Photocatalytic Activation of Aryl(trifluoromethyl) Diazos to Carbenes for High-Resolution Protein Labeling with Red Light.

State-of-the-art methods in photoproximity labeling center on the targeted generation and capture of short-lived reactive intermediates to provide a snapshot of local protein environments. Diazirines are the current gold standard for high-resolution proximity labeling, generating short-lived aryl(trifluoromethyl) carbenes. Here, we present a method to access aryl(trifluoromethyl) carbenes from a stable diazo source via tissue-penetrable, deep red to near-infrared light (600-800 nm). The operative mechanism of this activation involves Dexter energy transfer from photoexcited osmium(II) photocatalysts to the diazo, thus revealing an aryl(trifluoromethyl) carbene. The labeling preferences of the diazo probe with amino acids are studied, showing high reactivity toward heteroatom-H bonds. Upon the synthesis of a biotinylated diazo probe, labeling studies are conducted on native proteins as well as proteins conjugated to the Os photocatalyst. Finally, we demonstrate that the conjugation of a protein inhibitor to the photocatalyst also enables selective protein labeling in the presence of spectator proteins and achieves specific labeling of a membrane protein on the surface of mammalian cells via a two-antibody photocatalytic system.

Extracellular vesicles released by cancer-associated fibroblast-induced myeloid-derived suppressor cells inhibit T-cell function.

Myeloid cells are known to play a crucial role in creating a tumor-promoting and immune suppressive microenvironment. Our previous study demonstrated that primary human monocytes can be polarized into immunosuppressive myeloid-derived suppressor cells (MDSCs) by cancer-associated fibroblasts (CAFs) in a 3D co-culture system. However, the molecular mechanisms underlying the immunosuppressive function of MDSCs, especially CAF-induced MDSCs, remain poorly understood. Using mass spectrometry-based proteomics, we compared cell surface protein changes among monocytes, in vitro differentiated CAF-induced MDSCs, M1/M2 macrophages, and dendritic cells, and identified an extracellular vesicle (EV)-mediated secretory phenotype of MDSCs. Functional assays using an MDSC/T-cell co-culture system revealed that blocking EV generation in CAF-induced MDSCs reversed their ability to suppress T-cell proliferation, while EVs isolated from CAF-induced MDSCs directly inhibited T-cell function. Furthermore, we identified fructose bisphosphatase 1 (FBP1) as a cargo protein that is highly enriched in EVs isolated from CAF-induced MDSCs, and pharmacological inhibition of FBP1 partially reversed the suppressive phenotype of MDSCs. Our findings provide valuable insights into the cell surface proteome of different monocyte-derived myeloid subsets and uncover a novel mechanism underlying the interplay between CAFs and myeloid cells in shaping a tumor-permissive microenvironment.

Dual fluorescent labeling of GLP-1R in live cells via enzymatic tagging and bioorthogonal chemistry.

To study GPCR conformational dynamics in live cells, here we report an integrated approach combining enzymatic SNAP-tagging with bioorthogonal chemistry for dual fluorescent labeling of GLP-1R. The resulting GLP-1R conformational biosensors permit a FRET-based analysis of the receptor subdomain movement in response to ligand stimulation in live cells.

Cancer-Associated Fibroblasts Promote Immunosuppression by Inducing ROS-Generating Monocytic MDSCs in Lung Squamous Cell Carcinoma.

Cancer-associated fibroblasts (CAF) represent a functionally heterogeneous population of activated fibroblasts that constitutes a major component of tumor stroma. Although CAFs have been shown to promote tumor growth and mediate resistance to chemotherapy, the mechanisms by which they may contribute to immune suppression within the tumor microenvironment (TME) in lung squamous cell carcinoma (LSCC) remain largely unexplored. Here, we identified a positive correlation between CAF and monocytic myeloid cell abundances in 501 primary LSCCs by mining The Cancer Genome Atlas data sets. We further validated this finding in an independent cohort using imaging mass cytometry and found a significant spatial interaction between CAFs and monocytic myeloid cells in the TME. To delineate the interplay between CAFs and monocytic myeloid cells, we used chemotaxis assays to show that LSCC patient-derived CAFs promoted recruitment of CCR2+ monocytes via CCL2, which could be reversed by CCR2 inhibition. Using a three-dimensional culture system, we found that CAFs polarized monocytes to adopt a myeloid-derived suppressor cell (MDSC) phenotype, characterized by robust suppression of autologous CD8+ T-cell proliferation and IFNγ production. We further demonstrated that inhibiting IDO1 and NADPH oxidases, NOX2 and NOX4, restored CD8+ T-cell proliferation by reducing reactive oxygen species (ROS) generation in CAF-induced MDSCs. Taken together, our study highlights a pivotal role of CAFs in regulating monocyte recruitment and differentiation and demonstrated that CCR2 inhibition and ROS scavenging abrogate the CAF-MDSC axis, illuminating a potential therapeutic path to reversing the CAF-mediated immunosuppressive microenvironment.

