Separation of 17 DL-amino acids and chiral sequential analysis of peptides by reversed-phase liquid chromatography after labeling with R(-)-4- (3-isothiocyanatopyrrolidin-1-yl)-7-(N, N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole.
Seventeen DL-amino acids labeled with a fluorescent chiral labeling reagent, R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N, N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole (R(-)-DBD-PyNCS), were separated by reversed-phase chromatography and detected fluorometrically at 550 nm (excitation at 460 nm). The reagent reacted with amino functional group in dl-amino acids under basic medium. The thiocarbamoyl derivatives were converted to thiohydantoin via thiazolinone in trifluoroacetic acid (TFA) solution. The epimerization ratios during the reaction of the cyclization were less than 37% in all dl-amino acids tested. The resulting thiohydantoin derivatives of individual dl-amino acids were completely separated with isocratic elutions using acidic mobile phase involving 0.1% TFA. The separations of the thiohydantoins yielded from acidic, basic, neutral, hydroxyl, and aromatic amino acids were good enough for the identification of dl-amino acid. The method using the reagent was adopted to identification of dl-amino acid sequences in eight peptides. The separation and identification of the thiohydantoin derivatives liberated from the peptides labeled were performed by the isocratic elutions. The applicability of the proposed procedure to sequential analysis of peptide was demonstrated with [D-Ala(2)]-leucine enkephalin, [D-Ala(2)]-deltorphin II, d-Phe-Met-Arg-Phe-amide, and Phe-D-Met-Arg-Phe-amide. D-Ala, D-Phe, and D-Met in the peptides were positively identified with the proposed procedures. [L-Ala(2)]-leucine enkephalin, beta-lipotropin, Asp-Ser-Asp-Pro-Arg, and Pro-Asp-Val-Asp-His-Val-Phe-Leu-Arg-Phe-amide were also analyzed as the references without D-amino acid.
Toyo'oka
T
T
Department of Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka, 422-8526, Japan. toyooka@ys2.u-shizuoka-ken.ac.jp
Tomoi
N
N
Oe
T
T
Miyahara
T
T
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005069:Evaluation Studies as Topic; D005456:Fluorescent Dyes; D017879:Isothiocyanates; D010069:Oxadiazoles; D010455:Peptides / Q000737:chemistry*; D020539:Sequence Analysis, Protein / Q000379:methods*; D013237:Stereoisomerism; D013867:Thiohydantoins
C107817:4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole; D000596:Amino Acids; D005456:Fluorescent Dyes; D017879:Isothiocyanates; D010069:Oxadiazoles; D010455:Peptides; D013867:Thiohydantoins