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熾炎槍炎烈風(fēng)金蟲 (正式寫手)
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Multicomponent Reactions for de Novo Synthesis of BODIPY Probes:
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Fluorescent probes are chemical entities of enormous importance in biomedical research and medical imaging. In the context of fluorescence live cell imaging, they enable realtime tracking of biomolecules, metabolites and cells under physiological conditions without altering regular cellular functions.1 The 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) scaffold has played a pivotal role in fluorescent probe development, and it is one of the most exploited fluorophores due to its excellent photophysical properties.2 Amine and carboxylic acid-derivatized BODIPY dyes are readily available, and have been conjugated to numerous biomolecules to develop fluorescent compounds to enable biological interrogation. This approach has rendered a wide variety of BODIPY-based cell imaging probes.3 Combinatorial strategies have recently expanded the chemical diversity of the BODIPY core. These strategies employ efficient and stepwise reactions (e.g., Knoevenagel condensation, ‘click’ chemistry) to implement structural diversification into a presynthesized BODIPY scaffold.4 Subsequent high-throughput screenings of the resulting libraries have significantly accelerated the discovery of new fluorescent probes.5 We envisioned that the use of multicomponent reactions (MCRs)6 for the synthesis of BODIPY fluorescent probes would lead to novel complexstructures that are difficult to prepare by conventional synthetic strategies. MCRs can increase the chemical diversity of BODIPY dyes with the formation of unusual C−C bonds and give BODIPY compounds with unexplored chemical connectivity and potentially new features as imaging probes. Our group and others have described MCRs to prepare complex fluorescent molecules based on 2,6-cyanodianilines,7 isoquinolines,8 naphthalimides,9 benzoazepines10 and imidazoles. 11 Balakirev and co-workers recently reported the combinatorial exploitation of three component Ugi MCRs in droplet arrays to successfully discover new fluorophores with drug-like properties.12 Whereas these examples proved the suitability of MCRs to generate de novo fluorescent structures, they employed scaffolds with inherent limitations as fluorophores (e.g., short emission wavelengths, low extinction coefficients, poor quantum yields, compromised cell permeability). The adaptation of MCRs to the highly fluorescent and cell permeable BODIPY scaffold provides a practical platform to develop novel compounds with unexpected features as cell imaging fluorescent probes. Since the most versatile MCRs are based on isonitrile chemistry,13 we prepared an isonitrile- BODIPY scaffold (3) compatible with several MCRs (Scheme 1). Compound 3 was prepared in a two-step reaction from the BODIPY aniline 1, obtained by reduction of the corresponding nitro compound.14 The BODIPY aniline 1 was formylated with HCO2Et to render the formamide 2, which was subsequently dehydrated under standard conditions with POCl3 to afford the isonitrile 3. Notably, the isonitrile functional group did not affect the fluorescent properties of the BODIPY core (9) (Figure S1 and Table S1 in Supporting Information (SI)). To the best of our knowledge, this is the first report of an isonitrilefunctionalized BODIPY dye and its subsequent derivatization using MCRs. We employedas the starting material for a number of isonitrile-based MCRs, namely Passerini,15 Bienaymé−Blackburn− Groebcke,16 and three variants of the Ugi-MCR17 (Scheme 1). We performed a Bienaymé−Blackburn−Groebcke MCR with 3, α-aminopyridine, and 4-chlorobenzaldehyde toobtain the azaindole 4. A conventional Passerini reaction of 3 with propanal and 2,6-dimethyl-4-nitrobenzoic acid rendered the adduct 5. A four-component Ugi MCR with isobutylaldehyde, benzylamine, and benzoic acid afforded compound 6, whereas the β-amino acid variation led to the β-lactam 8. The adduct 7 was obtained with a variant of the Ugi MCR using formaldehyde and diethylamine. Notably, all adducts (4−8) were isolated in good yields (see details and characterization data in SI) and retained the characteristic fluorescent properties of the BODIPY core (Figure S1 and Table S1 in SI). The synthesis of this collection of BODIPY adducts confirms that the reactivity of the isonitrile group in different MCRs is not hampered by the BODIPY structure. Altogether, the results validate isonitrile-based MCRs as a synthetic platform for the diversification of the BODIPY scaffold toward fluorescent conjugates that might be difficult to prepare by conventional strategies.We assessed the cell permeability of compounds 3−8 by imaging their localization in live A549 cells together with different intracellular trackers (Figures S2 and S3 in SI) and observed that all adducts readily entered cells at concentrations in the nanomolar range. While most adducts stained the cytoplasm and some lysosomes, compound 7 exhibited a distinctive, vesicle-like staining pattern (Figure 1). We examined the subcellular localization of 7 in different cell lines by colocalization with LysoTracker Red, a fluorescent dye that labels acidic organelles (Figures S4 and S5 in SI). The similar staining patterns of 7 and Lysotracker Red indicated that 7 is an acidotropic fluorescent molecule with bright fluorescence emission in subcellular acidic environments. Compound 7 has a pKa of 5.76 ± 0.07 (Figure S6 in SI) andhigher sensitivity to pH than LysoTracker Red (Figure S7 in SI). We envisaged that these remarkable properties of 7 as a cell permeable fluorescent probe for acidic microenvironments may be applied to imaging phagosomal acidification in macrophages. Macrophages are immune cells with key roles in inflammation and tissue homeostasis. Macrophages ingest pathogens and particles by phagocytosis. During the course of phagocytosis the maturation and fusion of endosomes leads to a progressive phagosomal acidification.18 Most currently used probes for activated macrophages target the recognition of enzymes (e.g., cathepsins) or cell surface receptors (e.g., folate, integrins).19 Bogyo and co-workers recently described fluorescent probes tomonitor legumain activity in the acidic organelles of activated macrophages.20We observed that compound 7 was nontoxic to macrophages at 500 nM, even at long incubation times (Figure S10 in SI). We also studied whether the treatment with compound 7 affected the secretion of TNF-α and IL-6, two major cytokines released by macrophages. As shown in Figure 3, there were no significant differences in the levels of TNF-α and IL-6 secreted by nontreated and 7-treated macrophages before or after stimulation with liposaccharide S (LPS). These results validate 7 as a fluorescent probe to image phagocytic macrophages without impairing their normal function. On account of these observations, compound 7 was named as PhagoGreen. |
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