EMDee: an enzymatic method for determining enantiomeric excess.

The techniques of combinatorial chemistry have recently been applied to the discovery of new asymmetric catalysts for a variety of organic transformations.1 These techniques are useful for increasing the efficiency by which chiral ligands for metal catalysts are discovered since the design of these ligands is largely an empirical process, and there are currently no simple rules for reliably producing catalysts that give high stereoselectivity.2 Using combinatorial methods, it is straightforward to generate thousands of potential asymmetric catalysts. However, screening these catalysts for stereoselectivity is currently the bottleneck in the process because it is time-consuming to measure the enantiomeric excess of so many samples. The most common analytical techniques for measuring ee, such as chiral GC and HPLC, generally require tens of minutes per analysis. Thus, they can be used to analyze hundreds of samples at a time, especially when employing an autosampler, but they become impractical for analyzing the thousands of catalysts that can be generated through combinatorial synthesis. To bring the full power of combinatorial chemistry to bear on the problem of asymmetric catalysis, it will be necessary to develop new methods for the high throughput screening of enantiomeric excess. Several such methods have been reported in the recent literature. They rely on techniques that include kinetic resolution in conjunction with reaction microarrays3a or electrospray mass spectrometry (ESMS),3b the use of isotopically labeled pseudo-enantiomers with ESMS detection,3c IR detection of reactions using enantiomerically pure starting materials,3d capillary array electrophoresis using chiral host molecules,3e and HPLC using CD detection.3f In this communication we report on an enzymatic method for determining enantiomeric excess (EMDee) that can be used for the high throughput screening of asymmetric catalysts. In this method an enzyme is used to selectively process one enantiomer of a product from a catalytic reaction. We have chosen the addition of diethylzinc to benzaldehyde as a test-bed for demonstrating EMDee. This is a well-studied reaction for which a variety of enantioselective catalysts have been developed.4 The reaction yields 1-phenylpropanol 3, and the (S) enantiomer of this product can be oxidized to the corresponding ketone using the (S)-aromatic alcohol dehydrogenase from Thermoanaerobium sp. (Scheme 1). The rate of the enzymatic oxidation can be conveniently monitored by observing formation of NADPH by UV spectroscopy at 340 nm. (S)-3 is a good substrate for the enzyme with a KM value of 6.4 ( 1.1 mM, while (R)-3 is an inhibitor with a Ki value of 6.0 ( 1.5 mM. The Michaelis-Menten equation provides a direct relationship between the rate of this enzymatic oxidation and the concentrations of (S)-3 and (R)-3. Therefore, the rate of this oxidation can be used as a direct measure of the enantiomeric excess of samples of 1-phenylpropanol. We have examined the rate of the enzyme-catalyzed oxidation of a number of samples of 1-phenylpropanol that range in composition from 100% (S)-3 to 100% (R)-3. Figure 1 shows that there is an excellent correlation between sample % ee and reaction rate. Thus, EMDee is a reliable and fast method for determining the % ee of samples across the full range of stereochemical outcomes for the reaction of diethylzinc with benzaldehyde in the presence of chiral catalysts. To demonstrate the high throughput nature of this assay, 100 samples were analyzed in a 384-well format using a UV/fluorescence plate reader.5 The sample volume in these runs was 100 μL, and each sample contained 1 μmol of 1-phenylpropanol. Data from a 30 min window were used to calculate the relative rates of these 100 reactions. On the basis of the data from these runs along with the data shown in Figure 1, we estimate that EMDee can be used to determine the % ee of samples with an accuracy of approximately (10%. Thus, the method will be most useful as a preliminary high throughput screening technique for identifying the relatively small number of catalysts out of a library that show high stereoselectivity. Once these catalysts have been identified, their stereoselectivity can be measured more precisely, but more slowly, using chiral GC or HPLC. To observe how EMDee functions with the products from actual catalytic reactions, we have examined the solvent dependence for the addition of diethylzinc to benzaldehyde using the previously reported catalysts 5 and 6.4e It is known that catalyst