The development of a new class of protease inhibitors and EMDee: A new high-throughput enzymatic method for the determination of enantiomeric excess.

摘要

The further development of a new class of protease inhibitors is described. These inhibitors contain a cyclohexanone moiety as the electrophilic isostere. The cyclohexanone scaffold offers the ability to add appendages to the P as well as the P' diversity space within these inhibitors. The methodology for the solid phase synthesis of inhibitors that interact with the S2 and S2' was developed. It was found that inhibition constants against cathepsin B for this class of inhibitors with and without the additional P2' appendage were 6 and 24 mM, respectively.; The solid phase synthesis protocol allowed for the construction of a library of inhibitors that contained both P2 and P2' functionality. Each of these two positions contain one of 20 amino acids, and in total the library consisted of 400 individual members. The library was assayed for activity against plasmin, cathepsin B, papain, kallikrein and urokinase. The library and subsequent deconvolution assays against these enzymes provided biding profiles of the two subsites, S2 and S2' . Most notably plasmin prefers Trp at the S2 subsite and aromatic as well as hydrophobic amino acids within the S2' binding pocket. A lead compound was generated from the library that has an inhibition constant of 5 muM against plasmin.; The rational development of these second generation type inhibitors, that interact with the S as well as the S' subsites, is also described. These rationally designed inhibitors contain an alkylamino P1 appendage as well as P2 and P2' appendages, and were assayed against plasmin, thrombin, trypsin and kallikrein. These inhibitors have inhibition constants in the 20 muM range against plasmin.; The remainder of this thesis describes a new method for high-throughput screening of asymmetric catalysts. This method utilizes the enzyme ( S)-aromatic alcohol dehydrogenase to oxidize (S)-1-phenylpropanol to the corresponding ketone. This oxidation is coupled to the reduction of the required cofactor, NADP+. The rate of this enzymatic reaction is directly related to the enantiomeric excess of the phenylpropanol. The ee, as determined by the new EMDee (Enzymatic Method for Determining Enantiomeric Excess) procedure, was validated using conventional GC with a chiral stationary phase.