Detection of DBD-carbamoyl amino acids in amino acid sequence and D/L configuration determination of peptides with fluorogenic Edman reagent 7-[(N,N-dimethylamino)sulfonyl]-2,1,3-benzoxadiazol-4-yl isothiocyanate.
A method for amino acid sequence and D/L configuration identification of peptides by using fluorogenic Edman reagent 7-[(N, N-dimethylamino)sulfonyl]-2,1,3-benzoxadiazol-4-yl isothiocyanate (DBD-NCS) has been developed. This method was based on the Edman degradation principle with some modifications. A peptide or protein was coupled with DBD-NCS under basic conditions and then cyclized/cleaved to produce DBD-thiazolinone (TZ) derivative by BF3, a Lewis acid, which could significantly suppress the amino acid racemization. The liberated DBD-TZ amino acid was hydrolyzed to DBD-thiocarbamoyl (TC) amino acid under a weakly acidic condition and then oxidized by NaNO2/H+ to DBD-carbamoyl (CA) amino acid which was a stable and had a strong fluorescence intensity. The individual DBD-CA amino acids were separated on a reversed-phase high-performance liquid chromatography (RP-HPLC) for amino acid sequencing and their enantiomers were resolved on a chiral stationary-phase HPLC for identifying their D/L configurations. Combination of the two HPLC systems, the amino acid sequence and D/L configuration of peptides could be determined. This method will be useful for searching D-amino-acid-containing peptides in animals.
Huang
Y
Y
Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113, Japan.
Matsunaga
H
H
Toriba
A
A
Santa
T
T
Fukushima
T
T
Imai
K
K
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis* / Q000737:chemistry*; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004355:Drug Stability; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007202:Indicators and Reagents; D017879:Isothiocyanates; D010069:Oxadiazoles; D010455:Peptides / Q000737:chemistry; D017421:Sequence Analysis / Q000379:methods; D012997:Solvents; D013237:Stereoisomerism
D000596:Amino Acids; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D017879:Isothiocyanates; D010069:Oxadiazoles; D010455:Peptides; D012997:Solvents; C079705:7-N,N-dimethylaminosulfonyl-4-(2,1,3-benzoxadiazolyl)isothiocyanate