Fibroblast activation protein (FAP) is overexpressed in a variety of cancers, making it an attractive target for bifunctional chelator-based radiopharmaceuticals. This study initially aimed to assess the effect of chelator structure on the biodistribution of 203Pb/212Pb-labeled FAP inhibitor (FAPI) bioconjugates. However, suboptimal in vivo biodistribution and imaging results suggested the bioconjugate was unstable. RadioHPLC analysis of urine samples suggest the thiourea bond, formed during conjugation between an amine on the biomolecule, and an isothiocyanate-functionalized chelator, is unstable in vivo, resulting in detachment of the radiometal-chelator complex from the targeting vector, resulting in poor tumor accumulation. To determine whether this instability was specific to the FAPI system, a peptide-based (Cyclic melanocyte stimulating hormone, CycMSH) bioconjugate targeting the melanocortin-1 receptor was synthesized using the same thiourea linkage. Identical metabolites were observed, supporting the hypothesis that thiourea bonds are unstable in vivo with this theranostic isotope pair. Subsequently, the effect of bioconjugation chemistry, specifically thiourea and amide bonds, on the stability and biodistribution of 203Pb/212Pb-labeled bioconjugates was assessed. Modifying the bioconjugation linker to be an amide bond, formed by utilizing a chelate containing an active ester instead of an isothiocyanate, led to significantly improved in vitro and in vivo stability, as demonstrated by radioHPLC and biodistribution and imaging studies in both models. These findings highlight the importance of the choice of bioconjugation chemistry in the development of lead-based radiopharmaceuticals and emphasize the importance of selecting stable linkages to ensure optimal radiometal retention and tumor targeting.
Over the last half century, molecularly targeted bioconjugates have revolutionized nuclear medicine. An array of vectors, ranging from small molecule peptidomimetics to macromolecular immunoglobulins, has been radiolabeled with radionuclides to create probes that have been deployed in both the laboratory and the clinic for positron emission tomography (PET), single photon emission computed tomography (SPECT), and radiopharmaceutical therapy. Generally speaking, these compounds have four constituent parts: (i) a targeting vector; (ii) a radionuclide; (iii) a labeling moiety, such as a chelator for radiometals or a prosthetic group for radiohalogens; and (iv) a linker that connects the vector and the labeling moiety. Each of these parts is critical to the performance of the radiopharmaceutical, but the importance of the former─the linker─can be lost in the shadow of its flashier teammates. Strictly speaking, the linker's job is simple: stably connect the vector and the radiolabeling moiety so that they do not become detached in vivo. However, recent years have been a witness to increasing efforts to exploit the properties of linkers to improve the pharmacokinetic profiles of radiopharmaceuticals. Along these lines, the most common strategies are predicated on changing the structure of the linker to alter the hydrophobicity and bioavailability of the probe, but several other innovative approaches have emerged as well, including those that rely upon stimuli for the cleavage of the linker. In this review, we will offer a systematic and critical discussion of the ways in which radiopharmaceutical chemists have leveraged linker chemistry to optimize the in vivo performance of probes for nuclear imaging and therapy, with a particular emphasis on nascent methodologies. We will also explore the lessons that other fields, most notably the development of antibody-drug conjugates, can offer the radiopharmaceutical community with respect to the design and implementation of new linker technologies.
Plant virus-derived materials are finding applications in science, engineering, and technology-with some candidates advancing toward commercialization, scalable manufacturing methods are needed. We recently established ultrafiltration/diafiltration (UF/DF) for purification of cowpea mosaic virus (CPMV) and tested here the broader applicability of this process by purifying cowpea chlorotic mottle virus (CCMV) and its bioconjugates. We optimized extraction, ultrafiltration and ion exchange chromatography for CCMV: acidic buffers (pH 4.0) were ideal for extraction, removing ∼80% of plant host cell proteins while recovering nearly 100% of CCMV. Membranes with a 1000 kDa and 300 kDa molecular mass cut-off were best suited for ultrafiltration, allowing to remove impurities, both larger and smaller than CCMV, alongside bulk water. When combining these steps, the same level of purity (>99%) as the contemporary centrifugation process was achieved with only 7 instead of 13 process steps, thus shortening the processing time from ∼2-3 days to ∼7 h. The ultrafiltration process was also compatible with virus purification by PEG-precipitation, allowing to harness the advantages of both methods. Importantly, UF/DF enabled purification of CCMV bioconjugates, not only shortening the processing time and improving the recovery up to ∼95%, but also removing free dye more efficiently compared to centrifugation.
