Using enzyme inhibition as a high throughput method to measure the enantiomeric excess of a chiral sulfoxide

Article abstract of DOI:10.1021/ol0521681

(Chemical Equation Presented) The enantiomeric excess of methyl p-tolyl sulfoxide can be determined in a high throughput format by measuring its ability to inhibit the alcohol dehydrogenase catalyzed oxidation of ethanol. The two enantiomers of the sulfox

Full text of DOI:10.1021/ol0521681

ORGANIC
LETTERS
2005
Vol. 7, No. 22
5099-5102
Using Enzyme Inhibition as a High
Throughput Method to Measure the
Enantiomeric Excess of a Chiral
Sulfoxide
Christopher M. Sprout and Christopher T. Seto*
Department of Chemistry, Brown UniVersity, 324 Brook Street,
ProVidence, Rhode Island 02912
Christopher_Seto@brown.edu
Received September 8, 2005
ABSTRACT
The enantiomeric excess of methyl p-tolyl sulfoxide can be determined in a high throughput format by measuring its ability to inhibit the
alcohol dehydrogenase catalyzed oxidation of ethanol. The two enantiomers of the sulfoxide have very different inhibition constants for the
enzyme. Thus, the initial rate of ethanol oxidation in the presence of the sulfoxide is correlated with the sulfoxide enantiomeric excess.
High throughput methods for measuring the enantiomeric
excess (ee) of small molecules will have a significant impact
on a variety of chemical problems, including the discovery
of new enantioselective catalysts and the characterization and
screening of chiral molecules for biological activity. Over
the last several years, a number of such methods have been
described in the literature that rely on chromatographic,
spectroscopic, chemical, or biological tools to quantitate the
concentrations of two enantiomers in a sample.1,2 We recently
reported a biological technique, called EMDee for an
Enzymatic Method for Determining ee, that uses an enzyme
as an analytical tool to measure ee.3,4 An enzyme is
introduced into a sample of unknown stereochemical com-
position, where it catalyzes a reaction using only one of the
enantiomers as a substrate. The initial rate of the enzyme-
catalyzed reaction is correlated with the ee of the sample.
We developed two examples of the assay. The first uses an
(2) For selected reports, see: (a) Reetz, M. T.; Becker, M. H.; Klein,
H.-W.; Sto¨ckigt, D. Angew. Chem., Int. Ed. 1999, 38, 1758. (b) Taran, F.;
Gauchet, C.; Mohar, B.; Meunier, S.; Valleix, A.; Renard, P. Y.; Cre´minon,
C.; Grassi, J.; Wagner, A.; Mioskowski, C. Angew. Chem., Int. Ed. 2002,
41, 124. (c) Jarvo, E. R.; Evans, C. A.; Copeland, G. T.; Miller, S. J. J.
Org. Chem. 2001, 66, 5522. (d) Korbel, G. A.; Lalic, G.; Shair, M. D. J.
Am. Chem. Soc. 2001, 123, 361. (e) Mei, X.; Wolf, C. J. Am. Chem. Soc.
2004, 126, 14736. (f) Li, Z.-B.; Lin, J.; Pu, L. Angew. Chem., Int. Ed. 2005,
44, 1690. (g) Tielmann, P.; Boese, M.; Luft, M.; Reetz, M. T. Chem.sEur.
J. 2003, 9, 3882. (h) Zhu, L.; Zhong, Z.; Anslyn, E. V. J. Am. Chem. Soc.
2005, 127, 4260. (i) Folmer-Anderson, J. F.; Lynch, V. M.; Anslyn, E. V.
J. Am. Chem. Soc. 2005, 127, 7986. (j) Chen, Y.; Shimizu, K. D. Org.
Lett. 2002, 4, 2937. (k) Berkowitz, D. B.; Bose, M.; Choi, S. Angew. Chem.,
Int. Ed. 2002, 41, 1603.
(1) For reviews, see: (a) Reetz, M. T. Angew. Chem., Int. Ed. 2002, 41,
1335. (b) Reetz, M. T. Angew. Chem., Int. Ed. 2001, 40, 284. (c) Finn, M.
