HPLC separation of DL-amino acids derivatized withN ²-(5-fluoro-2,4-dinitrophenyl)-L-amino acid amides

Summary

Neutral, acidic, and basic protein DL-amino acids (DL-AA) have been separated by HPLC as diastereomeric derivatives obtained after derivatization withN ²-(5-fluoro-2,4-dinitrophenyl)-L-alanine amide (FDNP-Ala-NH₂, “Marfey's reagent”). HPLC was performed on 3 μm Spherisorb ODS II as the stationary phase with gradient elution using mixtures of triethylammonium phosphate buffer (pH 3) and acetonitrile. The differences in retention times (Δt_R) of diastereomers were compared with those obtained by derivatization of DL-AA with the novel FDNP-reagents FDNP-Val-NH₂, FDNP-Phe-NH₂, and FDNP-Pro-NH₂. FDNP-reagents were synthesized by reaction of 1,5-difluoro-2,4-dinitrobenzene with 0.5 equivalent of the respective L-AA amide in mixtures of aqueous NaHCO₃ and acetone at 40–50°C. All FDNP-reagents made possible the resolution of DL-AA. However, FDNP-Val-NH₂, gave the largest Δt_R-values in most cases. Large Δt_R-values mainly arise by the formation of an intramolecular hydrogen bond between the carboxy and carboxamide group in the L-L diastereomers and the non-formation of this hydrogen bond in the D-L diastereomer (the first letter refers to the configuration of the AA to be analysed, the second to that of the reagent AA amide) as well as by the low conformational freedom of amino acid residues in diastereomers.