Porcine circovirus type 2 (PCV2) is one of the major pathogens currently threatening the global swine industry. The capsid protein (Cap) of PCV2 is a key antigen for developing subunit vaccines. In this study, we established a bioconjugation strategy using chitosan oligosaccharide (COS) to construct a covalently linked PCV2d Cap-COS complex via thiol-maleimide click chemistry. Immunization and challenge experiments in mice demonstrated that when formulated with JLC-3 adjuvant, the PCV2d Cap-COS complex induced stronger and more durable specific immune responses, significantly improved the average daily weight gain postchallenge, and effectively alleviated histopathological damage in the lungs and kidneys. These findings indicate that the PCV2d Cap-COS complex enhances both antigen structure and immunogenicity, offering a novel strategy and experimental evidence for developing high-efficacy PCV2d subunit vaccines.
Site-specifically attaching biorthogonal handles to proteins is an essential tool for chemical biology research and diverse applications, including imaging, protein immobilization, and the development of next-generation therapeutics such as antibody drug-conjugates. Among the available strategies, enzymatic post-translational modification of short protein tags offers precision, stability, and modularity. However, complex substrate syntheses, the requirement of long recognition tags, and limited reaction efficiencies pose remaining limits for application. Here, we present ADDing, a straightforward enzymatic method for functionalizing proteins with click chemistry handles using the flavin transferase ApbE. We discovered that, given a dedicated adenine diphosphate derivative (ADD) substrate, ApbE attaches a phosphoribosyl moiety bearing bioorthogonal handles to DxxxGAT amino acid motifs. ADDing benefits from a streamlined workflow, due to simple enzymatic substrate synthesis from NAD+ and inexpensive precursors, allowing one-pot click handle generation and protein conjugation. ADDing enables rapid, high-yield functionalization of proteins featuring the recognition tag at either terminus or internal loops, and is compatible with copper and copper free azide-alkyne cycloaddition. We demonstrate its broad applicability in a wide variety of protein functionalizations, including fluorescent labeling, protein-protein, protein-DNA conjugation, and protein immobilization. This versatile technology thus holds great potential for chemical biology and therapeutic development.
The ability to track therapeutic cells is critical for advancing adoptive cell therapy. Positron emission tomography (PET) offers sensitive, quantitative imaging, but improved strategies for cell labeling remain needed. Here, we report a metabolic glycoengineering approach that installs azide groups onto the Jurkat T lymphocyte surface using the canonical tetraacetylated N-azidoacetylmannosamine (Ac4ManNAz). Azide-bearing cells were functionalized via strain-promoted azide-alkyne cycloaddition (SPAAC-based ligation) with a dual-clickable trancyclooctene (TCO)-bearing dibenzocyclooctyne (DBCO) derivative (sulfo-DBCO-PEG4-TCO), enabling subsequent inverse electron-demand Diels-Alder (IEDDA, TCO-tetrazine ligation) radiolabeling at cell-surface TCO moieties using an aluminum [18F]fluoride-tetrazine (Al[18F]F-Tz) tracer. Labeling conditions were optimized to achieve suitable cell-associated activity while maintaining good viability. We evaluated Al[18F]F-Tz pharmacokinetics, in vitro fluorine-18-labeled Jurkat cells, and a proof-of-concept pretargeting strategy in athymic nude mice. In vitro fluorine-18-labeled cells exhibited predictable trafficking and biodistribution over the imaging period, supporting the feasibility of MGE-based bioorthogonal radiolabeling for PET cell tracking. In contrast, pretargeted imaging requires further optimization and was dominated by the hepatic and intestinal signal consistent with hepatobiliary clearance of Al[18F]F-Tz. Overall, these findings underscore the potential of MGE-mediated bioorthogonal radiolabeling as a nongenetic platform for PET tracking of immune cells and provide a foundation for further development of pretargeted approaches for adoptively transferred cells.
