Design and Expeditious Synthesis of Quinoline-Pyrene-Based Ratiometric Fluorescent Probes for Targeting Lysosomal pH

Intracellular pH plays a significant role in many pathological and physiological processes. A series of quinoline-pyrene probes were synthesized in one-step fashion through an oxonium-ion-triggered alkyne carboamination sequence involving C−C, C−O and C−N bond formation for intracellular pH sensing. The quinoline-pyrenes showed significant red shifts at low pH. Fluorescence lifetime decay measurements of the probes showed decreases in lifetime at pH 4. The probes showed excellent selectivity in the presence of various potential interfering agents such as amino acids and cations/anions. Furthermore, the probes were found to show completely reversible emission behaviour in the window between pH 4 and 7. A morpholine-substituted quinoline-pyrene probe efficiently stained lysosomes with high Pearson correlation coefficients (0.86) with Lysotracker Deep Red DND-99 as a reference. A co-localization study of the probe with Lysotracker DND-99 showed selective intracellular targeting and a shift in fluorescence emission due to acidic lysosomal pH.     

Fluorescent Probes for HOCl Imaging

Small-molecule fluorescent probes for hypochlorous acid (HOCl), one of the poorly understood reactive oxygen species (ROS), help to unveil HOCl functions in health and disease. Numerous small-molecule HOCl fluorescent probes have been developed in the past decade. Nevertheless, only a portion of them demonstrated their practical applications in biomedical research because of common problems in selectivity, sensitivity, chemostability, and photostability, etc. The problems could be addressed by a combination of rational probe design and careful selection of fluorophore templates. In this review, we describe several classes of representative HOCl fluorescent probes based on their fluorophore templates, and we discuss their design strategies, photophysical properties, and biological applications. A comprehension of their strengths, weaknesses, and common uses will facilitate the development of ideal HOCl assays and the discovery of novel biological processes.     

Clickable Polyamine Derivatives as Chemical Probes for the Polyamine Transport System

Polyamines are essential for cell growth and differentiation, but their trafficking by the polyamine transport system is not fully understood. Herein, the synthesis of several azido‐derivatized polyamines for easy conjugation by click chemistry is described. Attachment of a 4,4‐difluoro‐4‐bora‐3a,4a‐diaza‐s‐indacene (BODIPY) dye gave fluorescent polyamine probes, which were tested in cell culture. The linear probe series showed superior cellular uptake compared with that of probes in which the dye was attached to a branch on one of the central amines. Interestingly, the linear probes accumulated rapidly in cancer cells (MCF‐7), but not in nontumorigenic cells (MCF‐10A). The fluorescent polyamine probes are therefore applicable to the study of polyamine trafficking, whereas the azido polyamines may be further utilized to transport cargo into cancer cells by exploiting the polyamine transport system.     

Alkoxy Tetrazine Substitution at a Boron Center: A Strategy for Synthesizing Highly Fluorogenic Hydrophilic Probes

Strongly fluorogenic boron dipyrromethene (BODIPY)–tetrazine probes have been obtained by introducing an alkoxy tetrazine fragment at the boron center. The fluorescence signal from these probes strongly increases by up to 225‐fold after reaction with bioorthogonal coupling partners, and the hydrophilicity of probes is improved, such that they are suitable for live‐cell imaging.     

Design Principles for Cationic,Astrocyte-Targeted Probes

The brain's astrocytes play key roles in normal and pathological brain processes. Targeting small molecules to astrocytes in the presence of the many other cell types in the brain will provide useful tools for their visualization and manipulation. Herein, we explore the functional consequences of synthetic modifications to a recently described astrocyte marker composed of a bright rhodamine-based fluorophore and an astrocyte-targeting moiety. We altered the nature of the targeting moiety to probe the dependence of astrocyte targeting on hydrophobicity, charge, and pK_a when exposed to astrocytes and neurons isolated from the mouse cortex. We found that an overall molecular charge of +2 and a targeting moiety with a heterocyclic aromatic amine are important requirements for specific and robust astrocyte labeling. These results provide a basis for engineering astrocyte-targeted molecular tools with unique properties, including metabolite sensing or optogenetic control.     

An Expanded Set of Fluorogenic Sulfatase Activity Probes

Fluorogenic probes that are activated by an enzymatic transformation are ideally suited for profiling enzyme activities in biological systems. Here, we describe two fluorogenic enzyme probes, 3‐O‐methylfluorescein‐sulfate and resorufin‐sulfate, that can be used to detect sulfatases in mycobacterial lysates. Both probes were validated with a set of commercial sulfatases and used to reveal species‐specific sulfatase banding patterns in a gel‐resolved assay of mycobacterial lysates. The fluorogenic probes described here are suitable for various assays and provide a starting point for creating new sulfatase probes with improved selectivity for mycobacterial sulfatases.     

