Recent Progress of Small-Molecule Ratiometric Fluorescent Probes for Peroxynitrite in Biological Systems

Peroxynitrite (ONOO⁻) as a major reactive oxygen species plays important roles in cellular signal transduction and homeostatic regulation. Precise detection of ONOO⁻ in biological systems is vital for exploring its physiological and pathological function. Among numerous detection methods, fluorescence imaging technology using fluorescent probes offers some advantages, including simple operation, high sensitivity and selectivity, as well as real-time and nondestructive detection. In particular, ratiometric fluorescent probes, in which the built-in calibration of the two emission bands prevents interference from the biological environment, have been extensively employed to monitor the fluctuation of bioactive species. In this review, we will discuss small-molecule ratiometric fluorescent probes for ONOO⁻ in live cells or in vivo, which involves chemical structures, response mechanisms, and biological applications. Moreover, the challenges and future prospects of ONOO⁻-responsive ratiometric fluorescent probe are also proposed.     

Supramolecular Assembly of TPE-Based Glycoclusters with Dicyanomethylene-4H-pyran (DM) Fluorescent Probes Improve Their Properties for Peroxynitrite Sensing and Cell Imaging

Two red-emitting dicyanomethylene-4H-pyran (DM) based fluorescent probes were designed and used for peroxynitrite (ONOO⁻) detection. Nevertheless, the aggregation-caused quenching effect diminished the fluorescence and restricted their further applications. To overcome this problem, tetraphenylethylene (TPE) based glycoclusters were used to self-assemble with these DM probes to obtain supramolecular water-soluble glyco-dots. This self-assembly strategy enhanced the fluorescence intensity, leading to an enhanced selectivity and activity of the resulting glyco-dot comparing to DM probes alone in PBS buffer. The glyco-dots also exhibited better results during fluorescence sensing of intracellular ONOO⁻ than the probes alone, thereby offering scope for the development of other similar supramolecular glyco-systems for chemical biological studies.     

Thiomaleimide Functionalization for Selective Biological Fluorescence Detection of Peroxynitrite as Tested in HeLa and RAW 264.7 Cells

The role of fluorescent molecules in diagnosis, treatment as well as in biomedical research has great current medicinal significance and is the focus of concentrated effort across the scientific research spectrum. Related research continues to reveal new practical sensing systems that bear enhanced features for interfacing of substituted molecules with biological systems. As part of an effort to better understand chalcogenide systems, a new dithiomaleimide BODIPY ( BDP‐NGM ) probe has been designed, synthesized and characterized. The fluorescence of BDP‐NGM was quenched by the incorporation of [3,4‐bis (phenylthio)] on the maleimide‐4‐phenyl moiety which is, in turn, placed at the meso ‐position of the BODIPY system. The probe shows a turn‐on fluorescence response upon reaction with ONOO⁻; mass spectral evidence reveals peaks in agreement with products involving oxidation of the sulfur groups to sulfone groups. An about 18.0‐fold emission intensity enhancement was found. By comparison, the emission signal from another ROS/RNS, superoxide, gave a modest turn on signal (≈5.0‐fold). The reaction is complete within 10 min, judging from the monitoring of the turn‐on fluorescence process; the detection limit was found to be 0.4 μm . BDP‐NGM can be used for the detection of ONOO⁻ under both acidic and basic conditions. Live cell imaging showed that the current probe can be used for the selective detection of ONOO⁻ in living systems.     

过氧亚硝酸的细胞生物学效应及其分析方法研究进展

对过氧亚硝酸的细胞生物学效应做了简单阐述，全面回顾和总结了近年来用于过氧亚硝酸测定的各种分析方法，并对各种方法的优缺点及其适用范围进行了比较．过氧亚硝酸的相关研究已取得很大进展，但其在线准确检测仍存在一定困难．     

Peroxynitrite: <Emphasis Type="BoldItalic">In vivo</Emphasis> and <Emphasis Type="BoldItalic">In vitro</Emphasis> synthesis and oxidant degradative action on biological systems regarding biomolecular injury and inflammatory processes

This review summarizes all significant data regarding peroxynitrite chemistry, the ways of its synthetic preparation as well as the degradative action of this species on biomolecules, in particular glycosaminoglycans, among which the hyaluronan degradation by peroxynitrite has recently been the subject of greater interest than ever before. The complex chemical behavior of a peroxynitrite molecule is strongly influenced by a few factors; conformational structural forms, active intermediates release, presence of CO₂ and trace transition metals, different reaction conditions, as well as the rules of kinetics. Special attention was focused on monitoring of the kinetics of the degradative action of peroxynitrite in or without the presence of residual hydrogen peroxide on high-molar-mass hyaluronan.     

