Proton:  A Major Factor for the Racemization and the Dehydration at the Cyclization/Cleavage Stage in the Edman Sequencing Method

Abstract

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.

Fluorogenic and fluorescent labeling reagents with a benzofurazan skeleton

“…Recently, the peptides or proteins containing D-amino acids were found, and therefore the method for the amino acid sequence and D/L-configuration determination were studied . At the cyclization and cleavage reaction, the use of boron trifluoride-etherate (BF 3 ) instead of TFA suppresses the racemization of DBD-TZ derivatives (Matsunaga et al, 1996a) and the method for the amino acid sequence and D/L-configuration determination were established using DBD-NCS (Matsunaga et al, 1996b). The reports on the comparison of DBD-NCS with other benzofurazan isothiocyanates reported that DBD-NCS was the most suitable reagent for the detection of TZ derivatives (Matsunaga et al, 1997;Toriba et al, 1999).…”

A manual sequence method of peptides and phosphopeptides using 4-(1′-cyanoisoindolyl)phenylisothiocyanate

“…This difference in detection sensitivity should be caused by the efficiency of the reaction, loss of the peptides on the membranes and/or absorption of the CIP-TZ-amino acids on the membranes, since the present system involves solid-phase reaction, washing with and extraction steps. Most importantly, there was no evidence for the dephosphorylation or degradation of phosphorylated CIP-TZ amino acids as has been reported in conventional Edman procedures [27].…”

Amino acid sequence and D/L‐configuration determination methods for D‐amino acid‐containing peptides in living organisms

“…The racemization at the a-carbon position occurred in a protic solvent such as TFA via keto-enol tautomerizations (Matsunaga et al, 1996). To suppress the racemization, BF 3 , an aprotic Lewis acid, was used instead of TFA in the cyclization/cleavage step.…”

“…To suppress the racemization, BF 3 , an aprotic Lewis acid, was used instead of TFA in the cyclization/cleavage step. When BF 3 was used as the cyclization/cleavage reagent, the racemization ratios of amino acid residues were less than 2.5% during the Edman procedure (Matsunaga et al, 1996). Also, the racemization was affected by the structure of the Edman reagents.…”

“…DBD-TZamino acids were enantiometrically separated on the modified b-CD chiral stationary phase (Matsunaga et al, 1996). The generated DBD-TZ-amino acids were converted to more stable fluorescent derivatives.…”