Spirohexene-Tetrazine Ligation Enables Bioorthogonal Labeling of Class B G Protein-Coupled Receptors in Live Cells.

A new bioorthogonal reactant pair, spiro[2.3]hex-1-ene (Sph) and 3,6-di(2-pyridyl)-s-tetrazine (DpTz), for the strain-promoted inverse electron-demand Diels-Alder cycloaddition, that is, tetrazine ligation, is reported. As compared to the previously reported strained alkenes such as trans-cyclooctene (TCO) and 1,3-disubstituted cyclopropene, Sph exhibits balanced reactivity and stability in tetrazine ligation with the protein substrates. A lysine derivative of Sph, SphK, was site-selectively incorporated into the extracellular loop regions (ECLs) of GCGR and GLP-1R, two members of class B G protein-coupled receptors (GPCRs) in mammalian cells with the incorporation efficiency dependent on the location. Subsequent bioorthogonal reactions with the fluorophore-conjugated DpTz reagents afforded the fluorescently labeled GCGR and GLP-1R ECL mutants with labeling yield as high as 68%. A multitude of functional assays were performed with these GPCR mutants, including ligand binding, ligand-induced receptor internalization, and ligand-stimulated intracellular cAMP accumulation. Several positions in the ECL3s of GCGR and GLP-1R were identified that tolerate SphK mutagenesis and subsequent bioorthogonal labeling. The generation of functional, fluorescently labeled ECL3 mutants of GCGR and GLP-1R should allow biophysical studies of conformation dynamics of this important class of GPCRs in their native environment in live cells.

Sequence-Specific 2-Cyanobenzothiazole Ligation.

The use of small, natural chemical reporters in conjunction with catalyst-free bioorthogonal reactions will greatly streamline protein labeling in a cellular environment with minimum perturbation to their function. Here we report the discovery of a 2-cyanobenzothiazole (CBT)-reactive peptide tag, CX10R7, from a cysteine-encoded peptide phage library using the phage-assisted interrogation of reactivity method. Fusion of CX10R7 with a protein of interest allows site-specific labeling of the protein with CBT both in vitro and on the surface of E. coli cells. Mutagenesis studies indicated that the reactivity and specificity of CX10R7 are attributed to the sequence environment, in which the residues surrounding cysteine help to stabilize the ligation product.

Synthesis and Site-Specific Incorporation of Red-Shifted Azobenzene Amino Acids into Proteins.

A series of red-shifted azobenzene amino acids were synthesized in moderate-to-excellent yields via a two-step procedure in which tyrosine derivatives were first oxidized to the corresponding quinonoidal spirolactones followed by ceric ammonium nitrate-catalyzed azo formation with the substituted phenylhydrazines. The resulting azobenzene-alanine derivatives exhibited efficient trans/cis photoswitching upon irradiation with a blue (448 nm) or green (530 nm) LED light. Moreover, nine superfolder green fluorescent protein (sfGFP) mutants carrying the azobenzene-alanine analogues were expressed in E. coli in good yields via amber codon suppression with an orthogonal tRNA/PylRS pair, and one of the mutants showed durable photoswitching with the LED light.

A genetically encoded alkyne directs palladium-mediated protein labeling on live mammalian cell surface.

The merging of site-specific incorporation of small bioorthogonal functional groups into proteins via amber codon suppression with bioorthogonal chemistry has created exciting opportunities to extend the power of organic reactions to living systems. Here we show that a new alkyne amino acid can be site-selectively incorporated into mammalian proteins via a known orthogonal pyrrolysyl-tRNA synthetase/tRNACUA pair and directs an unprecedented, palladium-mediated cross-coupling reaction-driven protein labeling on live mammalian cell surface. A comparison study with the alkyne-encoded proteins in vitro indicated that this terminal alkyne is better suited for the palladium-mediated cross-coupling reaction than the copper-catalyzed click chemistry.

Synthesis of cell-permeable stapled BH3 peptide-based Mcl-1 inhibitors containing simple aryl and vinylaryl cross-linkers.

