Bioorthogonal Chemistry Research Articles

In-situ construction of fluorescent probes for lysosomes imaging and lysosomal pH ratiometric detection based on bioorthogonal fluorescence

Based on a novel fluorescent bioorthogonal reaction between 4-(6-Methyl-1,2,4,5-tetrazin-3-yl) benzoic acid (TE) and norbornenes (NC), we reported a convenient strategy to in-situ construct fluorescent probes directly in live cells for lysosomal studies. Once the cells were treated with morpholine modified norbornene (MP-NC), the intracellular lysosomes were decorated fast as functionalizing substrate for bioorthogonal counterparts to react. With the introduction of TE, lysosomes-targeted probe LP1 was in-situ constructed directly in live cells within 10 min and emitted bioorthogonal fluorescence (515 nm). In a similar way, probe LP2 was assembled inside lysosomes rapidly. The probe emitted green bioorthogonal fluorescence (480 nm) and red rhodamine fluorescence (590 nm) in acidic lysosomal environment. A linear response (R = 0.9702) of the fluorescence intensity ratio (I590/I480) was found in pH range of 3.9–5.3. Ratiometric detection of lysosomal pH was successfully performed. Our modular assembling strategy to construct fluorescent probes presented excellent convenience and flexibility. We hoped our work to expand the research toolbox of bioorthogonal chemistry and to benefit more fluorescent applications in biological systems.

Cobalt protoporphyrin-induced nano-self-assembly for CT imaging, magnetic-guidance, and antioxidative protection of stem cells in pulmonary fibrosis treatment.

Mesenchymal stem cells (MSCs) transplantation is a promising approach for pulmonary fibrosis (PF), however it is impeded by several persistent challenges, including the lack of long-term tracking, low retention, and poor survival of MSCs, as well as the low labeling efficiency of nanoprobes. Herein, a cobalt protoporphyrin IX (CoPP) aggregation-induced strategy is applied to develop a multifunctional nano-self-assembly (ASCP) by combining gold nanoparticle (AuNPs), superparamagnetic iron oxide nanoparticles (SPIONs), and CoPP through a facile solvent evaporation-driven approach. Since no additional carrier materials are employed during the synthesis, high loading efficiency of active ingredients and excellent biocompatibility are achieved. Additionally, facile modification of the ASCPs with bicyclo[6.1.0]nonyne (BCN) groups (named as ASCP-BCN) enables them to effectively label MSCs through bioorthogonal chemistry. The obtained ASCP-BCN could not only help to track MSCs with AuNP-based computed tomography (CT) imaging, but also achieve an SPIONs-assisted magnetic field based improvement in the MSCs retention in lungs as well as promoted the survival of MSCs via the sustained release of CoPP. The in vivo results demonstrated that the labeled MSCs improved the lung functions and alleviated the fibrosis symptoms in a bleomycin–induced PF mouse model. Collectively, a novel ASCP-BCN multifunctional nanoagent was developed to bioorthogonally-label MSCs with a high efficiency, presenting a promising potential in the high-efficient MSC therapy for PF.

Live-Cell Imaging of Sterculic Acid-a Naturally Occurring 1,2-Cyclopropene Fatty Acid-by Bioorthogonal Reaction with Turn-On Tetrazine-Fluorophore Conjugates.

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.

Live‐Cell Imaging of Sterculic Acid—a Naturally Occurring 1,2‐Cyclopropene Fatty Acid—by Bioorthogonal Reaction with Turn‐On Tetrazine‐Fluorophore Conjugates**

AbstractIn 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.

Spatial specific delivery of combinational chemotherapeutics to combat intratumoral heterogeneity.

Hypoxia-induced intratumoral heterogeneity poses a major challenge in tumor therapy due to the varying susceptibility to chemotherapy. Moreover, the spatial distribution patterns of hypoxic and normoxic tissues makes conventional combination therapy less effective. In this study, a tumor-acidity and bioorthogonal chemistry mediated in situ size transformable nanocarrier (NP@DOXDBCO plus iCPPAN3) was developed to spatially deliver two combinational chemotherapeutic drugs (doxorubicin (DOX) and PR104A) to combat hypoxia-induced intratumoral heterogeneity. DOX is highly toxic to tumor cells in normoxia state but less toxic in hypoxia state due to the hypoxia-induced chemoresistance. Meanwhile, PR104A is a hypoxia-activated prodrug has less toxic in normoxia state. Two nanocarriers, NP@DOXDBCO and iCPPAN3, can cross-link near the blood vessel extravasation sites through tumor acidity responsive bioorthogonal click chemistry to enhance the retention of DOX in tumor normoxia. Moreover, PR104A conjugated to the small-sized dendritic polyamidoamine (PAMAM) released under tumor acidity can penetrate deep tumor tissues for hypoxic tumor cell killing. Our study has demonstrated that this site-specific combination chemotherapy is better than the traditional combination chemotherapy. Therefore, spatial specific delivery of combinational therapeutics via in situ size transformable nanocarrier addresses the challenges of hypoxia induced intratumoral heterogeneity and provides insights into the combination therapy.

