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Tumors growing in metabolically challenged environments, such as glioblastoma in the brain, are particularly reliant on crosstalk with their tumor microenvironment (TME) to satisfy their high energetic needs. To study the intricacies of this metabolic interplay, we interrogated the heterogeneity of the glioblastoma TME using single-cell and multi-omics analyses and identified metabolically rewired tumor-associated macrophage (TAM) subpopulations with pro-tumorigenic properties. These TAM subsets, termed lipid-laden macrophages (LLMs) to reflect their cholesterol accumulation, are epigenetically rewired, display immunosuppressive features, and are enriched in the aggressive mesenchymal glioblastoma subtype. Engulfment of cholesterol-rich myelin debris endows subsets of TAMs to acquire an LLM phenotype. Subsequently, LLMs directly transfer myelin-derived lipids to cancer cells in an LXR/Abca1-dependent manner, thereby fueling the heightened metabolic demands of mesenchymal glioblastoma. Our work provides an in-depth understanding of the immune-metabolic interplay during glioblastoma progression, thereby laying a framework to unveil targetable metabolic vulnerabilities in glioblastoma., Competing Interests: Declaration of interests L.A., D.J.K., A.K., and J.E. are inventors on European patents P091147NL and P099572EP describing relevant claims and their therapeutic potential., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cells rely heavily on the uptake of exogenous nutrients for survival, growth, and differentiation. Yet quantifying the uptake of small molecule nutrients at the single cell level is difficult. Here we present a new approach to studying the nutrient uptake in live single cells using Inverse Electron-Demand Diels Alder (IEDDA) chemistry. We have modified carboxyfluorescein-diacetate-succinimidyl esters (CFSE)-a quenched fluorophore that can covalently react with proteins and is only turned on in the cytosol of a cell following esterase activity-with a tetrazine. This tetrazine serves as a second quencher for the pendant fluorophore. Upon reaction with nutrients modified with an electron-rich or strained dienophile in an IEDDA reaction, this quenching group is destroyed, thereby enabling the probe to fluoresce. This has allowed us to monitor the uptake of a variety of dienophile-containing nutrients in live primary immune cell populations using flow cytometry and live-cell microscopy., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Affinity-based probes are valuable tools for detecting binding interactions between small molecules and proteins in complex biological environments. Metalloproteins are a class of therapeutically significant biomolecules which bind metal ions as part of key structural or catalytic domains and are compelling targets for study. However, there is currently a limited range of chemical tools suitable for profiling the metalloproteome. Here, we describe the preparation and application of a novel, photoactivatable affinity-based probe for detection of a subset of previously challenging to engage metalloproteins. The probe, bearing an 8-mercaptoquinoline metal chelator, was anticipated to engage several zinc metalloproteins, including the 26S-proteasome subunit Rpn11. Upon translation of the labelling experiment to mammalian cell lysate and live cell experiments, proteomic analysis revealed that several metalloproteins were competitively enriched. The diazirine probe SMK-24 was found to effectively enrich multiple components of the minichromosome maintenance complex, a zinc metalloprotein assembly with helicase activity essential to DNA replication. Cell cycle analysis experiments revealed that HEK293 cells treated with SMK-24 experienced stalling in G0/G1 phase, consistent with inactivation of the DNA helicase complex. This work represents an important contribution to the library of cell-permeable chemical tools for studying a collection of metalloproteins for which no previous probe existed., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The allylic trans-cyclooctene (TCO) functionality facilitates powerful control over the spatiotemporal activity of bio-active molecules, enabling precision targeting of druglike and imaging modalities. However, the introduction of this function onto molecules remains chemically challenging, particularly for peptides. Modification with TCOs of this important class of biomolecules remains a challenge, primarily due to the sensitivity of the TCO group to the strong acids typically used in global deprotection during solid phase peptide synthesis. Here, we present a novel synthetic approach to site-selectively introduce TCO-groups in peptides. Our approach utilizes azide groups to mask amine functions, enabling selective introduction of the TCO on a single lysine residue. Staudinger reduction of the azides back to the corresponding amines proceeds without disturbing the sensitive TCO. We show that using our method, we can produce TCO-inactivated antigenic peptides of previously unseen complexity., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Antibody-mediated delivery of immunogenic viral CD8 + T-cell epitopes to redirect virus-specific T cells toward cancer cells is a promising new therapeutic avenue to increase the immunogenicity of tumors. Multiple strategies for viral epitope delivery have been shown to be effective. So far, most of these have relied on a free C-terminus of the immunogenic epitope for extracellular delivery. Here, we demonstrate that antibody-epitope conjugates (AECs) with genetically fused epitopes to the N-terminus of the antibody can also sensitize tumors for attack by virus-specific CD8 + T cells. AECs carrying epitopes genetically fused at the N-terminus of the light chains of cetuximab and trastuzumab demonstrate an even more efficient delivery of the T-cell epitopes compared to AECs with the epitope fused to the C-terminus of the heavy chain. We demonstrate that this increased efficiency is not caused by the shift in location of the cleavage site from the N - to the C-terminus, but by its increased proximity to the cell surface. We hypothesize that this facilitates more efficient epitope delivery. These findings not only provide additional insights into the mechanism of action of AECs but also broaden the possibilities for genetically fused AECs as an avenue for the redirection of multiple virus-specific T cells toward tumors.

