Bioconjugate Chemistry最新文献

Glycosylated self-assembling molecules enable the construction of nanostructures whose surfaces are decorated with saccharides under aqueous conditions, reminiscent of biological systems at both the molecular and cellular levels. Such glyco-nanostructures thus hold great promise as soft materials for biological applications. Herein, we report the facile synthesis of glycosylated diarylmethyloxy-triazines, which serve as new building blocks for fabricating self-assembled nanostructures in aqueous media and forming supramolecular hydrogels. The triazine scaffold enables the protecting-group-free synthesis of glycosylated compounds with reasonable β-anomer selectivity. Notably, both the substituents on the aryl groups attached to the triazine core and the geometry of the glycosidic linkages in the disaccharides strongly influence the hydrogelation behavior.

The aberrant self-assembly of amyloid β (Aβ) peptides into toxic oligomers is a central event in Alzheimer’s disease, motivating the development of molecular systems capable of modulating or degrading these species. Porphyrins and porphyrin−peptide bioconjugates have emerged as promising photoactive platforms due to their known ability to generate the highly oxidant singlet oxygen (¹O₂) upon light excitation. Still, the mechanisms underlying their oxidative effects on Aβ remain unclear. Here, we investigate the photophysical properties and the ¹O₂-mediated oxidation capability of two porphyrin−peptide conjugates, Ph-Pep and its Zn-metallated analogue ZnPh-Pep, both incorporating the Aβ recognition motif KLVFF. Steady-state and time-resolved spectroscopic studies reveal the good preservation of the photophysical and photochemical properties of the porphyrin chromophore units after their conjugation with the peptide appendage. Population of the long-lived triplet state of both compounds under visible-light excitation results in an effective generation of ¹O₂. High-resolution mass spectrometry shows that the ¹O₂ photogenerated by ZnPh-Pep induces extensive oxidation, yielding peroxidic and hydroperoxidic species, and provides evidence of partial Aβ42 degradation. In contrast, Ph-Pep leads to a rapid loss of detectable Aβ signal, suggesting the formation of higher-order aggregates rather than peptide cleavage. These distinct outcomes highlight the roles of Zn²⁺ coordination and peptide conjugation in modulating the ¹O₂-driven pathways. Overall, this study provides a mechanistic insight into designing photoactivatable porphyrin-based constructs capable of degrading or modulating toxic Aβ assemblies, opening intriguing prospects for light-mediated therapeutic strategies for neurodegenerative diseases.

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cell carcinoma and remains challenging to treat at advanced stages due to immune evasion and inefficient intracellular delivery of therapeutic agents. In this study, we synthesized a modified generation 5 (G5) poly(amidoamine) dendrimer (G5PBA) to co-deliver small-interfering RNA targeting programmed death-ligand 1 (siPD-L1) and the antitumor peptide Tat-HSPB1, forming the G5PBA/siPD-L1/Tat-HSPB1 nanocomplex. The agarose gel retardation assay demonstrated that G5PBA efficiently condensed siPD-L1. Furthermore, the peptide encapsulation efficiency and loading capacity were measured, confirming the loading of Tat-HSPB1 into G5PBA/siPD-L1. Flow cytometry showed that G5PBA enhanced the cellular uptake of siPD-L1, while confocal microscopy demonstrated that G5PBA promoted lysosomal escape in Caki-1 cells. Western blot analysis further confirmed that G5PBA effectively reduced the PD-L1 protein expression. In cellular experiments, CCK-8, EdU, and wound-healing assays demonstrated that G5PBA/siPD-L1/Tat-HSPB1 inhibited the viability and migration of ccRCC cells. G5PBA/siPD-L1/Tat-HSPB1 suppressed tumor growth, downregulated PD-L1 expression, enhanced CD4⁺ and CD8⁺ T-cell infiltration, and elevated serum levels of IL-2, IL-6, TNF-α, and IFN-γ in mice. Major organs exhibited no obvious histopathological abnormalities upon H&E staining. Serum ALT, AST, and BUN levels showed no significant changes compared with the PBS group, suggesting the preliminary safety profile of G5PBA/siPD-L1/Tat-HSPB1. Collectively, the nanocomplex achieved efficient peptide/siPD-L1 co-delivery and demonstrated significant antitumor activity. This system thus provides a promising strategy and experimental basis for local administration of ccRCC.

