Proton: a major factor for the racemization and the dehydration at the cyclization/cleavage stage in the Edman...

The racemization of the liberated 7-[(N,N-dimethylamino)sulfonyl]-4-(2,1,3-benzoxadiazolyl)-thiazoli none (DBD-TZ) amino acid during the cyclization/cleavage reaction with trifluoroacetic acid (TFA) in the Edman sequencing procedure has been carefully investigated, and evidence is presented to show conclusively that the racemization is caused by the replacement of a hydrogen atom by TFA. The fluorescent reagent 7-[N,N-dimethylamino)sulfonyl]-4-(2,1,3-benzoxadiazolyl) isothiocyanate (DBD-NCS) was used for amino acid sequencing, and DBD-TZ amino acid was used for sequence and configuration determination. DBD-thiocarbamoylated peptides were cyclized and cleaved with deuterated TFA, and the protonated pseudomolecular ions (M-d1 + H)+ of DBD-TZ amino acids were detected by LC/MS. Furthermore, in the reaction kinetics study, we confirmed that the replacement reaction by TFA correlated sufficiently with the racemization of DBD-TZ amino acids. For the purpose of retaining D/L-amino acid configuration in sequencing, we used an aprotic acid, i.e., the Lewis acid boron trifluoride (BF3), for the cyclization/cleavage reaction. When we used BF3, the derivatized DBD-TZ amino acid was scarcely racemized under cyclization/cleavage conditions. Using this method, amino acid sequencing of D-Phe-Met-Arg-Phe-amide could be performed, retaining the D/L-configuration of the amino acid residues.