Although globular protein-polymer bioconjugates in solution have been shown to self-assemble into many of the same nanostructures observed for traditional coil-coil block copolymers, there are also key differences that appear to be universal across bioconjugate systems with different protein and polymer blocks. This suggests that factors originating from the coarse-grained shape of the molecules may play a key role in many of these behaviors. Here, coarse-grained molecular dynamics simulations of dumbbells consisting of a hard sphere, representing the protein, and a soft sphere, representing the polymer, were used to investigate the physics underlying self-assembly. This highly coarse-grained model captured many of the most notable features of the protein-polymer bioconjugate phase diagram, including compositional asymmetry and a lyotropic reentrant order-disorder transition. The hard sphere block was found to be an important determinant for phase diagram asymmetry, with the rigidity of the hard sphere prohibiting the formation of spheres and inverse phases. Furthermore, entropically driven hard sphere ordering at high concentrations appears to be correlated with a restriction in the rearrangement of the dumbbells into a well-ordered nanostructure, leading to the reentrant order-disorder transition into a weakly ordered phase. The insights from this model can inform the design of biomaterials that incorporate globular protein-polymer block bioconjugates.
Peptide nucleic acid (PNA) is a synthetic mimic of DNA where the deoxyribose-phosphodiester backbone is replaced with N-(2-aminoethyl) glycine units. The lack of deoxyribose-phosphodiester bonds enhances enzymatic stability and improves binding affinity of PNA with complementary DNA and RNA strands. To enhance target binding, conformational stability, and pharmacological activity, several chemical modifications have been introduced into PNA. Modified PNAs have demonstrated promising preclinical potential as antisense and anti-gene agents, supporting their use in diverse biomedical applications. The limited in vivo biodistribution and cellular uptake of PNA have significantly hindered its clinical development. Enhancing PNA biodistribution using nanoformulations and bioconjugate-based delivery strategies has resulted in substantial in vivo pharmacological effects. Further, with advancements in chemistry and delivery techniques, PNA holds promise in treating genetic diseases, metabolic disorders, cancers, and infectious diseases. This review summarizes PNA's pharmacological mechanisms, chemical modifications, delivery strategies, and therapeutic applications while addressing limitations for clinical translation.
Understanding the spatiotemporal dynamics of protein synthesis and degradation is important for establishing how cells maintain protein homeostasis. Conventional methods for detecting newly synthesized proteins include metabolic labeling with radioactive [35S]methionine (Met) or the incorporation of l-azidohomoalanine (AHA) or l-homopropargylglycine followed by fluorescent labeling via copper(I)-catalyzed click chemistry. However, these methods typically require cell fixation, making them unsuitable for live-cell imaging. Here, we describe a fluorescence imaging technique to monitor newly synthesized proteins in living cells by utilizing a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction, in which l-AHA-containing proteins are labeled with fluorescent dyes conjugated to dibenzocyclooctyne (DBCO). We synthesized orange-emitting tetramethylrhodamine (TAMRA)-DBCO and far-red-emitting silicon rhodamine (SiR)-DBCO. TAMRA-DBCO enabled the visualization of newly synthesized proteins and their time-dependent degradation throughout the entire cell. SiR-DBCO was similarly effective, but was mainly distributed to the cytoplasm. The time-dependent decrease of TAMRA-DBCO fluorescence intensity in living cells was suppressed by lysosomal enzyme inhibitors and a proteasome inhibitor, suggesting that newly synthesized proteins are degraded via both pathways. Moreover, imaging of drug-induced senescent cells with TAMRA-DBCO suggested that senescent cells have a lower protein degradation ability than nonsenescent cells. These methods should be useful for investigating protein homeostasis in living cells.
