Synthesis, Spectral Properties And Application Of Cy5 Dyes With Different Substituents On The Pentamethine Chain

Polymethine cyanine fluorescence dyes are of importance for their large molar extinction coefficient and wide absorption and emission tenability with the variation of the length of the conjugated chain, which has received more and more attention in bio-applications, such as DNA probes, protein labeling reagents, ion and small molecular sensors, imaging in live cells, tissues and targeting in live bodies. Pentamethine cyanine dyes (Cy5) are a kind of near-infrared (NIR) dyes with shortest wavelengths. In the dissertation, we mainly investigated the spectral properties, photo stabilities and bio-applications of Cy5 dyes by the introduction of different substituent groups into the central position (a-position) and side position (?-position) of conjugated chains.Firstly, several Cy5 dyes with different substituent groups (such as induced electro withdrawing groups (bromo-or chloro-group), conjugated electro withdrawing group (?f aldehyde or?a dicyanovinyl group) and conjugated electrodonating group (vinyl group)) at the central position has been synthesized, on the basis of having a knowledge of the existing work, the results demonstrated that there were two group of absorption and emission peaks by the introduction of aldehyde group at the central position with large pseudo-stokes shift, and furthermore, the photostability of Cy5 was largely improved. Whereas, compared with the other dyes, the fluorescent quantum yields of?f and?a were decreased to a large extent. In nonviscous or low-viscosity solutions, the central aldehyde group or dicyanonvinyl group can rotate around the C-C bond freely to relax the excited dye in the form of non-radiation. The fluorescence intensities at two peaks perform ratiometry increase when environmental viscosity is increased gradually. The theoretical calculation demonstrated that the transition of an electron is transferred from second excited state or first excited state to ground state was dramatically influenced by the viscosity of solutions. With the increase of viscosity of solution, the fluorescence intensity at each peak was differently increased, performing ratiometrically changes. The dyes can be structurally composed of two fluorephores with intersecting conjugation mode. On the other hand, the fluorescence lifetime at the longer peaks can be evidently lengthened with the increase of viscosity, which can be used to detect the viscosity by dual modes with higher reliability and accuracy.Secondly, a new synthetic route of Cy5 dyes with different amino groups at sideward ((3-position) position has been developed, not reported as far as we know. Compared with traditional Cy5 dyes, the new dyes has two absorption peaks and only one emission peak, both of which greatly blue shift, and large stokes shift as more than 60 nm was obtained. As calculated results demonstrated that, at ground state, the charge of amino group at?-position badly transferred to the conjugated chain, resulting from strongly coupling interaction between amino group and the adjacent carbon at polymethine chain. The conjugation of the polymethine chain was broken, resulting in shorter absorption. The charge would transfer from two end phenyl groups to the amino group by absorption of a photon, companying the changes of the configurations of amino groups because of decoupling between amino group and the carbon, the conjugation of the whole polymethine chain was rejoined to generate stable excited state with lower energy gap and longer emission. As far as the change of amino configuration concerned, there are three changes for different amino groups at?-position as follows:1) when the primary amine was connected to the?-position, solvent molecules can induce pseudo-Jahn-Teller (PJT) change of amino configuration; 2) When the amino group at?-position was tertiary amine, such as dimethyl amine, the twist intramolecular charge transfer (TICT) would dominate main channel to reduce coupling; and 3) for secondary amines, the above two effects can be observed simultaneously. The high viscosity of the solvents can prohibit the changes of the amino configuration to observe the shorter emissions, supporting the results of theoretical calculations.The fluorescence of these dyes was sensitive to polarity, and it would change much more with the variation of solvents than the increase of viscosity. The fluorescence quantum yields in DCM were more than 100 times higher than that in water, which can be used to detect the hydrophobic environments. The fluorescence of dyes would increase differently with the variation of hydrophobic cavities on the surface of proteins. The fluorescence intensity of IVc can be greatly and linearly increased upon addition of proteins, and the detection of limit of the dyes was as high as up to 20 ng in post staining protein detection by SDS-PAGE. Finally, it is live cell membrane permeable for these dyes to image the hydrophobic microenvironments.