Introduction
A new high-throughput screening method for determining enantiomeric excess is described.
This method, which we call EMDee, utilizes a lipase to process the products of an asymmetric
reaction. The enzyme converts one of the product enantiomers to the corresponding alcohol, and this
process is coupled to detection by UV spectroscopy. We have previously described how alcohol
dehydrogenases can be used to measure the ee of chiral alcohols that are obtained via the addition of
diethylzinc to benzaldehyde in the presence of chiral catalysts.1
In this abstract, we widen the scope of EMDee by focusing on the determination of ee of
chiral allylic acetates using lipases. The rate of acetic acid production during the lipase catalyzed
acetate hydrolysis is proportional to the concentration of the enantiomer that is the substrate. The
protonation of a pH indicator by the acetic acid that is produced can be conveniently followed by
observing a decrease in absorbance at 405 nm.
The Michaelis-Menten equation provides a direct relationship between the rate of this
enzymatic acetate hydrolysis and the concentration of one of the allylic acetate enantiomers.
Therefore, the rate of acetic acid production can be used as a direct measure of the enantiomeric
excess of allylic acetate products that are obtained from asymmetric reactions.
Background and Significance
The development of effective asymmetric catalysts is currently one of the most important
topics in organic chemistry. So far, many stereoselective catalysts have been developed, but much
research must be done towards further refining the existing catalysts and discovering new ones.
One problem in this area is that the design of potent and selective catalysts is still an
empirical process. Combinatorial chemistry can be useful to systematically synthesize catalyst
candidates and has the potential for increasing the efficiency by which chiral catalysts are discovered.
A number of groups have used combinatorial chemistry techniques to discover efficient catalysts.2
To date, the analysis of ee is the rate-limiting step in the combinatorial discovery of catalysts
for asymmetric synthesis. Currently, conventional methods like chiral GC or chiral HPLC are being
used to analyze the % ee of reaction products. These techniques require the serial analysis of
individual reactions, and therefore the analysis time increases linearly with library size. Another
drawback is the necessity to purify products in order to prevent contamination of the columns. It is
possible to generate a large number of catalyst candidates using combinatorial chemistry techniques,
but they can not be analyzed in a timely manner using conventional methods. Thus, the catalyst
libraries have usually been limited to less than one hundred catalysts, and have been analyzed by
standard methods. Several approaches to the high-throughput analysis of enantioselective catalysts
have been described,3 but additional work is necessary because no single high-throughput ee assay is
universal.
Results and Discussion
We have previously established the principal of EMDee utilizing alcohol dehydrogenases to
measure the % ee of chiral benzyl alcohols.1 This work has been featured in two recent articles in
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Chemical and Engineering News,4 and reviewed by Reetz.5 Our current interest is now to widen the
scope of EMDee by showing that it is applicable to other types of compounds besides alcohols.
For this purpose, we chose chiral allylic acetates as the compounds of interest, because they
are useful intermediates in synthetic chemistry. These compounds can be made by Pd-catalyzed
kinetic resolution6 and de-racemization reactions.7 We have employed the lipase from Pseudomonas
cepacia for determining the ee values of allylic acetates. The lipase catalyzes the acetate hydrolysis
of only one enantiomer, the (R)-allylic acetate, producing acetic acid as the side product (Figure 1).
The acetic acid produced during the reaction is followed by employing p-nitrophenoxide as a pH
indicator. The indicator is protonated to p-nitrophenol, resulting in a decrease in absorbance at 405
nm.8 The BES buffer and the indicator we used have the same pKas, thus acetic acid formation
causes a linear decrease in absorbance.
The kinetic studies we performed show that the (R)-allylic acetate is a substrate for this
enzyme with a Km of 2.21 mM, and the (S)-allylic acetate is an inhibitor with a Ki of 1.31 mM. We
have examined the rate of enzyme-catalyzed hydrolysis of a number of samples of (E)-3-acetyl-1phenyl-1-butene (2, Figure 3) that range in composition from 100% (S) to 100% (R) (Figure 2). We
found out that there is excellent correlation between sample % ee and reaction rate. Thus, EMDee is
a reliable and a fast method for determining the % ee of samples across the full range of
stereochemical outcomes for reactions having allylic acetates as products.