氧合血红蛋白与亚硝酸根、过氧化亚硝酸离子反应的动力学研究

利用快速混合停流时间分辨光谱研究了氧合血红蛋白与亚硝酸根的反应．研究表明氧合血红蛋白和NO2^-的反应历程比较特殊，根据实验结果提出了合理的反应机理．同时与氧合血红蛋白和过氧化亚硝酸离子的反应进行了对比，发现氧合血红蛋白和过氧化亚硝酸离子的反应具有双相反应动力学的特征，其中间体在第一步快速反应的过程中形成．     

Theoretical Studies on the Isomerization of Peroxynitrite to Nitrate Mediated by Peroxynitrous Acid

The conversion of peroxynitrite （ONOO-） to nitrate （NO3-） mediated by peroxynitrous acid （ONOOH） has been investigated at the CCSD/6-311G（d）//B3LYP/6-31 1＋G（d,p） level. Two kinds of pathways for the title reaction were found. The results show that the energy barrier of isomerization through pathway 1 is around 25 kcal/mol in the gas phase. This value is significantly lower than that of isomerization without any catalysts. Thus, it indicates that ONOOH definitely makes the conversion from ONOO- to NO3- feasible. Although pathway 2 does not decrease the energy barrier of this isomerization, peroxynitric acid （O2NOOH） was obtained; moreover, this is a new pathway for this formation. In view of the results that peroxynitrate anion can decompose into nitrite and dioxygen, we conclude that our results are consistent with the experimental observation that nitrate, nitrite, and dioxygen are the main final products of the decay of peroxynitrite around pH 7.     

Formation of 8-nitroguanine and 8-oxoguanine due to reactions of peroxynitrite with guanine

Reactions of peroxynitrite with guanine were investigated using density functional theory (B3LYP) employing 6-31G** and AUG-cc-pVDZ basis sets. Single point energy calculations were performed at the MP2/AUG-cc-pVDZ level. Genuineness of the calculated transition states (TS) was tested by visually examining the vibrational modes corresponding to the imaginary vibrational frequencies and applying the criterion that the TS properly connected the reactant and product complexes (PC). Genuineness of all the calculated TS was further ensured by intrinsic reaction coordinate (IRC) calculations. Effects of aqueous media were investigated by solvating all the species involved in the reactions using the polarizable continuum model (PCM). The calculations reveal that the most stable nitro-product complex involving the anion of 8-nitroguanine and a water molecule i.e. 8NO(2)G(-) + H(2)O can be formed according to one reaction mechanism while there are two possible reaction mechanisms for the formation of the oxo-product complex involving 8-oxoguanine and anion of the NO(2) group i.e. 8OG + NO(2)(-). The calculated relative stabilities of the PC, barrier energies of the reactions and the corresponding enthalpy changes suggest that formation of the complex 8OG + NO(2)(-) would be somewhat preferred over that of the complex 8NO(2)G(-) + H(2)O. The possible biological implications of this result are discussed.     

Optical probes for detection and quantification of neutrophils’ oxidative burst. A review

Neutrophils, also known as polymorphonuclear leukocytes (PMN), are the most common type of white blood cells, comprising about 50-70% of all white blood cells. In the event of inflammatory processes, neutrophils display increased mobility, tissue influx ability, prolonged life span, and an increased phagocytic capacity, constituting the initial participants in the cellular defense of the organism. One of the most important defense systems of neutrophils corresponds to their ability to mediate a strong oxidative burst through the formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). While oxidative burst is important for the elimination of invading microorganisms, the overproduction of ROS and RNS or the impairment of endogenous antioxidant defenses may result to detrimental effects to the host. The nature and the extent of ROS and RNS production by neutrophils in response to different stimuli is, consequently, a matter of extensive research, with scientific reports showing an enormous variability on the detection methodologies employed. This review attempts to provide a critical assessment of the most common approaches to identify and quantify reactive species formed during the neutrophils’ oxidative burst. The detection mechanisms and performance, as well as advantages and limitations of the different methodologies, are scrutinized, focusing on the use of fluorimetric, chemiluminometric and colorimetric probes.