A novel cyan-emitting fluorescent α-amino acid: synthesis, photophysical characterization and live-cell imaging properties

Abstract

Small-molecule-based fluorescent probes, particularly fluorescent analogs of α-amino acids (FAAs), have become valuable tools for labeling proteins of interest and visualizing biological processes in living cells. In this study, we report the design, synthesis, and characterization of a novel cyan-emitting fluorescent α-amino acid, 4-dibenzothiophen-4-yl-L-phenylalanine (DBT-FAA). DBT-FAA was synthesized in good yield via a facile 3-step procedure involving modified Suzuki–Miyaura cross-coupling and characterized by spectroscopic methods. The compound exhibits strong fluorescence in a broad range of aqueous and polar solvents, with a major emission peak at ∼424–445 nm and secondary shoulders at longer wavelengths (∼448–583 nm), along with a high quantum yield (Φ = 0.74 in DMSO at λ_ex = 380 nm and λ_em = 400–800 nm). DBT-FAA also demonstrates excellent photostability, showing strong resistance to photobleaching under aqueous conditions, which is essential for prolonged live-cell imaging. Confocal fluorescence microscopy confirmed that DBT-FAA is efficiently taken up by human HeLa cells, likely through an endocytic pathway, without observable cytotoxicity under the conditions studied. Once inside the cells, DBT-FAA initially accumulates in lysosomes and subsequently translocates to mitochondria with extended incubation time, suggesting dynamic intracellular redistribution. The robust optical properties, photostability, and cellular compatibility of DBT-FAA highlight its potential as a versatile fluorescent amino acid probe for real-time visualization of intracellular processes, offering promising applications in studying protein dynamics, molecular interactions, and cellular metabolism relevant to biochemistry, drug discovery, and protein engineering.