Fluorescent organic molecules are widely used in various applications, including biological imaging. However, many highly fluorescent molecules are large, potentially disrupting the bioactivity of the compounds they label. This necessitates the development of compact fluorophores that maintain bioactivity. The researchers highlight the need for small fluorescent labeling reagents that don't interfere with the biological activity of the target molecule. They explain that even when dealing with interactions between large biomolecules, the addition of a large fluorescent label can disrupt the binding affinity. Therefore, the development of a compact fluorescent labeling reagent is crucial for mechanistic studies of biologically active molecules, enabling visualization of their behavior within cells and tissues without compromising their inherent functionality. This paper focuses on the development of such a reagent based on the 1,3a,6a-triazapentalene (TAP) skeleton, aiming to achieve specific labeling of aliphatic thiols with minimal size impact on the target molecule. The researchers intend to demonstrate the utility of this new reagent by applying it to fluorescence imaging of a small molecule drug in living cells.

The introduction cites several works demonstrating the use of fluorescent organic molecules in biological imaging, sensing, and light-emitting devices. It mentions previous research on 1,3a,6a-triazapentalene (TAP) derivatives and their fluorescence properties, emphasizing that these derivatives could be further compacted to address the molecular size problem associated with commonly used fluorescent molecules. The review highlights the limitations of large fluorescent molecules in impacting the biological activity of the tagged molecule. The literature supports the need for smaller, less disruptive fluorescent probes.

The researchers synthesized a novel vinyl ketone analog of TAP, termed TAP-VK1 (2a), as a potential compact fluorescent labeling reagent. The synthesis started with 2-methoxycarbonyl-1,3a,6a-triazapentalene (4), proceeding through hydrolysis and condensation with N,O-dimethylhydroxylamine to yield Weinreb amide (5). The subsequent reaction with isobutenylmagnesium bromide yielded the desired TAP-VK1 (2a). The reactivity of TAP-VK1 with thiols was then investigated. Optimization studies revealed that the reaction of TAP-VK1 with various aliphatic thiols, in the presence of tetramethylguanidine (TMG) as a base, proceeded smoothly to afford 1,4-adducts in high yields. Significantly, other nucleophiles did not react. After thiol addition, a noteworthy blue-shift in the fluorescence maximum and a substantial increase in fluorescence intensity were observed. The reaction conditions were optimized for both organic and aqueous solvents. Reactions with various thiols were performed to assess the selectivity and efficiency of TAP-VK1, including those with additional functional groups like alcohols, amines, and triazoles. Reactions were also carried out with peptides, including glutathione and an octapeptide with multiple reactive functional groups. Finally, the labeling of human serum albumin (HSA) was also demonstrated. For cell imaging studies, a water-soluble peptide (7n) containing a membrane-permeable R8 peptide was labeled with TAP-VK1 (resulting in 3n), and the resulting compound was applied to A549 cells. The fluorescence imaging of Captopril, a small molecule ACE inhibitor drug, was performed in vascular endothelial cells (MBEC4). ACE inhibitory activity assays were conducted to compare the activity of Captopril and its TAP-VK1 derivative. Confocal microscopy was used to visualize the labeled compounds within cells. Computational details (TD-DFT calculations) are provided to explain the observed spectral changes. The authors also include detailed procedures for chemical synthesis, cell culture, confocal imaging, fluorescence measurements, computational methods, and ACE activity assays.

TAP-VK1 (2a) was successfully synthesized and shown to react smoothly and specifically with various aliphatic thiols in both organic and aqueous solutions. The reaction yielded 1,4-adducts with high yields and showed a remarkable increase in fluorescence intensity (up to 10 times) after thiol addition. A significant blue shift in the emission maximum was also observed upon thiol reaction. The labeling reaction proved highly thiol-specific, with no reaction observed with other nucleophiles such as amines or alcohols, even in the presence of multiple functional groups within the same molecule. The method successfully labeled various peptides, including glutathione, an octapeptide (7m), and HSA. The labeling of Captopril with TAP-VK1 retained the drug's potent ACE inhibitory activity, unlike labeling with fluorescein. Fluorescence imaging of TAP-VK1 labeled R8 peptide and Captopril in A549 and MBEC4 cells respectively successfully visualized the location of these molecules inside the cells. The fluorescently labeled Captopril showed accumulation in ACE, as evidenced by co-localization with anti-ACE antibody staining. TAP-VK1 demonstrated superior efficiency and photostability compared to coumarin, NBD, and ABD labels in terms of cellular imaging and retention of the Captopril’s biological function. The researchers concluded that the compactness of TAP-VK1 minimizes interference with biological activity, enabling successful imaging of small bioactive compounds.

The results address the research question by demonstrating the successful development and application of a compact, thiol-specific fluorescent labeling reagent. The significant increase in fluorescence intensity upon thiol reaction, coupled with the high specificity, makes TAP-VK1 a powerful tool for biological imaging. The retention of biological activity in labeled Captopril, a small molecule drug, validates the compact nature of the label. The successful imaging of Captopril inside cells showcases the utility of TAP-VK1 for studying the behavior of small biologically active molecules. The findings are relevant to the field of chemical biology, offering a solution to the limitations of existing fluorescent labeling techniques for small molecules. The superior performance of TAP-VK1 compared to other labels strengthens the impact of this study.

This research successfully developed TAP-VK1, a compact and thiol-specific fluorescent labeling reagent. Its high reactivity, selectivity, and fluorescence enhancement properties make it a valuable tool for biological imaging, particularly of small molecules. The successful imaging of Captopril without loss of activity demonstrates its potential for mechanistic studies. Future research may focus on modifying TAP-VK1 to enhance fluorescence intensity in aqueous environments and explore its application to a wider range of bioactive compounds and cellular processes.

The study primarily focused on aliphatic thiols; the reactivity with aromatic thiols needs further investigation. While the method showed excellent thiol specificity, the reaction conditions in water (sodium phosphate buffer) could be further optimized to increase the isolated yield. The intracellular fluorescence intensity could be improved by incorporating hydrophobic substituents into the TAP-VK1 structure to enhance fluorescence in aqueous environments. The study primarily employed two cell lines; validating the findings in other cell types and in vivo would strengthen the conclusion.