Chemical labeling methods for proteins are highly researched. Herein, we introduced β-carbonyl sulfonium compounds for selective cysteine modification in proteins within biological systems. Structural tuning led to sulfonium-based probes with high reactivity and selectivity. These probes show excellent biocompatibility, cell uptake, and specificity towards cysteine profiling in live cells.

The application scope of various bioconjugation technologies has been expanded through the implementation of photoredox catalytic bioconjugation technology, establishing a novel biomolecular framework with exceptional residue selectivity. Herein, we report a facile acylation reaction utilizing a thioacid and a photoexcited riboflavin tetraacetate (RFTA) catalyst under visible light (450 nm). The optimal reaction conditions were obtained using a high-throughput microfluidic-based chemical robotic system and GPR model. We have successfully modified a range of proteins and antibodies and utilized the functional handle to attach diverse biological molecules, demonstrating the versatility and generality of our approach. In addition, this photoredox catalytic reaction was also successfully employed in peptide modification, protein labeling, and antibody conjugation. In our work, we contribute to the photoredox catalytic lysine acylation in proteins and peptides. The optimal reaction conditions were obtained using a high-throughput microfluidic-based chemical robotic system and GPR model.

The copper-free azide-alkyne cycloaddition wasbroadly appliedin numerous research fields. Herein, we report a facile Cu-free clickreaction utilizing fluoride-responsive azide and alkynyl pyridiniumcycloaddition at ambient temperatures in aqueous media. The reactivityof alkynyl pyridinium was successfully masked by a silyl-protectinggroup at the alkyne group, and the deprotection could be readily achievedwith the addition of F-, which renders the reactivity.The substrates were readily synthesized and proven to be stable atthe bench. This bioorthogonal fluoride-responsive click reaction wasthen successfully employed in peptide modification, protein labeling,and cell imaging, suggesting its potential in various applications.

Cell surface proteins (CSPs) are valuable targets for therapeutic agents, but achieving highly selective CSP enrichment in cellular physiology remains a technical challenge. To address this challenge, we propose a newly developed sulfo-pyridinium ester (SPE) cross-linking probe, followed by two-step imaging and enrichment. The SPE probe showed higher efficiency in labeling proteins than similar NHS esters at the level of cell lysates and demonstrated specificity for Lys in competitive experiments. More importantly, this probe could selectively label the cell membranes in cell imaging with only negligible labeling of the intracellular compartment. Moreover, we successfully performed this strategy on MCF-7 live cells to label 425 unique CSPs from 1162 labeled proteins. Finally, we employed our probe to label the CSPs of insulin-cultured MCF-7, revealing several cell surface targets of key functional biomarkers and insulin-associated pathogenesis. The above results demonstrate that the SPE method provides a promising tool for the selective labeling of cell surface proteins and monitoring transient cell surface events.

Histone lysine crotonylation (Kcr) is one newly discovered acylation modification and regulates numerous pathophysiological processes. The binding affinity between Kcr and its interacting proteins is generally weak, which makes it difficult to effectively identify Kcr-interacting partners. Changing the amide of crotonyl to an ester increased reactivity with proximal cysteines and retained specificity for Kcr antibody. We then designed the probe "H3g27Cr" by incorporating the ester functionality into a H3K27 peptide. Using this probe, we successfully identified multiple Kcr-interacting partners including STAT3, which had not reported previously. Further experiments suggested that STAT3 possibly could form complexes with Histone deacetylase HDACs to downregulate the acetylation and crotonylation of Histone H3K27. Our unique design provided intriguing tools to further explore Kcr-interacting proteins and elucidate their working mechanisms.

Over the past 20 years, great efforts have been invested in developing site-specific approaches to protein modification to dissect protein functions directly and accurately. Here, we report a proximity-triggered group transfer strategy from a sulfonium warhead to a Cysteine (Cys) residue of the target protein. With a guiding ligand, cargoes could be transferred selectively from a sulfonium center onto the Cys residue in the vicinity of their binding interface. The successful thalidomide transfer of sulfonium 1-X could be applied intracellularly for epidermal growth factor receptor degradation, highlighting the potential of group transfer strategy as a suite of chemical biology studies, including cell imaging, protein profiling, and protein degradation by simply employing different transferrable groups. [GRAPHICS] .

The ligand-directed (LD) chemistry provides powerful tools for site-specific modification of proteins. We utilized a peptide with an appended methionine (Met) as a ligand; then, the Met thioether was modified into sulfonium which enabled a proximity induced group transfer onto protein cysteine in the vicinity upon peptide-target binding. The sulfonium warhead could be easily constructed with unprotected peptides, and the transferable group scope was conducted on model protein PDZ and its ligand peptides. In addition, a living cell labeling was successfully achieved.

A biomimetic method has been established for the chemo-selective desulfurization of cysteinyl peptides and proteins in aqueous media. The derivatives of biocatalytic cofactors, flavins, were found to be efficient photosensitizers in a thiyl-radical-mediated desulfurization of Cys. The reaction was conducted in an ultrafast manner with both polypeptides and proteins.

Histidine (His, H) undergoes various post-translational modifications (PTMs) and plays multiple roles in protein interactions and enzyme catalyzed reactions. However, compared with other amino acids such as Lys or Cys, His modification is much less explored. Herein we describe a novel visible-light-driven thioacetal activation reaction which enables facile modification on histidine residues. An efficient addition to histidine imidazole N3 under biocompatible conditions was achieved with an electrophilic thionium intermediate. This method allows chemo-selective modification on peptides and proteins with good conversions and efficient histidine-proteome profiling with cell lysates. 78 histidine containing proteins were for the first time found with significant enrichment, most functioning in metal accumulation in brain related diseases. This facile His modification method greatly expands the chemo-selective toolbox for histidine-targeted protein conjugation and helps to reveal histidine's role in protein functions.

Herein, we report the first facile Cu-free click reaction between alkynyl sulfonium and azide at ambient temperatures in aqueous media. DFT computations indicate that the sulfonium group is the key factor to gaining reactivity by stabilizing LUMO+1 and influencing the charge distribution of the triple bond. Sulfonium alkynes can be easily synthesized and scaled up, and most of them are biocompatible. We prepared candidate molecules and tested their use in multiple proof-of-concept biological applications.

A novel nanobody-drug conjugate (NDC) was constructed by incorporating an amphipathic peptide, GALA, which improved the cytotoxicity by one to two orders of magnitude. Mechanistic studies demonstrate that tethering to lipids induces GALA to form a helix, which dramatically enhances endocytosis. Our work provides a general strategy not only for improving the anti-cancer efficacy of protein-drug conjugates but also for increasing the efficiency of other types of endocytosis-dependent cell delivery.

A novel nanobody-drug conjugate (NDC) was constructed by incorporating an amphipathic peptide, GALA, which improved the cytotoxicity by one to two orders of magnitude. Mechanistic studies demonstrate that tethering to lipids induces GALA to form a helix, which dramatically enhances endocytosis. Our work provides a general strategy not only for improving the anti-cancer efficacy of protein-drug conjugates but also for increasing the efficiency of other types of endocytosis-dependent cell delivery.