Bioorthogonal Quinone Methide Decaging Enables Live-Cell Quantification of Tumor-Specific Immune Interactions.

Effective antitumor immunity hinges on the specific engagement between tumor and cytotoxic immune cells, especially cytotoxic T cells. Although investigating these intercellular interactions is crucial for characterizing immune responses and guiding immunotherapeutic applications, direct and quantitative detection of tumor-T cell interactions within a live-cell context remains challenging. We herein report a photocatalytic live-cell interaction labeling strategy (CAT-Cell) relying on the bioorthogonal decaging of quinone methide moieties for sensitive and selective investigation and quantification of tumor-T cell interactions. By developing quinone methide-derived probes optimized for capturing cell-cell interactions (CCIs), we demonstrated the capacity of CAT-Cell for detecting CCIs directed by various types of receptor-ligand pairs (e.g., CD40-CD40L, TCR-pMHC) and further quantified the strengths of tumor-T cell interactions that are crucial for evaluating the antitumor immune responses. We further applied CAT-Cell for ex vivo quantification of tumor-specific T cell interactions on splenocyte and solid tumor samples from mouse models. Finally, the broad compatibility and utility of CAT-Cell were demonstrated by integrating it with the antigen-specific targeting system as well as for tumor-natural killer cell interaction detection. By leveraging the bioorthogonal photocatalytic decaging chemistry on quinone methide, CAT-Cell provides a sensitive, tunable, universal, and noninvasive toolbox for unraveling and quantifying the crucial but delicate tumor-immune interactions under live-cell settings.

Bioinspired Flexible Hydrogelation with Programmable Properties for Tactile Sensing.

Tactile sensing requires integrated detection platforms with distributed and highly sensitive haptic sensing capabilities along with biocompatibility, aiming to replicate the physiological functions of the human skin and empower industrial robotic and prosthetic wearers to detect tactile information. In this regard, short peptide-based self-assembled hydrogels show promising potential to act as bioinspired supramolecular substrates for developing tactile sensors showing biocompatibility and biodegradability. However, the intrinsic difficulty to modulate the mechanical properties severely restricts their extensive employment. Herein, by controlling the self-assembly of 9-fluorenylmethoxycarbonyl-modifid diphenylalanine (Fmoc-FF) through introduction of polyethylene glycol diacrylate (PEGDA), wider nanoribbons are achieved by untwisting from well-established thinner nanofibers, and the mechanical properties of the supramolecular hydrogels can be enhanced 10-fold, supplying bioinspired supramolecular encapsulating substrate for tactile sensing. Furthermore, by doping with poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) and 9-fluorenylmethoxycarbonyl-modifid 3,4-dihydroxy-l-phenylalanine (Fmoc-DOPA), the Fmoc-FF self-assembled hydrogels can be engineered to be conductive and adhesive, providing bioinspired sensing units and adhesive layer for tactile sensing applications. Therefore, the integration of these modules results in peptide hydrogelation-based tactile sensors, showing high sensitivity and sustainable responses with intrinsic biocompatibility and biodegradability. The findings establish the feasibility of developing programmable peptide self-assembly with adjustable features for tactile sensing applications.

Bioorthogonal photocatalytic proximity labeling in primary living samples.

In situ profiling of subcellular proteomics in primary living systems, such as native tissues or clinic samples, is crucial for understanding life processes and diseases, yet challenging due to methodological obstacles. Here we report CAT-S, a bioorthogonal photocatalytic chemistry-enabled proximity labeling method, that expands proximity labeling to a wide range of primary living samples for in situ profiling of mitochondrial proteomes. Powered by our thioQM labeling warhead development and targeted bioorthogonal photocatalytic chemistry, CAT-S enables the labeling of mitochondrial proteins in living cells with high efficiency and specificity. We apply CAT-S to diverse cell cultures, dissociated mouse tissues as well as primary T cells from human blood, portraying the native-state mitochondrial proteomic characteristics, and unveiled hidden mitochondrial proteins (PTPN1, SLC35A4 uORF, and TRABD). Furthermore, CAT-S allows quantification of proteomic perturbations on dysfunctional tissues, exampled by diabetic mouse kidneys, revealing the alterations of lipid metabolism that may drive disease progression. Given the advantages of non-genetic operation, generality, and spatiotemporal resolution, CAT-S may open exciting avenues for subcellular proteomic investigations of primary samples that are otherwise inaccessible.