Gram-negative bacterial resistance continues to pose a major therapeutic challenge, largely due to outer membrane impermeability and biofilm-associated tolerance mechanisms. In this study, we report the rational design, synthesis, and mechanistic characterization of bile acid-phenothiazine (BA-PTZ) conjugates as membrane-targeted antibacterial hybrids. Triazole and Schiff base linkers were strategically incorporated to systematically tune electronic properties and optimize amphiphilic balance. The synthesized conjugates exhibited markedly enhanced antibacterial activity compared to the parent phenothiazine (PTZ). Notably, cholic acid triazole-phenothiazine (CTPTZ) and cholyhydrazide Schiff base-phenothiazine (ChHSB-PTZ) displayed IC50 values of 32.34 ± 3.7 and 34.76 ± 3.5 μM, respectively, against Serratia marcescens (S. marcescens), along with improved potency against Escherichia coli (E. coli). Quantitative antibiofilm assays demonstrated strain-dependent efficacy, with ChHSB-PTZ effectively inhibiting E. coli biofilms (IC50 = 62.49 ± 1.3 μM) and CTPTZ exhibiting superior activity against S. marcescens biofilms (IC50 = 79.74 ± 2.2 μM). Cytotoxicity profiling in mammalian cells revealed tunable selectivity, particularly for triazole-linked derivatives. Density functional theory calculations and global reactivity descriptor analysis established correlations between electronic softness, electrophilicity, and antibacterial performance. Complementary docking studies indicated enhanced binding affinity of the conjugates toward Gram-negative membrane-associated proteins relative to PTZ alone. Intracellular ROS assessment excluded oxidative stress-mediated cytotoxicity, supporting a membrane-associated mechanism modulated by electronic properties. Collectively, these results validate BA-PTZ conjugation as a rational design strategy to overcome Gram-negative permeability barriers and provide structure-guided principles for the optimization of membrane-targeted antimicrobial bioconjugates.
An interaction observed in an anti-drug antibody (ADA) bridging ELISA between the drug and the anti-digoxin (aDIG) conjugated horseradish peroxidase (HRP) led to increased assay background across several commercial aDIG-HRP sources. Understanding the source of this interaction will help inform reagent qualities to be considered in production, evaluation, and selection for bioanalytical assays. This work evaluates commercial sources of aDIG-HRP through bridging ELISA, binding ELISA, SPR, and chromatography to determine what characteristics of aDIG-HRP lead to the interaction observed in a bridging ELISA. Three of four commercial aDIG-HRP sources evaluated exhibit drug binding and size heterogeneity with large (>250 kDa) species. SE-UHPLC of one source localizes this binding capability to the >6 MDa species. Under typical HRP conjugation conditions, HRP can conjugate to itself, and these larger species can bind to drug. The conjugation of HRP to aDIG may lead to large species linked to HRP self-conjugation that can interfere with bioassays if not carefully controlled. Therefore, it is important to evaluate commercial reagents for consistency, availability, and critical quality when incorporating a new lot/vendor for better understanding of unexpected assay performance and effectively support reagent and assay life cycle management. Some therapeutics induce an unwanted immune response in patients that presents as anti-drug antibodies (ADA). These reactions are monitored in clinical trials using an ADA assay. In these assays, ADA presence is indicated using a horseradish peroxidase (HRP) linked (or conjugated) detection antibody. In this work, the researchers linked unwanted assay signal to interactions occurring between the HRP-conjugate and the drug, causing the assay to not perform as intended. The unique chemistry used to conjugate HRP to the detection antibody causes these interactions. Additionally, HRP can conjugate to itself as well as the detection antibody, leading to large extended molecules. These extended HRP conjugates are able to interact with the drug, resulting in unwanted ADA assay signal. Interestingly, one commercial vendor’s HRP-conjugate doesn’t exhibit these interactions. In this case, careful control over the HRP conjugation reaction or further purification of the conjugate can overcome these issues. Together, this work shows that careful production and purification of HRP-based detection reagents can improve assay performance. Additionally, this work also points to the importance of careful evaluation of assay reagents to achieve optimal assay performance.
Oxime ligation, a chemoselective coupling between carbonyl compounds and aminooxy groups, enables conjugation under mild and aqueous conditions. Here, we integrate this reaction with degradable polymers through a one-pot synthesis of aminooxy-functionalized poly(ε-caprolactone) (PCL) oligomers. The bifunctional initiator 6-(tert-butyloxycarbonylaminooxy)-1-hexanol was employed for simultaneous ε-caprolactone ring-opening polymerization and in situ Boc-deprotection catalyzed by methanesulfonic acid (MSA). Optimized conditions afforded >95% conversion for both reactions, while maintaining well-defined oligomer structures (DP = 5-20). The resulting NH3+-O-PCL oligomers underwent rapid and efficient oxime ligation with a diverse set of aldehydes and ketones, achieving >90% conversion within 1-2 h at room temperature. Reactions proceeded effectively even in mixed aqueous solvents (H2O/MeOH/CHCl3 = 1:3:1), for example, with the reducing ends of glucose and xylose. This work establishes a straightforward, water-tolerant synthetic platform for preparing "click-ready" degradable polymers, enabling broad integration with biobased and functional substrates.
