Spectral-Luminescent Properties of Products of Interaction of Polyfluorinated Pyrazoline-Containing Pyrylium Dyes with Bovine Serum Albumin and Amino Acids

Abstract

Objective: Studies of the reaction ability of fluorescent dyes to produce labelled proteins and amino acids are important for bioengineering and biomedicine, particularly for cell visualization by bioimaging and the study of the structure of labelled proteins by biophysical methods. Pyrylium dyes can interact with the amino groups of proteins to form luminescent products, which allows them to be used in the field of proteomics. It is interesting to study the pyrylium conjugates that contain both a polyfluorinated fragment responsible for increased lipophilicity of the protein conjugates and a pyrazoline fragment responsible for anticancer activity. Methods: The pyrylium dyes that contain the pyrazoline fragment and dialkylamino substituents (piperidino-, dibutylamino-, and 4-hydroxypiperidino-) in the donor part of the polyfluorinated aromatic ring have been synthesized by the Knoevenagel condensation reaction. The reaction of pyrylium dyes with compounds containing the primary amino group has been performed to obtain the pyridinium dyes by the ANRORC (Addition of Nucleophiles, Ring Opening and Ring Closure) Mechanism. Results and Discussion: The ability of the pyrуlium dyes to react with bovine serum albumin (BSA) and amino acids, such as Lys, Arg, Cys, and Phe to form pyridinium luminophore has been demonstrated. The spectral-luminescent properties of the resulting luminophores have been investigated. The product of the reaction of the pyrуlium dye (Е)-2,6-dimethyl-4-(4-{3-phenyl-5-[2,3,5,6-tetrafluorо-4-(piperidine-1-yl)phenyl]-4,5-dihydro-1Н-pуrazol-1-yl}-styryl)pyrylium tetrafluoroborate with Lys has been isolated, and its structure has been confirmed by NMR spectroscopy. The binding site of the pyrylium dyes with BSA, i.e., the ε-amino group of Lys, has been determined. Together with the pyridinium luminophores, hydrolysis products are formed in aqueous solutions, which are not bound to the protein and absorb in the short wavelength region. The amount of the pyrylium dye bound to BSA has been calculated to be 2 : 1.The synthesized pyrylium dyes react with BSA in the phosphate buffer-methanol mixture (pH 7.4) 3–4 orders of magnitude faster than the well-known julolidine dye Py-1. The relative reaction rates of (Е)-2,6-dimethyl-4-(4-{3-phenyl-5-[2,3,5,6-tetrafluorо-4-(4-hydroxypiperidine-1-yl)phenyl]-4,5-dihydro-1Н-pуrazol-1-yl}styryl) pyrуlium tetrafluoroborate with amino acids have been evaluated to be as follows: Lys > Cys >> Phe ≥ Arg. Conclusions: The synthesized polyfluoro pyrylium-pyrazoline dyes have the application perspective in the field of bioimaging, proteomics, and biomedicine due to the high conjugation rate and efficiency with BSA and amino acids.