Direct Chiral Separation of N-Fluorenylmethoxycarbonyl α-Amino Acids by HPLC for Determination of Enantiomeric Purity

Abstract

N-Protected α-amino acids have been widely used as important chiral building blocks in the fields of pharmaceutical chemistry and biochemistry. The fluorenylmethoxycarbonyl (FMOC) group is one of the most commonly used protecting groups for α-amino acids and provides the advantages of high sensitivity in fluorescence detection. Especially, the FMOC protecting group is attractive, because it is frequently used for amino protecting groups in solid phase synthesis and combinatorial chemistry. Owing to the importance of optical purity of N-FMOC α-amino acids, convenient and accurate methods to determine the enantiopurity of these compounds have been required and developed. Several methods for the liquid chromatographic separation of the enantiomers of N-FMOC α-amino acids have been reported using CSPs derived from cyclodextrin, macrocyclic antibiotics and cellulose derivatives as well as small molecules derived brush-type CSPs. Although the enantioseparation of some FMOC secondary amino acids has been performed on 1-naphthylethyl carbamoylated βcyclodextrin bonded CSP with good resolution, most FMOC α-amino acids enantiomers have not been resolved on βand γ-cyclodextrin bonded CSPs. It has been reported that nine N-FMOC α-amino acids enantiomers were separated on macrocyclic antibiotics derived CSP. Brushtype CSPs derived from α-amino acids and cinchona derivatives including Whelk-O CSP have been employed to resolve some FMOC α-amino acids enantiomers. Also, the enantioresolution of two FMOC α-amino acid type enantiomers has been reported on cellulosic-type CSP under reversed-phase conditions. For resolution of N-FMOC αamino acids, however, polysaccharide-derived CSPs have not been applied under normal phase conditions and the enantiomer separation of a series of these analytes has not been well investigated. In this study, the liquid chromatographic resolution of N-FMOC protected α-amino acids for determination of their enantiomeric purity was performed on polysaccharide-derived CSPs, Chiralcel OD and Chiralpak AD under normal phase conditions. Table 1 and Table 2 show liquid chromatographic results for the separation of the enantiomers of N-FMOC α-amino acids on Chiralcel OD and Chiralpak AD. Chiralcel OD and/ or Chiralpak AD afford fairly good enantioselectivities for the resolution of N-protected FMOC α-amino acids under normal phase conditions. Chiralcel OD affords comparable or better enantioselectivities for the resolution of N-FMOC α-amino acids than Chiralpak AD except N-FMOC protected glutamine and tyrosine analytes (entries 7 and 17). All investigated N-FMOC α-amino acids enantiomers were base-line separated on Chiralcel OD or Chiralpak AD. For example, while N-FMOC glutamine, norvaline, phenylalanine and tyrosine enantiomers were partially separated on Chiralcel OD (Rs 1.48). Therefore, Chiralcel OD and Chiralpak AD might be complementarily used for the separation of the enantiomers of N-FMOC α-amino acids. The consistent elution order of the resolved N-FMOC α-amino acids is not observed on Chiralcel OD and Chiralpak AD. As shown in Table 1, the elution of the (D)isomers prior to the (L)-isomers for resolution of N-FMOC α-amino acids examined except five analytes is observed on Table 1. Enantiomer Separation of N-FMOC Protected α-Amino Acids on Chiralcel OD