A novel strategy of designing neutrophil elastase fluorescent probe based on self-immolative group and its application in bioimaging
Introduction
Neutrophil elastase (NE, EC 3.4.21.37), a 29 kDa serine protease consisting of 267 amino acid residues, is widely existed in animals, plants and bacteria [[1], [2], [3], [4]]. In the human body, NE is mainly secreted by neutrophils released from bone marrow. And the catalytic active center of NE is composed of three key amino acid residues (His57, Asp102, Ser195), which has a powerful catalytic ability to the hydrolysis of amide bonds in proteins, and enables it to degrade various extracellular matrix proteins in complex biological systems, such as elastin, laminin, fibronectin, etc. [[5], [6], [7]]. Overexpression of NE may degrade some key proteins excessively, which leads to several serious lung diseases, including acute lung injury [8,9], chronic obstructive lung disease [[10], [11], [12]], acute respiratory distress syndrome [[13], [14], [15]], and so on. In addition, NE can promote tumor growth, invasion and metastasis, which is considered to be an independent indicator of lung cancer in tumor tissue [[16], [17], [18]]. Therefore, NE is regarded as a critical biomarker for the diagnosis and treatment of lung-related diseases. Consequently, developing an effective tool for the detection of NE levels in vitro and in vivo is of great meaning for the diagnosis of lung cancer.
In recent years, the rapid developing fluorescent probe technology is widely used for analysis and detection of ions, organic small molecules and biomacromolecules, which can be extensively applied to environment, food and medicine fields [[19], [20], [21], [22], [23]]. Especially in the medicine fields, it is usually used for tracking of biomarkers to diagnose or treat related diseases [[24], [25], [26], [27], [28]]. Nevertheless, few probes have been successfully used to detect NE levels in vitro and in vivo [[29], [30], [31], [32], [33], [34], [35]]. In 2013, we reported the first small-molecule-based NE fluorescent probe by using pentafluoropropionyl as the highly selective recognition group and amide bond as the linker, which can be cracked by NE [33]. Subsequently, some NE fluorescent probes were constructed by connected pentafluoropropionyl and amino-containing fluorophores, such as coumarins, rhodamines, hemicyanine and etc. [[36], [37], [38]]. All of these fluorophores must contain amino groups to construct amide bond linkers, which greatly restricts the development of NE fluorescent probes. Therefore, developing a novel design strategy to utilize other fluorophores (such as hydroxyl-containing fluorophores) to construct new NE fluorescent probes is of great importance.
In this work, we rationally design a novel NE fluorescent probe HFC-NE, using the self-immolative group as the linker to connect pentafluoropropionyl recognition group and hydroxyl-containing fluorophores HFC[39]. According to the probe design strategy, self-immolative group has two key functions: i) converting the hydroxyl groups in HFC to amino groups to construct the catalytic site of the amide bond; ii) improving the catalysis efficient due to the small steric hindrance from only one benzene ring in the self-immolative group when cleaved by NE, comparing with those amino-containing fluorophores. As expected, self-immolative group based HFC-NE can sensitively and selectively detect NE levels with a low fluorescence background in water solution, confirming that HFC-NE is able to be cleaved via NE catalysis. Furthermore, compared with our previous NE probe, HFC-NE has much stronger fluorescence enhancement (176-fold), higher affinity for NE (Km = 2.85 ± 0.060 μM) and catalytic efficiency (kcat/Km = 4.58 μM−1 min−1), indicating that the modification of self-immolative linker can significantly improve the response performance of NE fluorescent probe. More importantly, HFC-NE is successfully used for precisely real-time tracking endogenous NE levels in living cells and zebrafish, revealing that self-immolative group is an efficient tool to expand the application of the fluorophores.
Section snippets
Materials and equipments
All reagents and solvents were purchased from licensed manufactures. UV–vis absorption and fluorescence spectra were measured using a Spectra Max M2 Microplate reader. The structure of the intermediates and the final product HFC-NE was confirmed by 1H NMR, 13C NMR and HR-MS. Living Cells and zebrafish imaging were obtained by inverted fluorescence microscope (Olympus IX71, Japan).
Synthesis routs of HFC-NE
Synthesis of intermediate 1.4-Aminobenzyl alcohol (1.00 g, 8.09 mmol) was dissolved in 15 mL anhydrous DMF, then
Spectral response of probe HFC-NE toward NE
To verify the response ability of HFC-NE against NE, the ultraviolet and fluorescence spectra were initially investigated. As shown in Fig. S6a, HFC-NE had an abroad absorption peak with maximum value at 355 nm. After treated with NE, the absorption peak red shifted to 380 nm, which was consistent with the maximum absorption of the fluorophore HFC. With the excitation wavelength as 380 nm, the fluorescence spectra of HFC-NE to NE were further studied. As shown in Fig. S6b, free HFC-NE showed
Conclusion
In summary, we proposed a novel design strategy to rational construct a NE probe HFC-NE which connected hydroxyl-containing fluorophore HFC and pentafluoropropionyl recognition group via a self-immolative linker. Surprisingly, HFC-NE was successfully used to detect NE activity via cleaving HFC and self-immolative linker, which was verified by the HPLC and HRMS and confirmed the design strategy. Furthermore, HFC exhibited excellent response ability to NE, such as large Stokes shift (125 nm), low
CRediT authorship contribution statement
Xiang Li: Methodology, Data curation, Writing – original draft. Yun Guo: Methodology, Data curation. Yuan Qiu: Methodology, Writing – original draft. Xiaogang Luo: Investigation, Software. Genyan Liu: Supervision, Project administration. Yunfeng Han: Funding acquisition. Qi Sun: Investigation, Formal analysis, Data curation, Funding acquisition. Qingjian Dong: Writing – original draft, Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was funded by the National Natural Science Foundation of China (21804102), Hubei key Laboratory of Novel Reactor and Green Chemistry Technology of Wuhan Institute of Technology (NRG202107), Special Projects of the Central Government in Guidance of Local Science and Technology Development in Hubei Province (2020ZYYD040), the second batch of the Key Research and Development Project of Hubei Province (2020BAB073), Open Project of Key Laboratory of Novel Biomass-Based Environmental and
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