Rationally designed an innovative proximity labeling near-infrared fluorogenic probe for imaging of peroxynitrite in acute lung injury-海南省生物材料与医疗器械工程研究中心

Rationally designed an innovative proximity labeling near-infrared fluorogenic probe for imaging of peroxynitrite in acute lung injury

发布时间：2025-03-09 发布者：浏览次数：

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a: Key Laboratory of Emergency and Trauma of Ministry of Education, Department of Radiotherapy, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, Haikou 571199, China

b: Key Laboratory of Hainan Trauma and Disaster Rescue, Key Laboratory of Haikou Trauma, Engineering Research Center for Hainan Bio-Smart Materials and Bio-Medical Devices, College of Emergency and Trauma, Hainan Medical University, Haikou 571199, China

Received 8 April 2024, Revised 30 May 2024, Accepted 2 June 2024, Available online 3 June 2024, Version of Record 9 March 2025.

https://doi.org/10.1016/j.cclet.2024.110082

Abstract

Acute lung injury (ALI) is a serious clinical condition with a high mortality rate. Oxidative stress and inflammatory responses play pivotal roles in the pathogenesis of ALI. ONOO− is a key mediator that exacerbates oxidative damage and microvascular permeability in ALI. Accurate detection of ONOO− would facilitate early diagnosis and intervention in ALI. Near-infrared fluorescence (NIRF) probes offer new solutions due to their sensitivity, depth of tissue penetration, and imaging capabilities. However, the developed ONOO− fluorescent probes face problems such as interference from other reactive oxygen species and easy intracellular diffusion. To address these issues, we introduced an innovative self-immobilizing NIRF probe, DCI2F-OTf, which was capable of monitoring ONOO− in vitro and in vivo. Importantly, leveraging the high reactivity of the methylene quinone (QM) intermediate, DCI2F-OTf was able to covalently label proteins in the presence of ONOO−, enabling in situ imaging. In mice models of ALI, DCI2F-OTf enabled real-time imaging of ONOO− levels and found that ONOO− was tightly correlated with the progression of ALI. Our findings demonstrated that DCI2F-OTf was a promising chemical tool for the detection of ONOO−, which could help to gain insight into the pathogenesis of ALI and monitor treatment efficacy.

Graphical abstract

An innovative self-immobilizing NIRF probe DCI2F-OTf was capable of monitoring ONOO− in vitro and in vivo. DCI2F-OTf was able to covalently label proteins in the presence of ONOO−, enabling in situ imaging. In mice models of ALI, DCI2F-OTf enabled real-time imaging of ONOO− levels and found that ONOO− was tightly correlated with the progression of ALI.

Image, graphical abstract

Keywords

Near-infrared

Peroxynitrite

Proximity labeling

In situ bioimaging

Acute lung injury

Acute lung injury (ALI) refers to the injury of alveolar epithelial cells and capillary endothelial cells caused by various non-cardiogenic factors, with a high mortality rate [1]. Clinically, ALI can rapidly progress to acute respiratory distress syndrome, a more severe form of lung injury. Oxidative stress and exaggerated inflammatory responses are recognized as pivotal contributors to the pathogenesis and exacerbation of ALI [2,3]. In biological systems, the rapid diffusion of nitric oxide (NO) and superoxide anions produces endogenous ONOO−, a potent reactive species generated at a rate hundreds of times faster than the rate of NO binding to heme protein. ONOO− readily engages in reactions with proteins, lipids, or nucleic acids to promote oxidation or nitration of biomolecules that can lead to cellular damage. For instance, ONOO− can alter the structure of proteins through the formation of nitrifying proteins, thus affecting their physiological function [[4], [5], [6], [7], [8]]. ONOO− acts as a signaling molecule to participate in cell signal transduction and various physiological processes, including gene expression regulation and apoptosis [9]. On one hand, elevated levels of ONOO− disrupt the lung's antioxidant defense system, exacerbating oxidative stress and cellular damage; on the other hand, increased ONOO− levels significantly enhance the permeability of the lung microvascular wall, which ultimately impairs lung function [[10], [11], [12], [13], [14]]. Thus, the development of innovative tools for the detection of ONOO− to accurately diagnose ALI will help to track disease progression in time for early intervention and improve patient survival.

Fluorescent probes have emerged as a powerful tool for visualizing diverse physiological and pathological processes due to their high sensitivity, non-invasiveness, and real-time imaging capabilities. Particularly, near-infrared fluorescent (NIRF) probes offer superior optical penetration, less photodamage, and lower background fluorescence. In recent years, a variety of ONOO−-fluorescent probes have been constructed leveraging diverse reaction moieties, including unsaturated double bonds, boronic esters, boronic acids, hydrazines, and so on [[15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25]]. However, available ONOO−-fluorescent probes encounter two primary challenges: (1) susceptible to potential interference from high concentrations of reactive oxygen species (e.g., hypochlorite, hydrogen peroxide); (2) prone to diffuse from the reaction site, thereby decreasing imaging accuracy and signal-to-noise ratio. Hence, it is of great importance to design ONOO−-activated, proximity protein-captured, self-immobilizing NIRF probes at the cellular or in vivo level.

