The object of investigation: synthesis, spectral and fluorescence, protolytic, photophysical, photochemical properties and ways of practical application of norsquaraines. Object of the research is to create new pH-sensitive long-wavelength dyes based on symmetrical and asymmetrical norsquaraines, including those substituted in cyclobutenone ring. Methods: NMR 1H, X-ray diffraction analysis, high performance liquid chromatography (HPLC), elemental analysis, electronic absorption and fluorescence stationary and time-resolved spectroscopy and quantum-chemical calculations by semi-empirical methods (MNDO, AM1, PM3). Theoretical and practical results: the regularities of the reactions of squaric acid and its derivatives with indolenines were found, systematic series of symmetrical and asymmetrical norsquaraines as well as their dicyanomethylene, barbituric, and alkylamino derivatives were developed. The possibility of monosquaraines with hydrogen atom at the nitrogen atom of indolene ring synthesis was demonstrated. Asymmetric norsquaraines were first obtained. It was shown that the molecules of norsquaraines were characterized by intramolecular hydrogen bonds, higher planarity and greater conformational rigidity when compared to squaraines. These particularities result in their spectral-luminescent and photophysical properties. The influence of molecular structure of norsquaraines on their spectral-luminescent, protolytic, photophysical and photochemical properties was revealed in free state and when bound to protein. pH- Sensitive long-wave fluorescent labels and probes based on norsquaraines for biomedical applications based on measuring the intensity, polarization and fluorescence lifetime were developed. Novelty: a preparative method of synthesis of norsquaraines which were at first applicated as pH-sensitive long-wavelength fluorescent labels and probes for biomedical applications based on measuring the intensity, polarization and lifetime of fluorescence. These dyes cover a wider spectral range as compared to previously known norcyanines and have better photostability. Alkylaminonorsquaraines are the first pH-sensitive long-wavelength dyes in which both, protonated and deprotonated forms have fluorescence, which improves the accuracy of the bioanalytical measurements. Norsquaraine dyes, methods for their synthesis and application are defended by U.S. Pat. Degree of implementation: norsquaraine dyes are used by Corporation PerkinElmer (USA) and other organizations for drugs screening and biomedical applications. Sphere of application: research work, biomedical, pharmaceutical research, molecular and cell biology, analytical and bioanalytical chemistry.