Clickable Biomaterials for Modulating Neuroinflammation.

Crosstalk between the nervous and immune systems in the context of trauma or disease can lead to a state of neuroinflammation or excessive recruitment and activation of peripheral and central immune cells. Neuroinflammation is an underlying and contributing factor to myriad neuropathologies including neurodegenerative diseases like Alzheimer’s disease and Parkinson’s disease; autoimmune diseases like multiple sclerosis; peripheral and central nervous system infections; and ischemic and traumatic neural injuries. Therapeutic modulation of immune cell function is an emerging strategy to quell neuroinflammation and promote tissue homeostasis and/or repair. One such branch of ‘immunomodulation’ leverages the versatility of biomaterials to regulate immune cell phenotypes through direct cell-material interactions or targeted release of therapeutic payloads. In this regard, a growing trend in biomaterial science is the functionalization of materials using chemistries that do not interfere with biological processes, so-called ‘click’ or bioorthogonal reactions. Bioorthogonal chemistries such as Michael-type additions, thiol-ene reactions, and Diels-Alder reactions are highly specific and can be used in the presence of live cells for material crosslinking, decoration, protein or cell targeting, and spatiotemporal modification. Hence, click-based biomaterials can be highly bioactive and instruct a variety of cellular functions, even within the context of neuroinflammation. This manuscript will review recent advances in the application of click-based biomaterials for treating neuroinflammation and promoting neural tissue repair.

Enhancing adoptive T cell therapy for solid tumor with cell-surface anchored immune checkpoint inhibitor nanogels

The efficacy of Adoptive Cell Therapy (ACT) for solid tumor is still mediocre. This is mainly because tumor cells can hijack ACT T cells' immune checkpoint pathways to exert immunosuppression in the tumor microenvironment. Immune Checkpoint Inhibitors such as anti-PD-1 (aPD1) can counter the immunosuppression, but the synergizing effects of aPD1 to ACT was still not satisfactory. Here we demonstrate an approach to safely anchor aPD1-formed nanogels onto T cell surface via bio-orthogonal click chemistry before adoptive transfer. The spatial-temporal co-existence of aPD1 with ACT T cells and the responsive drug release significantly improved the treatment outcome of ACT in murine solid tumor model. The average tumor weight of the group treated by cell-surface anchored aPD1 was only 18 % of the group treated by equivalent dose of free aPD1 and T cells. The technology can be broadly applicable in ACTs employing natural or Chimeric Antigen Receptor (CAR) T cells.

From Bench to Cell: A Roadmap for Assessing the Bioorthogonal "Click" Reactivity of Magnetic Nanoparticles for Cell Surface Engineering.

In this work, we report the use of bioorthogonal chemistry, specifically the strain-promoted click azide–alkyne cycloaddition (SPAAC) for the covalent attachment of magnetic nanoparticles (MNPs) on living cell membranes. Four types of MNPs were prepared, functionalized with two different stabilizing/passivation agents (a polyethylene glycol derivative and a glucopyranoside derivative, respectively) and two types of strained alkynes with different reactivities: a cyclooctyne (CO) derivative and a dibenzocyclooctyne (DBCO) derivative. The MNPs were extensively characterized in terms of physicochemical characteristics, colloidal stability, and click reactivity in suspension. Then, the reactivity of the MNPs toward azide-modified surfaces was evaluated as a closer approach to their final application in a living cell scenario. Finally, the DBCO-modified MNPs, showing superior reactivity in suspension and on surfaces, were selected for cell membrane immobilization via the SPAAC reaction on the membranes of cells engineered to express azide artificial reporters. Overall, our work provides useful insights into the appropriate surface engineering of nanoparticles to ensure a high performance in terms of bioorthogonal reactivity for biological applications.

Exploiting Meltable Protein Hydrogels to Encapsulate and Culture Cells in 3D.

