A Simple Procedure for Selective Hydroxylation of L-Proline and L-Pipecolic Acid with Recombinantly Expressed Proline Hydroxylases

Due to their diverse regio- and stereoselectivities, proline hydroxylases provide a straightforward access to hydroxprolines and other hydroxylated cylic amino acids, valuable chiral building blocks for chemical synthesis, which are often not available at reasonable expense by classical chemical synthesis. As yet, the application of proline hydroxylases is limited to a sophisticated industrial process for the production of two hydroxyproline isomers. This is mainly due to difficulties in their heterologues expression, their limited in vitro stability and complex product purification procedures. Here we describe a facile method for the production of cis-3-, cis-4- and trans-4-proline hydroxylase, and their application for the regio- and stereoselective hydroxylation of L-proline and its six-membered ring homologue L-pipecolic acid. Since in vitro catalysis with these enzymes is not very efficient and conversions are restricted to the milligram scale, an in vivo procedure was established, which allowed a quantitative conversion of 6 mM L-proline in shake flask cultures. After facile product purification via ion exchange chromatography, hydroxyprolines were isolated in yields of 35-61% (175-305 mg per flask). L-Pipecolic acid was converted with the isolated enzymes to prove the selectivities of the reactions. In transformations with optimized iron(II) concentration, conversions of 17-68% to hydroxylated products were achieved. The regio-and stereochemistry of the products was determined by NMR techniques. To demonstrate the applicability of the preparative in vivo approach for non-physiological substrates, L-pipecolic acid was converted with an E. coli strain producing trans-4-proline hydroxylase to trans-5-hydroxy-L-pipecolic acid in 61% yield. Thus, a synthetically valuable group of biocatalysts was made readily accessible for application in the laboratory without a need for special equipment or considerable development effort.