Antibody-mediated delivery of immunogenic epitopes to redirect virus-specific CD8 + T-cells towards cancer cells is an emerging and promising new therapeutic strategy. These so-called antibody-epitope conjugates (AECs) rely on the proteolytic release of the epitopes close to the tumor surface for presentation by HLA class I molecules to eventually redirect and activate virus-specific CD8 + T-cells towards tumor cells. We fused the immunogenic EBV-BRLF1 epitope preceded by a protease cleavage site to the C-terminus of the heavy and/or light chains of cetuximab and trastuzumab. We evaluated these AECs and found that, even though all AECs were able to redirect the EBV-specific T-cells, AECs with an epitope fused to the C-terminus of the heavy chain resulted in higher levels of T-cell activation compared to AECs with the same epitope fused to the light chain of an antibody. We observed that all AECs were depending on the presence of the antibody target, that the level of T-cell activation correlated with expression levels of the antibody target, and that our AECs could efficiently deliver the BRLF1 epitope to cancer cell lines from different origins (breast, ovarian, lung, and cervical cancer and a multiple myeloma). Moreover, in vivo, the AECs efficiently reduced tumor burden and increased the overall survival, which was prolonged even further in combination with immune checkpoint blockade. We demonstrate the potential of these genetically fused AECs to redirect the potent EBV-specific T-cells towards cancer in vitro and in vivo., (© 2023. The Author(s).)

System-level analysis of single-cell data is rapidly transforming the field of immunometabolism. Given the competitive demand for nutrients in immune microenvironments, there is a need to understand how and when immune cells access these nutrients. Here, we describe a new approach for single-cell analysis of nutrient uptake where we use in-cell biorthogonal labeling of a functionalized amino acid after transport into the cell. In this manner, the bona fide active uptake of glutamine via SLC1A5/ASCT2 could be quantified. We used this assay to interrogate the transport capacity of complex immune subpopulations, both in vitro and in vivo. Taken together, our findings provide an easy sensitive single-cell assay to assess which cells support their function via SLC1A5-mediated uptake. This is a significant addition to the single-cell metabolic toolbox required to decode the metabolic landscape of complex immune microenvironments., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Bioorthogonal deprotections are readily used to control biological function in a cell-specific manner. To further improve the spatial resolution of these reactions, we here present a lysosome-targeted tetrazine for an organelle-specific deprotection reaction. We show that trans -cyclooctene deprotection with this reagent can be used to control the biological activity of ligands for invariant natural killer T cells in the lysosome to shed light on the processing pathway in antigen presenting cells. We then use the lysosome-targeted tetrazine to show that long peptide antigens used for CD8 + T cell activation do not pass through this organelle, suggesting a role for the earlier endosomal compartments for their processing.

Therapeutic antibody-epitope conjugates (AECs) are promising new modalities to deliver immunogenic epitopes and redirect virus-specific T-cell activity to cancer cells. Nevertheless, many aspects of these antibody conjugates require optimization to increase their efficacy. Here we evaluated different strategies to conjugate an EBV epitope (YVL/A2) preceded by a protease cleavage site to the antibodies cetuximab and trastuzumab. Three approaches were taken: chemical conjugation (i.e. a thiol-maleimide reaction) to reduced cysteine side chains, heavy chain C-terminal enzymatic conjugation using sortase A, and genetic fusions, to the heavy chain (HC) C-terminus. All three conjugates were capable of T-cell activation and target-cell killing via proteolytic release of the EBV epitope and expression of the antibody target was a requirement for T-cell activation. Moreover, AECs generated with a second immunogenic epitope derived from CMV (NLV/A2) were able to deliver and redirect CMV specific T-cells, in which the amino sequence of the attached peptide appeared to influence the efficiency of epitope delivery. Therefore, screening of multiple protease cleavage sites and epitopes attached to the antibody is necessary. Taken together, our data demonstrated that multiple AECs could sensitize cancer cells to virus-specific T cells., Competing Interests: BB, RGH, PP, and JS are (former) employees of Genmab and have ownership interests (including stock, patents, warrants etc.). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that this study received funding from Genmab. Genmab had the following involvement in the study: study design, materials supply, data collection and analysis, reviewing and revision of the manuscript, and decision to publish., (Copyright © 2023 van der Wulp, Gram, Bleijlevens, Hagedoorn, Araman, Kim, Drijfhout, Parren, Hibbert, Hoeben, van Kasteren, Schuurman, Ressing and Heemskerk.)

Uptake and processing of antigens by antigen presenting cells (APCs) is a key step in the initiation of the adaptive immune response. Studying these processes is complex as the identification of low abundant exogenous antigens from complex cell extracts is difficult. Mass-spectrometry based proteomics - the ideal analysis tool in this case - requires methods to retrieve such molecules with high efficiency and low background. Here, we present a method for the selective and sensitive enrichment of antigenic peptides from APCs using click-antigens; antigenic proteins expressed with azidohomoalanine (Aha) in place of methionine residues. We here describe the capture of such antigens using a new covalent method namely, alkynyl functionalized PEG-based Rink amide resin, that enables capture of click-antigens via copper-catalyzed azide-alkyne [2 + 3] cycloaddition (CuAAC). The covalent nature of the thus formed linkage allows stringent washing to remove a-specific background material, prior to retrieval peptides by acid-mediated release. We successfully identified peptides from a tryptic digest of the full APC proteome containing femtomole amounts of Aha-labelled antigen, making this a promising approach for clean and selective enrichment of rare bioorthogonally modified peptides from complex mixtures., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