There is a growing realization that 3D cell culture better mimics complex in vivo environments than 2D, lessening aberrant cellular behaviors and ultimately improving the outcomes of experiments. Chemically crosslinked hydrogels which imitate natural extracellular matrix (ECM) are proven cell culture platforms, but the encapsulation of cells within these hydrogel networks requires bioorthogonal crosslinking chemistries which can be cytotoxic, synthetically demanding, and costly. Capsular antigen fragment 1 (Caf1) is a bacterial, polymeric, fimbrial protein which can be genetically engineered to imitate ECM. Furthermore, it can, reversibly, thermally interconvert between its polymeric and monomeric forms even when chemically crosslinked within a hydrogel network. It is demonstrated that this meltable feature of Caf1 hydrogels can be utilized to encapsulate neonatal human dermal fibroblasts at a range of cell densities (2×105 -2×106 cellsmL-1 of hydrogel) avoiding issues with chemical cytotoxicity. These hydrogels supported cell 3D culture for up to 21d, successfully inducing cellular functions such as proliferation and migration. This work is significant because it further highlights the potential of simple, robust, Caf1-based hydrogels as a cell culture platform.

Bioorthogonally activatable cyanine dye with torsion-induced disaggregation for in vivo tumor imaging

Advancement of bioorthogonal chemistry in molecular optical imaging lies in expanding the repertoire of fluorophores that can undergo fluorescence signal changes upon bioorthogonal ligation. However, most available bioorthogonally activatable fluorophores only emit shallow tissue-penetrating visible light via an intramolecular charge transfer mechanism. Herein, we report a serendipitous “torsion-induced disaggregation (TIDA)” phenomenon in the design of near-infrared (NIR) tetrazine (Tz)-based cyanine probe. The TIDA of the cyanine is triggered upon Tz-transcyclooctene ligation, converting its heptamethine chain from S-trans to S-cis conformation. Thus, after bioorthogonal reaction, the tendency of the resulting cyanine towards aggregation is reduced, leading to TIDA-induced fluorescence enhancement response. This Tz-cyanine probe sensitively delineates the tumor in living mice as early as 5 min post intravenous injection. As such, this work discovers a design mechanism for the construction of bioorthogonally activatable NIR fluorophores and opens up opportunities to further exploit bioorthogonal chemistry in in vivo imaging.

Development of [18F]AmBF3 Tetrazine for Radiolabeling of Peptides: Preclinical Evaluation and PET Imaging of [18F]AmBF3-PEG7-Tyr3-Octreotide in an AR42J Pancreatic Carcinoma Model.

Radiolabeled peptides have emerged as highly specific agents for targeting receptors expressed in tumors for therapeutic and diagnostic purposes. Peptides developed for positron emission tomography (PET) are typically radiolabeled using prosthetic groups or bifunctional chelators for fast “kit-like” incorporation of the radionuclide into the structure. A novel [18F]alkylammoniomethyltrifluoroborate ([18F]AmBF3) tetrazine (Tz), [18F]AmBF3-Tz, was developed for the [18F]fluorination of trans-cyclooctene (TCO)-modified biomolecules using Tyr3-octreotides (TOCs) as model peptides. [18F]AmBF3-Tz (Am = 15.4 ± 9.2 GBq/μmol, n = 14) was evaluated in healthy mice by ex vivo biodistribution and PET/computed tomography (CT), where the radiolabel in the prosthetic group was found stable in vivo, indicated by the low bone uptake in tibia (0.4 ± 0.1% ID/g, t = 270 min). TCO-TOCs tailored with polyethylene glycol (PEG) linkers were radiolabeled with [18F]AmBF3-Tz, forming two new tracers, [18F]AmBF3-PEG4-TOC (Am = 2.8 ± 1.8 GBq/μmol, n = 3) and [18F]AmBF3-PEG7-TOC (Am of 6.0 ± 3.4 GBq/μmol, n = 13), which were evaluated by cell uptake studies and ex vivo biodistribution in subcutaneous AR42J rat pancreatic carcinoma tumor-bearing nude mice. The tracer demonstrating superior behavior ex vivo, the [18F]AmBF3-PEG7-TOC, was further evaluated with PET/CT, where the tracer provided clear tumor visualization (SUVbaseline = 1.01 ± 0.07, vs SUVblocked = 0.76 ± 0.04) at 25 min post injection. The novel AmBF3-Tz demonstrated that it offers potential as a prosthetic group for rapid radiolabeling of biomolecules in mild conditions using bioorthogonal chemistry.

