Development of an amino acid sequence and D/L-configuration determination method of peptide with a new fluorescence Edman reagent, 7-methylthio-4-(2,1,3-benzoxadiazolyl) isothiocyanate.
On the basis of the relationship between the fluorescence characteristics of the benzofurazan compounds and the Hammett constants (sigma p), a new fluorescence Edman reagent, 7-methylthio-4-(2,1,3-benzoxadiazolyl) isothiocyanate (MTBD-NCS) was designed and synthesized. MTBD-thiohydantoin (TH)-amino acid derivatives produced by the Edman sequencing method gave fluorescence, whereas other degradation byproducts such as MTBD-thiocarbamoyl (TC)- or carbamoyl (CA)-amino acids did not fluoresce. MTBD-NCS was applicable as an Edman sequencing reagent to the simultaneous determination of both the sequence and D/L-configuration of amino acids in peptides. Boron trifluoride (BF3) and HC1/methanol were adopted as the cyclization/cleavage and conversion reagents to suppress the amino acid residue racemization. The MTBD-TH-amino acids were separated on a reversed-phase column for amino acid sequencing, and their enantiomers were resolved on two types of polysaccharide-based chiral stationary phases for D/L-configuration determination. The method was successfully applied to the sequence and D/L-configuration determination of D-amino acid-containing peptide [D-Ala2]-deltorphin II.
Toriba
A
A
Graduate School of Pharmaceutical Sciences, University of Tokyo, Japan.
Adzuma
K
K
Santa
T
T
Imai
K
K
D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry; D007202:Indicators and Reagents; D017879:Isothiocyanates / Q000138:chemical synthesis* / Q000737:chemistry; D010069:Oxadiazoles / Q000138:chemical synthesis* / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D011487:Protein Conformation; D020539:Sequence Analysis, Protein / Q000379:methods*; D013237:Stereoisomerism
C406762:7-methylthio-4-(2,1,3-benzoxadiazolyl)isothiocyanate; D000596:Amino Acids; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D017879:Isothiocyanates; D010069:Oxadiazoles; D010455:Peptides