The Design, Synthesis And Biological Application Of Two-color Fluorescent Probes For Simultaneously Detecting Two Biologically Active Molecules

Biologically active molecules play essential roles in cell signaling transduction and occurrence and development of disease, including reactive oxygen species(ROS),metal ions and H+. For example, the cellular “redox” signaling mediated by hydrogen peroxide(H2O2) is closely related to normal processes and disease progression, such as angiogenesis, oxidative stress, aging, and cancer. Zinc(Zn2+) is an essential element that is required for a structural component and enzymatic activity of a great number of proteins. It also functions as an intracellular signaling molecule with a huge impact on the behaviors of the cells. Interactions of active molecules and relationships in concentration variation constitute molecule communication, which is closely related to the development of major diseases. It is well established that mitochondria act as a signaling organelle, in which many small molecules are involved in diverse physiological responses, including H2O2 and Zn2+. Fluorescence imaging acted as a method for the dynamic and in situ analysis is widely concerned,especially for detection of bioactive molecules in subcellular level. Although extensive attention has been given to the roles of mitochondrial H2O2 and Zn2+ in cell signaling regulation, coordinated regulation of these two molecules has been little studied. Thus, the simultaneous detection of two kinds of active molecules has an important role in study of the molecular mechanisms of some associated diseases.In order to solve the problem of simultaneous imaging with two biologically active molecules in mitochondria, we carried out two aspects of investigation:First, we designed and synthesized two fluorescent probes termed PF-H2O2 and Cy-O-DPA for simultaneous determining of H2O2 and Zn2+ concentration fluctuations in mitochondria. The probe PF-H2O2 was consisted of three parts. Merocyanine with a lipophilic cationic was served as the fluorescent chromophore and targeting group.Boricacidester was a H2O2-sensitive reporter. The electronic system of PF-H2O2 changed to “donor-acceptor” from “acceptor-acceptor” which making the fluorescence intensity enhanced. The probe detected H2O2 with high selectivity by naked eye and achieved a dynamic detection of intracellular H2O2. PF-H2O2 showed the linear fluorescence change over a range of H2O2 concentration from 1.0×10-7 to5.0×10-5 M, and the linear regression equation was F = 514.606 [H2O2](μΜ) +8735.48, with a correlation coefficient of 0.9960. The detection limit was calculated tobe 18 nM. Cy-O-DPA was designed and synthesized based on PET. The probe Cy-O-DPA was consisted of three parts. Cyanine with a lipophilic cationic was served as fluorescent chromophore and targeting group. DPA which could combine Zn2+ was a Zn2+-sensitive reporter. The probe realized instantaneous detection of Zn2+.Cy-O-DPA showed the linear fluorescence change over a range of Zn2+ concentration from 1.0×10-7 to 5.0×10-6 M, and the linear regression equation was F = 1083.17[Zn2+](μΜ) + 4485.26, with a correlation coefficient of 0.9901. The detection limit was calculated to be 11 nM. These two probes have high sensitivity and selectivity as well as distinguished excitation and emission spectra, which realizing the detection of collaborative concentration fluctuations of H2O2 and Zn2+ in mitochondria. Also we studied a mitochondrial love-hate triangle related to H2O2 、 Zn2+and apoptosis.PF-H2O2 and Cy-O-DPA will become excellent imaging probes to study biological effect of H2O2 and Zn2+, and provide an ideal tool for revealing the molecular mechanisms of disease associated with coordinated changes of H2O2 and Zn2+ in mitochondria.Second, we designed and synthesized a fluorescent probes termed PHS for imaging of mitochondrial H2 S concentration fluctuations in biological systems. By combining with Cy-O-DPA, the association of H2 S and Zn2+ was explored in mitochondria. The probe PHS was consisted of three parts. Merocyanine with a lipophilic cationic was served as fluorescent chromophore and targeting group.Dinitrophenyl ether was selected as the H2 S reactive site. Reaction with H2 S, the probe formed strong push-pull structure, which enhanced the fluorescence intensity.The probe detected H2 S in mitochondria with high selectivity by naked eye. PHS showed the linear fluorescence change over a range of NaHS concentration from 1.0×10-6 to 1.0×10-4 M, and the linear regression equation was F = 550.720 × [NaHS](μM) +18222.6, with a correlation coefficient of 0.9977. The detection limit was calculated to be 26 nM. These two probes are expected to achieve fluorescence imaging of synergies with H2 S and Zn2+. Moreover this multicolor imaging method will provide an ideal tool for revealing molecular biological mechanisms associated with H2 S and Zn2+.