Derivatization with fluorogenic benzofurazan reagents of amino acid enantiomers and their separation on a pirkle column

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Abstract
L‐ and D‐Amino acids (Leu or Phe) were derivatized with fluorogenic reagents, 4‐fluoro‐7‐nitro‐2,1,3‐benzoxadiazole (NBD‐F), 4‐(N,N‐dimethylaminosulfonyl)‐7‐fluoro‐2,1,3‐benzoxadiazole (DBD‐F), 4‐aminosulfonyl‐7‐fluoro‐2,1,3‐benzoxadiazole (ABD‐F) and 5‐(N,N‐dimethylamino)naphthalene‐1‐sulfonylchloride (DNS‐Cl), and separated on a Pirkle type column, Sumichiral OA 2500 (S) ((S)‐1‐naphthylglycyl‐3,5‐dinitrophenylamide silica gel) with a mobile phase of 20 mM ammonium acetate in methanol. The fluorometric detection of the derivatives was made at 530 nm, 590 nm and 530 nm with excitation at 470 nm, 450 nm, 450 nm and 350 nm, respectively. The former three derivatives of the enantiomers were separated well from each other; The as for each NBD‐, DBD‐ and ABD‐derivative of L‐ and D‐Leu were 1.10, 1.11 and 1.10, respectively. However, the DNS derivatives of L‐and D‐Leu were not separated (separation factor, α = 1.0). All NBD‐ and ABD‐derivatives of L‐ and D‐Phe were also well separated (αs were 1.18, 1.17 and 1.16, respectively), while DNS‐L‐ and ‐D‐Phe were barely separated (α = 1.04). These data suggest that the 2,1,3‐benzoxadiazole (benzofurazan) moiety is very effective and preferable to the dimethylaminonaphthalene sulfonyl (DNS) structure for the separation of enantiomers of amino acids derivatized with benzofurazan reagents. No recemization of each enantiomer occurred during the derivatization reaction with NBD‐F, DBD‐F and ABD‐F at pH 8.0 and 60°C for 2 min, at pH 9.3 and 60°C for 30 min and pH 9.3 and 60°C for 60 min, respectively, meaning that all benzofurazan type fluorogenic reagents are useful for sensitive determination of amino acid enantiomers.

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