The peptide spanning residues 35 to 55 of the protein myelin oligodendrocyte glycoprotein (MOG) has been studied extensively in its role as a key autoantigen in the neuroinflammatory autoimmune disease multiple sclerosis. Rodents and nonhuman primate species immunized with this peptide develop a neuroinflammatory condition called experimental autoimmune encephalomyelitis, often used as a model for multiple sclerosis. Over the last decade, the role of citrullination of this antigen in the disease onset and progression has come under increased scrutiny. We recently reported on the ability of these citrullinated MOG35-55 peptides to aggregate in an amyloid-like fashion, suggesting a new potential pathogenic mechanism underlying this disease. The immunodominant region of MOG is highly conserved between species, with the only difference between the murine and human protein, a polymorphism on position 42, which is serine in mice and proline for humans. Here, we show that the biophysical and biochemical behavior we previously observed for citrullinated murine MOG35-55 is fundamentally different for human and mouse MOG35-55. The citrullinated human peptides do not show amyloid-like behavior under the conditions where the murine peptides do. Moreover, we tested the ability of these peptides to stimulate lymphocytes derived from MOG immunized marmoset monkeys. While the citrullinated murine peptides did not produce a proliferative response, one of the citrullinated human peptides did. We postulate that this unexpected difference is caused by disparate antigen processing. Taken together, our results suggest that further study on the role of citrullination in MOG-induced experimental autoimmune encephalomyelitis is necessary., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Dextran-based hydrogels are promising therapeutic materials for drug delivery, tissue regeneration devices, and cell therapy vectors, due to their high biocompatibility, along with their ability to protect and release active therapeutic agents. This report describes the synthesis, characterization, and application of a new dynamic covalent dextran hydrogel as an injectable depot for peptide vaccines. Dynamic covalent crosslinks based on double Michael addition of thiols to alkynones impart the dextran hydrogel with shear-thinning and self-healing capabilities, enabling hydrogel injection. These injectable, non-toxic hydrogels show adjuvant potential and have predictable sub-millimolar loading and release of the peptide antigen SIINFEKL, which after its release is able to activate T-cells, demonstrating that the hydrogels deliver peptides without modifying their immunogenicity. This work demonstrates the potential of dynamic covalent dextran hydrogels as a sustained-release material for the delivery of peptide vaccines.

In the field of lipid research, bioorthogonal chemistry has made the study of lipid uptake and processing in living systems possible, whilst minimising biological properties arising from detectable pendant groups. To allow the study of unsaturated free fatty acids in live cells, we here report the use of sterculic acid, a 1,2-cyclopropene-containing oleic acid analogue, as a bioorthogonal probe. We show that this lipid can be readily taken up by dendritic cells without toxic side effects, and that it can subsequently be visualised using an inverse electron-demand Diels-Alder reaction with quenched tetrazine-fluorophore conjugates. In addition, the lipid can be used to identify changes in protein oleoylation after immune cell activation. Finally, this reaction can be integrated into a multiplexed bioorthogonal reaction workflow by combining it with two sequential copper-catalysed Huisgen ligation reactions. This allows for the study of multiple biomolecules in the cell simultaneously by multimodal confocal imaging., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Bioorthogonal chemistry combines well with activity-based protein profiling, as it allows for the introduction of detection tags without significantly influencing the physiochemical and biological functions of the probe. In this work, we introduced methyltetrazinylalanine (MeTz-Ala), a close mimic of phenylalanine, into a dipeptide fluoromethylketone cysteine protease inhibitor. Following covalent and irreversible inhibition, the tetrazine allows vizualisation of the captured cathepsin activity by means of inverse electron demand Diels Alder ligation in cell lysates and live cells, demonstrating that tetrazines can be used as live cell compatible, minimal bioorthogonal tags in activity-based protein profiling., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Protein glycosylation is a key post-translational modification important to many facets of biology. Glycosylation can have critical effects on protein conformation, uptake and intracellular routing. In immunology, glycosylation of antigens has been shown to play a role in self/non-self distinction and the effective uptake of antigens. Improperly glycosylated proteins and peptide fragments, for instance those produced by cancerous cells, are also prime candidates for vaccine design. To study these processes, access to peptides bearing well-defined glycans is of critical importance. In this review, the key approaches towards synthetic, well-defined glycopeptides, are described, with a focus on peptides useful for and used in immunological studies. Special attention is given to the glycoconjugation approaches that have been developed in recent years, as these enable rapid synthesis of various (unnatural) glycopeptides, enabling powerful carbohydrate structure/activity studies. These techniques, combined with more traditional total synthesis and chemoenzymatic methods for the production of glycopeptides, should help unravel some of the complexities of glycobiology in the near future.

