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Bioorthogonal late-stage diversification of amino acids and peptides bears enormous potential for drug discovery and molecular imaging. Despite major accomplishments, these strategies largely rely on traditional, lengthy prefunctionalization methods, heavily involving precious transition-metal catalysis. Herein, we report on a resource-economical manganese(I)-catalyzed C–H fluorescent labeling of structurally complex peptides ensured by direct alkynylation and alkenylation manifolds. This modular strategy sets the stage for unraveling structure-activity relationships between structurally discrete fluorophores towards the rational design of BODIPY fluorogenic probes for real-time analysis of immune cell function. Bioorthogonal diversification of peptides is generally dependent on impractical prefunctionalization methods. Here, the authors develop a manganese(I)-catalyzed C–H fluorescent labeling with BODIPY probes, which enables the development of activatable fluorophores to image cell function.

Fluorescent antimicrobial peptides are promising structures for in situ , real-time imaging of fungal infection. Here we report a fluorogenic probe to image Aspergillus fumigatus directly in human pulmonary tissue. We have developed a fluorogenic Trp-BODIPY amino acid with a spacer-free C-C linkage between Trp and a BODIPY fluorogen, which shows remarkable fluorescence enhancement in hydrophobic microenvironments. The incorporation of our fluorogenic amino acid in short antimicrobial peptides does not impair their selectivity for fungal cells, and enables rapid and direct fungal imaging without any washing steps. We have optimized the stability of our probes in human samples to perform multi-photon imaging of A. fumigatus in ex vivo human tissue. The incorporation of our unique BODIPY fluorogen in biologically relevant peptides will accelerate the development of novel imaging probes with high sensitivity and specificity. Functionalizing antimicrobial peptides with fluorescent groups is a useful strategy for imaging infection, but the tag can alter the performance of the probe. Here, the authors report a spacer-free method to directly functionalise an amino acid with a fluorogenic group and prepare peptide-based imaging agents for fungal infection.

Immunotherapy promotes the attack of cancer cells by the immune system; however, it is difficult to detect early responses before changes in tumor size occur. Here, we report the rational design of a fluorogenic peptide able to detect picomolar concentrations of active granzyme B as a biomarker of immune-mediated anticancer action. Through a series of chemical iterations and molecular dynamics simulations, we synthesize a library of FRET peptides and identify probe H5 with an optimal fit into granzyme B. We demonstrate that probe H5 enables the real-time detection of T cell-mediated anticancer activity in mouse tumors and in tumors from lung cancer patients. Furthermore, we show image-based phenotypic screens, which reveal that the AKT kinase inhibitor AZD5363 shows immune-mediated anticancer activity. The reactivity of probe H5 may enable the monitoring of early responses to anticancer treatments using tissue biopsies. Granzyme B is found in activated T cells and can be used as a marker of T cell activation. Here, the authors generate a fluorescent probe that can detect Granzyme B levels in tumours, and has the potential to be used as a biomarker of response to immunotherapy.

Programmed cell death or apoptosis is a central biological process that is dysregulated in many diseases, including inflammatory conditions and cancer. The detection and quantification of apoptotic cells in vivo is hampered by the need for fixatives or washing steps for non-fluorogenic reagents, and by the low levels of free calcium in diseased tissues that restrict the use of annexins. In this manuscript, we report the rational design of a highly stable fluorogenic peptide (termed Apo-15 ) that selectively stains apoptotic cells in vitro and in vivo in a calcium-independent manner and under wash-free conditions. Furthermore, using a combination of chemical and biophysical methods, we identify phosphatidylserine as a molecular target of Apo-15 . We demonstrate that Apo-15 can be used for the quantification and imaging of drug-induced apoptosis in preclinical mouse models, thus creating opportunities for assessing the in vivo efficacy of anti-inflammatory and anti-cancer therapeutics. Programmed cell death or apoptosis is an essential biological process that is impaired in some diseases and can be used to assess the effectiveness of drugs. Here the authors design Apo-15 as a fluorogenic peptide for the detection and real-time imaging of apoptotic cells.