Genetically encoded bioorthogonal tryptophan decaging in living cells.

Tryptophan (Trp) plays a critical role in the regulation of protein structure, interactions and functions through its π system and indole N-H group. A generalizable method for blocking and rescuing Trp interactions would enable the gain-of-function manipulation of various Trp-containing proteins in vivo, but generating such a platform remains challenging. Here we develop a genetically encoded N1-vinyl-caged Trp capable of rapid and bioorthogonal decaging through an optimized inverse electron-demand Diels-Alder reaction, allowing site-specific activation of Trp on a protein of interest in living cells. This chemical activation of a genetically encoded caged-tryptophan (Trp-CAGE) strategy enables precise activation of the Trp of interest underlying diverse important molecular interactions. We demonstrate the utility of Trp-CAGE across various protein families, such as catalase-peroxidases and kinases, as translation initiators and posttranslational modification readers, allowing the modulation of epigenetic signalling in a temporally controlled manner. Coupled with computer-aided prediction, our strategy paves the way for bioorthogonal Trp activation on more than 28,000 candidate proteins within their native cellular settings.

Decaging-to-labeling: Development and investigation of quinone methide warhead for protein labeling.

Biomolecule labeling in living systems is crucial for understanding biological processes and discovering therapeutic targets. A variety of labeling warheads have been developed for multiple biological applications, including proteomics, bioimaging, sequencing, and drug development. Quinone methides (QMs), a class of highly reactive Michael receptors, have recently emerged as prominent warheads for on-demand biomolecule labeling. Their highly flexible functionality and tunability allow for diverse biological applications, but remain poorly explored at present. In this regard, we designed, synthesized, and evaluated a series of new QM probes with a trifluoromethyl group at the benzyl position and substituents on the aromatic ring to manipulate their chemical properties for biomolecule labeling. The engineered QM warhead efficiently labeled proteins both in vitro and under living cell conditions, with significantly enhanced activity compared to previous QM warheads. We further analyzed the labeling efficacy with the assistance of density functional theory (DFT) calculations, which revealed that the QM generation process, rather than the reactivity of QM, contributes more predominantly to the labeling efficacy. Noteworthy, twelve nucleophilic residues on the BSA were labeled by the probe, including Cys, Asp, Glu, His, Lys, Asn, Gln, Arg, Ser, Thr, Trp and Tyr. Given their high efficiency and tunability, these new QM warheads may hold great promise for a broad range of applications, especially spatiotemporal proteomic profiling for in-depth biological studies.

Research on the dentification of Panax ginseng and Panax quinquefolium using catalysed hairpin assembly technology.

INTRODUCTION: Panax ginseng and Panax quinquefolium are traditional Chinese herb medicines and similar in morphology and some chemical components but differ in drug properties, so they cannot be mixed. However, the processed products of them are often sold in the form of slices, powder, and capsules, which are difficult to identify by traditional morphological methods. Furthermore, an accurate evaluation of P. ginseng, P. quinquefolium and the processed products have not been conducted. OBJECTIVE: This study aimed to establish a catalysed hairpin assembly (CHA) identification method for authenticating products made from P. ginseng and P. quinquefolium based on single nucleotide polymorphism (SNP) differences. METHOD: By analysing the differences of SNP in internal transcribed spacer 2 (ITS2) in P. ginseng and P. quinquefolium to design CHA-specific hairpins. Establish a sensitive and efficient CHA method that can identify P. ginseng and P. quinquefolium, use the sequencing technology to verify the accuracy of this method in identifying Panax products, and compare this method with high-resolution melting (HRM). RESULTS: The reaction conditions of CHA were as follows: the ratio of forward and reverse primers, 20:1; hairpin concentration, 5 ng/µL. Compared with capillary electrophoresis, this method had good specificity and the limit of detection was 0.5 ng/µL. The result of Panax product identification with CHA method were coincidence with that of the sequencing method; the positive rate of CHA reaction was 100%. CONCLUSION: This research presents an effective identification method for authenticating P. ginseng and P. quinquefolium products, which is helpful to improve the quality of Panax products.