Bioluminescence (BL) is widely used as an optical readout in bioassays and molecular imaging applications. In this study, we present a systematic approach to develop an advanced multiplex imaging system using novel coelenterazine (CTZ) analogs, termed the "G-series." We synthesized a library of 20 CTZ analogs with diverse functional groups at the C-2 and C-8 positions of the imidazopyrazinone backbone and classified them into five categories. These analogs were characterized with respect to luciferase specificity, BL stability, emission spectra, kinetics, and computationally predicted binding modes. Most of the G-series CTZ analogs were found to be water-soluble and exhibited excellent specificity for NanoLuc from Oplophorus luciferase, while G1 alone showed high specificity for ALuc16 derived from a group of copepod luciferases, together with unique emission colors including blue, green, and orange. The applicability of the G-series CTZ analogs for multiplex imaging was further evaluated using single-chain BL probes and by in vivo imaging of xenografts in living mice. The results showed that substrate G6 facilitates specific BL signals (red emission) from the single-chain probe in response to estrogen antagonists. The mice xenografted with subcutaneous (s.c.) tumors expressing ALuc16 and NanoLuc were imaged with excellent signal-to-background ratios using G1 and G20, respectively. This study demonstrates that the G-series CTZ analogs constitute a versatile and tunable molecular imaging platform with superior optical properties for both in vitro and in vivo applications.
This study presents a DNA-compatible synthesis of diverse N-fused imidazopyridines via a catalyst-free Ugi-type multicomponent reaction using TMSCN as a functional isonitrile equivalent. The desilylation activation occurs efficiently in water without additional catalysts. The method exhibits a broad substrate scope for aldehydes and heterocyclic amidines and excellent chemoselectivity, underscoring its utility for constructing privileged heteroaromatic scaffolds in DNA-encoded library technology.
Aminoglycosides have long served as indispensable antibacterial agents, yet their clinical utility has diminished due to toxicity and pervasive resistance with the advent of superbugs. Over the past decade, however, a renewed chemical interest transformed these classical antibiotics into versatile molecular scaffolds through various covalent modifications. Among these the lipidation, a strategy that fundamentally reprograms their biological behavior is of particular interest. By installing hydrophobic or amphiphilic domains onto the polycationic 2-deoxystreptamine framework, aminoglycosides acquire a new mode of action that diverges strikingly from ribosomal targeting. Lipidated aminoglycosides emerge as potent membrane-active agents capable of overcoming multidrug resistance, penetrating biofilms, eradicating persister cells, and displaying broad-spectrum antifungal activity. In parallel, their intrinsic RNA affinity and enriched cationic functionality enable efficient condensation with nucleic acids, endosomal escape, and the formation of self-assembled nanostructures. This positions lipidated aminoglycosides as promising candidates for nonviral DNA, siRNA, and mRNA delivery. This review focuses on the chemical logic, methods, and mechanistic insights that underpin the evolution of lipidated aminoglycosides from early acylated derivatives to modern amphiphilic, guanidinium-linked, sterol conjugated, and ionizable aminoglycoside lipids.
The development of DNA-encoded library (DEL) technology is contingent upon robust and DNA-compatible reactions to expand accessible chemical space. Tandem transformations, which combine functional group interconversion and scaffold construction in one step, are particularly attractive for streamlining on-DNA synthesis. Herein, we report a copper-mediated tandem reaction conducted under mild, aqueous conditions that enables the in situ reduction of nitro groups followed by reductive amination with aldehydes. This DNA-compatible protocol efficiently furnishes secondary amines directly from nitro substrates, circumventing the need for prereduction. Moreover, the methodology can be extended to o-nitroaniline derivatives, providing efficient one-pot access to benzimidazole scaffolds through tandem nitro reduction and cyclization with aldehydes. Compared to conventional stepwise sequences requiring isolated intermediates, this strategy provides a more streamlined and atom-economical route for constructing privileged pharmacophores directly on DNA.
