DEVELOPMENT AND OPTIMIZATION OF A NOVEL ENZYMATIC KINETIC RESOLUTION AS PART OF THE DYNAMIC KINETIC RESOLUTION OF AMIDES WITH A REMOTE STEREOCENTER

Abstract

Optical purity is an essential feature of many compounds in the pharmaceutical industry. Chiral centers which lie beyond the alpha position of a functional group, called remote stereocenters, are a common feature of these compounds, making their resolution of great interest to the field. Although the field is underdeveloped, it has been previously shown that enzymes are incredibly capable of setting remote stereocenters. Biocatalytic kinetic resolutions are commonly used strategies in industry for the creation of highly enantioenriched compounds from racemic starting material, but they are limited to maximum yields of 50 %. The dynamic kinetic resolution of a racemic compound offers a solution to this drawback, since it enables a maximum theoretical yield of 100 %. Unfortunately, there are very few DKRs which target remote stereocenters, limiting the scope of DKRs to compounds with proximal stereocenters. In order to address such limitation, this project seeks to develop the dynamic kinetic resolution of amides with remote stereocenters using racemization via 1,5-HAT. The work presented herein focuses on the development of the biocatalytic kinetic resolution which accompanies this racemization to create a novel pathway to optically pure primary amines with remote stereocenters. An enzyme was identified which orchestrates the kinetic resolution of the model substrate with 51 % conversion and an enantiomeric ratio of 91:1, affording an E- value of 22. Additionally, preliminary results of protein engineering via site-saturation mutagenesis led to the identification of promising mutants which provide guidance for further optimization.