本文選題：四鏈體DNAs + 拓?fù)洚悩?gòu)酶　； 參考：《山西大學(xué)》2014年碩士論文

【摘要】：本論文設(shè)計(jì)合成了兩種新穎的含有香豆素基團(tuán)和苯并噻唑基團(tuán)的三聯(lián)吡啶衍生物(1)和三聯(lián)吡啶衍生物(2),并通過(guò)多種實(shí)驗(yàn)手段探究了目標(biāo)化合物1與人端粒(h-telo、i-motif)和原癌基因啟動(dòng)子(c-myc、c-kit2)四鏈體DNAs的相互作用,同時(shí)測(cè)試了目標(biāo)化合物1對(duì)端粒酶、拓?fù)洚悩?gòu)酶I、癌細(xì)胞增殖及其細(xì)胞周期進(jìn)程的影響。實(shí)驗(yàn)結(jié)果如下：1.在FRET-melting實(shí)驗(yàn)中,當(dāng)目標(biāo)化合物1的濃度增加到3 rtM時(shí),Fh-teloT、 Fc-mycT和Fc-kit2T的熔點(diǎn)溫度變化(△Tm)值達(dá)到了15.0、13.6和9.3。C,這表明目標(biāo)化合物1能夠穩(wěn)定h-telo、c-myc和c-kit2 G-四鏈體DNAs,且對(duì)h-tclo G-四鏈體DNA的穩(wěn)定效果好。PCR-Stop實(shí)驗(yàn)進(jìn)一步證明了目標(biāo)化合物1能夠在104 M濃度較好地抑制三種G-四鏈體DNAs的PCR擴(kuò)增產(chǎn)物。UV-melting實(shí)驗(yàn)證實(shí)目標(biāo)化合物1也能夠穩(wěn)定人端粒i-motif DNA結(jié)構(gòu),但是不能穩(wěn)定ct-DNA。2.競(jìng)爭(zhēng)FRET-melting實(shí)驗(yàn)結(jié)果表明,在25倍過(guò)量的雙螺旋ds26存在時(shí),Fc-mycT和Fc-kit2T的△%值沒(méi)有明顯的變化,說(shuō)明目標(biāo)化合物1優(yōu)先與四鏈體結(jié)合；而在50倍過(guò)量的雙螺旋ds26存在時(shí),Fc-kit2T的△Tm值減小幅度較大,說(shuō)明當(dāng)雙螺旋DNA的濃度增大時(shí),目標(biāo)化合物1對(duì)Fc-kit2T的選擇性降低；在10倍過(guò)量雙螺旋ds26存在下,FA-teloT的ATm值從9.6℃減小到4.6℃,說(shuō)明相對(duì)于雙螺旋ds26,目標(biāo)化合物1對(duì)Fh-teloT的選擇性較差。FID實(shí)驗(yàn)結(jié)果也顯示,目標(biāo)化合物1不能有效置換出與三個(gè)四鏈體DNAs結(jié)合的TO,可能是由于二者在DNA上的作用位點(diǎn)不同。3.紫外可見(jiàn)吸收滴定實(shí)驗(yàn)結(jié)果表明目標(biāo)化合物1能夠與h-telo、c-myc、c-kit2和i-motif四鏈體DNAs以及雙螺旋ct-DNA相互作用且結(jié)合常數(shù)Kb約為105 M-1。CD實(shí)驗(yàn)結(jié)果表明：在無(wú)陽(yáng)離子存在下,目標(biāo)化合物1不能誘導(dǎo)h-telo DNA形成四鏈體結(jié)構(gòu)；在100 mM K+條件下,目標(biāo)化合物1能夠誘導(dǎo)雜交構(gòu)象的h-teloG-四鏈體DNA逐漸地形成反平行構(gòu)象。且目標(biāo)化合物1只對(duì)c-myc和c-kit2G-四鏈體DNAs的構(gòu)象稍有擾動(dòng),而且?guī)缀醪挥绊慽-motif四鏈體DNA的構(gòu)象。4. TRAP實(shí)驗(yàn)證實(shí)：目標(biāo)化合物1能夠在10-5M濃度有效地抑制人端粒酶的活性,表明目標(biāo)化合物1是一種潛在的端粒酶活性抑制劑。5.凝膠遷移率實(shí)驗(yàn)結(jié)果證實(shí)：目標(biāo)化合物1沒(méi)有使質(zhì)粒pBR322 DNA發(fā)生切割。瓊脂糖凝膠電泳實(shí)驗(yàn)結(jié)果顯示,目標(biāo)化合物1能夠以濃度依賴(lài)型方式顯著地抑制Topo I調(diào)控的質(zhì)粒pBR322 DNA的松弛。且對(duì)三聯(lián)吡啶衍生物而言,香豆素基團(tuán)的引入和Pt(Ⅱ)的配位提高了它的Topo I抑制活性。6.MTT實(shí)驗(yàn)結(jié)果證實(shí)：目標(biāo)化合物1能夠以濃度依賴(lài)型方式抑制HepG2和MCF7癌細(xì)胞的增殖,其IC50值分別為3.66和1.54 μM。細(xì)胞周期測(cè)試結(jié)果表明,目標(biāo)化合物1將HepG2癌細(xì)胞的細(xì)胞周期進(jìn)程阻滯于S期,同時(shí)將MCF7癌細(xì)胞的細(xì)胞周期進(jìn)程阻滯于G0/G1和S期。
[Abstract]:In this paper, we have designed and synthesized two novel tripyridine derivatives containing coumarin group and benzothiazole group. The interaction of DNAs in c-myctropho-c-kit2) of oncogene promoter. The effects of target compound 1 on telomerase, topoisomerase I, cancer cell proliferation and cell cycle progression were also tested. The results of the experiment are as follows: 1. In the FRET-melting experiment, When the concentration of target compound 1 was increased to 3 rtM, the melting point temperature (Tm) of Fh-teloT, Fc-mycT and Fc-kit2T reached 15.0 ~ 13.6 and 9.3.Crespectively, which indicated that target compound 1 could stabilize h-teloc-myc and c-kit2 G- quaternary DNA, and had a stable effect on h-tclo G-quad DNA. The result of Gaohao. PCR-Stop further proved that target compound 1 could inhibit the PCR amplification products of three kinds of G- quadruplex DNAs at 104m concentration. UV-melting experiment confirmed that target compound 1 could also stabilize the structure of human telomere i-motif DNA, and the target compound 1 could stabilize the structure of human telomere i-motif DNA. But ct-DNA. 2. The results of competitive FRET-melting experiments showed that the% values of Fc-mycT and Fc-kit2T did not change significantly in the presence of 25 times excess double helix ds26, indicating that target compound 1 preferentially binds to the quadruplex. In the presence of 50 times of double helix ds26, the TM value of Fc-kit2T decreased greatly, which indicated that the selectivity of target compound 1 to Fc-kit2T decreased when the concentration of double helix DNA increased. In the presence of 10 times excess double helix ds26, the ATm value of FA-teloT decreased from 9.6 鈩，

本文編號(hào)：1885548