Boron-dipyrromethene (BODIPY) dyes have been extensively investigated in recent years for a variety of bioanalytical and bioimaging applications. The success of these applications relies on the stability of BODIPYs, particularly under acidic conditions. In this work, the stability of a series of 4,4′-disubstituted BODIPYs (−F, −CN, −Ph, −Me, −OMe) toward addition of excess trifluoroacetic acid (TFA) was studied systematically and comprehensively through ¹¹B and ¹H NMR, UV–vis, fluorescence, thin layer chromatography, mass spectrometry, and infrared. The results indicate that 4,4′-dicyano-BODIPY 2 is the most stable among this series and remains unchanged even 3 days after addition of excess TFA. On the other hand, 4,4′-dimethyl-BODIPY 3 and 4,4′-dimethoxy-BODIPY 5 are the least stable, toward addition of TFA, and the 4,4′-diphenyl and 4,4′-difluoro-BODIPYs 1 and 4 were found to have intermediate stability. The experimental analysis and comparison with theoretical calculations indicate that the 4,4′-dicyano-BODIPY 2 has the greater aromaticity of the series, as evaluated by the BLA parameter, decreased charge on boron, and upon TFA addition it forms an unusually stable BODIPY 2···TFA complex. On the other hand, all other BODIPYs decompose within hours after TFA addition. Computational modeling demonstrates that 4,4′-dicyano substitution increases aromaticity and stabilizes the B–N bond, resulting in the most stable compound from the series studied.

A series of α-amino acid–BODIPY derivatives were synthesized using commercially available N-Boc-l-amino acids, via boron functionalization under mild conditions. The mono-linear, mono-spiro, and di-amino acid–BODIPY derivatives were obtained using an excess of basic (histidine, lysine, and arginine), acidic (aspartic acid), polar (tyrosine, serine), and nonpolar (methionine) amino acid residues, in yields that ranged from 37 to 66%. The conformationally restricted mono-spiro- and di-amino acid–BODIPYs display strong absorptions in the visible spectral region with high molar extinction coefficients and significantly enhanced fluorescence quantum yields compared with the parent BF₂–BODIPY. Cellular uptake and cytotoxicity studies using the human HEp2 cell line show that both the presence of an N,O-bidentate spiro-ring and basic amino acids (His and Arg) increase cytotoxicity and enhance cellular uptake. Among the series of BODIPYs tested, the spiro-Arg- and spiro-His-BODIPYs were found to be the most cytotoxic (IC₅₀ ∼ 22 μM), while the spiro-His-BODIPY was the most efficiently internalized, localizing preferentially in the cell lysosomes, ER, and mitochondria.