We report the synthesis of a series of distance-matching aryl and vinylaryl cross-linkers for constructing stapled peptides containing cysteines at i,i+7 positions. Langevin dynamics simulation studies helped to classify these cross-linkers into two categories: the rigid cross-linkers with narrower S-S distance distribution and the flexible cross-linkers with wider S-S distance distribution. The stapled Noxa BH3 peptides with the flexible distance-matching cross-linkers gave the highest degree of helicity as well as the most potent inhibitory activity against Mcl-1. However, the stapled peptides with the highest hydrophobicity showed the most efficient cellular uptake. Together, this work illustrates the divergent nature of binding affinity and cellular uptake, and the vital importance of choosing appropriate cross-linkers in constructing stapled peptides with the drug-like properties.

Storable N-phenylcarbamate palladacycles for rapid functionalization of an alkyne-encoded protein.

Here we report the synthesis of storable N-phenylcarbamate palladacycles that showed robust reactivity in the cross-coupling reaction with an alkyne-encoded protein with a second-order rate constant approaching 19 770 ± 930 M(-1) s(-1).

Fast and sequence-specific palladium-mediated cross-coupling reaction identified from phage display.

Fast and specific bioorthogonal reactions are highly desirable because they provide efficient tracking of biomolecules that are present in low abundance and/or involved in fast dynamic process in living systems. Toward this end, classic strategy involves the optimization of substrate structures and reaction conditions in test tubes, testing their compatibility with biological systems, devising synthetic biology schemes to introduce the modified substrates into living cells or organisms, and finally validating the superior kinetics for enhanced capacity in tracking biomolecules in vivo--a lengthy process often mired by unexpected results. Here, we report a streamlined approach in which the "microenvironment" of a bioorthogonal chemical reporter is exploited directly in biological systems via phage-assisted interrogation of reactivity (PAIR) to optimize not only reaction kinetics but also specificity. Using the PAIR strategy, we identified a short alkyne-containing peptide sequence showing fast kinetics (k2=13,000±2000 M(-1) s(-1)) in a palladium-mediated cross-coupling reaction. Site-directed mutagenesis studies suggested that the residues surrounding the alkyne moiety facilitate the assembly of a key palladium-alkyne intermediate along the reaction pathway. When this peptide sequence was inserted into the extracellular domain of epidermal growth factor receptor (EGFR), this reactive sequence directed the specific labeling of EGFR in live mammalian cells.

Photoclick chemistry: a fluorogenic light-triggered in vivo ligation reaction.

The ability to use chemical reactivity to monitor and control biomolecular processes with a spatial and temporal precision motivated the development of light-triggered in vivo chemistries. To this end, the photoinduced tetrazole-alkene cycloaddition, also termed 'photoclick chemistry' offers a very rapid chemical ligation platform for the manipulation of biomolecules and matrices in vivo. Here we outline the recent developments in the optimization of this chemistry, ranging from the search for substrates that offer two-photon photoactivatability, superior reaction kinetics, and/or genetic encodability, to the study of the reaction mechanism. The applications of the photoclick chemistry in protein labeling in vitro and in vivo as well as in preparing 'smart' hydrogels for 3D cell culture are highlighted.

Bioorthogonal chemistry: strategies and recent developments.

The use of covalent chemistry to track biomolecules in their native environment-a focus of bioorthogonal chemistry-has received considerable interest recently among chemical biologists and organic chemists alike. To facilitate wider adoption of bioorthogonal chemistry in biomedical research, a central effort in the last few years has been focused on the optimization of a few known bioorthogonal reactions, particularly with respect to reaction kinetics improvement, novel genetic encoding systems, and fluorogenic reactions for bioimaging. During these optimizations, three strategies have emerged, including the use of ring strain for substrate activation in the cycloaddition reactions, the discovery of new ligands and privileged substrates for accelerated metal-catalysed reactions, and the design of substrates with pre-fluorophore structures for rapid "turn-on" fluorescence after selective bioorthogonal reactions. In addition, new bioorthogonal reactions based on either modified or completely unprecedented reactant pairs have been reported. Finally, increasing attention has been directed toward the development of mutually exclusive bioorthogonal reactions and their applications in multiple labeling of a biomolecule in cell culture. In this feature article, we wish to present the recent progress in bioorthogonal reactions through the selected examples that highlight the above-mentioned strategies. Considering increasing sophistication in bioorthogonal chemistry development, we strive to project several exciting opportunities where bioorthogonal chemistry can make a unique contribution to biology in the near future.