Synthesis, spectroscopic properties, and biological applications of eight novel chlorinated fluorescent proteins-labeling probes.

Eight novel chlorinated fluorescent proteins-labeling probes with a linker and reactive group were prepared in 7 steps by the reaction of chlorinated resorcinols with 3, 6-dichloro-4-carboxyphthalic anhydride in the presence of methanesulfonic acid. Structures of target compounds and intermediates were determined via IR, MS, (1)H NMR and element analysis. The spectral properties of the chlorinated fluoresceins were studied. These fluorescent probes showed absorbance peaks at 508-536 nm and fluorescence peaks at 524-550 nm. It was found that they have absorption and emission maxima at long wavelengths and high fluorescence quantum yields. Emission spectra of chlorinated fluoresceins shifted towards long wavelength with increase in chlorine. The probes were used for fluorescence imaging of cells in order to investigate whether they can conjugate to cells. The fluorescence imaging of living cells showed that they were localized in cell nucleus. However, they were localized in cytosol of chemically fixed cells. These probes will be useful reagents for the preparation of stable fluorescent conjugates.