Herein, we report the synthesis and biological evaluation of the first phospholipid prodrug of gedatolisib, a dual PI3K/mTOR inhibitor, and liposomal nanoparticles based on this conjugate. The liposomes were designed for selective cleavage by phospholipase A₂, enabling the release of the active drug from the gedatolisib−phospholipid conjugate. Compared with free gedatolisib, the liposomal formulation demonstrated cytotoxicity that was 2−3 orders of magnitude lower across several cancer cell lines. In vivo studies using the EMT6 breast cancer model showed that the liposomal prodrug exhibited superior antitumor efficacy while being substantially less hepatotoxic than free gedatolisib, which displayed negligible antitumor activity. This phospholipid conjugation strategy represents a promising approach for potentially improving the therapeutic index of gedatolisib.

Ultrasound-triggered site-specific molecular chemistry has emerged as a transformative strategy for cancer therapy, yet precise spatiotemporal control of their activity remains a critical bottleneck. Endogenous stimulus-responsive strategies are hampered by tumor heterogeneity, while exogenous physical triggers provide superior controllability. This article proposes that ultrasound-triggered site-specific chemistry, termed “acoustic programming”, offers a transformative solution. By integrating focused ultrasound deep-tissue penetration with molecular-level chemical control, acoustically programmable bioconjugates achieve deep-tissue, on-demand therapeutic activation. We survey recent advances in sono-labile linkers, ultrasound-triggered charge reversal, and sono-chemically activated prodrugs, and critically evaluate emerging proof-of-concept and in vivo validated applications in ultrasound-caged PROTACs, immunomodulators, and antibody-drug conjugates. This paradigm shift from “ultrasound-responsive materials” to “ultrasound-responsive molecules” positions acoustic programming as a core platform technology for next-generation precision oncology.

DNA-encoded chemical libraries (DELs) accelerate ligand discovery by combining combinatorial synthesis with parallel affinity selection of DNA-tagged compounds. Conventional DEL selection protocols involve the immobilization of protein targets on a solid support such as magnetic beads. Several washes are performed on the beads after library incubation to remove non-binder library members and to enrich for binding compounds. However, excessive washing could also deplete weak or rapidly dissociating binders. Here, we systematically study the impact of the number of washing steps on DEL hit enrichment during screening of a newly synthesized library named THPP-DEL and based on a central 2-amino-3-(4-(1,2,3,6-tetrahydropyridin-4-yl)phenyl)propanoic acid scaffold and containing ∼8 million compounds. THPP-DEL was screened against Carbonic Anhydrase IX (CAIX), Prostate-Specific Membrane Antigen (PSMA), and Human Serum Albumin (HSA) by systematically varying the number of washing steps from 0 to 20. High-throughput sequencing revealed enriched chemotypes against the three targets, with affinities ranging from low nanomolar to low micromolar concentration values. For all hits, total counts and enrichment factors depended on washing conditions and stabilized after three washing steps. The results presented in this paper define practical selection conditions that preserve micromolar binders, control background levels, and reduce unnecessary hit resynthesis of false positives.

Antibody-drug conjugates (ADCs) based on Fc antigen-binding fragments (Fcabs) promise improved tumor penetration while retaining the FcRn-mediated long half-life. Building on prior spheroid studies, we directly compared a 50 kDa HER2-targeted Fcab-MMAE ADC with a matched 150 kDa control molecule (aDIG-Fcab-MMAE) consisting of the same HER2-targeting Fcab ADC equipped with additional non-targeting anti-digoxigenin (aDIG) Fab arms and trastuzumab Fab- and IgG-based ADCs (50 kDa T-Fab-MMAE and 150 kDa T-IgG-MMAE) using the same linker-payloads. Site-specific conjugation yielded high-purity DAR2 species with excellent serum stability. In vitro cytotoxicity assays revealed that Fcab-MMAE and aDIG-Fcab-MMAE exhibited lower potency compared to T-Fab-MMAE and T-IgG-MMAE, correlating with their approximately 10-fold lower binding affinity for HER2. A pharmacokinetic study in mice showed similar exposure for the Fcab-MMAE, its 150 kDa control, and a higher exposure for T-IgG-MMAE ADC, while the T-Fab-MMAE ADC cleared rapidly. In an NCI-N87 gastric cancer SCID mouse xenograft model, T-IgG-MMAE demonstrated superior efficacy, while Fcab-MMAE and aDIG-Fcab-MMAE showed similar efficacy despite their size difference, with T-Fab-MMAE showing lower efficacy. These data indicate that at similar exposure, reduced scaffold size alone may not improve in vivo efficacy.

