Amine-Reactive BODIPY Dye: Spectral Properties and Application for Protein Labeling

Abstract

A boron-dipyrromethene (BODIPY) derivative reactive towards amino groups of proteins (<b>NHS-Ph-BODIPY</b>) was synthesized. Spectroscopic and photophysical properties of amine-reactive <b>NHS-Ph-BODIPY</b> and its non-reactive precursor (<b>COOH-Ph-BODIPY</b>) in a number of organic solvents were investigated. Both fluorescent dyes were characterized by green absorption (521-532 nm) and fluorescence (538-552 nm) and medium molar absorption coefficients (46,500-118,500 M^-1·cm^-1) and fluorescence quantum yields (0.32 - 0.73). Solvent polarizability and dipolarity were found to play a crucial role in solvent effects on <b>COOH-Ph-BODIPY</b> and <b>NHS-Ph-BODIPY</b> absorption and emission bands maxima. Quantum-chemical calculations were used to show why solvent polarizability and dipolarity are important as well as to understand how the nature of the substituent affects spectroscopic properties of the fluorescent dyes. <b>NHS-Ph-BODIPY</b> was used for fluorescent labeling of a number of proteins. Conjugation of <b>NHS-Ph-BODIPY</b> with bovine serum albumin (BSA) resulted in bathochromic shifts of absorption and emission bands and noticeable fluorescence quenching (about 1.5 times). It was demonstrated that the sensitivity of BSA detection with <b>NHS-Ph-BODIPY</b> was up to eight times higher than with Coomassie brilliant blue while the sensitivity of PII-like protein PotN (PotN) detection with <b>NHS-Ph-BODIPY</b> and Coomassie brilliant blue was almost the same. On the basis of the molecular docking results, the most probable binding sites of <b>NHS-Ph-BODIPY</b> in BSA and PotN and the corresponding binding free energies were estimated.