亚细胞器靶向性和固定性荧光探针的设计合成和应用研究

发布时间：2018-01-29 19:59

本文关键词： Snap-tag 近红外荧光染料荧光探针荧光成像荧光寿命成像　出处：《大连理工大学》2017年博士论文　论文类型：学位论文

【摘要】：随着光学显微成像技术的迅速发展,荧光染料和荧光探针在生物医学领域应用越来越广泛;然而大多数荧光探针亚细胞器靶向性不稳定,发射波长较短,易受细胞自发荧光干扰。因此,开发具有亚细胞器稳定靶向和近红外荧光探针成为了研究热点。本文设计合成Snap-tag融合蛋白靶向和亚细胞器稳定靶向的荧光探针,及一系列近红外荧光染料。研究了其化学和光物理性质,探索其在荧光成像及检测方面的应用。1、从O6-鸟嘌呤出发,设计合成了 Snap-tag靶向通用中间体PYBG和ADBG。通过PYBG和ADBG分别与罗丹明、萘酰亚胺、氟硼吡咯的Click反应和Sonogashira偶联,得到 PYBG-TMR、PYBG-BODIPY、ADBG-Np-N、ADBG-Np-N+、ADBG-Np-OH,PYBG-SF、PYBG-NO、PYBG-ClO 和 PYBG-Vis。荧光染料 PYBG-TMR、PYBG-BODIPY和ADBG-Np-OH都高效特异性标记表达在细胞核、线粒体、细胞质基质中的Snap-tag蛋白。荧光探针PYBG-NO在活细胞内成功响应信号分子NO;荧光探针PYBG-ClO在活细胞内成功响应次氯酸;荧光探针PYBG-Vis成功探测细胞内不同亚细胞器的粘度差异。2、设计合成了能够与蛋白反应的粘度探针Cl-N-Vis和M-Vis-A,比率型氧气敏感探针M-O-A。其中Cl-N-Vis和M-Vis-A的荧光寿命随着粘度变化呈现线性关系。探针Cl-N-Vis定位在脂滴中,通过荧光寿命成像方法展示了肝细胞(HL7702细胞)的脂滴粘度。探针M-Vis-A通过醛基与蛋白质上氨基的化学反应键合在线粒体上,并通过荧光寿命成像原位指示SMMC7721细胞病理状态下线粒体的粘度变化。探针M-O-A通过苄氯蛋白质上巯基的化学反应键合在线粒体上,并通过荧光比率成像原位的指示细胞内氧气浓度变化。3、结合不对称罗丹明和菁类染料,设计合成近红外荧光染料N-NIR、S-NIR和K-NIR,近红外pH荧光探针pH-A-NIR和pH-B-NIR;通过预留溴原子,得到衍生平台染料Br-N-NIR。在甲醇中N-NIR和S-NIR分别有0.4、0.21的荧光量子产率和10万以上的摩尔消光系数;荧光染料K-NIR有接近800nm的最大吸收波长。通过Br-N-NIR得到可用于Snap-tag标记的近红外底物PYBG-NIR,并实现PYBG-NIR特异性标记细胞核、线粒体、细胞质基质中的Snap-tag蛋白。pH响应探针pH-A-NIR(pKa=7.2)和pH-B-NIR(pKa=6.1)有接近中性pKa和较高荧光量子产率。pH探针成功实现探测活细胞内pH变化。
[Abstract]:With the rapid development of optical microscopic imaging technology, fluorescent dyes and fluorescent probes are more and more widely used in the field of biomedicine. However, most fluorescent probes have unstable suborganelles, short emission wavelengths and are susceptible to cellular autofluorescence interference. The development of suborganele-stable and near-infrared fluorescent probes has become a hot topic. In this paper, we designed and synthesized Snap-tag fusion protein targeting and suborganelle stable targeting fluorescent probes. And a series of near infrared fluorescent dyes. The chemical and photophysical properties of the dyes were studied, and their applications in fluorescence imaging and detection were explored, starting from O6-guanine. PYBG and ADBG, the universal intermediates of Snap-tag targeting, were designed and synthesized with Rhodamine and naphthalimide by PYBG and ADBG, respectively. The Click reaction of Fluoropyrrole was coupled with Sonogashira to obtain PYBG-BODIPYN ADBG-Np-N. ADBG-Np-N ADBG-Np-OHG PYBG-SFN PYBG-NO. PYBG-ClO and PYBG-Vis. fluorescent dye PYBG-TMR. Both PYBG-BODIPY and ADBG-Np-OH are highly specific and expressed in nuclei and mitochondria. Snap-tag protein in cytoplasmic matrix. Fluorescent probe PYBG-NO successfully responded to signal molecule no in living cells. Fluorescence probe PYBG-ClO successfully responded to hypochloric acid in living cells. Fluorescence probe PYBG-Vis was successfully used to detect the viscosity difference of different suborganelles in cells. The viscosity probes Cl-N-Vis and M-Vis-A were designed and synthesized to react with protein. Ratio oxygen sensitive probe M-O-A.The fluorescence lifetime of Cl-N-Vis and M-Vis-A showed a linear relationship with the change of viscosity, and the probe Cl-N-Vis was located in the lipid droplet. The lipid droplet viscosity of hepatocyte HL-7702 was demonstrated by fluorescence lifetime imaging. The probe M-Vis-A was bonded to mitochondria by chemical reaction of aldehyde group with amino group on protein. Fluorescence lifetime imaging in situ indicated the changes of mitochondrial viscosity in SMMC7721 cells. The probe M-O-A was bound to mitochondria by the chemical reaction of mercapto group on benzyl chloride protein. Near-infrared fluorescent dyes N-NIRS-NIR and K-NIR were designed and synthesized by fluorescence ratio imaging in situ indicating the change of oxygen concentration in the cell. Combined with asymmetric Rhodamine and cyanine dyes, N-NIRS-NIR and K-NIR were synthesized. Near infrared pH fluorescence probe pH-A-NIR and pH -B-NIR; By reserving bromine atoms, the derivative platform dye Br-N-NIR was obtained. The N-NIR and S-NIR in methanol were 0.4 and 0.4 respectively. The fluorescence quantum yield and molar extinction coefficient above 100,000; The fluorescence dye K-NIR has a maximum absorption wavelength of nearly 800nm. The near infrared substrate PYBG-NIR, which can be used for Snap-tag labeling, was obtained by Br-N-NIR. The specific labeling of nucleus and mitochondria by PYBG-NIR was realized. Snap-tag protein in cytoplasmic matrix. PH-A-NIRP pKa7. 2) and pH-B-NIRI pKaV6. 1). Close to neutral pKa and high fluorescence quantum yield. Ph probe was successfully used to detect the pH changes in living cells.
【学位授予单位】：大连理工大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：O657.3

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