Hydroxypyridyl-substituted 1,2,4,5-tetrazines have recently been developed as advanced scissors for bioorthogonal click-to-release chemistry. In contrast to the otherwise characteristic pink color of 1,2,4,5-tetrazines, these compounds were observed to give yellow solutions in methanol and buffered aqueous media. This study shows that the solvatochromism of hydroxypyridyl-tetrazines has its origin in varying protonation states of the phenolic OH group and reveals the impact of deprotonation on bioorthogonal click reactivity. We demonstrate that the rate of the Diels-Alder cycloaddition is significantly affected by the pH of the aqueous solution, thereby providing critical insights for the development of molecular tools for bioorthogonal bond cleavage.

Ovarian cancer remains a lethal malignancy due to chemoresistance and toxicity, which limits the dose of chemotherapy that can be used, necessitating the development of more targeted therapies such as antibody-drug conjugates (ADCs). However, conventional ADCs suffer from heterogeneity. This study aimed to develop a stable, homogeneous ADC by utilizing a bifunctional dibromomaleimide (DBM) linker to cross-link antibody cysteine residues via disulfide-bridging. A DBM linker was synthesized from 3,4-dibromofuran-2,5-dione and bound to the cytotoxic agent triptolide. This triptolide payload was then conjugated to trastuzumab via site-specific disulfide rebridging to yield a homogeneous trastuzumab-triptolide conjugate to target human epithelial growth factor receptor 2 (HER2) on ovarian cancer cells. The study evaluated the ADC's efficacy against SKOV-3 (high HER2 expression) and OVCAR-8 (low HER2 expression) cell lines. The results showed that the ADC was slightly more potent in SKOV-3 cells, yielding lower IC50 values compared to OVCAR-8. Mechanistic studies of the ADC via flow cytometry revealed that the ADC induced significant apoptosis and cell cycle arrest, characterized by a concentration-dependent increase in Caspase-3/7 expression and distinct alterations in cell population distribution. Furthermore, ADC treatment led to a concentration-dependent decrease in HER2 levels in SKOV-3 cells, confirming successful targeting. The study demonstrates that converting conventional maleimides into bifunctional DBM linkers allows the production of a homogeneous ADC via disulfide-bridging. This approach offers a promising strategy for developing potent anticancer therapeutics with improved selectivity for HER2-overexpressing ovarian cancers.

Maleimides are widely utilized in bioconjugation because they efficiently and selectively react with thiols. Owing to their favorable reaction kinetics, maleimides have found broad applications in bioconjugate chemistry, including the synthesis of antibody-drug conjugates for cancer therapy, fluorescence labeling of proteins, or functionalization of materials. As increasingly complex platforms are developed, multivalent linkers are being incorporated more frequently into synthetic strategies. Although the chemistry of monovalent maleimides is well understood, the chemoselectivity of multivalent maleimide linkers has not been fully established. To investigate differences in the chemoselectivity of mono- and bivalent maleimides, we performed bioconjugation of mal-PEG11-mal or mPEG12-mal with the physalis mottle virus-like particle. Through a series of chemical assays and characterization studies, we demonstrate that while monovalent maleimides are highly selective for cysteine modifications, bivalent maleimides exhibit cross-reactivity toward both lysine and cysteine residues. Overall, this study highlights how linker valency can influence the chemoselectivity of maleimide-based conjugation chemistry.

5-Fluorouracil (5-FU) is a chemotherapeutic drug that is widely used to treat gastrointestinal cancers (e.g., hepatocellular carcinoma). Unfortunately, its systemic toxicity limits its clinical utility; therefore, new means for its targeted delivery at the pathological site are highly sought after to enhance therapeutic efficacy. In this work, we describe a 5-FU covalent conjugate with the self-assembling, heterochiral tripeptide DLeu-Phe-Phe (lFF) via a redox-sensitive linker (5-FU-SS-lFF). The conjugate yields supramolecular hydrogels with rheological properties similar to those of lFF hydrogels and enables 5-FU release under reducing conditions, such as those present in the tumor microenvironment. We demonstrate the selectivity of the drug release under such conditions over a period of 24 h, with consequent significant cytotoxicity on cancer cells, as demonstrated on hepatocellular carcinoma spheroids. Overall, this study opens new opportunities for peptide-based supramolecular hydrogels as versatile and smart vehicles for the selective release of anticancer drugs under tumor-like reducing conditions.

