Characterization of labelling and de‐labelling reagents for detection and recovery of tyrosine residue in peptide

Abstract

This paper characterized the labelling and de‐labelling reagents for reversible labelling of tyrosine (Tyr)‐containing peptide, which involves detection and recovery. The phenolic hydroxyl group (‐OH) in Tyr structure reacted with 4‐fluoro‐7‐nitro‐2,1,3‐benzoxadiazole (NBD‐F), 4‐(N,N‐dimethylaminosulfonyl)‐7‐fluoro‐2,1,3‐benzoxadiazole (DBD‐F), and 1‐fluoro‐2,4‐dinitrobenzene (DNFB) under mild conditions at room temperature at pH 9.3. The labels in the resulting derivatives were removed with the treatment of nucleophiles, such as thiols (cysteine, N‐acetyl‐L‐cysteine and dithiothreitol) and amines (dimethylamine, methylamine, diethylamine, ethylamine and pyrrolidine). The de‐labelling reactions of NBD‐labelled N‐acetyl‐L‐tyrosine (N‐AcTyr) with the nucleophiles produced N‐AcTyr, accompanied by NBD‐nucleophile. Although DBD‐F and DNFB also successfully labeled the ‐OH group in N‐AcTyr, the efficiency of CO bond cleavage and recovery of N‐AcTyr by the nucleophiles was relatively low compared with NBD‐label. Among the de‐labelling reagents, N‐acetyl‐L‐cysteine and dimethylamine were recommended for the elimination of NBD moiety, with respect to the reaction rate, the side reaction, and the yield of recovery. The proposed procedure, which includes the labelling with NBD‐F and the removal of NBD moiety by the nucleophiles, was successfully applied to the reversible labelling of N‐terminal amine‐blocked peptides, i.e. N‐AcTyr‐Val‐Gly, Z‐Glu‐Tyr, Z‐Phe‐Tyr, N‐Formyl‐Met‐Leu‐Tyr, and N‐AcArg‐Pro‐Pro‐Gly‐Phe‐Ser‐Pro‐Tyr‐Arg. Copyright © 2003 John Wiley & Sons, Ltd.