In order to investigate the functional group tolerance of the labelling method, tetrapeptides 1 containing various unprotected natural amino acids were then engaged in the reaction (Scheme 2a). Reproducibility of the reaction on larger scale (20 instead of 1 µmol) was first confirmed by full conversion of AFYA-NH2 (1a) to the corresponding O-VBX 3a, which was isolated in 64% yield following HPLC purification and characterized by 1 H, C and 2-dimensional NMR spectroscopy. A sterically hindered leucine residue did not affect the reactivity and O-VBX bioconjugate 3b was obtained in 65% isolated yield. High chemoselectivity was achieved in presence of tryptophan, however peptide 3c could only be isolated as a 1:2 mixture with N3-VBX 4. No side reactions occurred in presence of the nucleophilic and basic arginine (3d), histidine (3e) and lysine (3f) residues. When cysteine-containing tetramer 1g was submitted to the reaction conditions, fast addition of the cysteine residue was observed, followed by reaction of the tyrosine, leading to a doubly labelled S-and O-VBX product 3g in 40% isolated yield. Due to the inherent higher nucleophilicity of thiols, the functionalization of tyrosine in presence of free cysteine has usually not been reported, except for a few exceptions with selected biomolecules. Serine (3h), methionine (3i) and aspartic acid (3j) did not affect the reactivity, whereas a slightly lower yield due to incomplete conversion was observed for asparagine (3k). Finally, the position of tyrosine in the tetramer was varied (1l-1n). All peptides were successfully labelled to give products 3l-n, including N-and C-terminal tyrosine residues (3m and 3n).