Fluorescent peptides with excellent target specificity are useful imaging probes. This protocol describes the synthesis of a tryptophan-based fluorogenic amino acid (Fmoc-Trp(C 2 -BODIPY)-OH) and its incorporation into peptides for live-cell imaging. Fluorescent peptides are valuable tools for live-cell imaging because of the high specificity of peptide sequences for their biomolecular targets. When preparing fluorescent versions of peptides, labels must be introduced at appropriate positions in the sequences to provide suitable reporters while avoiding any impairment of the molecular recognition properties of the peptides. This protocol describes the preparation of the tryptophan (Trp)-based fluorogenic amino acid Fmoc-Trp(C 2 -BODIPY)-OH and its incorporation into peptides for live-cell fluorescence imaging—an approach that is applicable to most peptide sequences. Fmoc-Trp(C 2 -BODIPY)-OH contains a BODIPY (4,4-difluoro-4-bora-3a,4a-diaza- s -indacene) fluorogenic core, which works as an environmentally sensitive fluorophore, showing high fluorescence in lipophilic conditions. It is attached to Trp via a spacer-free C–C linkage, resulting in a labeled amino acid that can mimic the molecular interactions of Trp, enabling wash-free imaging. This protocol covers the chemical synthesis of the fluorogenic amino acid Fmoc-Trp(C 2 -BODIPY)-OH (3–4 d), the preparation of the labeled antimicrobial peptide BODIPY-cPAF26 by solid-phase synthesis (6–7 d) and its spectral and biological characterization as a live-cell imaging probe for different fungal pathogens. As an example, we include a procedure for using BODIPY-cPAF26 for wash-free imaging of fungal pathogens, including real-time visualization of Aspergillus fumigatus (5 d for culturing, 1–2 d for imaging).

The concept that dimeric protein complexes in synapses can sequentially replace their subunits has been a cornerstone of Francis Crick’s 1984 hypothesis, explaining how long-term memories could be maintained in the face of short protein lifetimes. However, it is unknown whether the subunits of protein complexes that mediate memory are sequentially replaced in the brain and if this process is linked to protein lifetime. We address these issues by focusing on supercomplexes assembled by the abundant postsynaptic scaffolding protein PSD95, which plays a crucial role in memory. We used single-molecule detection, super-resolution microscopy and MINFLUX to probe the molecular composition of PSD95 supercomplexes in mice carrying genetically encoded HaloTags, eGFP, and mEoS2. We found a population of PSD95-containing supercomplexes comprised of two copies of PSD95, with a dominant 12.7 nm separation. Time-stamping of PSD95 subunits in vivo revealed that each PSD95 subunit was sequentially replaced over days and weeks. Comparison of brain regions showed subunit replacement was slowest in the cortex, where PSD95 protein lifetime is longest. Our findings reveal that protein supercomplexes within the postsynaptic density can be maintained by gradual replacement of individual subunits providing a mechanism for stable maintenance of their organization. Moreover, we extend Crick’s model by suggesting that synapses with slow subunit replacement of protein supercomplexes and long-protein lifetimes are specialized for long-term memory storage and that these synapses are highly enriched in superficial layers of the cortex where long-term memories are stored.

