Design of Fluorescent Probes for Bioorthogonal Labeling of Carbonylation in Live Cells

Hazel Erkan,Dilek Telci,Ozlem Dilek

doi:10.1038/s41598-020-64790-y

Hazel Erkan, Dilek Telci + Show 1 more

Open Access

https://doi.org/10.1038/s41598-020-64790-y

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Abstract

With the rapid development of chemical biology, many diagnostic fluorophore-based tools were introduced to specific biomolecules by covalent binding. Bioorthogonal reactions have been widely utilized to manage challenges faced in clinical practice for early diagnosis and treatment of several tumor samples. Herein, we designed a small molecule fluorescent-based biosensor, 2Hydrazine-5nitrophenol (2Hzin5NP), which reacts with the carbonyl moiety of biomolecules through bioorthogonal reaction, therefore can be utilized for the detection of biomolecule carbonylation in various cancer cell lines. Our almost non-fluorescent chemical probe has a fast covalent binding with carbonyl moieties at neutral pH to form a stable fluorescent hydrazone product leading to a spectroscopic alteration in live cells. Microscopic and fluorometric analyses were used to distinguish the exogenous and endogenous ROS induced carbonylation profile in human dermal fibroblasts along with A498 primary site and ACHN metastatic site renal cell carcinoma (RRC) cell lines. Our results showed that carbonylation level that differs in response to exogenous and endogenous stress in healthy and cancer cells can be detected by the newly synthesized bioorthogonal fluorescent probe. Our results provide new insights into the development of novel bioorthogonal probes that can be utilized in site-specific carbonylation labeling to enhance new diagnostic approaches in cancer.

Highlights

‘Oxidative stress’ is interrelated with an imbalance in the level of reactive oxygen species (ROS) and antioxidants metabolism in the cellular system
Secondary protein carbonylation is a consequence of covalent adduction either between advanced lipid peroxidation end products (ALEs) or advanced glycation end products (AGEs) with amino acid residues[7]
Synthesis and Structural Characterization of 2-Hydrazine-5-nitrophenol (2Hzin5NP) and its hydrazone products. 2Hzin5NP (Scheme 1) was synthesized by diazotization of 2-Amino-5-nitrophenol as described Portoghese et al During organic synthesis, all reactions were monitored by thin layer chromatography (TLC) which provided Rf value for each sample

Summary

Introduction

‘Oxidative stress’ is interrelated with an imbalance in the level of reactive oxygen species (ROS) and antioxidants metabolism in the cellular system. The increased level of ROS and disturbed redox signaling results in the oxidative damage on biomolecules in living systems. Alcohol dehydrogenases, carbonyl reductases and aldo-keto reductases are capable of reducing carbonyl moiety on proteins[9] Another enzymatic decarbonylation mechanism based on activation of Lon protease, which is activated under stress conditions such as high level of H2O2 and participates in the degradation of carbonylated proteins[10]. Biotin hydrazide probes are designed as DNPH alternatives which permit the application in immunoblotting, spectrophotometric and MS analysis to detect carbonylation of proteins, lipids and glycans[19]. Photostability and solubility of fluorescent probes allow efficiently tracking of a single protein, cytotoxicity of chemical probes is the major limitation in live cell labeling

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