Most lectins bind carbohydrate ligands with relatively low affinity, making the identification of optimal ligands challenging. Here we introduce a point accumulation in nanoscale topography (PAINT) super-resolution microscopy method to capture weak glycan-lectin interactions at the single-molecule level in living cells (Glyco-PAINT). Glyco-PAINT exploits weak and reversible sugar binding to directly achieve single-molecule detection and quantification in cells and is used to establish the relative k on and k off rates of a synthesized library of carbohydrate-based probes, as well as the diffusion coefficient of the receptor-sugar complex. Uptake of ligands correlates with their binding affinity and residence time to establish structure-function relations for various synthetic glycans. We reveal how sugar multivalency and presentation geometry can be optimized for binding and internalization. Overall, Glyco-PAINT represents a powerful approach to study weak glycan-lectin interactions on the surface of living cells, one that can be potentially extended to a variety of lectin-sugar interactions., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The functional activity and differentiation potential of cells are determined by their interactions with surrounding cells. Approaches that allow unbiased characterization of cell states while at the same time providing spatial information are of major value to assess this environmental influence. However, most current techniques are hampered by a tradeoff between spatial resolution and cell profiling depth. Here, we develop a photocage-based technology that allows isolation and in-depth analysis of live cells from regions of interest in complex ex vivo systems, including primary human tissues. The use of a highly sensitive 4-nitrophenyl(benzofuran) cage coupled to a set of nanobodies allows high-resolution photo-uncaging of different cell types in areas of interest. Single-cell RNA-sequencing of spatially defined CD8 + T cells is used to exemplify the feasibility of identifying location-dependent cell states. The technology described here provides a valuable tool for the analysis of spatially defined cells in diverse biological systems, including clinical samples., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Bacterial infections are still one of the leading causes of death worldwide; despite the near-ubiquitous availability of antibiotics. With antibiotic resistance on the rise, there is an urgent need for novel classes of antibiotic drugs. One particularly troublesome class of bacteria are those that have evolved highly efficacious mechanisms for surviving inside the host. These contribute to their virulence by immune evasion, and make them harder to treat with antibiotics due to their residence inside intracellular membrane-limited compartments. This has sparked the development of new chemical reporter molecules and bioorthogonal probes that can be metabolically incorporated into bacteria to provide insights into their activity status. In this review, we provide an overview of several classes of metabolic labeling probes capable of targeting either the peptidoglycan cell wall, the mycomembrane of mycobacteria and corynebacteria, or specific bacterial proteins. In addition, we highlight several important insights that have been made using these metabolic labeling probes.

Proteolysis is fundamental to many biological processes. In the immune system, it underpins the activation of the adaptive immune response: degradation of antigenic material into short peptides and presentation thereof on major histocompatibility complexes, leads to activation of T-cells. This initiates the adaptive immune response against many pathogens. Studying proteolysis is difficult, as the oft-used polypeptide reporters are susceptible to proteolytic sequestration themselves. Here we present a new approach that allows the imaging of antigen proteolysis throughout the processing pathway in an unbiased manner. By incorporating bioorthogonal functionalities into the protein in place of methionines, antigens can be followed during degradation, whilst leaving reactive sidechains open to templated and non-templated post-translational modifications, such as citrullination and carbamylation. Using this approach, we followed and imaged the post-uptake fate of the commonly used antigen ovalbumin, as well as the post-translationally citrullinated and/or carbamylated auto-antigen vinculin in rheumatoid arthritis, revealing differences in antigen processing and presentation., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Cathepsin S is a lysosomal cysteine protease highly expressed in immune cells such as dendritic cells, B cells and macrophages. Its functions include extracellular matrix breakdown and cleavage of cell adhesion molecules to facilitate immune cell motility, as well as cleavage of the invariant chain during maturation of major histocompatibility complex II. The identification of these diverse specific functions has brought the challenge of delineating cathepsin S activity with great spatial precision, relative to related enzymes and substrates. Here, the development of a potent and highly selective two-step activity-based probe for cathepsin S and the application in multicolor bio-orthogonal correlative light-electron microscopy is presented. LHVS, which has been reported as a selective inhibitor of cathepsin S with nanomolar potency, formed the basis for our probe design. However, in competitive activity-based protein profiling experiments LHVS showed significant cross-reactivity toward Cat L. Introduction of an azide group in the P2 position expanded the selectivity window for cathepsin S, but rendered the probe undetectable, as demonstrated in bio-orthogonal competitive activity-based protein profiling. Incorporation of an additional azide handle for click chemistry on the solvent-exposed P1 position allowed for selective labeling of cathepsin S. This highlights the influence of click handle positioning on probe efficacy. This probe was utilized in multicolor bio-orthogonal confocal and correlative light-electron microscopy to investigate the localization of cathepsin S activity at an ultrastructural level in bone marrow-derived dendritic cells. The tools developed in this study will aid the characterization of the variety of functions of cathepsin S throughout biology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 van Dalen, Bakkum, van Leeuwen, Groenewold, Deu, Koster, van Kasteren and Verdoes.)

The protein myelin oligodendrocyte glycoprotein (MOG) is a key component of myelin and an autoantigen in the disease multiple sclerosis (MS). Post-translational N-glycosylation of Asn 31 of MOG seems to play a key role in modulating the immune response towards myelin. This is mediated by the interaction of Lewis-type glycan structures in the N-glycan of MOG with the DC-SIGN receptor on dendritic cells (DCs). Here, we report the synthesis of an unnatural Lewis X (Le X )-containing Fmoc-SPPS-compatible asparagine building block (SPPS=solid-phase peptide synthesis), as well as asparagine building blocks containing two Le X -derived oligosaccharides: LacNAc and Fucα1-3GlcNAc. These building blocks were used for the glycosylation of the immunodominant portion of MOG (MOG 31-55 ) and analyzed with respect to their ability to bind to DC-SIGN in different biological setups, as well as their ability to inhibit the citrullination-induced aggregation of MOG 31-55 . Finally, a cytokine secretion assay was carried out on human monocyte-derived DCs, which showed the ability of the neoglycopeptide decorated with a single Le X to alter the balance of pro- and anti-inflammatory cytokines, inducing a tolerogenic response., (© 2020 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Correlative light and electron microscopy (CLEM) entails a group of multimodal imaging techniques that are combined to pinpoint to the location of fluorescently labeled molecules in the context of their ultrastructural cellular environment. Here we describe a detailed workflow for STORM-CLEM, in which STochastic Optical Reconstruction Microscopy (STORM), an optical super-resolution technique, is correlated with transmission electron microscopy (TEM). This protocol has the advantage that both imaging modalities have resolution at the nanoscale, bringing higher synergies on the information obtained. The sample is prepared according to the Tokuyasu method followed by click-chemistry labeling and STORM imaging. Then, after heavy metal staining, electron microscopy imaging is performed followed by correlation of the two images. The case study presented here is on intracellular pathogens, but the protocol is versatile and could potentially be applied to many types of samples., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Bioorthogonal correlative light-electron microscopy (B-CLEM) can give a detailed overview of multicomponent biological systems. It can provide information on the ultrastructural context of bioorthogonal handles and other fluorescent signals, as well as information about subcellular organization. We have here applied B-CLEM to the study of the intracellular pathogen Mycobacterium tuberculosis ( Mtb ) by generating a triply labeled Mtb through combined metabolic labeling of the cell wall and the proteome of a DsRed-expressing Mtb strain. Study of this pathogen in a B-CLEM setting was used to provide information about the intracellular distribution of the pathogen, as well as its in situ response to various clinical antibiotics, supported by flow cytometric analysis of the bacteria, after recovery from the host cell ( ex cellula ). The RNA polymerase-targeting drug rifampicin displayed the most prominent effect on subcellular distribution, suggesting the most direct effect on pathogenicity and/or viability, while the cell wall synthesis-targeting drugs isoniazid and ethambutol effectively rescued bacterial division-induced loss of metabolic labels. The three drugs combined did not give a more pronounced effect but rather an intermediate response, whereas gentamicin displayed a surprisingly strong additive effect on subcellular distribution., Competing Interests: The authors declare the following competing financial interest(s): The abstract figure was designed by bergdesigns.nl., (© 2020 American Chemical Society.)

