High-performance liquid chromatographic resolution of (R, S)-α-alkyl-α-amino acids as diastereomeric derivatives

A number (27) of racemicα-alkyl-α-amino acids (AAA) were derivatized either witho-phthaldialdehyde (OPA) in combination withN-t-butoxycarbonyl-L-cysteine (Boc-Cys) orN-acetyl-L-cysteine (Ac-Cys), or withN (2)-(5-fluoro-2,4-dinitrophenyl)-L-alanine amide (Marfey's reagent). The resolution of the diastereoisomers formed was investigated by reversed-phase (C18) high-performance liquid chromatography (HPLC) using gradient elution conditions employing sodium phosphate buffers of pH 7.2 together with acetonitrile, and fluorescence detection at 344 nm (excitation) and 443 nm (emission) for the OPA/Boc-Cys or OPA/Ac-Cys derivatives. For the diastereomers formed by derivatization with Marfey's reagent triethylammonium phosphate buffers of pH 3.0 (pH 7.2 for acidic AAA) together with acetonitrile, and u.v. detection at 340 nm were used. Whereas with Marfey's reagent all diastereomers of AAA showed complete, or almost complete, resolution, only 8, or 11, respectively of the diastereomers formed by derivatization with OPA/Boc-Cys or OPA/Ac-Cys were resolved under the chromatographic conditions used.