Synthesis, Properties Of Short-wavelength Cyanine Dyes As Well As Their Application In Biomedical Areas

Cyanine dyes present typical optical properties and act as the most important organic functional dyes in many processes of technological interest like sensitizers in photography, optical recording materials in laser disks, sensitizers in solar cell, pH fluorescence probe, nonlinear optical materials etc. A very attractive additional feature of short-wavelength cyanine dyes is the affinity for biological structures, especially for DNA/RNA. Besides, they are nuclear membrane permeant, tolerated by living cells, photostable, and low cell cytotoxicity. Therefore, they have been utilized in many fields, such as gel staining, DNA sequencing, and flow cytometry, cell imaging, biological macromolecules labeling, etc. However, the types and application areas of the commercial live cell fluorescent dyes are limited, which are expensive and their popularization in clinical analysis are limited. In order to explore the types and application areas of the fluorescent dyes with excellent performance, the short-wavelength cyanine dyes are as the target synthesis compound in the paper. The short-wavelength cyanine dyes are used in live cell imaging and flow cytometry. The possibility of the dyes as the potential fluorescence probe is discussed. The paper presents the structure features, properties and application of cyanine dyes, and the preparation of dimethine cyanine dyes, styryl hemicyanine dyes and monomethine merocyanine dyes in recent years.The main research included several aspects as the follows:1. Seven short-wavelength cyanine dyes (six novel compounds) were designed and synthesized by microwave irradiation method. The products were identified by 1H NMR, IR, UV-Vis spectroscopy and elemental analysis. Based on the microwave power and irradiation time, the reaction activity for quaternary salts was discussed. It could be found that the sequence of the reaction acitivity of 1H-indole-3-carbaldehyde and various heterocyclic quaternary salts was:quinoline quaternary≈2-methyl pyridine qunaternary salt≈4-methyl pyridine qunaternary salt> naphthothiazole quaternary salt≈benzothiazole quaternary salt> benzindole quaternary salt> benzimidazole quaternary salt. The dyes absorbed in the region 376.0～484.0 nm and had molar extinction coefficients of 1.8×104～5.7×104 L-mol-1cm-1. Their fluorescence maxima were located at 512.6～548.6 nm, their fluorescence quantum yields were in the range of 0.0018～0.0285 in methanol, and their Stokes shifts were in the range of 53.6～104.6 nm. Theλmax had relationship with the basicity of heterocyclic nucleus, increasingλmax is considered to indicate decreasing basicity of heterocyclic nucleus. Moreover, theλmax was blue-shifted with increasing the solvent dielectric constant.2. Thirteen novel short-wavelength cyanine dyes were designed and synthesized by microwave irradiation and classical solvent methods. The products were identified by 1H NMR, IR, UV-Vis spectroscopy and elemental analysis. The different substituents on the reactivity of quaternary salts was investigated, the result showed that reaction would took easier with increasing the electron donating ability of the substiuent. For the same substituent, 6-substituted heterocyclic quaternary slats was easier than that of 7-substituted heterocyclic quaternary when reaction with 1H-indole-3-carbaldehyde. The absorption and fluorescence properties of prepared dimethine cyanine dyes in the DNA/BSA presence were investigated, and the dyes had good affinity for DNA. Significant enhancement of the fluorescence intensity was observed for all the dye. The quantum yields of DNA-dyes increased up to 68.8 times compared with quantum yield of free dyes.3. Four monomethine merocyanine dyes (three novel compounds) were synthesized by one-pot method. The products were identified by1H NMR, IR, UV-Vis spectroscopy and elemental analysis. The dyes absorbed in the region 17.0～429.0 nm and had molar extinction coefficients of 3.3×104～4.5×104 L·mol-1cm-1. Their fluorescence maxima were located at 476.0～511.6 nm, their fluorescence quantum yields were in the range of 0.0008～0.0024 in methanol. and their Stokes shifts were in the range of 59.0-82.6 nm. The crystal structures and packing modes of four monomethine merocyanine dyes were analyzed by X-ray diffraction, which revealed molecular conformation, intra-and intermolecular interactions in solid state of the dyes.4. B3LYP/6-31G* and PBE1PBE/6-31G* levels were used for investigating the absorption spectra of seven indole dimethine cyanine dyes. The effect of solvent on the absorption and emission spectra of the dyes was taken into account using the polarizable continuum model PCM. The result showed that PBE1PBE/6-31G*(PCM) level gave more reliable description of the absorption characters. Based on the main torsion angles, it could be found that the better planar structural rigidity and the largerπconjugated system of the molecular, the stronger fluorescence it appeared. The lowest energy absorption in dyes was due to the first dipole-allowedπ→π* transition from HOMO to LUMO with largest oscillator strength. From the molecular orbitals, it could be found that the HOMO→LUMO absorption transition involved the intra electron displacement.CIS/PCM level was used for investigating the emisson spectra of seven quinoline dimethine cyanine dyes on the S1 geometry. The result showed that the average relative deviation is 1.7%, the method could be used for predicting the emission spectra of the dyes. The molar extinction coefficients were calculated by Swizard program, and the shape of simulated absorption spectra are in a principal agreement the experimental spectra.5. A further objective of this study was to explore the potential applications of the twenty prepared cyanine dyes as fluorescent probes in live cell imaging of H929 myeloma cells and sperms. The result showed that dyes A8,A11,A28 and A31 showed excellent fluorescence in the concentration of 1uM-5uM, and the dyes showed nuclear membrane permeant, low cell cytotoxicity, tolerated by living cells, little background interference, little photobleaching and good affinity for DNA. The dyes represented thus the potential cell-permeant fluorescence probes.We also investigated effect of the twenty dyes on counterstaining with PI in flow cytometry, the commercial SYBR Green I was taken as the contrastive probe. Another significant advantage of the dyes A8,A28 and A31 was their little remnant and no interference on the accuracy in flow cytometry when counterstaining with PI. However, SYBR Green I and PI existed in the dead cells simultaneously, which affect the accuracy obtained by flow cytometry. The short-wavelength dyes A8,A28 and A31 were able to replace SYBR Green I as fluorescent probes in flow cytometry.