Studying posttranslational modifications classically relies on experimental strategies that oversimplify the complex biosynthetic machineries of living cells. Protein glycosylation contributes to essential biological processes, but correlating glycan structure, underlying protein, and disease-relevant biosynthetic regulation is currently elusive. Here, we engineer living cells to tag glycans with editable chemical functionalities while providing information on biosynthesis, physiological context, and glycan fine structure. We introduce a non-natural substrate biosynthetic pathway and use engineered glycosyltransferases to incorporate chemically tagged sugars into the cell surface glycome of the living cell. We apply the strategy to a particularly redundant yet disease-relevant human glycosyltransferase family, the polypeptide N-acetylgalactosaminyl transferases. This approach bestows a gain-of-chemical-functionality modification on cells, where the products of individual glycosyltransferases can be selectively characterized or manipulated to understand glycan contribution to major physiological processes.

O-Linked α-<i>N</i>-acetylgalactosamine (O-GalNAc) glycans constitute a major part of the human glycome. They are difficult to study because of the complex interplay of 20 distinct glycosyltransferase isoenzymes that initiate this form of glycosylation, the polypeptide <i>N</i>-acetylgalactosaminyltransferases (GalNAc-Ts). Despite proven disease relevance, correlating the activity of individual GalNAc-Ts with biological function remains challenging due to a lack of tools to probe their substrate specificity in a complex biological environment. Here, we develop a "bump-hole" chemical reporter system for studying GalNAc-T activity in vitro. Individual GalNAc-Ts were rationally engineered to contain an enlarged active site (hole) and probed with a newly synthesized collection of 20 (bumped) uridine diphosphate <i>N</i>-acetylgalactosamine (UDP-GalNAc) analogs to identify enzyme<b>-</b>substrate pairs that retain peptide specificities but are otherwise completely orthogonal to native enzyme<b>-</b>substrate pairs. The approach was applicable to multiple GalNAc-T isoenzymes, including GalNAc-T1 and -T2 that prefer nonglycosylated peptide substrates and GalNAcT-10 that prefers a preglycosylated peptide substrate. A detailed investigation of enzyme kinetics and specificities revealed the robustness of the approach to faithfully report on GalNAc-T activity and paves the way for studying substrate specificities in living systems.

Because the backbone of most organic molecules is composed primarily of carbon-carbon bonds, the development of efficient methods for their construction is one of the central challenges of organic synthesis. Transition metal-catalyzed cross-coupling reactions between organic electrophiles and nucleophiles serve as particularly powerful tools for achieving carbon-carbon bond formation. Until recently, the vast majority of cross-coupling processes had used either aryl or alkenyl electrophiles as one of the coupling partners. In the past 15 years, versatile new methods have been developed that effect cross-couplings of an array of alkyl electrophiles, thereby greatly expanding the diversity of target molecules that are readily accessible. The ability to couple alkyl electrophiles opens the door to a stereochemical dimension-specifically, enantioconvergent couplings of racemic electrophiles-that substantially enhances the already remarkable utility of cross-coupling processes.

An asymmetric decarboxylative Csp(3)-Csp(2) cross-coupling has been achieved via the synergistic merger of photoredox and nickel catalysis. This mild, operationally simple protocol transforms a wide variety of naturally abundant α-amino acids and readily available aryl halides into valuable chiral benzylic amines in high enantiomeric excess, thereby producing motifs found in pharmacologically active agents.

Studying posttranslational modifications classically relies on experimental strategies that oversimplify the complex biosynthetic machineries of living cells. Protein glycosylation contributes to essential biological processes, but correlating glycan structure, underlying protein, and disease-relevant biosynthetic regulation is currently elusive. Here, we engineer living cells to tag glycans with editable chemical functionalities while providing information on biosynthesis, physiological context, and glycan fine structure. We introduce a non-natural substrate biosynthetic pathway and use engineered glycosyltransferases to incorporate chemically tagged sugars into the cell surface glycome of the living cell. We apply the strategy to a particularly redundant yet disease-relevant human glycosyltransferase family, the polypeptide N-acetylgalactosaminyl transferases. This approach bestows a gain-of-chemical-functionality modification on cells, where the products of individual glycosyltransferases can be selectively characterized or manipulated to understand glycan contribution to major physiological processes.

O-Linked α-<i>N</i>-acetylgalactosamine (O-GalNAc) glycans constitute a major part of the human glycome. They are difficult to study because of the complex interplay of 20 distinct glycosyltransferase isoenzymes that initiate this form of glycosylation, the polypeptide <i>N</i>-acetylgalactosaminyltransferases (GalNAc-Ts). Despite proven disease relevance, correlating the activity of individual GalNAc-Ts with biological function remains challenging due to a lack of tools to probe their substrate specificity in a complex biological environment. Here, we develop a "bump-hole" chemical reporter system for studying GalNAc-T activity in vitro. Individual GalNAc-Ts were rationally engineered to contain an enlarged active site (hole) and probed with a newly synthesized collection of 20 (bumped) uridine diphosphate <i>N</i>-acetylgalactosamine (UDP-GalNAc) analogs to identify enzyme<b>-</b>substrate pairs that retain peptide specificities but are otherwise completely orthogonal to native enzyme<b>-</b>substrate pairs. The approach was applicable to multiple GalNAc-T isoenzymes, including GalNAc-T1 and -T2 that prefer nonglycosylated peptide substrates and GalNAcT-10 that prefers a preglycosylated peptide substrate. A detailed investigation of enzyme kinetics and specificities revealed the robustness of the approach to faithfully report on GalNAc-T activity and paves the way for studying substrate specificities in living systems.

