The synthesis of a phosphonated acyclic bifunctional chelate L* for the labeling of biomaterial is described. L* is based on a pyridine backbone, functionalized in ortho positions by aminomethyl-bis-methylphosphonic acids, and, in the para position, by a side chain containing a reactive NHS carbamate function. The stability of L* in aqueous solutions at different pH values was studied by mass spectrometry, showing the activated function to be sensitive to hydrolysis above neutral pH. The reactivity of L* towards amine functions was tested using ethylamine under different conditions of pH and concentrations, and by the labeling of two reference peptides containing both an N-terminal amino function and a ε-amino group of a lysine residue in the backbone, and a supplementary thiol group of a cysteine residue for one of these two peptides. The results showed the coupling to be efficient at pH 8.0, with a total selectivity for the terminal amine function with respect to lysine and cysteine. The labeling was further performed on B28-13, a mouse monoclonal antibody specifically recognizing tenascin-C protein in human cancer. The labeled antibody was characterized by means of mass spectrometry and spectrofluorimetry, unraveling a labeling ratio of one chelate per antibody. Finally, the affinity of the labeled antibody towards its target was controlled by immunofluorescence staining experiments on human colon cancer biopsies, confirming the affinity of the labeled peptide for tenascin-C.
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