
determination of ee values in high-throughput screening. However,
this requires an additional step (derivatization) and an excess of a 1:1
mixture of the pseudo-enantiomeric reagent as well as the assumption
that conversion is complete without any enrichment of one of the
diastereomers.
moisture-sensitive, but remains stable for months at room
temperature under an inert atmosphere. Contact with traces
of air immediately results in conversion of the intensely
colored 1 into a yellow powder. Compound 1 is soluble in
polar organic solvents such as THF and pyridine, sparely
soluble in toluene, and insoluble in hexane.
[
11] Determination of conversion by ESI-MS using an internal standard:
a) S. Takayama, S. T. Lee, S.-C. Hung, C.-H. Wong, Chem. Commun.
1999, 127 ± 128; b) S. A. Gerber, C. R. Scott, F. Turecek, M. H. Gelb, J.
An X-ray diffraction study[ reveals that 1 is a centrosym-
3]
Am. Chem. Soc. 1999, 121, 1102 ± 1103.
[
3
12] Aliquots (10 mL) of 1mm 1:1 mixtures of 15 and 16 in CH OH were
metric dimer with a bent sandwich structural motif. As shown
injected into a Rheodyne port of an ESI-MS system (ESI-MS
conditions: MS: Hewlett Packard 5989B MS engine quadrupole mass
spectrometer equipped with a Hewlett Packard 59987A API electro-
spray source II with hexapole ion guide (Analytica of Branford) and
ChemStation data system; data acquisition: positive-ion mode scan
spectra; m/z 90 ± 300; step size m/z 0.1, unit resolution, gaussian mass
filter m/z 0.3; gaussian time filter 0.05 min; API source conditions:
potential difference between spray needle and first electrode
6
2�
10 12
7
in Figure 1, each U atom is h -bound to nido-C B H , h -
2
4�
bound to arachno-C B H , and coordinated to two B�H
2
10 12
bonds from the C B bonding face of the neighboring arachno-
2
5
4�
C B H
ligand. This results in a highly distorted tetrahedral
geometry at U with a cent(S)�U�cent(L) angle of 136.38
cent(S) and cent(L) are the centroids of the C B and C B
5
2
10 12
(
2
4
2
bonding faces, respectively). Compound 1 represents not only
�
2 2
5250 V, pressure of N nebulization gas: 4140 Torr, flow of N
�
1
� 1
7
4�
drying gas ca. 9 Lmin
(1508C), solvent flow 0.06 mLmin
,
the first metallacarborane containing a novel h -C B H
2
10 12
CH
3 2
OH/H O 8/2). ESI mass spectra were collected, and the ratios
ligand, but also the first organoactinide compound bearing a
of 15 and 16 were determined on the basis of absolute intensities of the
6
2�
h -C B H
10 12
ligand.
‡
‡
2
peaks of the corresponding sodium adducts ([15‡Na] and [16‡Na] ,
Figure 1). The ratios of the peak intensities (and thus the ee values of
the synthetic mixtures of 15 and 16) were obtained automatically using
a macro to submit the data from the m/z intensity table of each
measurement to an Excel spread sheet.
The average distance between U and a cage atom of the
C B4 bonding face in 1 (2.867(7) Š) is longer than that
2
between U and a cage atom of the C B bonding face in
2
3
[
[
13] All enzyme-catalyzed hydrolytic reactions and esterifications were
performed in deep-well microtiter plates (total volume 1.2 mL). For
ESI-MS analysis a defined volume of the resulting product mixtures
was extracted with diethyl ether. The extracts were automatically
transferred in microtiter plates and diluted with methanol to a final
concentration of 0.5 ± 2.0 mm. The microtiter plates were placed in an
automated sample manager (Scheme 2) equipped with a Rheodyne
port for the injections.
[
*] Prof. Z. Xie, Dr. C. Yan, Prof. Q. Yang, Prof. T. C. W. Mak
Department of Chemistry
The Chinese University of Hong Kong
Shatin NT, Hong Kong (China)
Fax: (‡852)26035057
E-mail: zxie@cuhk.edu.hk
14] M. T. Reetz, M. H. Becker, H.-W. Klein, D. Stöckigt, 1999, patent
application submitted.
[**] This work was supported by the Hong Kong Research Grants Council
Earmarked Grant CUHK 4183/97P and Direct Grant 2060147.
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