The inverse electron-demand Diels-Alder (IEDDA) pyridazine elimination is one of the key bioorthogonal bond-breaking reactions. In this reaction trans -cyclooctene (TCO) serves as a tetrazine responsive caging moiety for amines, carboxylic acids and alcohols. One issue to date has been the lack of synthetic methods towards TCO ethers from functionalized (aliphatic) alcohols, thereby restricting bioorthogonal utilization. Two novel reagents were developed to enable controlled formation of cis -cyclooctene (CCO) ethers, followed by optimized photochemical isomerization to obtain TCO ethers. The method was exemplified by the controlled bioorthogonal activation of the lac operon system in E. coli using a TCO-ether-modified carbohydrate inducer., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The poly-ADP-ribose polymerase (PARP) is a protein from the family of ADP-ribosyltransferases that catalyzes polyadenosine diphosphate ribose (ADPR) formation in order to attract the DNA repair machinery to sites of DNA damage. The inhibition of PARP activity by olaparib can cause cell death, which is of clinical relevance in some tumor types. This demonstrates that quantification of PARP activity in the context of living cells is of great importance. In this work, we present the design, synthesis and biological evaluation of photo-activatable affinity probes inspired by the olaparib molecule that are equipped with a diazirine for covalent attachment upon activation by UV light and a ligation handle for the addition of a reporter group of choice. SDS-PAGE, western blotting and label-free LC-MS/MS quantification analysis show that the probes target the PARP-1 protein and are selectively outcompeted by olaparib; this suggests that they bind in the same enzymatic pocket. Proteomics data are available via ProteomeXchange with identifier PXD018661., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

The inverse electron demand Diels-Alder pyridazine elimination reaction between tetrazines and allylic substituted trans-cyclooctenes (TCOs) is a key player in bioorthogonal bond cleavage reactions. Determining the rate of elimination of alkylamine substrates has so far proven difficult. Here, we report a fluorogenic tool consisting of a TCO-linked EDANS fluorophore and a DABCYL quencher for accurate determination of both the click and release rate constants for any tetrazine at physiologically relevant concentrations., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Toll-like receptors (TLRs) are key pathogen sensors of the immune system. Their activation results in the production of cytokines, chemokines, and costimulatory molecules that are crucial for innate and adaptive immune responses. In recent years, specific (sub)-cellular location and timing of TLR activation have emerged as parameters for defining the signaling outcome and magnitude. To study the subtlety of this signaling, we here report a new molecular tool to control the activation of TLR2 via "click-to-release"-chemistry. We conjugated a bioorthogonal trans-cyclooctene (TCO) protecting group via solid support to a critical position within a synthetic TLR2/6 ligand to render the compound unable to initiate signaling. The TCO-group could then be conditionally removed upon addition of a tetrazine, resulting in restored agonist activity and TLR2 activation. This approach was validated on RAW264.7 macrophages and various murine primary immune cells as well as human cell line systems, demonstrating that TCO-caging constitutes a versatile approach for generating chemically controllable TLR2 agonists.

