Synthesis and evaluation of new fluorescent derivatization reagents for resolution of chiral amines by RP-HPLC

Abstract

New fluorescent chiral derivatization reagents (i.e., DBD- trans-4-hydroxy- l-proline, DBD- cis-4-hydroxy- l-proline, DBD- cis-4-hydroxy- d-proline, and DBD- trans-3-hydroxy- l-proline) were synthesized from the reaction of 4-( N, N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole with corresponding hydroxy-prolines. These reagents reacted with chiral amine to produce a couple of diastereomers. The labeling efficiently proceeded in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and pyridine as the activation reagents. The reaction conditions are mild and no racemization occurred during both the reagent synthesis and the diastereomer formation (<0.4%). The resulting diastereomers fluoresce at around 560 nm (excitation at around 450 nm). Good linearity of the calibration curves was obtained in the range of 1–75 pmol and the detection limits on chromatogram were less than 1 pmol. The separability of the diastereomers was evaluated in terms of separation factor ( α) and resolution value (Rs). DBD- trans-4-hydroxy- l-proline was efficient for the resolution of dl-phenylalanine methylester; while DBD- cis-4-hydroxy- d(or l)-proline was excellent for the separation of 1-(1-naphtyl)ethylamines, as comparing with trans-4-hydroxy isomer. The reagents of cis-isomer seemed to be predominant for the resolution of hydrophobic enantiomers. On the other hand, trans-isomers were suitable for the separation of the racemic amines containing ester in the structure. With respect to the position of OH group, the effect seems to be less, judging from the results of DBD- trans-4-hydroxy- l-proline and DBD- trans-3-hydroxy- l-proline toward phenylalanine methylester. The results suggest that the separation is dependent upon both structures of the amines and the reagent used. Thus, the stereostructure, hydrophobicity and hydrogen bonding of the diastereomer, etc. seem to be affecting the separation.