Because the backbone of most organic molecules is composed primarily of carbon-carbon bonds, the development of efficient methods for their construction is one of the central challenges of organic synthesis. Transition metal-catalyzed cross-coupling reactions between organic electrophiles and nucleophiles serve as particularly powerful tools for achieving carbon-carbon bond formation. Until recently, the vast majority of cross-coupling processes had used either aryl or alkenyl electrophiles as one of the coupling partners. In the past 15 years, versatile new methods have been developed that effect cross-couplings of an array of alkyl electrophiles, thereby greatly expanding the diversity of target molecules that are readily accessible. The ability to couple alkyl electrophiles opens the door to a stereochemical dimension-specifically, enantioconvergent couplings of racemic electrophiles-that substantially enhances the already remarkable utility of cross-coupling processes.

An asymmetric decarboxylative Csp(3)-Csp(2) cross-coupling has been achieved via the synergistic merger of photoredox and nickel catalysis. This mild, operationally simple protocol transforms a wide variety of naturally abundant α-amino acids and readily available aryl halides into valuable chiral benzylic amines in high enantiomeric excess, thereby producing motifs found in pharmacologically active agents.

Alkylboron compounds are an important family of target molecules, serving as useful intermediates, as well as end points, in fields such as pharmaceutical science and organic chemistry. Facile transformation of carbon-boron bonds into a wide variety of carbon-X bonds (where X is, for example, carbon, nitrogen, oxygen, or a halogen), with stereochemical fidelity, renders the generation of enantioenriched alkylboronate esters a powerful tool in synthesis. Here we report the use of a chiral nickel catalyst to achieve stereoconvergent alkyl-alkyl couplings of readily available racemic α-haloboronates with organozinc reagents under mild conditions. We demonstrate that this method provides straightforward access to a diverse array of enantioenriched alkylboronate esters, in which boron is bound to a stereogenic carbon, and we highlight the utility of these compounds in synthesis.

In vitro, PD-AuNPs was associated with upregulated expression of the chondrogenic markers aggrecan (ACAN) (∼ 1.6-fold) and SRY-box transcription factor 9 (SOX9) (∼ 1.2-fold) compared with AuNPs alone. In vivo, PD-AuNPs improved repair outcomes in the defect model, showing higher scores for defect filling (3.7 vs. 2.3), osteochondral reconstitution (1.7 vs. 0.6), cell morphology (2.3 vs. 1.2), and matrix staining (3.3 vs. 1.3), along with enhanced ACAN and SOX9 expression. In the OA model, PD-AuNP was associated with reduced OA-related changes, including improved weight-bearing (49.5 % vs. 47.0 %), reduced joint swelling, and a markedly lower final OARSI score (2.7 vs. 12.8). They also increased ACAN and SOX9 while decreasing IL-6 and MMP-9 compared with AuNPs. Collectively, these results suggest that PD-AuNPs contribute to cartilage preservation and repair, offering a potential approach for improving cartilage regeneration strategies.

Bifunctional <i>trans</i>-cyclooctene (bTCO) with a carbamate or carbonate at the allylic position and tetrazine provide a promising bioorthogonal click chemistry pair for the click-to-release approach, successfully employed in various biotechnological applications. Herein, we demonstrate a simple and straightforward method to synthesize C₂TCO, a symmetrical bTCO derivative with two hydroxyl groups at the allylic positions. The efficiently synthesized C₂TCO at first was selectively functionalized with a fluorophore (C₂TCO-FL), and the conjugate was labeled onto monoclonal antibodies (Ab-C₂TCO-FL). The fluorophore of Ab-C₂TCO-FL was easily removed from the antibody through the mild treatment of tetrazine, enabling multicycle fluorescent bioimaging. Next, an antibody-drug conjugate targeting PD-L1 was prepared using the linker based on C₂TCO. The cytotoxic payload was efficiently released from the antibody upon tetrazine treatment, which induced cellular cytotoxicity.

Proteolysis-targeting chimeras (PROTACs) degrade target proteins through the ubiquitin-proteasome system. To date, PROTACs are primarily used to treat various diseases; however, they have not been applied in regenerative therapy. Herein, this work introduces MDM2-targeting PROTACs customized for application in bone regeneration. An MDM2-PROTAC library is constructed by combining Nutlin-3 and CRBN ligands with various linker designs. Through a multistep validation process, this work develops MDM2-PROTACs (CL144 and CL174) that presented potent degradation efficiency and a robust inductive effect on the biomineralization. Next, this work performs whole-transcriptome analysis to dissect the biological effects of the CL144, and reveals the upregulation of osteogenic marker genes. Furthermore, CL144 effectively induced bone regeneration in bone graft and ovariectomy (OVX) models after local and systemic administration, respectively. In the OVX model, the combination treatment with CL144 and alendronate induced a synergistic effect. Overall, this study demonstrates the promising role of MDM2-PROTAC in promoting bone regeneration, marking the first step toward expanding the application of the PROTAC technology.