Despite its potent anti-aging efficacy, the practical application of retinol (ROH) remains highly restricted by its poor photostability, low aqueous solubility, and concentration-dependent cytotoxicity. Herein, we report the synthesis of a hyaluronic acid−retinol (HA−ROH) conjugate to overcome these limitations and improve transdermal delivery. The successful conjugation was confirmed via proton nuclear magnetic resonance (¹H NMR) spectroscopy, high-performance liquid chromatography (HPLC) and Fourier-transform infrared spectroscopy (FT-IR). Notably, the HA−ROH conjugate exhibited remarkable photostability under UVA irradiation compared to free retinol. HA−ROH presented a sustained, hyaluronidase-responsive ROH release profile, indicating its potential as an enzyme-responsive controlled delivery system. Furthermore, the conjugation significantly mitigated the cytotoxicity of retinol in both HaCaT keratinocytes and human dermal fibroblasts. The conjugate suppressed matrix metalloproteinase-1 (MMP-1) expression more effectively than free ROH. In vitro cellular uptake and ex vivo skin permeation studies demonstrated enhanced transdermal delivery of the HA−ROH conjugate. Taken together, these results suggest that the HA−ROH conjugate is a highly promising, stable, and safe candidate for advanced dermatologic and cosmetic applications.

In vivo characterization and visualization of tumors often relies on radioactive tracers and expensive, complex equipment, which limits the frequency of analysis, throughput, and accessibility. Short-wave infrared (SWIR) fluorescence is a new nonradioisotopic imaging modality with flexible readouts at high depth and high signal-to-background contrast. Here, we report semiconductor quantum dots (QDs) with SWIR fluorescence engineered to selectively target tumor-associated macrophages (TAMs) by functionalization with the polysaccharide dextran (Dex). TAMs are often abundant in the tumor microenvironment and mediate immune suppression, angiogenesis, metastatic progression, and therapy resistance, making them important targets for in situ analysis. We compare QD-Dex probes with QDs coated with polyethylene glycol (QD-PEG) as nontargeted controls with equivalent fluorescence, hydrodynamic size, and electrostatic charge. In vitro, QD-Dex exhibited receptor-mediated uptake by cultured macrophages whereas QD-PEG uptake was only through noncompetitive passive processes. In vivo, SWIR imaging of immunocompetent mice bearing TAM-rich orthotopic 4T1 mammary tumors revealed tumor retention of QD-Dex after intravenous administration. Tumor signals for QD-PEG were much lower in comparison. Tumor signals for QD-Dex exceeded blood levels ∼2 h after injection and decayed substantially after 24 h, providing an appropriate time window for preclinical imaging studies in rodents. These findings establish baseline metrics for macrophage-targeted SWIR nanoprobes as a platform for preclinical in vivo tumor analysis without ionizing radiation.

Antibody drug conjugates (ADCs) are a key therapy in the cancer treatment arsenal. Critical to the success of the ADC is the release of the drug cargo at the desired site, and thus, enzymatically or chemically responsive linkers, connecting the drug to the antibody, play a central role. Linkers containing the peptide sequence Glu-Val-Cit are particularly important as cleavage of this peptide occurs by both murine and human cathepsin B enzymes. Additional linker design considerations include components for free drug release via the self-immolative moiety para-aminobenzyl alcohol (PABOH), copper-free click conjugation to an azide-functionalized antibody using dibenzocyclooctyne (DBCO), and aqueous solubility via a polyethylene glycol spacer (PEG). To facilitate widespread use and translation, we describe the 9-step gram-scale synthesis of the linker DBCO-PEG₄-Glu-Val-Cit-PABOH conjugated to monomethyl auristatin E (MMAE). Using only liquid-phase synthesis protocols and chromatography-free, precipitation-based intermediate isolations gives the drug-linker conjugate in 36% overall yield, and this method is an improvement over prior methods (yield < 1%). Finally, we prepare the corresponding MMAE-linked ADC and validate its target-dependent potency across ErbB2/Her2-high (MDA-MB-453) and ErbB2/Her2-low (MDA-MB-231) human breast cancer cell lines.