Supramolecular encapsulation is known to alter chemical properties of guest molecules. We have applied this strategy of molecular encapsulation to temporally control the catalytic activity of a stable copper(I)-carbene catalyst. Encapsulation of the copper(I)-carbene catalyst by the supramolecular host cucurbit[7]uril (CB[7]) resulted in the complete inactivation of a copper-catalyzed alkyne-azide cycloaddition (CuAAC) reaction. The addition of a chemical signal achieved the near instantaneous activation of the catalyst, by releasing the catalyst from the inhibited CB[7] catalyst complex. To broaden the scope of our on-demand CuAAC reaction, we demonstrated the protein labeling of vinculin with the copper(I)-carbene catalyst, to inhibit its activity by encapsulation with CB[7] and to initiate labeling at any moment by adding a specific signal molecule. Ultimately, this strategy allows for temporal control over copper-catalyzed click chemistry, on small molecules as well as protein targets., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Studying metal-protein interactions is key for understanding the fate of metallodrugs in biological systems. When a metal complex is not emissive and too weakly bound for mass spectrometry analysis, however, it may become challenging to study such interactions. In this work a synthetic procedure was developed for the alkyne functionalization of a photolabile ruthenium polypyridyl complex, [Ru(tpy)(bpy)(Hmte)](PF 6 ) 2 , where tpy = 2,2':6',2''-terpyridine, bpy = 2,2'-bipyridine, and Hmte = 2-(methylthio)ethanol. In the functionalized complex [Ru(HCC-tpy)(bpy)(Hmte)](PF 6 ) 2 , where HCC-tpy = 4'-ethynyl-2,2':6',2''-terpyridine, the alkyne group can be used for bioorthogonal ligation to an azide-labeled fluorophore using copper-catalyzed "click" chemistry. We developed a gel-based click chemistry method to study the interaction between this ruthenium complex and bovine serum albumin (BSA). Our results demonstrate that visualization of the interaction between the metal complex and the protein is possible, even when this interaction is too weak to be studied by conventional means such as UV-vis spectroscopy or ESI mass spectrometry. In addition, the weak metal complex-protein interaction is controlled by visible light irradiation, i.e ., the complex and the protein do not interact in the dark, but they do interact via weak van der Waals interactions after light activation of the complex, which triggers photosubstitution of the Hmte ligand.

Synthetic long peptide (SLP) vaccination is a promising new treatment strategy for patients with a chronic hepatitis B virus (HBV) infection. We have previously shown that a prototype HBV-core protein derived SLP was capable of boosting CD4 + and CD8 + T cell responses in the presence of a TLR2-ligand in chronic HBV patients ex vivo. For optimal efficacy of a therapeutic vaccine in vivo, adjuvants can be conjugated to the SLP to ensure delivery of both the antigen and the co-stimulatory signal to the same antigen-presenting cell (APC). Dendritic cells (DCs) express the receptor for the adjuvant and are optimally equipped to efficiently process and present the SLP-contained epitopes to T cells. Here, we investigated TLR2-ligand conjugation of the prototype HBV-core SLP. Results indicated that TLR2-ligand conjugation reduced cross-presentation efficiency of the SLP-contained epitope by both monocyte-derived and naturally occurring DC subsets. Importantly, cross-presentation was improved after optimization of the conjugate by either shortening the SLP or by placing a valine-citrulline linker between the TLR2-ligand and the long SLP, to facilitate endosomal dissociation of SLP and TLR2-ligand after uptake. HBV-core SLP conjugates also triggered functional patient T cell responses ex vivo. These results provide an import step forward in the design of a therapeutic SLP-based vaccine to cure chronic HBV., Competing Interests: Declaration of competing interest WJ Krebber, CJM Melief and GG Zom are employed by biotech company ISA Pharmaceuticals B.V. (Immune System Activation), aiming at development of synthetic peptide–based cancer vaccines. WJ Krebber, CJM Melief and GG Zom receive a salary from ISA Pharmaceuticals B.V., CJM Melief is in possession of stock appreciation rights. In addition, CJM Melief and WJ Krebber are inventors on numerous patents that are licensed to or owned by ISA Pharmaceuticals B.V. dealing with synthetic long peptide vaccines or dealing with the proprietary TLR ligand used in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Proteasome inhibitors are established therapeutic agents for the treatment of hematological cancers, as are anthracyclines such as doxorubicin. We here present a new drug targeting approach that combines both drug classes into a single molecule. Doxorubicin was conjugated to an immunoproteasome-selective inhibitor via light-cleavable linkers, yielding peptide epoxyketone-doxorubicin prodrugs that remained selective and active toward immunoproteasomes. Upon cellular uptake and immunoproteasome inhibition, doxorubicin is released from the immunoproteasome inhibitor through photoirradiation. Multiple myeloma cells in this way take a double hit: immunoproteasome inhibition and doxorubicin-induced toxicity. Our strategy, which entails targeting of a cytotoxic agent, through a covalent enzyme inhibitor that is detrimental to tumor tissue in its own right, may find use in the search for improved anticancer drugs.

Glycosylation plays a myriad of roles in the immune system: Certain glycans can interact with specific immune receptors to kickstart a pro-inflammatory response, whereas other glycans can do precisely the opposite and ameliorate the immune response. Specific glycans and glycoforms can themselves become the targets of the adaptive immune system, leading to potent antiglycan responses that can lead to the killing of altered self- or pathogenic species. This hydra-like set of roles glycans play is of particular importance in cancer immunity, where it influences the anticancer immune response, likely playing pivotal roles in tumor survival or clearance. The complexity of carbohydrate biology requires synthetic access to glycoproteins and glycopeptides that harbor homogeneous glycans allowing the probing of these systems with high precision. One particular complicating factor in this is that these synthetic structures are required to be as close to the native structures as possible, as non-native linkages can themselves elicit immune responses. In this Review, we discuss examples and current strategies for the synthesis of natively linked single glycoforms of peptides and proteins that have enabled researchers to gain new insights into glycoimmunology, with a particular focus on the application of these reagents in cancer immunology.

N-Azidoacetyl-d-glucosamine (GlcNAz) is a particularly useful tool in chemical biology as the azide is a metabolically stable yet accessible handle within biological systems. Herein, we report a practical synthesis of FmocAsn(N-Ac 3 GlcNAz)OH, a building block for solid phase peptide synthesis (SPPS). Protecting group manipulations are minimised by taking advantage of the inherent chemoselectivity of phosphine-mediated azide reduction, and the resulting glycosyl amine is employed directly in the opening of Fmoc protected aspartic anhydride. We show potential application of the building block by establishing it as a substrate for enzymatic glycan extension using sugar oxazolines of varying size and biological significance with several endo-β-N-acetylglucosaminidases (ENGases). The added steric bulk resulting from incorporation of the azide is shown to have no or a minor impact on the yield of enzymatic glycan extension.

