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

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.

Retro Diels–Alder Fragmentation of Fulvene–Maleimide Bioconjugates for Mass Spectrometric Detection of Biomolecules

“…A number of different photocleavable groups have been used as the basis for designing mass tags, including 6,11-dihydrothiochromeno­[4,3- b ]­indole, o -nitrobenzyl, triphenylmethyl (trityl), Ru­(II) polypyridine complex, phenacyl, and coumarin-based linkers. ,− Our linkers were based on the o -nitrobenzyl group, owing to their straightforward synthesis, appropriate absorption maxima (≈350 nm), and easily modified structure (for the general photocleavage mechanism see SI, Scheme S1). Starting from 4′-hydroxy-3′-methoxyacetophenone 1 , nitrobenzyl linker 6 was synthesized in seven steps (Scheme ).…”

Current Probes for Imaging Carbonylation in Cellular Systems and Their Relevance to Progression of Diseases

“…From a different perspective, one of the critical approaches to monitor oxidative stress–induced carbonylation processes was to use a novel fluorescent probe to tag carbonylation in live cancer cells. 51 Dilek group published a relevant work that described the turnoff and fluorescence strategy ( Figure 13 ) to understand the relationship of oxidative stress–induced carbonylation levels with cancer signaling pathways in renal cancer lines ( Figure 14 ).…”

“…(D) Solutions of 2Hzin5NP, its aliphatic hydrazone in methanol under room light (left) and long wavelength fluorescent light (right). 51 …”

A Novel Fluorogenic Assay for the Detection of Nephrotoxin-Induced Oxidative Stress in Live Cells and Renal Tissue

“… 10 , 11 Our approach was also validated by Vemula et al using a commercially available probe, 7-(diethylamino)coumarin-3-carbohydrazide (DCCH) 9 and very recently by others with a synthetic probe. 12 Since crucial prerequisites for identifying mild phenotypes of chemical toxicity are high sensitivity of the probe and its applicability in the renal system, this work is aimed at achieving these objectives. Leveraging our experience in probe development for biomolecule carbonyls, we have developed a new sensor, 4-trifluoromethyl-7-hydrazinyl-2 H -chromen-2-one (TFCH), that is particularly suited for detecting mild signs of nephrotoxin-induced carbonylation in live cells and living tissues.…”

“…Carbonylation is commonly detected using alpha-effect amines as reporter molecules in biochemical assays. , The conventional assays often require lengthy tedious downstream processing and harsh chemical components that can alter subcellular structures, thereby misrepresenting spatial distribution of carbonylated biomolecules . Moreover, end-point analyses of fixed cells or cell lysates were the only options until we demonstrated the first live cell compatible assay using synthetic probes, coumarin hydrazine (7-hydrazinyl-4-methyl-2 H -chromen-2-one, CH; see Figure S1 for structures) and benzocoumarin hydrazine (7-hydrazinyl-4-methyl-2 H -benzo­[ h ]­chromen-2-one, BzCH). , Our approach was also validated by Vemula et al using a commercially available probe, 7-(diethylamino)­coumarin-3-carbohydrazide (DCCH) and very recently by others with a synthetic probe . Since crucial prerequisites for identifying mild phenotypes of chemical toxicity are high sensitivity of the probe and its applicability in the renal system, this work is aimed at achieving these objectives.…”