New Fluorescent Probes Based On Antioxidant Peptides And Amino Acids For Detecting Reactive Oxygen Species In Living Cells

Various free radicals are generated in the course of biology metabolism. Superoxide anion radicals (O2-·), hydroxy peroxide (H2O2), hydroxyl radicals (·OH), singlet oxygen (1O2), ROO·and nitric oxide (NO) are some typical ones. They have important relation with aging and many diseases. It is reported that appropriate level of reactive oxygen species (ROS) could help to build biological immune system, while excess ROS have damages on organisms. In recent years, more and more studies focus on detecting reactive oxygen species and the detection methods have been developed. Among these methods, using fluorescent probes has many advantages such as high sensitivity, high selectivity and easy to operate. Besides, fluorescent probes can interact with reactive oxygen species to form strongly fluorescent substances. With the help of confocal microscopic imaging, visualizing and real-time detecting of reactive oxygen species can be achieved. Near-infrared (NIR) light at around 600-1000 nm is less absorbed than visible light by biological substances, and can penetrate more deeply into tissues. Moreover, it has the further advantage that autofluorescence is not observed upon NIR excitation. So NIR fluorescent bioimaging is one of the most fascinating fields in chemical biology.Based on the changes in spectrum characters of the fluorescent probes reacting with singlet oxygen (1O2), we designed and synthesized a series of new NIR fluorescent probes with high selectivity and high sensitivity to detect 1O2. The designed fluorescent probes were also applied to confocal imaging of 1O2 from living cells. The aspects of investigation are as follows:First, based on the scavenging activity of antioxidant amino acids on singlet oxygen, a new NIR fluorescent probe including histidine conjugated tricarbocyanine was designed and synthesized. Upon treament with 1O2, the weak-fluorescent probe was transformed into a fluorescent product (λex/em = 754/794 nm). The fluorimetric experiments of the probe showed that the sensitive fluorogenic reagent features a high selectivity for 1O2 over other intracellular ROS, metal ions and biological compounds. The fluorescence increase is proportional to the concentration of 1O2 in the range of 5.0×10-8-1.2×10-6 M. The limit of detection is 99 nm. Confocal microscopic imaging of 1O2 drived from living cells reveals that the probe is cell-permeable and intracellular 1O2-responsive.Second, a probe (Car-Cy) composed of cyanine (Cy-SO3-) and carnosine (Car) was designed and synthesized to detect 1O2 in vivo. The imidazole ring of the probe was the active center reacting with 1O2, causing the changes of fluorescence. Car-Cy was an excellent fluorescent probe, which possesses good water solubility and biological compatible, and it could be used for confocal microscopic imaging of living cells.