Effect of the Substituent Group at the Isothiocyanate Moiety of Edman Reagents on the Racemization and Fluorescence Intensity of Amino Acids Derivatized With 2,1,3-Benzoxadiazolyl Isothiocyanates

Abstract

It is shown that an electron-withdrawing or -donating group at the para-position of aromatic isothiocyanate significantly affects the racemization of 2,1,3-benzoxadiazolylthiazolinone (TZ) derivatives of amino acids, derivatized with newly synthesized benzoxadiazolyl isothiocyanates in Edman sequence analysis. A linear relationship between the logarithms of the TZ-amino acid enantiomer ratio and the para-substituent constants (s p ) for the isothiocyanate moiety was obtained, and the D/L configuration of the amino acid residue was retained with an isothiocyanate containing an electron-donating group at the para-position. The para-substitution effect on the racemization of phenylthiohydantoin (PTH) amino acids was also confirmed by several para-substituted phenylisothiocyanate (PITC) reagents, including nitro-PITC, chloro-PITC, PITC, methyl-PITC and methoxy-PITC. The relationship between the fluorescence intensity of the 2,1,3-benzoxadiazolyl TZ amino acid and s p was also demonstrated. When the isothiocyanate containing an electron-donating group was used, the fluorescence intensity of the TZ-amino acid decreased while retaining the D/L configuration of the amino acid residues.

Fluorogenic and fluorescent labeling reagents with a benzofurazan skeleton

“…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). DBD-NCS was also useful for the Edman sequence with the detection of carbamoyl (CA) derivatives.…”

Comparison of four fluorescence Edman reagents with benzofurazan structure for the detection of thiazolinone amino acid derivatives

“…In a previous paper, we reported that the fluorescence of the generated 2,1,3-benzoxadiazolyl-TZ-amino acids was more intense when a stronger electron-withdrawing group is present at the para position to the isothiocyanate moiety. 10 DBD-TZamino acid, which had the strongest electron-withdrawing para substituent at that time, gave the strongest fluorescence intensity. Therefore, in this study, two new benzofurazan fluorescence Edman reagents with stronger electron-withdrawing substituents, which have larger Hammett substituent constants (s p ) 12 than that of DBD-NCS, namely 7-phenylsulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (PSBD-NCS) and 7-methylsulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (MSBD-NCS), were synthesized and applied to sequencing analysis.…”

“…To overcome these disadvantages, we have recently reported a new Edman procedure using the fluorescence Edman reagent, 7-N,N-dimethylaminosulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (DBD-NCS), [9][10][11] in which DBD-TZ-amino acids generated by the cleavage/cyclization reaction were detected fluorimetrically and the step of the conversion reaction to THamino acid was eliminated to simplify the sequencing process. Furthermore, DBD-NCS itself did not fluoresce and thus the interfering peak derived from the excess reagents did not interfere with the detection of the TZ-amino acids.…”

Thiourea‐ and Amino‐Substituted Benzoxadiazole Dyes with Large Stokes Shifts as Red‐Emitting Probe Monomers for Imprinted Polymer Layers Targeting Carboxylate‐Containing Antibiotics

“…Subsequently, methylation of 1 b afforded the dialkyl derivative 1 c . The alkoxy nitro‐BD derivative 1 d was obtained following described procedures [29] …”

“…Subsequently, methylation of 1 b afforded the dialkyl derivative 1 c. The alkoxy nitro-BD derivative 1 d was obtained following described procedures. [29] Nitrobenzoxadiazoles 1 were further reduced to the amino analogues with iron powder and acetic acid as solvent. The reaction of the amino BDs with either benzoyl isothiocyanate or 4-nitrophenyl isothiocyanate provided the thiourea benzoxadiazoles 2 and 3, respectively, in good yields (see Experimental Section and Supporting Information for more details).…”