Enzyme-coupled ee screening systems

The kinetic resolution of (5, 7 )-l-methoxy-2-propylacetate was investigated using various commercially available hydrolases. The agreement between the apparent selectivity factor app and the actual value true determined by GC turned out to be excellent at low enantioselectivity E — 1.4— 13), but less so at higher enantioselec-tivity (20% variation at E— 80). This limitation may cause problems when attempting to enhance enantioselectivity beyond E — 50. 

Whenever the product concentration (e.g., of an alcohol) can be determined, the ee value is accessible by using an enzyme to catalyze a further reaction which can be monitored by UV/Vis spectroscopy. In the so-called EMDee method, a selective 

In what appears to be a particularly irmovative development in the area of UV/ Vis-based ee screening systems, the determination of the enantiomeric purity of chiral alcohols 9 is based on a new concept of using two enantioselective enzymes to modify the product (84). The method allows the determination of ee values independent of the concentration, which may be of significant advantage in directed evolution projects. It can be used in three different biocatalytic processes, namely biohydroxylation of alkanes, reductase-catalyzed reduction of ketones, and lipase-or esterase-catalyzed ester hydrolysis. 

The method was successfully applied to a series of alcohols 9. Of the 83 measured ee values, 79 turned out to be within 10% of the true value, and four resulted in an error of +12%. As the authors stated, the method has several distinctive features 84)  

Several other UV/Vis-based ee assays have been developed, but their general application in directed evolution remains to be demonstrated 66-70). One of them is a well- designed screening system based on enzyme immunoassays (85). The success of the ee assay depends upon the availability of specific antibodies that are easily raised for almost any chiral product of interest. In a given reaction, two antibodies are needed, one that measures the product concentration and the other that measures the amount of one of the enantiomers. About 1000 ee determinations are possible per day, the precision amounting to + 9% (85). 

---