β-Glucocerebrosidase (GBA) is the enzyme that degrades glucosylceramide in lysosomes. Defects in GBA that result in overall loss of enzymatic activity give rise to the lysosomal storage disorder Gaucher disease, which is characterized by the accumulation of glucosylceramide in tissue macrophages. Gaucher disease is currently treated by infusion of mannose receptor-targeted recombinant GBA. The recombinant GBA is thought to reach the lysosomes of macrophages, based on the impressive clinical response that is observed in Gaucher patients (type 1) receiving this enzyme replacement therapy. In this study, we used cyclophellitol-derived activity-based probes (ABPs) with a fluorescent reporter that irreversibly bind to the catalytic pocket of GBA, to visualize the active enzymes in a correlative microscopy approach. The uptake of pre-labeled recombinant enzyme was monitored by fluorescence and electron microscopy in human fibroblasts that stably expressed the mannose receptor. The endogenous active enzyme was simultaneously visualized by in situ labeling with the ABP containing an orthogonal fluorophore. This method revealed the efficient delivery of recombinant GBA to lysosomal target compartments that contained endogenous active enzyme., (© 2019 The Authors. Traffic published by John Wiley & Sons Ltd.)

Multiple sclerosis (MS) is an autoimmune disorder manifested via chronic inflammation, demyelination, and neurodegeneration inside the central nervous system. The progressive phase of MS is characterized by neurodegeneration, but unlike classical neurodegenerative diseases, amyloid-like aggregation of self-proteins has not been documented. There is evidence that citrullination protects an immunodominant peptide of human myelin oligodendrocyte glycoprotein (MOG 34-56 ) against destructive processing in Epstein-Barr virus-infected B-lymphocytes (EBV-BLCs) in marmosets and causes exacerbation of ongoing MS-like encephalopathies in mice. Here we collected evidence that citrullination of MOG can also lead to amyloid-like behavior shifting the disease pathogenesis toward neurodegeneration. We observed that an immunodominant MOG peptide, MOG 35-55 , displays amyloid-like behavior upon site-specific citrullination at positions 41, 46, and/or 52. These amyloid aggregates are shown to be toxic to the EBV-BLCs and to dendritic cells at concentrations favored for antigen presentation, suggesting a role of amyloid-like aggregation in the pathogenesis of progressive MS.

The inverse-electron-demand Diels-Alder/pyridazine elimination tandem reaction, in which the allylic substituent on trans-cyclooctene is eliminated following reaction with tetrazines, is gaining interest as a versatile bioorthogonal process. One potential shortcoming of such currently used reactions is their propensity to proceed faster and more efficiently at lower pH, a feature caused by the nature of the tetrazines used. Here, we present aminoethyl-substituted tetrazines as the first pH-independent reagents showing invariably fast elimination kinetics at all biologically relevant pH values., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

CD4 + T cell differentiation into multiple T helper (Th) cell lineages is critical for optimal adaptive immune responses. This report identifies an intrinsic mechanism by which programmed death-1 receptor (PD-1) signaling imparted regulatory phenotype to Foxp3 + Th1 cells (denoted as Tbet + iTreg PDL1 cells) and inducible regulatory T (iTreg) cells. Tbet + iTreg PDL1 cells prevented inflammation in murine models of experimental colitis and experimental graft versus host disease (GvHD). Programmed death ligand-1 (PDL-1) binding to PD-1 imparted regulatory function to Tbet + iTreg PDL1 cells and iTreg cells by specifically downregulating endo-lysosomal protease asparaginyl endopeptidase (AEP). AEP regulated Foxp3 stability and blocking AEP imparted regulatory function in Tbet + iTreg cells. Also, Aep -/- iTreg cells significantly inhibited GvHD and maintained Foxp3 expression. PD-1-mediated Foxp3 maintenance in Tbet + Th1 cells occurred both in tumor infiltrating lymphocytes (TILs) and during chronic viral infection. Collectively, this report has identified an intrinsic function for PD-1 in maintaining Foxp3 through proteolytic pathway., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Activation of a cytotoxic T-cell is a complex multistep process, and tools to study the molecular events and their dynamics that result in T-cell activation in situ and in vivo are scarce. Here, we report the design and use of conditional epitopes for time-controlled T-cell activation in vivo. We show that trans-cyclooctene-protected SIINFEKL (with the lysine amine masked) is unable to elicit the T-cell response characteristic for the free SIINFEKL epitope. Epitope uncaging by means of an inverse-electron demand Diels-Alder (IEDDA) event restored T-cell activation and provided temporal control of T-cell proliferation in vivo.