Differently Tagged Probes for Protein Profiling of Mitochondria

The mitochondrion is one of the most important organelles in the eukaryotic cell. Characterization of the mitochondrial proteome is a prerequisite for understanding its cellular functions at the molecular level. Here we report a proteomics method based on mitochondrion-targeting groups and click chemistry. In our strategy, three different mitochondrion-targeting moieties were each augmented with a clickable handle and a cysteine-reactive group. Fluorescence-based bioimaging and fractionation experiments clearly showed that most signals arising from the labels were localized in the mitochondria of cells, as a result of covalent attachment between probe and target proteins. The three probes had distinct profiling characteristics. Furthermore, we successfully identified more than two hundred mitochondrial proteins. The results showed that different mitochondrion-targeting groups targeted distinct proteins with partial overlap. Most of the labeled proteins were localized in the mitochondrial matrix and inner mitochondrial membrane. Our results provide a tool for chemoproteomic analysis of mitochondrion-related proteins.     

有机pH荧光探针研究进展

细胞内p H在许多细胞生物学过程中发挥着至关重要的作用,异常的p H变化会导致细胞功能紊乱、生长和分裂的突变,还可能引发癌症和阿尔茨海默综合症等疾病。因此,研究细胞内p H的变化具有非常重要的理论和实践意义。荧光分析法具有操作简便、灵敏度高、选择性好、实时检测以及对生物体损伤小等优点而得到了广泛地研究和应用。双光子荧光探针技术相对于单光子荧光技术具有长波吸收、短波发射、高度的三维空间选择性、大的穿透深度、避免荧光漂白和光致毒以及降低组织自发荧光干扰等特点,在生命科学领域显示出了广阔的应用前景。介绍了有机单光子和双光子p H荧光探针的研究现状,同时对有机p H荧光探针未来的发展趋势进行了展望。     

Glycosyl‐Modified Diporphyrins for in Vitro and in Vivo Fluorescence Imaging

The application of probes for optical imaging is becoming popular as they have high safety and good biocompatibility. We prepared two kinds of glycosyl‐modified diporphyrins, and their potentials as fluorescent probes were tested for the first time. After preparation of the glycosyl‐modified porphyrin monomers, Ag‐promoted coupling of the monomers was used to obtain glucose‐modified porphyrin dimer (GPD) and lactose‐modified porphyrin dimer (LPD). The strong interaction between the two porphyrin rings achieves red‐shifted emission, and thus circumvents autofluorescence and light‐scattering in biological samples. Although the glycosylation improves solubility, it also yielded selective attachment to cell membranes, and to chorions of early developmental‐stage zebrafish. Patch‐clamp experiments revealed the biocompatibility and low toxicity of GPD and LPD. Moreover, an in vivo imaging experiment provided direct evidence that zebrafish chorion contains sugar‐binding proteins. The modification and derivatization make porphyrins potential bioimaging probes for specific optical imaging.     

细胞Zn2+荧光探针研究进展

对近年来测定细胞内游离Zn2 的荧光探针进行较详细地评述,主要包括喹啉类、荧光素及其衍生物类、肽和蛋白类荧光探针;并对其化学规律、性质和优缺点进行了讨论。     

基于生命体系中氨基键合标记的荧光探针研究进展

氨基是生物分子化学结构中常见的基团。通常可利用荧光探针的静电吸引、疏水作用和化学键合的方式对其进行荧光标记。本文综述了基于化学键合作用的荧光探针及其常见的键合类型和染料母体结构。有48篇参考文献。     

Fast-Response Fluorogenic Probe for Visualizing Hypochlorite in Living Cells and in Zebrafish

A fast-response fluorogenic probe—compound D1 —for monitoring hypochlorite (ClO⁻), based on specific ClO⁻ cleavage of a C=N bond and producing results observable to the naked eye, has been developed. The response of the probe to ClO⁻ increases linearly, and the fluorescence intensity was heightened by a factor of about 25. D1 responses to ClO⁻, with high selectivity and sensitivity, were observable by naked eye within 10 s. D1 can not only detect levels of hypochlorite in vitro, such as in urine, but is also capable of monitoring hypochlorite content under extremely cold conditions, as low as −78 °C. Meanwhile, its good biocompatibility permitted the use of D1 to detect intracellular ClO⁻ by confocal microscopy. Moreover, D1 was successfully applied to monitor exogenous and endogenous ClO⁻ in zebrafish through fluorescence imaging.     

Design,Synthesis, and Biological Evaluation of Novel Fluorescent Probes Targeting the 18-kDa Translocator Protein

A series of fluorescent probes from the 6-chloro-2-phenylimidazo[1,2-a]pyridine-3-yl acetamides ligands featuring the 7-nitro-2-oxa-1,3-diazol-4-yl (NBD) moiety has been synthesized and biologically evaluated for their fluorescence properties and for their binding affinity to the 18-kDa translocator protein (TSPO). Spectroscopic studies including UV/Vis absorption and fluorescence measurements showed that the synthesized fluorescent probes exhibit favorable spectroscopic properties, especially in nonpolar environments. In vitro fluorescence staining in brain sections from lipopolysaccharide (LPS)-injected mice revealed partial colocalization of the probes with the TSPO. The TSPO binding affinity of the probes was measured on crude mitochondrial fractions separated from rat brain homogenates in a [¹¹C]PK11195 radioligand binding assay. All the new fluorescent probes demonstrated moderate to high binding affinity to the TSPO, with affinity (K_i) values ranging from 0.58 nM to 3.28 μM. Taking these data together, we propose that the new fluorescent probes could be used to visualize the TSPO.