Computationally Guided Approach to Produce Ratiometric Probes Operating in the Red to Near-infrared Region to Accurately Determine pH Levels within Organelles

摘要 利用计算机引导的方法，这个项目将开发比率荧光探针 工作在红色到近红外区，准确测定细胞外和细胞内的pH值 跨细胞膜和不同细胞器的变化，如溶酶体、线粒体、内质 网状结构，高尔基体，并在有丝分裂过程中监测线粒体向溶酶体的输送 由营养饥饿、缺氧和药物治疗引起。探测器的光谱化学属性将是 在合成前确定，以便判断在所需光谱范围内的适用性。探测器将包括 连接到近红外染料中的近红外荧光团以实现对pH的比率响应 基于pH调制的变化导致探针的π结合变化。红色到近红外线 荧光团将包括BODIPY染料、传统的近红外Xanthene染料及其衍生物，其中 黄原烯核心的中心氧原子将被其他元素取代，如硅、S和螺内酯 以及螺内酰胺分子开关，可以调节到与不同的 将使用正在研究的细胞器。针对不同细胞器和细胞膜的特异性靶向 通过引入溶酶体特异的吗啉、线粒体特异的三苯基膦、高尔基体 装置特有的苯磺酰胺、内质网特有的对甲苯磺酰胺或细胞 膜上特异的两亲性两性离子残基通过低聚(乙二醇基)间隔基到荧光 探测器。基于螺内酰胺开关的红色至近红外比例荧光探针将具有pka 值在4.5到6.2之间，仅显示中性和碱性条件下近红外荧光团的荧光 PH条件自罗多尔部分在略微碱性条件下关闭螺内酰胺环结构 在线粒体内。溶酶体的酸性环境会触发螺内酰胺开关的打开 罗多尔基团，显著增强了荧光团和罗多尔基团之间的π偶联， 从而产生对pH变化的比率荧光响应。这些过程也将通过以下方式描述 在进行合成前的理论计算。这些探头将具有近乎于 红外成像(包括组织深度穿透、细胞损伤最小、无生物干扰 自动荧光)，以及具有自校准功能的比率成像，以克服 仅基于强度的荧光探针。此外，该探针还将具有良好的水溶性、高 稳定性好，细胞通透性好，生物相容性好，细胞内保留率高，选择性高， 和敏感性，以及对pH变化的快速和可逆反应。七名本科生将协助 在每一年的提案中进行这些实验，这将使他们能够学习有价值的合成 以及在这一领域的定性研究技能。本科生还将进行理论研究 使用GASSUS程序进行计算，以确定探头的吸收特性。

Abstract Employing a computationally guided approach, this project will develop ratiometric fluorescent probes operating in the red to the near-infrared region to accurately determine extracellular and intracellular pH changes across cell membranes and in different organelles, such as lysosomes, mitochondria, the endoplasmic reticulum, the Golgi apparatus, and to monitor mitochondrial delivery to the lysosomes during mitophagy caused by nutrient starvation, hypoxia, and drug treatment. Spectrochemical attributes of probes will be determined before synthesis in order to judge applicability in the desired spectral range. Probes will consist of near-infrared fluorophores linked into near-infrared rhodol dyes to achieve ratiometric responses to pH changes based on pH modulation resulting in π-conjugation changes of the probes. Red to near-infrared fluorophores will include BODIPY dyes, traditional near-infrared xanthene dyes, and their derivatives, where the central oxygen atom of xanthene cores will be replaced by other elements such as Si, S, and N. Spirolactone and spirolactam molecular switches that can be tuned to appropriate pKa values matching the different organelles under study will be used. Specific targeting to different organelles and cell membranes will be achieved by introducing lysosome-specific morpholine, mitochondria-specific triphenylphosphonium, Golgi apparatus-specific benzenesulfonamide, endoplasmic reticulum-specific p-toluene sulfonamide, or cell membrane-specific amphilphilic zwitterion residues through oligo(ethylene glycol) spacers to the fluorescent probes. The ratiometric red to near-infrared fluorescent probes based on spirolactam switches will have pKa values from 4.5 to 6.2 and will only show fluorescence of near-infrared fluorophores under neutral and basic pH conditions since rhodol moieties have closed spirolactam ring structures under the slightly basic conditions within mitochondria. A lysosomal acidic environment will trigger the opening of the spirolactam switches on the rhodol moieties, significantly enhancing π-conjugation between the fluorophore and rhodol moieties, resulting in ratiometric fluorescence responses to pH changes. These processes will also be delineated via theoretical calculations before syntheses are conducted. The probes will have the combined advantages of near- infrared imaging (including deep tissue penetration, minimum cell damage, lack of interference from biological autofluorescence), and ratiometric imaging with a self-calibration feature to overcome systematic errors of intensity-only based fluorescent probes. In addition, the probes will possess good water solubility, high stability, excellent cell permeability, good biocompatibility, excellent intracellular retention, high selectivity, and sensitivity, as well as fast and reversible responses to pH changes. Seven undergraduate students will assist in conducting these experiments for each year of the proposal which will allow them to learn valuable synthetic and characterization research skills in this area. Undergraduate students will also conduct theoretical calculations, using the program Gaussian, to determine the absorptive properties of the probes.