Near Infrared Light-Triggered Photocatalytic Decaging for Remote-Controlled Spatiotemporal Activation in Living Mice.

Spatiotemporal manipulation of biological processes in living animals using noninvasive, remote-controlled stimuli is a captivating but challenging endeavor. Herein, we present the development of a biocompatible photocatalytic technology termed CAT-NIR, which uses external near infrared light (NIR, 740 nm) to trigger decaging reactions in living mice. The Os(II) terpyridine complex was identified as an efficient NIR photocatalyst for promoting deboronative hydroxylation reactions via superoxide generation in the presence of NIR light, resulting in the deprotection of phenol groups and the release of bioactive molecules under living conditions. The validation of the CAT-NIR system was demonstrated through the NIR-triggered rescue of fluorophores, prodrugs as well as biomolecules ranging from amino acids, peptides to proteins. Furthermore, by combining genetic code expansion and computer-aided screening, CAT-NIR could regulate affibody binding to the cell surface receptor HER2, providing a selective cell tagging technology through external NIR light. In particular, the tissue-penetrating ability of NIR light allowed for facile prodrug activation in living mice, enabling noninvasive, remote-controlled rescue of drug molecules. Given its broad adaptability, this CAT-NIR system may open new opportunities for manipulating the functions of bioactive molecules in living animals using external NIR light with spatiotemporal resolution.

Optical Control of Protein Functions via Genetically Encoded Photocaged Aspartic Acids.

Site-specific protein decaging by light has become an effective approach for in situ manipulation of protein activities in a gain-of-function fashion. Although successful decaging of amino acid side chains of Lys, Tyr, Cys, and Glu has been demonstrated, this strategy has not been extended to aspartic acid (Asp), an essential amino acid residue with a range of protein functions and protein-protein interactions. We herein reported a genetically encoded photocaged Asp and applied it to the photocontrolled manipulation of a panel of proteins including firefly luciferase, kinases (e.g., BRAF), and GTPase (e.g., KRAS) as well as mimicking the in situ phosphorylation event on kinases. As a new member of the increasingly expanded amino acid-decaging toolbox, photocaged Asp may find broad applications for gain-of-function study of diverse proteins as well as biological processes in living cells.

Deficiency of BAP1 inhibits neuroblastoma tumorigenesis through destabilization of MYCN.

The transcription factor MYCN is frequently amplified and overexpressed in a variety of cancers including high-risk neuroblastoma (NB) and promotes tumor cell proliferation, survival, and migration. Therefore, MYCN is being pursued as an attractive therapeutic target for selective inhibition of its upstream regulators because MYCN is considered a "undruggable" target. Thus, it is important to explore the upstream regulators for the transcription and post-translational modification of MYCN. Here, we report that BRCA1-associated protein-1 (BAP1) promotes deubiquitination and subsequent stabilization of MYCN by directly binding to MYCN protein. Furthermore, BAP1 knockdown inhibits NB tumor cells growth and migration in vitro and in vivo, which can be rescued partially by ectopic expression of MYCN. Importantly, depletion of BAP1 confers cellular resistance to bromodomain and extraterminal (BET) protein inhibitor JQ1 and Aurora A kinase inhibitor Alisertib. Furthermore, IHC results of NB tissue array confirmed the positive correlation between BAP1 and MYCN protein. Altogether, our work not only uncovers an oncogenic function of BAP1 by stabilizing MYCN, but also reveals a critical mechanism for the post-translational regulation of MYCN in NB. Our findings further indicate that BAP1 could be a potential therapeutic target for MYCN-amplified neuroblastoma.

