Molecular Rotors of BOSCHIBAs Derived From α-Amino Acids as Fluorescent Target of Gram-Positive Bacteria

Fluorescent Gram-positive staining probes have become excellent and novel methods for flow cytometry and fluorescence microscopy. However, there are still few commercially available materials of this type. Recently, boron Schiff bases (BOSCHIBAs) have shown advantages such as low cytotoxicity, good cell staining capability, low-cost synthesis, tunable optical properties, and simple synthesis procedures. Herein, we report an effective multi-component synthesis of two new fluorescent molecular rotors (FMRs) derived from amino acids (1: phenylalanine and 2: tryptophan) and benzene 1,4-diboronic acid. The FMRs were fully characterized by nuclear magnetic resonance (NMR; ¹H, ¹³C, and ¹¹B), and high-resolution mass spectrometry (HRMS). The molecular structure by X-ray diffraction of rotor 1 is reported. Compound 1 exhibited higher fluorescence intensity compared with the tryptophan-derived compound 2. However, compound 2 showed high sensitivity to viscosity changes. Density functional theory (DFT) calculations were performed to understand the optical properties and photophysical mechanisms, considering changes in selected dihedral angles in the first excited state (S₁). According to time-dependent density functional theory (TD-DFT), molecular excitation promotes an intramolecular charge transfer (ICT) process that induces structural reorganization in the excited state. An increase in solvent viscosity affects the photoinduced conformational changes, favoring radiative deactivation in specific conformations. Both compounds showed low cytotoxicity in human and bacterial cells at concentrations ranging from 0.1 to 5 μg/mL and 2 μg/mL, respectively. Fluorescent bioimaging assays revealed that human cells (HUVEC, HeLa, and erythrocytes) did not show a staining pattern, in contrast to E. coli and B. subtilis, demonstrating that compounds 1 and 2 exhibited affinity and specificity for bacterial cells.