Methylene quinone (QM) is an important class of electrophilic intermediates with high reactivity in chemical reactions. In biological applications, the QM released from the reaction of a chemical probe with physiologically reactive species is easily trapped by nucleophilic groups in nearby protein molecules [[26], [27], [28], [29]]. This strategy has been widely used in the design and synthesis of in situ labeling probes for hydrogen peroxide [30], neuraminidases [31], β-galactosidase [32], nitroreductase [33], γ-glutamyl transpeptidase [34], and alkaline phosphatase [35]. Inspired by these pioneering efforts, we herein disclosed a novel activatable self-immobilizing NIRF probe DCI2F-OTf for the detection of ONOO− in vitro and in vivo. In this design, dicyanoisophorone derivatives (DCI) were adopted as a NIRF signaling unit due to their excellent stability, large Stoke shift, and ease of synthesis, while trifluoromethane sulfonate was employed as the reactive moiety for ONOO− owing to its ultra-high selectivity. The molecular design of DCI2F-OTf facilitated its implementation for precise imaging of ONOO− in cells and mice model of ALI. NIRF imaging allowed DCI2F-OTf to successfully visualize changes in ONOO− levels in both cells and mice model of ALI. Furthermore, DCI2F-OTf could serve as an indicator to tracer the protective effect of sulforaphane (SFN) on lipopolysaccharide (LPS)-induced ALI in mice. These findings suggested that DCI2F-OTf could be used as a promising tool to obtain information about oxidative stress-related diseases and to track the effects of drug therapy.

Two DCI-type fluorescence probes DCI2F-OTf and DCI-OTf were designed and synthesized. The chemical structures of all compounds were comprehensively characterized by nuclear magnetic resonance (NMR) and high-resolution mass spectroscopy (HRMS) (Figs. S1–S6 in Supporting information). Of note, the fluorescence response of DCI-OTf to ONOO− was minimal and spectrally blue-shifted to 525 nm compared to DCI2F-OTf, whereas DCI2F-OTf exhibited excellent selectivity, sensitivity, and NIRF emission characteristics (Fig. S8 in Supporting information). We hypothesized that this disparity could be attributed to the electron-withdrawing difluoromethyl (-CHF2) in the ortho position of the hydroxyl group, which was able to effectively reduce the pKa value of DCI2F-OTf, enhance its reactivity toward ONOO−, and lower the detection limit of ONOO−. It was widely known that ONOO− itself was oxidizing and nucleophilic. In this design, the sulfur-centered atom of the positively charged trifluoromethanesulfonyl group was initially attacked by the nucleophile ONOO−, and then underwent a nucleophilic addition-elimination reaction, which cleaved the sulfur-oxygen bond to delocalize the trifluoromethanesulfonyl group, forming the key intermediate DCI2F. Subsequently, the DCI2F underwent intramolecular rearrangement to generate the QM, which was captured by nucleophilic groups (e.g., hydroxyl, amine, and sulfhydryl groups) around the active site of the protein within cells, facilitating in situ imaging (Scheme 1). To elucidate the reaction mechanism of DCI2F-OTf with ONOO−, the mixture of DCI2F-OTf reacting with ONOO− was analyzed by HRMS. The results revealed a mass peak at m/z = 361.1372, which was attributed to DCI-H2O and different from the mass peak of DCI2F-OTf at m/z = 472.0889. The plausible reaction mechanism involved the reaction of DCI2F-OTf with ONOO− to release QM, followed by nucleophilic attack by H2O in the mixed system to form the product DCI-H2O, consistent with the mechanism of protein capture of QM (Fig. S7 in Supporting information).

Scheme 1

Scheme 1. Schematic illustration of dual sensing and labeling NIRF probe DCI2F-OTf for ONOO−.

Fig 5

Fig. 5. (a) NIRF images of control and LPS-stimulated mice after intratracheal drip injection of DCI2F-OTf (200 μmol/L, 45 μL). (b) NIRF imaging and photographs of dissected major organs (1: heart, 2: kidney, 3: liver, 4: spleen, 5: lung) from mice in panel (a). (c) The histogram presents the average radiant efficiency of mice in panel (a). (d) The histogram presents the average radiant efficiency of the lung in panel (b). (e) H&E staining histological images for lung tissue in control and LPS-stimulated mice. Scale bar: 50 μm. (f–h) Serum levels of inflammatory factors TNF-α, IL-6, and IL-1β in control and LPS-stimulated mice. (i) The protein levels of iNOS, COX-2, NF-κB, and IL-6 were detected by Western blot analysis. (j–m) The relative protein expression level of iNOS, COX-2, NF-κB, and IL-6 in (i). (n, o) Sections of lung tissue were immunostained with an antibody that detected 3-NT. Scale bar: 50 μm. (p) NIRF images of mice fresh lung tissue sections from ALI mice in the LPS6 group. The confocal Z-axis scan images at 0, 5, 10, 15, 20, 25, 30, 35, 40, and 50 µm penetration depths. λex = 488 nm; red channel: λem = 600–700 nm. Data are presented as mean ± SD (n = 3).

In conclusion, we have designed a novel NIRF probe, DCI2F-OTf, for fluorescence imaging ONOO− in live cells and mice model of ALI. Under physiological conditions, DCI2F-OTf was highly sensitive and selective for ONOO− and yielded a linear response to ONOO over a wide concentration range from 0 to 70 μmol/L. The QM covalently labeled neighboring proteins released from the reaction of DCI2F-OTf with ONOO− could solve the problem of intracellular diffusion of the probe, enabling precise imaging of intracellular ONOO−. This strategy was superior to the vast majority of ONOO− probes that have been developed so far. The excellent sensing performance and NIRF emission characteristics enabled DCI2F-OTf to image ONOO− levels in mice and tissues with ALI. In vivo imaging results indicated that SFN significantly down-regulated the oxidative stress level, attenuating the severity of LPS-induced ALI, and could exert a protective role against ALI. Therefore, DCI2F-OTf was a valuable tool for studying the physiological function of ONOO− and was expected to facilitate the understanding of oxidative stress diseases such as ALI and the rapid screening of corresponding therapeutic drugs.

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