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

摘要 本计画将利用电脑导引的方法，发展比率荧光探针 在红光至近红外区域操作，以准确测定细胞外和细胞内pH 在细胞膜和不同细胞器中的变化，如溶酶体、线粒体、内质网、细胞膜和细胞膜上的变化。 网，高尔基体，并监测线粒体交付的溶酶体在线粒体自噬 营养缺乏缺氧和药物治疗引起的探针的光谱化学属性将是 在合成之前确定，以便判断在所需光谱范围内的适用性。探针将包括 近红外荧光团连接到近红外对苯二酚染料中，以实现对pH的比率响应 基于pH调节的变化导致探针的π-缀合变化。红光至近红外 荧光团将包括BODIPY染料、传统的近红外咕吨染料及其衍生物，其中 氧杂蒽核的中心氧原子将被其它元素如Si、S和N取代。螺内酯 和螺内酰胺分子开关，其可以被调节到与不同的分子匹配的适当的pKa值。 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将是 通过引入溶酶体特异性吗啉，Escherichia特异性三苯基膦，高尔基体， 器官特异性苯磺酰胺，内质网特异性对甲苯磺酰胺，或细胞 膜特异性两亲性两性酶残基通过寡聚（乙二醇）间隔区与荧光 probes.基于螺内酰胺开关的比率红色至近红外荧光探针将具有pKa 值从4.5到6.2，并且仅在中性和碱性下显示近红外荧光团的荧光 pH条件，因为在微碱性条件下，罗丹明部分具有封闭的螺内酰胺环结构 在线粒体内。溶酶体的酸性环境会触发螺旋内酰胺开关的打开， 对羟基苯甲酸部分，显著增强荧光团和对羟基苯甲酸部分之间的π-共轭， 导致对pH变化的比率荧光响应。这些过程也将通过 进行合成前的理论计算。这些探测器将具有近- 红外成像（包括深层组织穿透、最小细胞损伤、无生物干扰） 自发荧光），以及具有自校准特征的比率成像，以克服 仅基于强度的荧光探针。此外，该探针将具有良好的水溶性、高选择性、高选择性和高选择性。 稳定性、优异的细胞渗透性、良好的生物相容性、优异的细胞内滞留、高选择性， 和灵敏度，以及对pH值变化的快速和可逆反应。七名本科生将协助 在进行这些实验的每一年的建议，这将使他们能够学习有价值的合成 以及这方面的研究能力。本科生还将进行理论 计算，使用高斯程序，以确定探针的吸收特性。

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.