Synthesis And Biological Applications Of Thiazole Orange Derived Unsymmetrical Cyanine Dyes

Cyanine dyes own excellent spectral performance and simple method for synthesis and purification. Recently, with the significant application of fluorescence detection technologies in life sciences and medical diagnosis, cyanines as fluorescent labels for biomolecules such as proteins and nucleic acids, and as environmentally sensitive probes for reporting on local properties such as viscosity, polarity, pH, temperature and others are becoming the the focus of attention. In the dissertation, we mainly investigated the synthesis and biological applications of thiazole orange derived unsymmetrical cyanine dyes.To satisfy the great demand in the biomolecule fluorescent imaging and medical diagnostics, three trimethine thiazole orange (TO-3) derivative dyes Ⅱa-Ⅱc were synthesized. The fluorescence excitation/emission spectra of Ⅱa-Ⅱc were located in red light area (>620nm).Ⅱa-Ⅱc all show low intrinsic fluorescence (quantum yield<0.004) and the fluorescence quantum yields of them were significantly enhanced over90-fold upon binding to DNA. They are much sensitive than the UV excitated4’,6-diamidino-2-phenylindole (DAPI) and the DNA intercalator ethidium bromide (EB). Diethylamino-butyl substituted monocationic Ⅱa and ethyl substituted monocationic Ⅱb are permeant to live cells; Ⅱa showed nucleus specific staining with high sensitivity and can be applied for DNA quantification by flow cytometry. While triethylammonio-butyl substituted dicationic Ⅱc can only be used for DNA selective staining in fixed cells.On the basis of the single-emission fluorescent probes, rhodamine-cyanine composed FRET-based two color cassette Ⅲb and TO-3homodimmer Ⅲc have been developed. Ⅲb, with its two fluorescence emission signals (λem (RhB)=582nm; λem (TO3)=650nm), and the ratio thereof, allowed the modes of dye-binding with isolated nucleic acids (dsDNA, RNA, AT segments and GC segments) to be assessed.Ⅲb showed mitochondrially related structure imaging for live cells, and can also be used as a whole cell marker defining the regions of the nucleoli, nucleus, cytoplasm regions and boundaries between them in fixed cells. Ⅲb is expected to be applied to image-based high content screening (HCS) determination.TO-3homodimmer Ⅲc was almost non-fluorescent in solution and exhibited950-flod fluorescence enhancement when bound to DNA, achieving quantitative DNA detection with high sensitivity and selectivity. The emission detection limit was13.3ng/mL of dsDNA. The dramatic fluorescence enhancement of Ⅲc can be ascribed as two reasons:the dissociation of inter/intra-molecular H-aggregates upon binding to DNA, and the restriction of intramolecular twisting and torsion of monomer TO-3fluorophore in the minor groove of DNA.Based on fluorescence responses to nucleic acids and protein, seven thiazole orange (TO-3) derivatives dyes were designed. Among them, Ⅳa showed nucleoli and ribosomal RNA-specific fluorescence imaging; IVc showed nucleus-specific fluorescence imaging; Ⅳf and Ⅳg gave selective mitochondria or Golgi apparatus fluorescence imaging under different conditions. Due to the selective binding to nucleic acids or protein, the application of dyes Ⅳa, Ⅳf and Ⅳg to the automatic blood cell analysis system (five types of leukocytes and reticulocytes analysis) was the first domestic case. Besides, these dyes were very efficient, fast in staining (30-60s and less than2μM of incubation) and more sensitive than the commercially avaiable blood cell reagents.