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To observe how EMDee functions with the products from actual catalytic reactions, we have
examined the DMAP catalyzed acetylation of a number of alcohol samples. Assays are run in a 96- well
format and data from a 25-minute window is used to calculate the rates. The data show that there is a
good agreement between the ee values obtained with HPLC and the EMDee method (data not shown).
We have examined two allylic acetates (compounds 2 and 4) in this high-throughput ee screen.
In order to prepare the calibration curve for the enzymatic reactions, the synthesis of pure enantiomers
of both of the compounds was necessary. First, the racemic compounds were prepared following
previously published synthetic routes (Figure 3).9
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Rac-4 was subjected to stereoselective hydrolysis using the immobilized lipase from Candida
antarctica (Figure 4). The reaction was stopped at 45% conversion, and the product (R)-3 was
separated from the remaining acetate. Acetylation of alcohol (R)-3 gave (R)-4. An additional 10%
conversion of the remaining acetate using the lipase gave (S)-4 in enantiomerically pure form.
To prepare (S)-3, rac-3 was subjected to lipase catalyzed acetylation using 0.5 eq. of
isopropenyl acetate (Figure 5).10 This procedure resulted in (S)-3. Following the same routes, we
prepared the enantiomers (R)-1 and (R)-2 in >99% ee, and (S)-1 and (S)-2 with 98% ee. The ee values
of all enantiomers were determined by HPLC using a Chiralcel-OD column.
Conclusion
In summary, we have used EMDee to determine the ee values of allylic acetates in a highthroughput manner. The method can measure the % ee of samples that range from 100% S to 100%
R with an accuracy of approximately +/-10%. Furthermore, ~200 samples can be processed in one
hour.
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1
Abato P.; Seto C. T. J. Am. Chem. Soc. 2001, 123, 9206.
(a) Gilbertson, S. R.; Collibee, S. E.; Agarkov, A. J. Am. Chem. Soc. 2000, 122, 6522. (b) Brouwer,
A. J.; Linden, H.J.; Liskamp, R.M.J. J. Org. Chem. 2000, 65, 1750.
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(a) Korbel, G. A.; Lalic, G.; Shair, M. D. J. Am. Chem. Soc. 2001, 123, 361. (b) Reetz M. T.;
Becker, M. H.; Klein, H. -W.; Stockigt, D. Angew. Chem. Int. Ed. 1999, 38, 1758. (c) Reetz, M.T.;
Becker, M. H.; Kuhling, K. M.; Holzwarth, A. Angew. Chem. Int. Ed. 1998, 37, 2647. (d) Taran, F.;
Gauchet, C.; Mohar, B.; Meunier, S.; Valleix, A.; Renard, P. Y.; Creminon, C.; Grassi, J.; Wagner,
A.; Mioskowski, C. Angew. Chem. Int. Ed. 2002, 41, 124. (e) Chen, Y.; Shimizu, K. D. Org. Lett.,
2002, 17, 2937.
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(a) A Fast Way to Find Catalysts, Rebecca Rawls, Chemical & Engineering News, 80(21), May 27,
2002. (b) C&EN Cover Story – Taking a Measure of Chiral Riches, A. Maureen Rouhi, Chemical &
Engineering News, 80(23), June 10, 2002.
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Reetz, M. T. Angew. Chem. Int. Ed. 2002, 41,1335.
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Gilbertson, S. R.; Collibee, S. E.; Agarkov, A. J. Am. Chem. Soc. 2000, 122, 6522.
7
Trost, B. M.; Organ, M. G. J. Am. Chem. Soc. 1994, 116, 10320.
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Janes L. E.; Kazlauskas, R. J. J. Org. Chem. 1997, 62, 4560.
9
Hoppe, I.; Marsch, M.; Harms, K.; Boche, G.; Hoppe, D. Angew. Chem. Int. Ed. 1995, 34, 2158.
10
For a similar work, see Pchelka, B.K.; Loupy, A.; Plenkiewicz, J.; Blanco, L. Tetrahedron:
Asymmetry, 2000, 11, 2719.
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