VITAMIN-K DEPENDENT CARBOXYLATION - DETERMINATION OF THE STEREOCHEMICAL COURSE USING 4-FLUOROGLUTAMYL-CONTAINING SUBSTRATE

The stereochemical course of the vitamin K dependent carboxylation has been elucidated using a (4S)-4-fluoroglutamyl-containing pentapeptide as a substrate. The absolute configuration of the [C-13]-4-carboxy-4-fluoroglutamate obtained when the carboxylation was carried out with C-13-labeled sodium bicarbonate, was determined after reduction of the [C-13]-4-carboxy-4-fluoroglutamyl residue into 4-fluoro-5,5'-dihydroxyleucine, hydrolysis, lactonization, and peracetylation. The absolute configuration at C-4 was determined to be S by locating the C-13 label in the lactone ring of the trans isomeric lactone and in the hydroxymethyl group of the cis isomer following HPLC separation of both isomers and analysis by GC/MS/MS techniques. It follows that the vitamin K dependent carboxylation occurs with inversion of configuration.