Synthesis and Application of Affinity-Based Probes: Identification of Pregnenolone Binding Proteins and Study of Telomere-Directed DNA Alkylating Agents

• Main Authors: Tzu-Chiao Huang, 黃子樵
• Other Authors: Chao-Tsen Chen
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/21823495243268046594

碩士 === 國立臺灣大學 === 化學研究所 === 100 === 　　Affinity-based probes (AfBPs) have been developed recently by several research groups. Both of Cravatt and Yao groups independently reported the first AfBPs for metalloproteases. AfBPs achieves labelling by binding at a specific site of a protein followed by a non-specific covalent bond-forming event. The seminal works of AfBPs research enlighten us to design and synthesize probes for targeting different biomolecules of interest. 　　Embryonic cell movement is essential for morphogenesis and establishment of body shape. In the previous research reported by our group, showed the steroid hormone, pregnenolone, can preserve the abundance of microtubules, and thus effectively promotes the cell movement. In light of this study, we speculate that there might be pregnenolone binding proteins (PBPs), which can bind pregnenolone and further control the formation of microtubules. To verify our assumption, the photoreactive probes (P5C7b-O-NBPN) composed of a pregnenolone as the PBPs binding group, benzophenone as the photo cross-linker and a biotin as the reporter were designed and synthesized. It is hoped that by using this set of probes, PBPs could be identified and further purified prospectively. 　　On the other hand, telomeres are specialized DNA ends providing protection against genomic instability and cell senescence during cell division. The homeostasis of telomere is maintained by telomerase, which is selectively expressed in most tumors, and hence has a crucial role in cellular immortalization.38(?) The G-rich sequence of human telomeric DNA has a strong propensity to form the DNA G-quadruplex secondary structure, which can inhibit the activity of telomerase.53(?) Recently, 3,6-bis(1-methyl-4-vinylpyridium) carbazole diiodide (BMVC) was reported as a G-quartet stabilizer and a fluorescence probe. Preserving the key structural features of BMVC, a series of BMVC-mustard conjugates (BMVC-CnM; n = 2, 3, 6) were designed and synthesized in our group in attempts to develop telomere-directed DNA alkylating agents. The recognition between BMVC-CnM and G-quarduplex were analyzed and confirmed by various spectroscopic tools and DNA-PAGE studies previously. In this thesis, FRET analysis and DNA footprinting were applied to investigate the binding modes of BMVC-CnM with G-quarduplex. Furthermore, the TRAP-assay and cell images experimental results indicate BMVC-CnM have great potentials to be developed as anti-tumor agents.