G. Chirality 2002, 14, 534. (d) Tsukamoto, M.; Kagan, H. B. AdV. Synth.
Catal. 2002, 344, 453.
(3) Abato, P.; Seto, C. T. J. Am. Chem. Soc. 2001, 123, 9206.
(4) Onaran, M. B.; Seto, C. T. J. Org. Chem. 2003, 68, 8136.
10.1021/ol0521681 CCC: $30.25
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Published on Web 10/04/2005

alcohol dehydrogenase to analyze chiral alcohols, while the
second uses a lipase to analyze chiral esters. These and other
parallel methods have a significant speed advantage over the
traditional serial methods that have been used to measure
ee, such as chiral HPLC and GC, optical rotation measure-
ments, and NMR methods. As a result, these high throughput
ee screens are powerful tools that can be used, in conjunction
with combinatorial methods, to facilitate the discovery of
new enantioselective catalysts for a variety of chemical
transformations. Here, we report a new method that over-
comes one of the major limitations of the original EMDee
assay. In previous examples, the enzyme was required to
both bind and catalyze a reaction with the analyte. In our
current experiments, we remove the turnover restriction and
demonstrate that EMDee works well with analytes in which
the two enantiomers simply bind with different affinities to
the enzyme and, thus, inhibit its activity to a different extent.
These studies broaden the scope of the EMDee assay since
there are a variety of chiral compounds that are enzyme
inhibitors, and in most cases, the two enantiomers of these
potential analytes have different affinities for their enzyme
target.
We have chosen sulfoxide 1 as a proof of concept for this
new EMDee procedure. Chiral sulfoxides have important
applications in both chemistry and biology.5 They have been
used as chiral auxiliaries and as ligands for asymmetric
catalysis and also in medicinal chemistry as anti-ulcer drugs
and to prevent alcohol metabolism.5b,6 For example, the anti-
ulcer drug Nexium is a chiral sulfoxide. One of the most
efficient routes to prepare chiral sulfoxides is the asymmetric
catalytic oxidation of sulfides. While a number of enantio-
selective catalysts have been developed for this reaction, there
is room for improved oxidation catalysts that avoid over-
oxidation of the sulfide to the sulfone, that use lower ligand
loadings and nontoxic metals and that provide higher yields
and enantioselectivities.7 High throughput methods to
measure the ee of sulfoxides will facilitate the discovery
effort.8
The rate of the reaction was monitored by following the
conversion of the cofactor â-NAD to â-NADH by UV
spectroscopy. Both enantiomers of 1 are uncompetitive
inhibitors, but (S)-1 is a potent inhibitor with a Kii value of
33 ( 3 µM, while (R)-1 is a weak inhibitor with a Kii value
of 656 ( 66 µM. Because these are uncompetitive inhibitors,
Kii represents the dissociation constant of the sulfoxide from
the HLADH/â-NADH/sulfoxide ternary complex.9 Uncom-
petitive inhibition was confirmed using a combination of both
Dixon and Cornish-Bowden analyses.10
Since there is a 20-fold difference between the Kii values
of the two enantiomers, we can measure the ee of the chiral
sulfoxide by monitoring the initial rate of the HLADH-
catalyzed oxidation of ethanol in the presence of a known
concentration of this inhibitor. Reactions that are run in the
presence of a higher concentration of (S)-1 are slow, while
reactions with (R)-1 are fast. We used a modified form of
the Michaelis-Menten equation for uncompetitive inhibition
to correlate the initial rate of the enzymatic reaction in the
presence of (S)-1 and (R)-1 with the ee of the inhibitor (eq
1). When the total sulfoxide and substrate concentrations,
and the kinetic and inhibition constants are known, eq 1 can
be used to convert initial rate data into ee values for the
sulfoxide. In this particular example, the sulfoxide is an
uncompetitive inhibitor of the enzyme. However, EMDee
should be equally as effective with analytes that are competi-
tive inhibitors.