Natural peptides show high degrees of specificity in their biological action. However, their therapeutical profile is severely limited by their conformational freedom and metabolic instability. Stapled peptides constitute a solution to these problems and access to these structures lies on a limited number of reactions involving the use of non-natural amino acids. Here, we describe a synthetic strategy for the preparation of unique constrained peptides featuring a covalent bond between tryptophan and phenylalanine or tyrosine residues. The preparation of such peptides is achieved in solution and on solid phase directly from the corresponding sequences having an iodo-aryl amino acid through an intramolecular palladium-catalysed C–H activation process. Moreover, complex topologies arise from the internal stapling of cyclopeptides and double intramolecular arylations within a linear peptide. Finally, as a proof of principle, we report the application to this new stapling method to relevant biologically active compounds. Macrocyclic, constrained peptides show promise in therapeutic applications due to the stable and defined conformations that can be produced. Here, the authors report a method to form macrocyclic peptides through C–H activation on tryptophan and coupling with iodo-substituted aryl amino acids

The essential functions that cytokine/immune cell interactions play in tissue homeostasis and during disease have prompted the molecular design of targeted fluorophores to monitor their activity in real time. Whereas activatable probes for imaging immune-related enzymes are common, many immunological functions are mediated by binding events between cytokines and their cognate receptors that are hard to monitor by live-cell imaging. A prime example is interleukin-33 (IL-33), a key cytokine in innate and adaptive immunity, whose interaction with the ST2 cell-surface receptor results in downstream signaling and activation of NF-κB and AP-1 pathways. In the present work, we have designed a chemical platform to site-specifically introduce OFF-to-ON BODIPY fluorophores into full cytokine proteins and generate the first nativelike fluorescent analogues of IL-33. Among different incorporation strategies, chemical aminoacylation followed by bioorthogonal derivatization led to the best labeling results. Importantly, the BODIPY-labeled IL-33 derivativesunlike IL-33-GFP constructsexhibited ST2-specific binding and downstream bioactivity profiles comparable to those of the wild-type interleukin. Real-time fluorescence microscopy assays under no wash conditions confirmed the internalization of IL-33 through ST2 receptors and its intracellular trafficking through the endosomal pathway. We envision that the modularity and versatility of our BODIPY labeling platform will facilitate the synthesis of minimally tagged fluorogenic cytokines as the next generation of imaging reagents for real-time visualization of signaling events in live immune cells.

We previously showed that fluorizoline, a fluorinated thiazoline compound, binds to both subunits of the mitochondrial prohibitin (PHB) complex, PHB1 and PHB2, being the expression of these proteins required for fluorizoline-induced apoptosis in mouse embryonic fibroblasts. To investigate the conservation of this apoptotic mechanism, we studied the effect of PHB downregulation on fluorizoline activity on two human cell lines, HEK293T and U2OS. Then, we asked whether PHBs mediate the effect of fluorizoline in a multicellular organism. Interestingly, reduced levels of PHBs in the human cells impaired the induction of apoptosis by fluorizoline. We observed that fluorizoline has a detrimental dose-dependent effect on the development and survival of the nematode model Caenorhabditis elegans. Besides, such effects of fluorizoline treatment in living nematodes were absent in PHB mutants. Finally, we further explored the apoptotic pathway triggered by fluorizoline in human cell lines. We found that the BH3-only proteins NOXA, BIM and PUMA participate in fluorizoline-induced apoptosis and that the induction of NOXA and PUMA is dependent on PHB expression.

Immunosuppressants are clinically approved drugs to treat the potential rejection of transplanted organs and require frequent monitoring due to their narrow therapeutic window. Immunophilins are small proteins that bind immunosuppressants with high affinity, yet there are no examples of fluorogenic immunophilins and their potential application as optical biosensors for immunosuppressive drugs in clinical biosamples. In the present work, we designed novel diazonium BODIPY salts for the site-specific labeling of tyrosine residues in peptides via solid-phase synthesis as well as for late-stage functionalization of whole recombinant proteins. After the optimization of a straightforward one-step labeling procedure for immunophilins PPIA and FKBP12, we demonstrated the application of a fluorogenic analogue of FKBP12 for the selective detection of the immunosuppressant drug tacrolimus, including experiments in urine samples from patients with functioning renal transplants. This chemical methodology opens new avenues to rationally design wash-free immunophilin-based biosensors for rapid therapeutic drug monitoring.