Deuteration Degree-Controllable Methylation via a Cascade Assembly Strategy using Methylamine-Water as Methyl Source.

We present a novel and effective photocatalytic method for the methylation of ß-diketones with controllable degrees of deuterium incorporation via development of new methyl sources. By utilizing a methylamine-water system as the methyl precursor and a cascade assembly strategy for deuteration degree control, we synthesized methylated compounds with varying degrees of deuterium incorporation, showcasing the versatility of this approach. We examined a range of ß-diketone substrates and synthesized key intermediates for drug and bioactive compounds with varying degrees of deuterium incorporation, ranging from 0 to 3. We also investigated and discussed the postulated reaction pathway. This work demonstrates the utility of readily available reagents, methylamines and water, as a new methyl source, and provides a simple and efficient strategy for the synthesis of degree-controllable deuterium-labelled compounds.

Fmoc-diphenylalanine gelating nanoarchitectonics: A simplistic peptide self-assembly to meet complex applications.

9-fluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF), has been has been extensively explored due to its ultrafast self-assembly kinetics, inherent biocompatibility, tunable physicochemical properties, and especially, the capability of forming self-sustained gels under physiological conditions. Consequently, various methodologies to develop Fmoc-FF gels and their corresponding applications in biomedical and industrial fields have been extensively studied. Herein, we systemically summarize the mechanisms underlying Fmoc-FF self-assembly, discuss the preparation methodologies of Fmoc-FF hydrogels, and then deliberate the properties as well as the diverse applications of Fmoc-FF self-assemblies. Finally, the contemporary shortcomings which limit the development of Fmoc-FF self-assembly are raised and the alternative solutions are proposed, along with future research perspectives.

Photocatalytic Chemical Crosslinking for Profiling RNA-Protein Interactions in Living Cells.

The dynamic interactions between RNAs and proteins play crucial roles in regulating diverse cellular processes. Proteome-wide characterization of these interactions in their native cellular context remains desirable but challenging. Herein, we developed a photocatalytic crosslinking (PhotoCAX) strategy coupled with mass spectrometry (PhotoCAX-MS) and RNA sequencing (PhotoCAX-seq) for the study of the composition and dynamics of protein-RNA interactions. By integrating the blue light-triggered photocatalyst with a dual-functional RNA-protein crosslinker (RP-linker) and the phase separation-based enrichment strategy, PhotoCAX-MS revealed a total of 2044 RBPs in human HEK293 cells. We further employed PhotoCAX to investigate the dynamic change of RBPome in macrophage cells upon LPS-stimulation, as well as the identification of RBPs interacting directly with the 5' untranslated regions of SARS-CoV-2 RNA.

Bioorthogonally Activatable Base Editing for On-Demand Pyroptosis.

Pyroptosis is an inflammatory cell death form triggered by protease-mediated truncation and release of the N-terminal pore-forming domain of the gasdermin (GSDM) family proteins in various cell types. We report a Bioorthogonally ACtivatable Base editor (BaseBAC) for in situ and on-demand initiation of cell-type-specific pyroptosis. We first made the enzymatic activity of a cytosine base editor (CBE) switchable by establishing a bioorthogonal blockage on the PAM-interacting residue to control its DNA-binding ability. The resulting BaseBAC allowed in situ control of base editing on the GSDME gene that switched to the truncated expression of its N-terminal domain to activate pyroptosis. BaseBAC offers a general method for on-demand awakening of functional domains of self-inhibiting proteins and the corresponding cellular processes with high specificity in living systems.

