Novel Polymeric reagent for synthesizing 9-fluorenylmethoxycarbonyl L-prolinyl derivatives for chiral high-performance liquid chromatography of amino acids

Abstract

The synthesis of a novel polymer-supported 9-fluorenylmethoxycarbonyl-L-proline reagent for derivatizing nucleophiles is described. The new polymer, containing a 1-hydroxybenzotriazole activated ester, is highly reactive. Significant improvement in the ease of use and the rate of reaction with strong and weak nucleophiles is achieved. In this study, the utility of the solid-phase reagent for derivatizing amino acids for chiral high-performance liquid chromatography is described. Derivatization is accomplished simply by mixing a sample of the amino acid in acetonitrile with the polymer at room temperature for 10 min. Racemization of amino acids under the mild reaction conditions is not observed. Despite 9-fluorenylmethoxycarbonyl-L-prolinyl-D,L-amino acids are diastereoisomeric, the isomers are not separated by simple reversed-phase chromatography. Since these derivatives posses the necessary functional groups for metal chelation, mobile phases containing chiral Cu(II) complexes wee used to resolve the optical isomers. Excellent resolution of all the D and L enantiomers of natural amino acids was achieved by using Cu(II)-L-histidine methyl ester and Cu(II)-l-proline eluents on reversed phases columns with various concentrations of acetonitrile. The separated derivatives were detected in the low-nanogram range by fluorescence at 315 nm with excitation at 275 nm.