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
Y. Huang et al., 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, ANALYT BIOC, 270(2), 1999, pp. 257-267
A method for amino acid sequence and D/L configuration identification of pe
ptides 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 fluoresc
ence intensity. The individual DBD-CA amino acids were separated on a rever
sed-phase high-performance liquid chromatography (RP-HPLC) for amino acid s
equencing 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-containi
ng peptides in animals. (C) 1999 Academic Press.