Acute lung injury (ALI) involves inflammatory dysregulation and oxidative stress, processes in which cysteine (Cys), an endogenous sulfur-containing biothiol, participates through thiol redox homeostasis. We designed HD-Cys, an activatable hemicyanine probe, to visualize Cys in real-time in an LPS-induced murine ALI model. The reaction with Cys releases the emissive HD form and produces a pronounced near-infrared turn-on response. HD-Cys showed high selectivity, a detection limit of 160 nM, and a linear concentration-dependent response. Its performance was examined in BEAS-2B cells, serum, and mice with LPS-induced ALI. After probe administration, pulmonary fluorescence was stronger in ALI mice than in normal controls and decreased following N-ethylmaleimide (NEM)-mediated thiol depletion. Together, these findings establish HD-Cys as an experimental tool for tracking Cys-associated thiol redox changes during inflammatory lung injury.

Elucidating protein-protein interactions plays a crucial part in understanding disease mechanisms and advancing pharmacological research. Photocatalytic proximity labeling using antibody-catalyst conjugates enables the highly target-specific analysis of protein-protein interactions on the cell surface without altering the native cellular state through genetic manipulation. Here, we describe an extension of the deazaflavin-diazirine energy-transfer (DarT) labeling platform through the development and evaluation of trastuzumab-deazaflavin (Tra-dFl) conjugates for mapping the extracellular microenvironment of human epidermal growth factor receptor 2 (HER2). Four Tra-dFl conjugates were synthesized via azide-DBCO click chemistry, varying in PEG linker size and catalyst loading: Tra-PEG0-dFl, Tra-PEG6-dFl, Tra-PEG12-dFl, and Tra-bis-dFl, exhibiting a branched linker for dual attachment. Imaging and proteomic pulldown experiments revealed that linker size influences biotinylation efficiency and proteomic enrichment, resulting in Tra-PEG12-dFl emerging as the most effective construct, enabling the enrichment of cancer-associated cell surface proteins in HER2-positive SK-BR-3 cells. Evaluation of catalyst valency using a branched linker resulted in fewer enriched proteins, suggesting that linker architecture is a more critical parameter for conjugate performance than increased catalyst loading. Together, these findings provide guidelines for antibody-based deazaflavin conjugates and expand the applicability of DarT labeling for target-directed surfaceome mapping.

Shiga toxins (Stx) are key virulence factors of Shiga toxin-producing Escherichia coli (STEC), which are responsible for severe foodborne infections that can progress to hemolytic-uremic syndrome (HUS). Currently, no specific antitoxin therapies are available. In this study, we devised a glycoengineering strategy utilizing Functional-Spacer-Lipid (FSL) conjugates to create small extracellular vesicles (sEVs)-based decoy receptors for Shiga toxin type 1 (Stx1). sEVs isolated from human Caco-2 and HEK293T cells were functionalized with Gb3 trisaccharide (Galα1→4Galβ1→4Glc)-containing FSL conjugates, yielding Gb3-decorated vesicles displaying the Galα1→4Gal epitope. Characterization of FSL-modified sEVs confirmed that FSL incorporation did not adversely affect sEV morphology, size distribution, or surface charge. Western blotting and bead-assisted flow cytometry verified the presence of exosomal markers (CD9 and CD63) and the Gb3 epitope on modified vesicles. Gb3-tagged sEVs from both cell types exhibited high specificity in binding Stx1B, while control vesicles carrying Galili epitope (Galα1→3Galβ1→4GlcNAc), lacking Stx1B binding, demonstrated negligible binding. Gb3-expressing Caco-2 cell-based assays revealed that Gb3-decorated sEVs markedly reduced Stx1B binding to Caco-2 cells, indicating effective competition with cellular receptors. Furthermore, glycoengineered sEVs did not impair Caco-2 cell viability at concentrations sufficient for Stx1B sequestration. These findings establish FSL-mediated glycoengineering as a rapid and versatile approach for generating sEV-based decoy receptors that effectively bind Stx1B. Gb3-containing human sEVs may serve as an agent for neutralizing Stx1B and potentially other glycan-binding toxins, supporting the development of promising next-generation antitoxin therapeutics.

