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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.
Anal Biochem. 1999 Jun 01; 270(2):257-67.AB

Abstract

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.

Authors+Show Affiliations

Huang Y
Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113, Japan.
Matsunaga H
No affiliation info available
Toriba A
No affiliation info available
Santa T
No affiliation info available
Fukushima T
No affiliation info available
Imai K
No affiliation info available

MeSH

Amino Acid SequenceAmino AcidsAnimalsChromatography, High Pressure LiquidDrug StabilityFluorescent DyesHydrogen-Ion ConcentrationHydrolysisIndicators and ReagentsIsothiocyanatesOxadiazolesPeptidesSequence AnalysisSolventsStereoisomerism

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

10334843

Citation

Huang, Y, 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." Analytical Biochemistry, vol. 270, no. 2, 1999, pp. 257-67.
Huang Y, Matsunaga H, Toriba A, 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. Anal Biochem. 1999;270(2):257-67.
Huang, Y., Matsunaga, H., Toriba, A., Santa, T., Fukushima, T., & Imai, K. (1999). 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. Analytical Biochemistry, 270(2), 257-67.
Huang Y, 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. Anal Biochem. 1999 Jun 1;270(2):257-67. PubMed PMID: 10334843.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Huang,Y, AU - Matsunaga,H, AU - Toriba,A, AU - Santa,T, AU - Fukushima,T, AU - Imai,K, PY - 1999/5/21/pubmed PY - 1999/5/21/medline PY - 1999/5/21/entrez SP - 257 EP - 67 JF - Analytical biochemistry JO - Anal Biochem VL - 270 IS - 2 N2 - 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. SN - 0003-2697 UR - https://www.unboundmedicine.com/medline/citation/10334843/Detection_of_DBD_carbamoyl_amino_acids_in_amino_acid_sequence_and_D/L_configuration_determination_of_peptides_with_fluorogenic_Edman_reagent_7_[_NN_dimethylamino_sulfonyl]_213_benzoxadiazol_4_yl_isothiocyanate_ DB - PRIME DP - Unbound Medicine ER -

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