Abstract 296: Antigen-independent delivery of 4-1BB agonist to the tumor microenvironment improves immune response while reducing hepatotoxicity

Abstract Background: Clinical progress of α4-1BB has been impeded by hepatotoxicity. Current research is focused on improving targeted delivery of α4-1BB to the tumor using tumor-specific markers. However, targeted delivery of 4-1BB agonists to tumors faces challenges due to a lack of tumor biomarkers and effector T cells within tumors. To overcome these challenges, we developed antigen-independent targeting approach based on unnatural sugar-mediated metabolic glycoengineering and bio-orthogonal click chemistry to deliver α4-1BB to tumors. We utilized nanoparticles that can deliver a sugar analogue containing click-chemistry moiety to the tumor microenvironment. Method: The Ac4ManNAz was loaded in mPEG-PLGA nanoparticles (MazNP), and the loading efficiency was determined by HPLC. DBCO-functionalized α4-1BB (DBCO-α4-1BB) was synthesized via amine-NHS coupling reaction. The tumor inhibition efficiency was assessed on mice bearing different tumor models. Mice were either inoculated subcutaneously on the left flank (75,000 B16F10 cells) or injected on the left fourth mammary fat pad (100,000 4T1 cells). Mice were given MazNPs (IV) on day 5, 6, 10, and 11, and DBCO-α4-1BB (IV) and αPD-1 (IP) on day 8 and 13. T lymphocytes were analyzed by fluorescent IF staining. Serum liver enzyme was examined to evaluate hepatotoxicity of treatments. Liver pathology was assessed by H&E and CD8+ IHC staining. Results: The αPD1 plus DBCO-α4-1BB with MazNPs showed a 36.4% cure rate, compared to 0% of αPD1 plus DBCO-α4-1BB or αPD1 plus DBCO-α4-1BB with free Ac4ManNAz in B16F10 melanoma model. We then re-challenged the cured mice with 200,000 B16F10 cells and 50% of cured mice survived without any further treatment. In 4T1 breast cancer model, the survival data analyzed using the log-rank test showed that the median survival of αPD1 plus DBCO-α4-1BB with MazNP was increased by 71%, compared to the PBS, and 26% αPD1 plus α4-1BB or αPD1 plus DBCO-α4-1BB with free Ac4ManNAz. We showed that the involvement of NK and CD8+ T cells in the antitumor efficacy of αPD1 plus DBCO-α4-1BB with MazNP. Notably, a massive CD8+ T cell infiltration in the liver was observed in both αPD1 plus α4-1BB (29.8 ± 18.2%) and αPD1 plus DBCO-α4-1BB with Ac4ManNAz (22.1 ± 10.9), significantly higher than PBS control (0.9 ± 0.6%), while the percentage of CD8+ T cells in the MazNP treatment (5.4 ± 5.8%) was five times lower. Hepatotoxicity was further confirmed by serum liver enzyme analysis that ALT and AST levels were substantially elevated by αPD1 plus α4-1BB or αPD1 plus DBCO-α4-1BB with free Ac4ManNAz, as compared to PBS control group, while the MazNP-treated mice had normal serum ALT and AST levels except for one. Conclusion: Our findings demonstrated that antigen-independent targeted delivery of α4-1BB can improve anti-tumor immune responses while reducing hepatotoxicity. Citation Format: Hyesun Hyun, Bo Sun, Stephanie A. Montgomery, Teresa Griffin, Juanzhu Yan, Albert Wielgus, Yue Wang, Tian Zhang, Jianjun Cheng, Andrew Z. Wang. Antigen-independent delivery of 4-1BB agonist to the tumor microenvironment improves immune response while reducing hepatotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 296.

In vivo nanotracer for the detection of brain thrombi in an AD mouse model.

Background: Alzheimer’s disease (AD) is the most common cause of dementia. It is a multifactorial degenerative disease pathologically characterized by intracellular neurofibrillary tangles and extracellular deposition of amyloid. An early hemostatic dysregulation is also present and contributes to an increment in clot formation, leading to hypoperfusion, blood brain barrier disruption and neuronal loss. The detection of this prothrombotic state is of the upmost importance in diagnostic approaches to identify AD patients who would benefit from anticoagulation. Method: The aim of this project is to use an in vivo nanotracer for the detection of brain thrombi in an AD mouse model by fast pre-targeted positron emission tomography (PET) imaging. For that purpose, AD animals and their wild-type littermates were intravenously injected with the antiplatelet antibody against CD41 conjugated with transcyclooctene (TCO-antiCD41). Twenty-four hours later, [68Ga]core-doped iron oxide nanoparticles (NP) functionalized with tetrazine (TZ) were intravenously administered. TCO and TZ produce a rapid in vivo reaction by means of bioorthogonal chemistry, allowing to non-invasively evaluate platelets’ levels by PET. Two hours after [68Ga]NP-TZ injection, a static PET study of each mouse was acquired with a scanner for small animals (nanoScan® PET/CT, Mediso, USA). Finally, biodistribution assays of different organs after the PET study were performed. All PET images were analyzed by regions of interest and voxel-wise analyses. Conclusions: Our results provide a neuroimaging strategy to diagnose the prothrombotic state towards the personalization of anticoagulation treatment in AD patients.