While DNA-encoded libraries (DELs) are well recognized as valuable tools for the development of novel small-molecule therapeutics, their significant potential for developing new imaging probes initially received less attention. However, as DEL technology develops and novel screening modalities are introduced, several robust strategies for generating imaging probes from DEL screening campaigns have emerged. The current topical review aims to provide an overview of DEL technology as it relates to the discovery of new molecular probes and to present recent contributions that highlight innovative ways DELs are advancing the field. Approaches to harnessing DEL-derived probes for therapeutic applications, including their conversion into integrated theranostic modalities, will also be discussed.
Antibody-drug conjugates are a promising class of therapeutics that offer targeted cancer treatments with fewer side effects. Optimizing the mAb, linker, payload, conjugation site, and drug-to-antibody ratio (DAR) are crucial for developing ADC candidates. However, assessing the influence of each factor on the higher-order structure (HOS) of the ADC remains challenging due to their complexity and heterogeneity, and the development of high-throughput workflows capable of detecting subtle changes in structure and stability would be advantageous. Here, we used ion mobility-mass spectrometry (IM-MS) and collision induced unfolding (CIU) to investigate the impact of linker-payload (LP) structure and conjugation strategy on the HOS and stability of ADCs. Site-specific conjugation to engineered cysteines revealed that LPs with different structures differentially affect the gas-phase stability of the Fc domain, and a correlation between gas-phase stability (CIU50) and thermal stability (Tm) of the ADC was observed. Conjugation at interchain cysteine residues disrupted the HOS of the ADC to a greater extent compared to site-specific conjugates, as evidenced by higher root-mean-square deviation values. CIU data revealed differences in conformation and stability in ADCs with different DARs and different positional isomers with the same DAR. These findings demonstrate the utility of IM-MS and CIU as a high-throughput, information-rich assay for probing the HOS and stability of ADCs during their discovery and development.
Metabolic glycoengineering enables the installation of unnatural chemical motifs on cell-surface glycoconjugates by leveraging endogenous biosynthetic pathways, yet prior efforts have primarily emphasized covalent chemistries. In this work, high-affinity supramolecular recognition motifs are introduced onto mammalian cell surfaces by using N-acyl-modified mannosamine precursors bearing adamantane guests for cucurbit[7]uril (CB[7]) complexation. A series of adamantane-mannosamine derivatives with varied linker lengths are synthesized and evaluated for metabolic incorporation in Jurkat and MCF-7 cells. Efficient conversion to adamantane-modified sialic acids and presentation at the cell membrane is achieved only for the shortest linker architecture, as confirmed by fluorescence labeling, flow cytometry, and direct mass spectrometric detection of modified sialic acids. Incorporation efficiency exhibited a strong dependence on both linker length and incubation time, revealing steric constraints imposed by the sialic acid biosynthetic machinery. Exploiting this supramolecular handle, a CB[7]-drug conjugate showed enhanced cytotoxicity in metabolically labeled cells. These results establish metabolic glycoengineering as a viable strategy for installing noncovalent, high-affinity supramolecular recognition motifs on cell surfaces, expanding the scope of orthogonal targeting approaches in biological environments.
The extensive utilization of nanomaterials and aptamers in biosensing has positioned the construction of efficient, stable, and user-friendly nanoprobe systems as a pivotal strategy for rapid small-molecule detection. This study developed a dual-probe fluorescence detection system utilizing gold nanoparticles (AuNPs) and quantum dots (QDs) for ochratoxin A (OTA) analysis. The AuNP probes were functionalized via an innovative freeze-driven asymmetric conjugation approach, while a distinct freeze-driven labeling strategy was applied to QDs. Compared to conventional labeling protocols, the freeze-driven methodology reduced conjugation duration from several hours to merely 20 min and was successfully implemented for QD-aptamer conjugation for the first time, facilitating efficient dual-probe assembly. The complete process, spanning from probe preparation to OTA quantification, was accomplished within 1.5 h. Under optimized parameters, the biosensor demonstrated a linear detection range of 0.5-50 ng/mL, achieved a detection limit of 0.183 ng/mL, and exhibited satisfied specificity, reproducibility, and matrix interference resistance. Validation experiments across various herbal specimens confirmed its accuracy and practical applicability. This research not only establishes a rapid and efficient platform for OTA monitoring but also provides a generalized framework for developing nanomaterial-based biosensing systems.