Synthesis and Application of New Chiral Variants of Marfey's Reagent for Liquid Chromatographic Separation of the Enantiomers of α-Amino Acids

Two new structural variants of Marfey's reagent (1-fluoro-2,4-dinitrophenyl-5-L-alaninamide, FDNP-L-Ala-NH2) were synthesized by nucleophilic substitution of one fluorine atom in 1,5-difluoro-2,4-dinitrobenzene (DFDNB) by L-methioninamide or D-phenylglycinamide. The new variants FDNP-L-Met-NH2 and FDNP-D-Phg-NH2 were characterized and used for derivatization of twenty-six alpha-amino acids. The resulting diastereomeric derivatives were separated on a reversed-phase C-18 HPLC column using a linear gradient of acetonitrile and aqueous trifluoroacetic acid (TFA) and the results were compared with those obtained by use of Marfey's reagent. To determine and establish the efficiency of both the new variants, separation results were compared for diastereomers of five representative amino acids (Ala, Phe, Ser, Asp, and Asn) prepared with MR, FDNP-L-Phe-NH2, FDNP-L-Val-NH2, FDNP-L-Leu-NH2, and FDNP-L-Pro-NH2, experiments being performed under identical conditions. Both the new chiral reagents enabled better separation of the diastereomers; than Marfey's reagent for most of the amino acids.. The reagent FDNP-D-Phg-NH2 enabled excellent separation for serine and asparagine whereas other reagents failed or provided poor results.