Vmax [EtOH]
νi )
(1)
[(S)-1]
[(R)-1]
Km + [EtOH] 1 +
+
(
)
]
[
Kii (S)-1 Kii (R)-1
Figure 1 shows plots of the initial rate of the HLADH-
catalyzed reaction in the presence of 200 µM sulfoxide 1.
We analyzed 84 samples that ranged from 100% R to
100% S using a 96-well UV plate reader. Each sample
contained 36 nmol of sulfoxide, and initial rates were
measured over a 30 min window. The solid line in each plot
represents the theoretical curve generated using the modified
form of the Michaelis-Menten equation for uncompetitive
inhibition (eq 1). There is a good fit of the experimental
data to the theoretical curve, demonstrating that inhibition
rate profiles can be used to estimate ee of the sulfoxide
inhibitor.
The assay procedure is outlined in Scheme 1. Plapp has
shown that chiral sulfoxides are inhibitors of horse liver
alcohol dehydrogenase (HLADH).9 We examined sulfoxide
1 as an inhibitor of this enzyme using ethanol as the substrate.
Scheme 1. EMDee Procedure to Determine the Enantiomeric
Excess of Methyl p-Tolyl Sulfoxide by Measuring its Inhibition
of the Oxidation of Ethanol to Acetaldehyde Catalyzed by
HLADHa
To demonstrate the utility of this assay for the analysis of
products from catalytic enantioselective reactions, we screened
(5) (a) Ferna´ndez, I.; Khiar, N.Chem. ReV. 2003, 103, 3651. (b) Federsel,
H.-J. Chirality 2003, 15, S128. (c) Miller, J. A.; Gross, B. A.; Zhuravel,
M. A.; Jin, W.; Nguyen, S. T. Angew. Chem., Int. Ed. 2005, 44, 3885. (d)
Legros, J.; Dehli, J. R.; Bolm, C. AdV. Synth. Catal. 2005, 347, 19.
(6) (a) Chadha, V. K.; Leidal, K. G.; Plapp, B. V. J. Med. Chem. 1983,
26, 916. (b) Chadha, V. K.; Leidal, K. G.; Plapp, B. V. J. Med. Chem.
1985, 28, 36.
(7) For a catalyst that uses a nontoxic oxidant and metal, see: Legros,
J.; Bolm, C. Chem.sEur. J. 2005, 11, 1086.
(8) Reetz, M. T.; Daligault, F.; Brunner, B.; Hinrichs, H.; Deege, A.
Angew. Chem., Int. Ed. 2004, 43, 4078.
(9) Cho, H.; Plapp, B. V. Biochemistry 1998, 37, 4482.
(10) (a) Dixon, M. Biochem. J. 1953, 55, 170. (b) Cornish-Bowden, A.
Biochem. J. 1974, 137, 143.
a Assay conditions: total concentration of sulfoxide 1 ) 200 µM,
46 mM phosphate buffer (pH 7), [EtOH] ) 50 mM, [â-NAD] )
15 mM, monitored by UV spectroscopy at 340 nm over 30 min.
5100
Org. Lett., Vol. 7, No. 22, 2005

Figure 2. Comparison of ee values of sulfoxide (R)-1 measured
by chiral HPLC (black bars) and EMDee (gray bars). Samples of
sulfoxide 1 were obtained from the enantioselective oxidation of
sulfide 2 in the presence of the tartrate ester ligands listed on the
x-axis. Abbreviations for tartrate esters: DIT ) diisopropyl
L-tartrate, DBT ) di-n-butyl L-tartrate, DET ) diethyl L-tartrate,
DBnT ) dibenzyl L-tartrate, DMT ) dimethyl L-tartrate, (-)-DIT
) diisopropyl D-tartrate. The gray bars represent the average of
four separate EMDee measurements; the error bars correspond to
( one standard deviation from the mean. Negative values on the
y-axis correspond to the ee of (S)-1: [a] 10 mol % of the DBT
ligand was used in these reactions. [b] This sample was racemic
by chiral HPLC.
