Comparative Evaluation of Two Fluorescent Covalent Probes for Nucleophilic Amino Acid Sensing

New covalent probes 2 and 3 were designed by linking the 9,9-dimethyl-2-phenyl-9H-fluorene fluorophore to two electrophilic groups showing potentially different reactivities toward nucleophilic amino acid residues. The reactivities of probes 2 and 3 were characterized using dimethyl sulfoxide as the solvent in place of buffered aqueous solutions owing to the high lipophilicity of the probes that could produce aggregation phenomena. The two covalent probes revealed different reactivity profiles. In fact, the Morita-Baylis-Hillman adduct (MBHA) derivative 2 showed a relatively low selectivity in reacting with Nα-acetyl-L-lysine methyl ester, Nα-acetyl-L-histidine methyl ester, and N-acetyl-L-cysteine methyl ester, whereas the acrylanilide derivative 3 showed a peculiar chemo-selectivity toward N-acetyl-L-cysteine methyl ester. Photophysical characterization studies revealed that MBHA derivative 2 behaved as a fluorogenic probe displaying a certain sensitivity to the amino acid covalently linked. On the other hand, acrylanilide derivative 3 was found to increase its emission features after reacting with N-acetyl-L-cysteine methyl. Owing to its promising features, the capability of MBHA probe 2 of labeling proteins was finally tested with recombinant human serum albumin (HSA) by employing different techniques such as NMR spectroscopy, circular dichroism spectroscopy, SDS-PAGE, and computational docking simulations to obtain information about the interaction of probe 2 with HSA.