Spatiotemporal multiplexed immunofluorescence imaging of living cells and tissues with bioorthogonal cycling of fluorescent probes.

Cells in complex organisms undergo frequent functional changes, but few methods allow comprehensive longitudinal profiling of living cells. Here we introduce scission-accelerated fluorophore exchange (SAFE), a method for multiplexed temporospatial imaging of living cells with immunofluorescence. SAFE uses a rapid bioorthogonal click chemistry to remove immunofluorescent signals from the surface of labeled cells, cycling the nanomolar-concentration reagents in seconds and enabling multiple rounds of staining of the same samples. It is non-toxic and functional in both dispersed cells and intact living tissues. We demonstrate multiparameter (n ≥ 14), non-disruptive imaging of murine peripheral blood mononuclear and bone marrow cells to profile cellular differentiation. We also show longitudinal multiplexed imaging of bone marrow progenitor cells as they develop into neutrophils over 6 days and real-time multiplexed cycling of living mouse hepatic tissues. We anticipate that SAFE will find broad utility for investigating physiologic dynamics in living systems.

Spatial and temporal proteomics reveals the distinct distributions and dynamics of O-GlcNAcylated proteins.

SUMMARYProtein O-GlcNAcylation plays critical roles in many cellular events, and its dysregulation is related to multiple diseases. Integrating bioorthogonal chemistry and multiplexed proteomics, we systematically and site specifically study the distributions and dynamics of protein O-GlcNAcylation in the nucleus and the cytoplasm of human cells. The results demonstrate that O-GlcNAcylated proteins with different functions have distinct distribution patterns. The distributions vary site specifically, indicating that different glycoforms of the same protein may have different distributions. Moreover, we comprehensively analyze the dynamics of O-GlcNAcylated and non-modified proteins in these two compartments, respectively, and the half-lives of glycoproteins in different compartments are markedly different, with the median half-life in the cytoplasm being much longer. In addition, glycoproteins in the nucleus are more dramatically stabilized than those in the cytoplasm under the O-GlcNAcase inhibition. The comprehensive spatial and temporal analyses of protein O-GlcNAcylation provide valuable information and advance our understanding of this important modification.

Recent Advances in the Development of Tetrazine Ligation Tools for Pretargeted Nuclear Imaging.

Tetrazine ligation has gained interest as a bio-orthogonal chemistry tool within the last decade. In nuclear medicine, tetrazine ligation is currently being explored for pretargeted approaches, which have the potential to revolutionize state-of-the-art theranostic strategies. Pretargeting has been shown to increase target-to-background ratios for radiopharmaceuticals based on nanomedicines, especially within early timeframes. This allows the use of radionuclides with short half-lives which are more suited for clinical applications. Pretargeting bears the potential to increase the therapeutic dose delivered to the target as well as reduce the respective dose to healthy tissue. Combined with the possibility to be applied for diagnostic imaging, pretargeting could be optimal for theranostic approaches. In this review, we highlight efforts that have been made to radiolabel tetrazines with an emphasis on imaging.

Synthesis and ex vivo biodistribution of two 68Ga-labeled tetrazine tracers: Comparison of pharmacokinetics