Figure 1. (a) Correlation between the relative initial rate of the
HLADH-catalyzed oxidation of ethanol as a function of sulfoxide
ee from 100 to 0% ee (R)-1. (b) Correlation between the relative
initial rate of the HLADH-catalyzed oxidation of ethanol as a
function of sulfoxide ee from 0 to 100% ee (S)-1. The solid line in
each plot represents the theoretical curve generated using the
Michaelis-Menten equation for uncompetitive inhibition (eq 1).
Note that the y-axes in the two plots are drawn to different scales.
eq 1 was used to calculate ee values from the initial rate
data. All but one of the reactions was performed in duplicate.
The EMDee assays are generally in good agreement with
the ee values that were determined by HPLC, with a majority
of the experiments showing less than (10% difference
between the two measurements. Like the other EMDee
systems, this level of accuracy is sufficient for high
throughput screening of large libraries of potential catalysts
to identify those catalysts that give high levels of enantio-
selectivity. This much smaller subset of catalysts can then
be further analyzed by more accurate, but slower methods,
such as chiral HPLC. Interestingly, in our hands di-n-butyl
tartrate provided the highest level of enantioselectivity in
this reaction, slightly higher than that of the diethyl ester
that was reported by Kagan. One of the (-)-DIT samples
(far right of Figure 1) showed a large difference between
the ee values determined by EMDee and HPLC. This
difference is likely due to the fact that the EMDee assay
shows its lowest level of sensitivity with samples near 100%
ee (S)-1.
a series of tartrate esters as ligands in the enantioselective
oxidation reaction reported by Kagan (Scheme 2).11 These
Scheme 2. Enantioselective Oxidation of Sulfide 2 to
Sulfoxide 1
reactions use a stoichiometric amount of the tartrate ester as
a chiral controller. The unquenched reaction mixtures were
passed through a silica plug, and without further purification,
the crude products were analyzed by EMDee and HPLC
using a chiral column (Figure 2).12 For the EMDee analyses,
We would like to emphasize several important character-
istics of this and other EMDee assays. (1) All of the enzymes
that we use are commercially available and relatively
inexpensive. (2) The assays are straightforward to set up.
There is little development time since assay procedures for
many enzymes are available in the literature. (3) The only
piece of specialized equipment needed to implement the
assay in its high throughput format is a 96- or 384-well UV
(11) (a) Zhao, S. H.; Samuel, O.; Kagan, H. B. Tetrahedron 1987, 43,
5135. (b) Brunel, J.-M.; Diter, P.; Duetsch, M.; Kagan, H. B. J. Org. Chem.
1995, 60, 8086.
(12) This isolation procedure does not lead to enantiomeric fractionation
of sulfoxide 1. See: Diter, P.; Taudien, S.; Samuel, O.; Kagan, H. B. J.
Org. Chem. 1994, 59, 370.
Org. Lett., Vol. 7, No. 22, 2005
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or fluorescence plate reader. (4) The assays are high
throughput; 84 samples can be analyzed in 30 min. (5) All
of the EMDee assays that we have reported to date function
well with crude samples generated from catalytic asymmetric
reactions.
the highest ee values of both absolute configurations. Given
the variety of chiral molecules that are either inhibitors or
substrates for known enzymes, we believe that this method
should be a useful tool for speeding the discovery of new
asymmetric catalysts.
In these studies, we measured two kinetic constants (Vmax
and Km) and two inhibition constants (Kii for both (R)-1 and
(S)-1), so that we can use eq 1 to correlate relative rates
with sample ee. However, for screening applications, where
the goal is simply to identify the most stereoselective
catalysts out of a large library, these measurements are not
necessary. Samples that give the fastest and slowest initial
rates in the enzyme assays correspond to the compounds with
Supporting Information Available: Experimental details
for the assays, Cornish-Bowden and Dixon plots for both
enantiomers of sulfoxide 1, general procedure for the
enantioselective oxidation of compound 2. This material is
available free of charge via the Internet at http://pubs.acs.org.
OL0521681
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Org. Lett., Vol. 7, No. 22, 2005