Coiled coil peptide assemblies are useful scaffolds for biomolecular and materials design, but their function often depends on preserving the folded oligomeric state after chemical modification or exposure to demanding conditions. While many peptide-stapling strategies stabilize individual α-helices, approaches that covalently reinforce higher-order architectures remain less developed. Here, we introduce an "assemble-and-click" strategy for interhelical stapling of a designed tetramer coiled coil. Cysteine and vinyl sulfonamide handles were positioned at symmetry-related sites that are brought into proximity by coiled-coil assembly, enabling pH-triggered thiol-Michael coupling after tetramer formation. LC-MS supports rapid formation of covalently linked dimeric products, SEC-MALS indicates that the stapled peptide retains the expected tetrameric assembly state, and circular dichroism shows markedly enhanced thermal robustness, with no cooperative unfolding observed up to 90 °C under the conditions examined. This approach provides a chemically programmable route to reinforcing higher-order coiled coil assemblies while preserving their oligomeric architecture.

DNA-encoded library (DEL) technology has emerged as a powerful platform for small-molecule discovery, in which on-DNA reaction development plays a central role in determining accessible chemical space. Early on-DNA chemistry mainly focused on establishing robust DNA-compatible transformations under mild aqueous conditions but often generated structurally limited libraries. Recent advances in photochemistry, electrochemistry, biocatalysis, and complexity-generating reactions have substantially expanded the scope of accessible on-DNA transformations and enabled the incorporation of increasingly diverse and medicinally relevant scaffolds into DELs. In this Viewpoint, we discuss the recent progress and emerging trends in on-DNA reaction development, with particular emphasis on the transition from compatibility-driven chemistry toward function-oriented DEL synthesis. We further highlight current challenges and future opportunities for developing precision on-DNA chemistry to support next-generation ligand discovery.

This study investigates how the structural design of secondary PNA probes affects their ability to carry the cytotoxic agent DM1 in a novel affibody-PNA-based pretargeting approach directed at Human Epidermal growth factor Receptor 2 (HER2)-expressing cells. Six different DM1-conjugated secondary 8-mer PNA probes with strategic hydrophilic modifications were designed and evaluated. Surface plasmon resonance analysis confirmed that the modifications preserved hybridization to the HER2-targeting affibody-PNA primary probe, with all secondary probes showing similar binding kinetics (KD = 310-660 pM). Biodistribution studies in nontumor-bearing NMRI mice with lutetium-177 (177Lu)-labeled secondary probes revealed that probe modifications dramatically influenced organ distribution patterns. The secondary probes SP2, SP5, and SPc demonstrated favorable biodistribution profiles with reduced accumulation in critical organs, i.e., liver and kidneys. Receptor-mediated endocytosis was also affected by secondary probe design, showing an increase in cellular internalization for SP5 (20.4% after 24 h) compared to SP2 (12.1%). Our findings highlight how rational design can optimize in vivo pharmacokinetics while preserving binding properties essential for effective pretargeting, providing a foundation for developing novel targeted cytotoxic delivery systems with potentially improved therapeutic indexes.

The central roles that glycans play in cell communication, adhesion, signal transduction, and pathogen recognition have driven growing interest in understanding glycan-mediated protein recognition events. Because protein-glycan binding is governed by three-dimensional carbohydrate presentation, precise structural control over synthetic glycan assemblies is essential for understanding fundamental principles underlying the recognition of both natural and artificial glycans. Most conventional glycomimetics, however, often lack the precise structural organization characteristic of native glycans, which limits detailed understanding of the architectural parameters that dictate their function. Molecular inorganic glycan assemblies provide an emerging platform that provides control over glycan valency, spacing, and topology to achieve programmable recognition behavior. This Viewpoint highlights the development of metal-templated glycan complexes that afford well-defined architectures with tunable recognition behavior and biological function and also discusses developing opportunities at the interface of inorganic and organic chemistry, glycobiology, and biomolecular design.

