Derivatization with fluorogenic benzofurazan reagents of amino acid enantiomers and their separation on a Pirkle type column.
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-CI), 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, 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 alphas 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, alpha = 1.0). All NBD-, DBD- and ABD-derivatives of L- and D-Phe were also well separated (alphas were 1.18, 1.17 and 1.16, respectively), while DNS-L- and -D-Phe were barely separated (alpha = 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Imai
K
K
Branch Hospital Pharmacy, University of Tokyo, Japan.
Fukushima
T
T
D000596:Amino Acids / Q000737:chemistry* / Q000302:isolation & purification; D001572:Benzofurans / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003619:Dansyl Compounds / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D005470:Fluorometry; D010069:Oxadiazoles / Q000737:chemistry; D010080:Oxazoles / Q000737:chemistry; D013237:Stereoisomerism; D013449:Sulfonamides / Q000737:chemistry
D000596:Amino Acids; D001572:Benzofurans; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D010069:Oxadiazoles; D010080:Oxazoles; D013449:Sulfonamides; C046364:4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole; C060548:4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole; C029038:dansyl chloride