The imaging of intracellular pathogens inside host cells is complicated by the low resolution and sensitivity of fluorescence microscopy and by the lack of ultrastructural information to visualize the pathogens. Herein, we present a new method to visualize these pathogens during infection that circumvents these problems: by using a metabolic hijacking approach to bioorthogonally label the intracellular pathogen Salmonella Typhimurium and by using these bioorthogonal groups to introduce fluorophores compatible with stochastic optical reconstruction microscopy (STORM) and placing this in a correlative light electron microscopy (CLEM) workflow, the pathogen can be imaged within its host cell context Typhimurium with a resolution of 20 nm. This STORM-CLEM approach thus presents a new approach to understand these pathogens during infection., (© 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

One of the areas in which bioorthogonal chemistry-chemistry performed inside a cell or organism-has become of pivotal importance is in the study of host-pathogen interactions. The incorporation of bioorthogonal groups into the cell wall or proteome of intracellular pathogens has allowed study within the endolysosomal system. However, for the approach to be successful, the incorporated bioorthogonal groups must be stable to chemical conditions found within these organelles, which are some of the harshest found in metazoans: the groups are exposed to oxidizing species, acidic conditions, and reactive thiols. Here we present an assay that allows the assessment of the stability of bioorthogonal groups within host cell phagosomes. Using a flow cytometry-based assay, we have quantified the relative label stability inside dendritic cell phagosomes of strained and unstrained alkynes. We show that groups that were shown to be stable in other systems were degraded by as much as 79% after maturation of the phagosome.

Chemical tools and methods that report on G protein-coupled receptor (GPCR) expression levels and receptor occupancy by small molecules are highly desirable. We report the development of LEI121 as a photoreactive probe to study the type 2 cannabinoid receptor (CB 2 R), a promising GPCR to treat tissue injury and inflammatory diseases. LEI121 is the first CB 2 R-selective bifunctional probe that covalently captures CB 2 R upon photoactivation. An incorporated alkyne serves as ligation handle for the introduction of reporter groups. LEI121 enables target engagement studies and visualization of endogenously expressed CB 2 R in HL-60 as well as primary human immune cells using flow cytometry. Our findings show that strategically functionalized probes allow monitoring of endogenous GPCR expression and engagement in human cells using tandem photoclick chemistry and hold promise as biomarkers in translational drug discovery.

Progress made in peptide-based vaccinations to induce T-cell-dependent immune responses against cancer has invigorated the search for optimal vaccine modalities. Design of new vaccine strategies intrinsically depends on the knowledge of antigen handling and optimal epitope presentation in both major histocompatibility complex class I and -II molecules by professional antigen-presenting cells to induce robust CD8 and CD4 T-cell responses. Although there is a steady increase in the understanding of the underlying mechanisms that bridges innate and adaptive immunology, many questions remain to be answered. Moreover, we are in the early stage of exploiting this knowledge to clinical advantage. Several adaptations of peptide-based vaccines like peptide-adjuvant conjugates have been explored and showed beneficial outcomes in preclinical models; but in the clinical trials conducted so far, mixed results were obtained. A major limiting factor to unravel antigen handling mechanistically is the lack of tools to efficiently track peptide vaccines at the molecular and (sub)cellular level. In this mini-review, we will discuss options to develop molecular tools for improving, as well as studying, peptide-based vaccines.

Electron microscopy (EM) is traditionally employed as a follow-up to fluorescence microscopy (FM) to resolve the cellular ultrastructures wherein fluorescently labelled biomolecules reside. In order to translate the information derived from FM studies to EM analysis, biomolecules of interest must be identified in a manner compatible with EM. Although fluorescent signals can serve this purpose when FM is combined with EM in correlative light and electron microscopy (CLEM), the traditional immunogold labelling remains commonly used in this context. In order to investigate how much these two strategies relate, we have directly compared the subcellular localization of on-section fluorescence labelling with on-section immunogold labelling. In addition to antibody labelling of LAMP-1, bioorthogonal click labelling was used to localize soluble cysteine cathepsins or membrane-associated sialylated glycans. We reveal and characterize the existence of inherent discrepancies between the fluorescence signal and the distribution of gold particles in particular in the case of membrane-associated antigens., (© 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.)

Membrane-compound exchange is vital for cell-to-cell communication, yet quantification of this process is difficult. Here we present a method using flow cytometry in combination with bioorthogonal and fluorescent labelling techniques to quantify the amount of exchange of cholesterol and sialylated compounds between cells. We demonstrate that direct cell-cell contact is the likely mechanism of sterol-exchange and show that by manipulating the contact time between cells using complementary coiled-coil peptides results in an enhanced exchange rate of membrane components between cells.

The detection of infectious pathogens is essential for the induction of antimicrobial immune responses. The innate immune system detects a wide array of microbes using a limited set of pattern-recognition receptors (PRRs). One family of PRRs with a central role in innate immunity are the Toll-like receptors (TLRs). Upon ligation, these receptors initiate signaling pathways culminating in the release of pro-inflammatory cytokines and/or type I interferons (IFN-I). In recent years, it has become evident that the specific subcellular location and timing of TLR activation affect signaling outcome. The subtlety of this signaling has led to a growing demand for chemical tools that provide the ability to conditionally control TLR activation. In this review, we survey current models for TLR signaling in time and space, discuss how chemical tools have contributed to our understanding of TLR ligands, and describe how they can aid further elucidation of the dynamic aspects of TLR signaling., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Ligand-conjugated microparticles of iron oxide (MPIO) have the potential to provide high sensitivity contrast for molecular magnetic resonance imaging (MRI). However, the accumulation and persistence of non-biodegradable micron-sized particles in liver and spleen precludes their clinical use and limits the translational potential of MPIO-based contrast agents. Here we show that ligand-targeted MPIO derived from multiple iron oxide nanoparticles may be coupled covalently through peptide linkers that are designed to be cleaved by intracellular macrophage proteases. The synthesized particles possess potential characteristics for targeted MRI contrast agents, including high relaxivity, unappreciable sedimentation, clearance from circulation and no overt toxicity. Importantly, we demonstrate that these particles are rapidly degraded both in vitro and in vivo, and that the targeted probes can be used for detection of inflammation in vivo using MRI. This approach provides a platform for molecular MRI contrast agents that is potentially more suitable for translation to humans.