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Insulin, a peptide hormone, forms fibrils under aberrant physiological conditions leading to a reduction in its biological activity. To ameliorate insulin aggregation, we have synthesized a small library of oligopyridylamide foldamers decorated with different combination of hydrophobic side chains. Screening of these compounds for insulin aggregation inhibition using a Thioflavin-T assay resulted in the identification of a few hit molecules. The best hit molecule, BPAD2 inhibited insulin aggregation with an IC50 value of 0.9 μM. Mechanistic analyses suggested that BPAD2 inhibited secondary nucleation and elongation processes during aggregation. The hit molecules worked in a mechanistically distinct manner, thereby underlining the importance of structure-activity relationship studies in obtaining a molecular understanding of protein aggregation.

Emerging diseases require generating new vaccines, which can often be time consuming. An alternate method to boost host defense is by inducing nonspecific innate immune memory, called trained immunity, to develop novel prophylactics. Many molecules, most notably β-glucan, induce trained immunity, but their effects are often short-lived and uncontrolled. This lack of temporal control limits both the therapeutic ability of training and provides fundamental questions about its nature. To achieve temporal control of trained immunity, controlled release nanoparticles encapsulating only 3.5% of the standard dose of β-glucan to attain sustained release over a month are engineered. Nanoparticle-trained mice exhibit prolonged training effects and improve resistance to a B16F10 tumor challenge compared to mice that receive an equivalent amount of free β-glucan. The duration of trained immunity is further fine tuned by synthesizing nanoparticles composed of different molecular weights to modulate the release kinetics. These results demonstrate that dosing and temporal control can substantially alter the trained response to unanticipated levels. As such, this approach using sustained release platforms might lead to a novel prophylactic strategy for improved disease resistance against a wide variety of diseases.

We report a mechanistic study comparing the immune activation of conjugated Toll-like receptor (TLR) agonists and their unlinked mixtures. Herein, we synthesized a set of six linked dual agonists with different ligands, molecular structures, receptor locations, and biophysical characteristics. With these dimers, we ran a series of in vitro cell-based assays, comparing initial and overall NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation, cytokine expression profiles, as well as time-resolved TNF-α (Tumor Necrosis Factor alpha) expression. We show that initial activation kinetics, ligand specificity, and the dose of the agonist influence the activity of these linked TLR systems. These results can help improve vaccine design by showing how linked TLR agonists can improve their potency with the appropriate selection of key criteria.

Antigen-adjuvant conjugation is known to enhance antigen-specific T-cell production in vaccine models, but scalable methods are required to generate site-specific conjugation for clinical translation of this technique. We report the use of the cell-free protein synthesis (CFPS) platform as a rapid method to produce large quantities (> 100 mg/L) of a model antigen, ovalbumin (OVA), with site-specific incorporation of p-azidomethyl-l-phenylalanine (pAMF) at two solvent-exposed sites away from immunodominant epitopes. Using copper-free click chemistry, we conjugated CpG oligodeoxynucleotide toll-like receptor 9 (TLR9) agonists to the pAMF sites on the mutant OVA protein. The OVA-CpG conjugates demonstrate enhanced antigen presentation in vitro and increased antigen-specific CD8+ T-cell production in vivo. Moreover, OVA-CpG conjugation reduced the dose of CpG needed to invoke antigen-specific T-cell production tenfold. These results highlight how site-specific conjugation and CFPS technology can be implemented to produce large quantities of covalently-linked antigen-adjuvant conjugates for use in clinical vaccines.

The first palladium-catalyzed interceptive decarboxylative 1,4-addition of allyl carbonates with squarates is reported. Interestingly, the C-3 carbonyl group of N-substituted isatins undergoes smooth decarboxylative 1,2-addition with allyl carbonates. This transformation offers a straightforward method for the synthesis of spiro-oxepane-fused 2-oxindole.

This method usually proceeds under incomplete conversion resulting in low to moderate yields.