Ionizable lipid nanoparticles (LNPs) underpin today's RNA medicines by shielding nucleic acids in the bloodstream and transforming into membrane-active cationic nanoparticles inside acidifying endosomes. This behavior critically depends on the apparent pKa of LNPs, which can be tuned by altering the chemistry of lipids/lipidoids and the ratios of helper lipids. However, formulators typically assume that different RNA payloads exert identical effects on LNP ionization. In this study, we challenge this assumption and quantify how RNA modulates LNP pKa. LNP formulations with different ionizable lipidoids (SM-102, cKK-E12, ALC-0315, 306Oi10, XMAN6, and C12-200) were used to encapsulate various RNA cargos, which ranged from 21-nt to 4.5-kb mRNAs. Apparent pKa values were determined using a TNS fluorescence titration assay. RNA payload size impacted the apparent pKa of the LNPs by 0.1-0.5 pH units, the direction and magnitude of the change being dependent on the structure of the ionizable lipidoid. Moreover, same-length 21-nt siRNA and miRNA cargos produced a 0.15 pH-unit difference in XMAN6 LNPs, indicating that RNA structural features can modulate apparent pKa independently of payload size. With the monoamine lipidoid SM-102, short RNAs lowered the apparent pKa relative to mRNA-loaded particles, whereas the multiamine lipidoids cKK-E12 and XMAN6 exhibited a monotonic decrease in apparent pKa as RNA length increased, plateauing at ∼1.9 kb. The most pronounced discrepancies between apparent pKa values of lipidoid particles and corresponding LNPs (of ≤0.5 pH units) were observed with the triamine lipidoid XMAN6, pointing to a role of amino group multiplicity in the modulation of apparent pKa. These findings demonstrate that RNA payload size is an underappreciated lever for tuning LNP ionization behavior and that considering RNA payload size when conducting lipid/lipidoid library screens could enhance the development of future RNA-based therapeutics.

There is an unmet need to develop vehicles for delivering protein cargo and ribonucleoprotein complexes to cells. Virus capsids, or virus-like particles, have numerous advantages as self-assembling protein platforms, but there remains a need to develop robust methods for the specific internal packaging of cargo. In this work, we use the Hepatitis B Virus (HBV) capsid as our delivery vehicle. To specifically associate protein cargo with the capsid, we covalently attached a derivative of an HBV sulfamoylbenzamide (SBA) antiviral to green fluorescent protein (GFP). We show that SBA-tagged GFP can associate with HBV capsids, but efficient internal packaging requires engineering the assembly of capsids around a cargo. Under standard co-assembly conditions, SBA-tagged GFP predominantly displayed on the capsid exterior, reaching ∼40 GFP cargo proteins per capsid. We tested two strategies to drive encapsidation of protein cargo. First, remodeling the capsid interior was nominally effective. Conversely, His-tag/Ni2+-mediated clustering converts the cargo into a multivalent capsid-binding scaffold that templates assembly, enabling internal packaging of up to ∼30 cargo proteins per capsid. Based on capsid geometry, we suggest the practical limit is approximately 30 cargo proteins per T = 4 HBV capsid, indicating that this strategy approaches maximal loading. Together, these results provide a highly effective method for packaging proteins inside VLPs.

Herein, we demonstrate how Au(III) organometallic chemistry offers the potential to streamline the generation of constrained peptide tricycles. By leveraging the chemoselectivity and ultrafast kinetics of tetrametallic Au(III)-based aryl reagents toward cysteine (Cys) bioconjugation, we have constructed a small library of constrained peptide tricycles. These tricycles can be synthesized and isolated in 25-55% yields from various tetracysteine-containing linear peptides. In all cases, tricyclization conversion occurs quantitatively under five min at sub-mM concentrations of the target peptide and proceeds under varied distributions of the four Cys residues in the target peptide sequence. Furthermore, by constraining various linear peptides around the aryl core of tetraphenylethylene, a common solid-state luminogen, we generate fluorescent macrocycles with robust solution-state emissive profiles. We evaluated the innate affinity of these fluorescent hybrid peptides toward cells, finding that they behave as highly selective luminogenic cell markers of cellular debris.

With the increasing cancer incidence and death tolls worldwide, antibody-drug conjugates (ADCs) have emerged as a promising platform to selectively deliver cytotoxic payloads to cancerous tissues. This platform has the potential to spare patients from off-target effects compared to traditional chemotherapy. Despite the clinical success of some ADCs, many in the pipeline did not even reach the clinical stage. We turn our attention to their linker chemistry that connects the antibody and the drug molecules together. Despite their small size, linkers play a key role in governing the payload release kinetics and location. In this Viewpoint, we collect and discuss a plethora of linkers employed in ADC development, specifically focusing on advancements in antibody modification and linker cleavage chemistries. The choice of linker chemistry has evolved over time, going from stochastically attached non-stimuli-responsive to site-specifically modified cleavable linkers. Then we correlate the linker technologies to their pharmacokinetic outcomes in clinics, followed by a discussion of the upcoming "linker-free" technology for more facile and efficient antibody-conjugated targeted delivery.