MicroRNA expression is deregulated by aberrant methylation in B-cell acute lymphoblastic leukemia mouse model.

BACKGROUND: The expression of microRNAs (miRNAs) in the serum of B-cell acute lymphoblastic leukemia (B-ALL) patients is abnormal. Nevertheless, the underlying mechanism remains unclear. Recent studies indicate that the methylation state of circulating cell-free DNA (cfDNA) is different between cancer patients and healthy individuals. Therefore, we speculate that abnormal expression of miRNA may be associated with cfDNA methylation. METHODS: A green fluorescent protein (GFP) labeled B-ALL transplantation animal model was established to explore the relationship between the miRNA expression and cfDNA methylation of the related gene. Quantitative real-time PCR (qRT-PCR) was used to detect the expression levels of miRNAs. Further, cfDNA methylation levels of the related genes were evaluated through bisulfite sequencing polymerase chain reaction (BSP). RESULTS: The expression levels of miR-196b, miR-203, miR-34a-5p, miR-335-3p, miR-34b-5p, miR-615, miR-375-3p and miR-193b-5p in the serum of the model mice were significantly lower than those of the control group (P < 0.05). The methylation level of miR-196b promoter in cfDNA of the model group was significantly lower than that of the control group (P < 0.05), whereas no significant difference was noted in miR-203 promoter. The methylation levels of miR-196b and miR-203 coding region in cfDNA of the model group were significantly higher than those of the control group (P < 0.05). CONCLUSIONS: These results showed that CpG island hypermethylation in the miRNA coding region of cfDNA is related to the low expression of miR-196b and miR-203.

Bioorthogonal Photocatalytic Decaging-Enabled Mitochondrial Proteomics.

Spatiotemporally resolved dissection of subcellular proteome is crucial to our understanding of cellular functions in health and disease. We herein report a bioorthogonal and photocatalytic decaging-enabled proximity labeling strategy (CAT-Prox) for spatiotemporally resolved mitochondrial proteome profiling in living cells. Our systematic survey of the photocatalysts has led to the identification of Ir(ppy)2bpy as a bioorthogonal and mitochondria-targeting catalyst that allowed photocontrolled, rapid rescue of azidobenzyl-caged quinone methide as a highly reactive Michael acceptor for proximity-based protein labeling in mitochondria of live cells. Upon careful validation through in vitro labeling, mitochondria-targeting specificity, in situ catalytic activity as well as protein tagging, we applied CAT-Prox for mitochondria proteome profiling in living Hela cells as well as hard-to-transfect macrophage RAW264.7 cells with approximately 70% mitochondria specificity observed from up to 300 proteins enriched. Finally, CAT-Prox was further applied to the dynamic dissection of mitochondria proteome of macrophage cells upon lipopolysaccharide stimulation. By integrating photocatalytic decaging chemistry with proximity-based protein labeling, CAT-Prox offers a general, catalytic, and nongenetic alternative to the enzyme-based proximity labeling strategies for diverse live cell settings.

Unleashing the Power of Bond Cleavage Chemistry in Living Systems.

Bioorthogonal cleavage chemistry has been rapidly emerging as a powerful tool for manipulation and gain-of-function studies of biomolecules in living systems. While the initial bond formation-centered bioorthogonal reactions have been widely adopted for labeling, tracing, and capturing biomolecules, the newly developed bond cleavage-enabled bioorthogonal reactions have opened new possibilities for rescuing small molecules as well as biomacromolecules in living systems, allowing multidimensional controls over biological processes in vitro and in vivo. In this Outlook, we first summarized the development and applications of bioorthogonal cleavage reactions (BCRs) that restore the functions of chemical structures as well as more complex networks, including the liberation of prodrugs, release of bioconjugates, and in situ reactivation of intracellular proteins. As we embarked on this fruitful progress, we outlined the unmet scientific needs and future directions along this exciting avenue. We believe that the potential of BCRs will be further unleashed when combined with other frontier technologies, such as genetic code expansion and proximity-enabled chemical labeling.

