The Design, Synthsis And Application Of Small Molecule Bioactive Probes

Bioactive small molecule probes are important as versatile tools for targets identification and recognition. In this dissertation, the bioactive photo-affinity probes and fluorescent probes were designed and synthesized. Photo-affinity probe contains photo-affinity group and purification group, which has the capacity to covalent bind with the target after UV irradiation and then the target was separated and enriched. Fluorescent probe contains fluorescent group which is able to recognize the location of the target.Natural product(-)-arctigenin and its derivatives are evaluated to have the effect for both promoting glucose uptake in L6 skeletal muscle cells and reducing the blood glucose in db/db mouse model significantly. Mechanistic study of(-)-arctigenin showed that it was the agonist of AMPK signaling pathway, but its direct target(s) was still unknown. Activity based protein profiling was applied to identify the target of(-)-arctigenin. After structure-activity relationship study, 2-14 which exhibited excellent bioactivity was chosen as lead compound, which could be replaced for the benzyl group by photo-affinity groups. Photo-affinity group as diaziridine or benzophenone was introduced in the structure of 2-14 based on in situ strategy and diversity oriented synthesis. The photo-affinity probe 2P14 was achieved with good bioactivity in vitro(1.789@1.5 μM). Further target identification was studied based on 2P14, according to the fellowing protocol: incubation 2P14 with L6 skeletal muscle cells for the target binding, then UV irradiation to covalent bind 2P14 with its target, after the Huisgen reaction between alkyne and Biotin-azide, streptavidin was used to separate and enrich the target protein, further separated the protein sample by SDS-PAGE and selected special bands for the protein identification by LC-MS. After the analysis of the data, three proteins P06576、E9PN89 and P81605 were identified and more studies are still under test. For the target location ongoing, 10 fluorescent probes were synthesized, but none of them showed any bioactivity. From this work, the process of target identification was set up and the experimental conditions were optimized. Also 11 kinds of photo-affinity building blocks were synthesized and applied to achieve the high activity photo-affinity probe 3P5(IC50=153 nM) which has the selective antihepatocarcinoma activity like lead compound Yhhu-258.Novel BODIPY based fluorescent probes were synthesized during the previous work. The structure-fluorescence relationship shows that aniline is required for fluorescence self-quenching, the electronic donating groups are benefit for fluorescence quenching, the distance between BODIPY and aniline could turn on the probe’s fluorescence intensity. After fully understand of those properties, 4P28 was designed and synthesized for Aβ recognition. Evaluation of 4P28 showed that it had high affinity with Aβ fibril(Kd=3.5 nM), and could stain senile plaques in the brain slice from double transgenic mice(APPsw/PS1De9, 12 months old, male). 4P28 was also found as the first probe to stain senile plaques in brain slices in high signal-to-background ratio without the washing procedure. Further NIR probe 4P36 was synthesized and showed 2 fold of brain fluorescence signal ratio between the transgenic mice and the wild type mice in 5 to 45 minutes after the intravenous injection. The further evaluation of 4P36 is still under test.