Design, Synthesis And Application Of Two - Photon Fluorescence Probe

Over-generation of reactive oxygen species (ROS) is closely associated with organism physiology, pathology and aging. Recently, abundant evidence suggests many biological processes including signal transduction are mediated by ROS. Abundant evidence suggests that excessively produced ROS have been implicated in a wide variety of diseases, including degenerative disorders, ischemia-reperfusion (IR) injury in surgery. Superoxide anion (O2) is first generated among ROS and then convert into other ROS. H2O2was once viewed only as a marker for oxidative stress and damage events connected to disease and aging or as a killing agent released by immune cells in response to microbial invasion, but mounting new data suggest that H2O2serves as a messenger in normal cellular signal transduction. However, excessive generation of O2and H2O2could give rise to various diseases, such as cancer, diabetes and cardiovascular disease. Therefore, ROS plays an important role in biology. It is pressing to develop powerful tools for real-time monitor in cellular level for purpose of detailed acquaintance with ROS.Understanding the molecular interactions in biological systems is of fundamental importance. Various types of assay and imaging tools have been developed for studying diverse biological processes. Fluorescence imaging offers a powerful approach to monitor the levels of intracellular bioactive molecule owing to its high sensitivity. However, existing fluorescent probes of bioactive molecule used for cell imaging have some drawbacks. For instance, the small molecular fluorescent probes often suffer with interference from other ROS, delayed response time, irreversible reaction. Thus, alternative fluorogenic probes with distinct advantages, such as high specificity, instantaneous response and reversible interaction, remain in urgent pursuit for bioimaging in cells and in vivo.Along with conventional one-photon confocal microscopy, two-photon fluorescence microscopy (TFPM) based on the two-photon excitation combined with laser scanning technology has received increasing interest in recent years. As is well known, two-photon (TP) imaging possesses distinguished advantages including increased penetration depth and reduced specimen photodamage. In recent years many two-photon excitable fluorescent probes have been developed. The princeples for desiging two-photon fluorescence probe are as follows:one is coupling an analyte-specific binding or a reaction site to a two-photon excitable dye; another is constructing a new two-photon fluorescence dyes after the probe reation with the bioactive moleculer. However, TPFM applications have been dramatically limited by reason of the shortage of ideal two-photon (TP) fluorescent probe with high TP absorption cross section. Hence, it is urgent to design and synthesize two-photon fluorescence probes with larger two-photon absorption across sectionTherefore a series fluorescent two-photon (TP) probes have been developed. With these ideal tools, we visualize O2and H2O2fluctuations, and revealed the relationship between ROS and disease and aging.The main results of this dissertation were shown as follows:1. We construct a novel probe TCA ((N, N’)-di ((2E)-3-(3,4-dihydroxyphenyl) acrylic acyl)-1,3,5-triazine-2,4,6-triamine. TCA for O2was designed on the basis of a tripolycyanamide scaffold, covalently conjugating with two caffeic acid molecules (Scheme1). In the presence of O2, we hypothesized that caffeic acid residue of TCA can transform from pyrocatechol, an electron-donor, to benzoquinone, an electron-acceptor. By a computational study performed at the B3LYP/6-31G(d,p) level, we confirmed the charges on caffeic acid moiety of TCAO decreased, resulting from changing hydroxyl to carbonyl. Our results showed TCA combining merits of OP and TP fluorescence imaging features, for dynamically spying on fluctuations of O2selectively, instantaneously and reversibly. TCA converted into TCAO with a concomitant increase in OP fluorescence which is linear for O2concentrations ranging from1.0×10-8to2.0×10-5M (Figure S2), and the linear regression equation was F=271.0O2(μM)+3578, with a correlation coefficient of0.9950. The detection limit was calculated to be2.3nM. The probe can conveniently visualize the concentration changes of O2during reperfusion injury in hepatocytes, zebrafish and mice, by means of one-photon or two-photon imaging according to depths of various samples. As far as we know, no reports to date have been published on the use of dual-mode fluorescent probes to image O2 2. We report a new reversible fluorescent TP probe (PY-CA) with high TP absorption across section, which allow monitoring O2fluxes dynamically with excellent selectivity and sensitivity, as well as pH-independent fluorescence response. The guiding principle of the superior optical characters is to enhance TP absorption cross section of O2probe for better imaging performance. To do this, we chose symmetric styryl-pyrazine possessing high TP absorption cross section as the molecular framework, and conjugated two caffeic acid molecules (O2recognition group) at the both end of it. Besides, PY-CA shows the linear fluorescence change over a board range of O2concentration from0to1.0×10-5M, and the linear regression equation is F=1276.84[O2](μM)+547.07, with a correlation coefficient of0.997. The detection limit of3.2nM. PY-CA was successfully employed to visualize O2in living HepG2cells and tumor tissue (depth of900μm) in a mouse tumor model. Correlation between O2and lifespan drew from our imaging results indicate that O2of high level can shorten the life of Caenorhabditis elegans.3. We developed a new TP probe which can monitor O2fluxes and H2O2selectively with different colors at the same excited light. With the assistance of fluorescence microscope, it is promising to elucidate the relationship between O2and H2O2, and the role of them in the signal transduction in organism.4. We designed a new mitochondrial TP probe which can monitor O2fluxes and pH selectively and reversibly. This probe is promising to elucidate the relationship between O2and pH, the role of superoxide flash and O2/pH in the signal transduction in organism.