Pretargeted PET imaging allows the use of radiotracers labeled with short-living PET radionuclides for tracing drugs with slow pharmacokinetics. Recently, especially methods based on bioorthogonal chemistry have been under intensive investigation for pretargeted PET imaging. The pharmacokinetics of the radiotracer is one of the factors that determine the success of the pretargeted strategy. Here, we report synthesis and biological evaluation of two 68Ga-labeled tetrazine (Tz)-based radiotracers, [68Ga]Ga-HBED-CC-PEG4-Tz ([68Ga]4) and [68Ga]Ga-DOTA-PEG4-Tz ([68Ga]6), aiming for development of new tracer candidates for pretargeted PET imaging based on the inverse electron demand Diels-Alder (IEDDA) ligation between a tetrazine and a strained alkene, such as trans-cyclooctene (TCO). Excellent radiochemical yield (RCY) was obtained for [68Ga]4 (RCY > 96%) and slightly lower for [68Ga]6 (RCY > 88%). Radiolabeling of HBED-CC-Tz proved to be faster and more efficient under milder conditions compared to the DOTA analogue. The two tracers exhibited excellent radiolabel stability both in vitro and in vivo. Moreover, [68Ga]4 was successfully used for radiolabeling two different TCO-functionalized nanoparticles in vitro: Hepatitis E virus nanoparticles (HEVNPs) and porous silicon nanoparticles (PSiNPs).

Chemical editing of proteoglycan architecture.

Proteoglycans are heterogeneous macromolecular glycoconjugates that orchestrate many important cellular processes. While much attention has focused on the poly-sulfated glycosaminoglycan chains that decorate proteoglycans, other important elements of their architecture, such as core proteins and membrane localization, have garnered less emphasis. Hence, comprehensive structure-function relationships that consider the replete proteoglycan architecture as glycoconjugates are limited. Here we present an extensive approach to study proteoglycan structure and biology by fabricating defined semisynthetic modular proteoglycans that can be tailored for cell surface display. The expression of proteoglycan core proteins with unnatural amino acids permits bioorthogonal click chemistry with functionalized glycosaminoglycans for methodical dissection of the parameters required for optimal binding and function of various proteoglycan-binding proteins. We demonstrate that these sophisticated materials can recapitulate the functions of native proteoglycan ectodomains in mouse embryonic stem cell differentiation and cancer cell spreading while permitting the analysis of the contributing architectural elements toward function.

Deciphering the Structure and Formation of Amyloids in Neurodegenerative Diseases With Chemical Biology Tools.

Protein aggregation into highly ordered, regularly repeated cross-β sheet structures called amyloid fibrils is closely associated to human disorders such as neurodegenerative diseases including Alzheimer’s and Parkinson’s diseases, or systemic diseases like type II diabetes. Yet, in some cases, such as the HET-s prion, amyloids have biological functions. High-resolution structures of amyloids fibrils from cryo-electron microscopy have very recently highlighted their ultrastructural organization and polymorphisms. However, the molecular mechanisms and the role of co-factors (posttranslational modifications, non-proteinaceous components and other proteins) acting on the fibril formation are still poorly understood. Whether amyloid fibrils play a toxic or protective role in the pathogenesis of neurodegenerative diseases remains to be elucidated. Furthermore, such aberrant protein-protein interactions challenge the search of small-molecule drugs or immunotherapy approaches targeting amyloid formation. In this review, we describe how chemical biology tools contribute to new insights on the mode of action of amyloidogenic proteins and peptides, defining their structural signature and aggregation pathways by capturing their molecular details and conformational heterogeneity. Challenging the imagination of scientists, this constantly expanding field provides crucial tools to unravel mechanistic detail of amyloid formation such as semisynthetic proteins and small-molecule sensors of conformational changes and/or aggregation. Protein engineering methods and bioorthogonal chemistry for the introduction of protein chemical modifications are additional fruitful strategies to tackle the challenge of understanding amyloid formation.

Spatially resolved cell tagging and surfaceome labeling via targeted photocatalytic decaging

<h2>Summary</h2> Spatially resolved <i>in situ</i> tagging of the cell of interest is crucial for in-depth mechanistic dissection of multicellular architectures or processes. With continuing interest in bioorthogonal photocatalytic decaging chemistry, we herein report the extracellular-targeted photocatalytic decaging system (CAT-Ex) for spatially resolved cell tagging and surface proteome profiling under living conditions. An antibody-conjugated photocatalysis system was established and extensively validated, enabling photocatalytic decaging of biotin precursors and proximal quinone methide probes on target cells. Visible-light-controlled selective cell tagging in cell mixture as well as in primary cells from tumor xenografts were demonstrated. Spatially resolved membrane proteome profiling was further achieved by coupling quinone methide decaging chemistry with CAT-Ex, revealing a potential microdomain protein cluster surrounding the endogenous HER2 receptor. Finally, we expanded our strategy to photocatalytic prodrug decaging for selective tumor cell killing, establishing CAT-Ex as a general platform for diverse photo-controlled molecular manipulations on targeted cells with spatial-temporal precision.