Although cyclometalated IrIII complexes have emerged as promising photosensitizers for photodynamic therapy, some key drawbacks still hamper clinical translation, such as operability in the phototherapeutic window and reactive oxygen species (ROS) production efficiency and selectivity. In this work, a cyclometalated IrIII complex conjugated to a far‐red‐emitting coumarin, IrIII–COUPY, is reported with highly favourable properties for cancer phototherapy. IrIII–COUPY was efficiently taken up by HeLa cells and showed no dark cytotoxicity and impressive photocytotoxicity indexes after irradiation with green and blue light, even under hypoxia. Importantly, a clear correlation between cell death and intracellular generation of superoxide anion radicals after visible light irradiation was demonstrated. This strategy opens the door to novel fluorescent photodynamic therapy agents with promising applications in theragnosis.
Among the palette of previously described fluorescent organic molecules, coumarins are ideal candidates for developing cellular and molecular imaging tools due to their high cell permeability and minimal perturbation of living systems. However, blue-to-cyan fluorescence emission is usually difficult in in vivo applications due to the inherent toxicity and poor tissue penetration of short visible light wavelengths. Here, we introduce a new family of coumarin-based fluorophores, nicknamed COUPY, with promising photophysical properties, including emission in the far-red/near-infrared (NIR) region, large Stokes shifts, high photostability, and excellent brightness. COUPY fluorophores were efficiently synthesized in only three linear synthetic steps from commercially available precursors, with the N-alkylation of a pyridine moiety being the key step at the end of the synthetic route, as it allows for the tuning of the photophysical properties of the resulting dye. Owing to their low molecular weights, COUPY dyes show excellent cell permeability and accumulate selectively in nucleoli and/or mitochondria of HeLa cells, as their far-red/NIR fluorescence emission is easily detected at a concentration as low as 0.5 μM after an incubation of only 20 min. We anticipate that these coumarin scaffolds will open a way to the development of novel coumarin-based far-red to NIR emitting fluorophores with potential applications for organelle imaging and biomolecule labeling.
Replacement of electron-donating N,N-dialkyl groups with three- or four-membered cyclic amines (e.g., aziridine and azetidine, respectively) has been described as a promising approach to improve some of the drawbacks of conventional fluorophores, including low fluorescent quantum yields (Φ) in polar solvents. In this work, we have explored the influence of azetidinyl substitution on nonconventional coumarin-based COUPY dyes. Two azetidine-containing scaffolds were first synthesized in four linear synthetic steps and easily transformed into far-red/NIR-emitting fluorophores through N-alkylation of the pyridine moiety. Azetidine introduction in COUPY dyes resulted in enlarged Stokes' shifts with respect to the N,N-dialkylamino-containing parent dyes, but the Φ were not significantly modified in aqueous media, which is in contrast with previously reported observations in other fluorophores. However, azetidinyl substitution led to an unprecedented improvement in the photostability of COUPY dyes, and high cell permeability was retained since the fluorophores accumulated selectively in mitochondria and nucleoli of HeLa cells. Overall, our results provide valuable insights for the design and optimization of novel fluorophores operating in the far-red/NIR region, since we have demonstrated that three important parameters (Stokes' shifts, Φ, and photostability) cannot be always simultaneously addressed by simply replacing a N,N-dialkylamino group with azetidine, at least in nonconventional coumarin-based fluorophores.
Fluorophores based on organic molecules hold great potential for ligand-targeted imaging applications, particularly those operating in the optical window in biological tissues. In this work we have developed three straightforward solid-phase approaches based on amide-bond formation or Cu(I)-catalyzed azide-alkyne click (CuAAC) reaction for labelling octreotide peptide with far-red emitting coumarin-based COUPY dyes. First, the conjugatable versions of COUPY fluorophores incorporating the required functional groups (e.g., carboxylic acid, azide or alkyne) were synthesized and characterized. All of them were found fully compatible with Fmoc/tBu solid-phase peptide synthesis, which allowed the labeling of octreotide either through amide-bond formation or by CuAAC reaction. A near quantitative conversion was obtained after only 1 h of reaction at RT when using CuSO 4 and sodium ascorbate independently of the click chemistry approach used (azido-COUPY/alkynyl-peptide resin or alkynyl-COUPY/azido-peptide resin). COUPY-octreotide conjugates were found stable in cell culture medium as well as non-cytotoxic in HeLa cells, and their spectroscopic and photophysical properties were found similar to those of their parent coumarin dyes. Finally, the potential bioimaging applications of COUPY-octreotide conjugates were demonstrated by confocal microscopy through the visualization of living HeLa cells overexpressing somatostatin subtype-2 receptor.Owing to the potential of fluorophores based on organic molecules in ligand-targeted imaging applications, it is urgent to develop novel low molecular-weight fluorescent probes operating in the far-red and near-infrared (NIR) region, since only the use of non-toxic and high tissuepenetrating radiation will guarantee clinical translation in the next years. 3 Ideally, such fluorophores should be amenable to smart structural modifications to tune, on demand, photophysical and physicochemical properties, as well as to facilitate conjugation to a broad variety of targeting ligands (e.g., peptides, proteins, folic acid, monoclonal antibodies (mAb), etc.) by using efficient and chemoselective conjugation chemistries. Although many times forgotten, the biological properties of a targeting ligand should not be impaired by the fluorescent tag. This issue is particularly problematic in the case of short peptide sequences since uptake and subcellular localization may be strongly influenced by the fluorophore. 4 In the same way, some structural modifications of cyanine-based dyes have been reported to alter the mechanism of mAb when conjugated together, and non-specific hydrophobic interactions between Epidermal Growth Factor (EGF) receptor and the dye moiety in BODIPY-peptide conjugates were found to reduce peptide-receptor binding specificity. 5Hence, the choice of the fluorophore cannot be underestimated since constitutes a critical parameter in ligand-targeted imaging applications.Recently, we have developed a novel family of coumarin-based fluorophores, nicknamed COUPYs, with promising photoph...
This report shows how the net supramolecular chirality that emerged by spontaneous mirror-symmetry breaking (SMSB) at the mesoscale level can be transferred towards asymmetric solution chemistry.
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