Copper-Triggered Bioorthogonal Cleavage Reactions for Reversible Protein and Cell Surface Modifications.

Temporal and reversible control over protein and cell conjugations holds great potential for traceless release of antibody-drug conjugates (ADCs) on tumor sites as well as on-demand altering or removal of targeting elements on cell surface. We herein developed a bioorthogonal and traceless releasable reaction on proteins and intact cells to fulfill such purposes. A systematic survey of transition metals in catalyzing the bioorthogonal cleavage reactions revealed that copper complexes such as Cu(I)-BTTAA and dual-substituted propargyl (dsPra) or propargyloxycarbonyl (dsProc) moieties offered a bioorthogonal releasable pair for reversible blockage and rescue of primary amines and phenol alcohols on small molecule drugs, protein side chains, as well as intact cell surface. For proof-of-concept, we employed such Cu(I)-BTTAA/dsProc and Cu(I)-BTTAA/dsPra pairs as a "traceless linker" strategy to construct cleavable ADCs to unleash cytotoxic compounds on cancer cells in situ and as a "reversible modification" strategy for cell surface engineering. Furthermore, by coupling with the genetic code expansion strategy, we site-specifically modulated ligand-receptor interactions on live cell membranes. Together, our work expanded the transition-metal-mediated bioorthogonal cleavage tool kit from terminal decaging to internal-linker breakage, which offered a temporal and reversible conjugation strategy on therapeutic proteins and cells.

Reductive Cross-Coupling of Aldehydes and Imines Mediated by Visible Light Photoredox Catalysis.

Under photoredox catalysis conditions, the conventional electrophilic reactivity of ketimines is inverted to generate nucleophilic species. As a result, chemoselective cross-electrophile couplings between aldehydes and ketimines are achieved via umpolung reactivity of ketimines to furnish amino alcohols (44 examples with good to excellent yields). To illustrate the utility of the amino alcohol products, 1,2-dihydroindol-3-one-based fluorophores are easily synthesized using the coupling products. Finally, a plausible reaction pathway is discussed.

Visible light-promoted CO2 fixation with imines to synthesize diaryl α-amino acids.

Light-mediated transformations with CO2 have recently attracted great attention, with the focus on CO2 incorporation into C-C double and triple bonds, organohalides and amines. Herein is demonstrated visible light -mediated umpolung imine reactivity capable of engaging CO2 to afford α-amino acid derivatives. By employing benzophenone ketimine derivatives, CO2 fixation by hydrocarboxylation of C=N double bonds is achieved. Good to excellent yields of a broad range of α,α-disubstituted α-amino acid derivatives are obtained under mild conditions (rt, atmospheric pressure of CO2, visible light). A procedure that avoids tedious chromatographic purification and uses sustainable sunlight is developed to highlight the simplicity of this method.

Synergistic enzymatic and bioorthogonal reactions for selective prodrug activation in living systems.

Adverse drug reactions (ADRs) restrict the maximum doses applicable in chemotherapy, which leads to failure in cancer treatment. Various approaches, including nano-drug and prodrug strategies aimed at reducing ADRs, have been developed, but these strategies have their own pitfalls. A renovated strategy for ADR reduction is urgently needed. Here, we employ an enzymatic supramolecular self-assembly process to accumulate a bioorthogonal decaging reaction trigger inside targeted cancer cells, enabling spatiotemporally controlled, synergistic prodrug activation. The bioorthogonally activated prodrug exhibits significantly enhanced potency against cancer cells compared with normal cells. This prodrug activation strategy further demonstrates high tumour inhibition efficacy with satisfactory biocompatibility, pharmacokinetics, and safety in vivo. We envision that integration of enzymatic and bioorthogonal reactions will serve as a general small-molecule-based strategy for alleviation of ADRs in chemotherapy.