P73: Dynamic resolution of chiral 2-hydroxy acids by spinach glycolate oxidase and subsequent purification of the (R)-enantiomers using Simulated Moving Bed Chromatography

Optically active 2-hydroxy acids are important building blocks for asymmetric synthesis of numerous bioactive molecules (e.g., ACE inhibitors, A2 antagonists, Ca-channel blockers, beta-blockers) including glycols, halo esters and epoxides.  Glycolate oxidase (GO, EC 1.1.3.15) is a flavin mononucleotide-dependent enzyme, which catalyzes the oxidation of (S)-2-hydroxy carboxylic acids such as (S)-lactate to the corresponding 2-keto acids.  Spray-dried whole cells of Pichia pastoris expressing GO from spinach were used for the dynamic resolution of 2-hydroxy acids.  As GO is absolutely specific on (S)-enantiomer, GO catalyzed oxidation of racemic (RS)-2–hydroxy acids produced 2-keto acids, only from (S)-enantiomer, keeping (R)-isomer intact.  Non-selective reduction of 2-ketoacids using sodium borohydride produced (RS)-2–hydroxy acids to facilitate a one-pot dynamic process for production of (R)-2–hydroxy acids with high yield (> 95%).  GO could be recycled twice in this dynamic process with > 92% yield of the (R)-acids.  The entire “one pot – two step” process was carried out in water and at room temperature in order to make the process economical.  Finally, (R)-2-hydroxy acids were purified from the crude post-reaction mixtures by cation exchange using a Semba Octave ™ simulated moving bed chromatography (SMBC) system.  The initial SMBC conditions were determined using triangle theory based separation parameters, and optimized by adjusting those parameters based on the analysis of the purified fractions.  Continuous separation of the hydroxy acids from their 2-keto intermediates and other reaction components was achieved with yields >80% and purities >99%.