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 IC 50 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.
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.
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.
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.The development of efficient methods to construct organic frameworks continues as a challenge in both academic as well as industrial research applications. Since the ground-breaking work of Tsuji, 1 Saegusa, 2 and Trost, 3 palladium-catalyzed decarboxylative allylation has emerged as an important method for the construction of new carbon-carbon bonds. Intensive research efforts have been devoted to transition-metal-catalyzed intramolecular decarboxylative allylation by many research groups. 4 Recent advances toward interceptive decarboxylative allylation include the reactions of allyl-β-keto esters, 5 allylcarbonates, 6 allyl carbamates, 7 and allyl diphenylglycinate esters 8 with electrophilic olefins. Hayashi and coworkers have extended the palladium-catalyzed IDcA to malonate-derived valerolactones. 9 We have recently disclosed our success on palladiumcatalyzed allylation reactions of isatylidenes 10 and heptafulvenes. 11 Nucleophilic allylation of carbonyl groups have attracted attention of a number of organic chemists. 12 Yamamoto et al. reported the first palladium-catalyzed allylation of aldehydes using bis-π-allylpalladium intermediates. 13 Allylation of carbonyl groups using iridium, 14 indium, 15 zinc, 16 and other metal reagents 17 have been extensively studied. In 2004, Schaus and co-workers reported the palladium(0)-catalyzed decarboxylative aldol reaction of allyl-β-keto esters and aldehydes. 18 But there are only limited reports on palladium-catalyzed intermolecular decarboxylative coupling with carbonyl groups. 9c,8b This fact combined with our interest in allylation using π-allyl-palladium complex prompted us to investigate its reactivity on activated ketones such as squarates and isatins. Herein, we report the catalytic interceptive decarboxylative 1,4-addition of allylcarbonates with squarates and 1,2-addition of carbonyl group of acenaphthenequinone and isatins.Squarates, fascinating and versatile C-4 synthons, offer the prospect of serving as useful starting materials for the synthesis of a wide variety of compounds. 19 The monoaddition and two-fold addition to the carbonyl group of squarates by organolithium and Grignard reagents to synthesize polycyclic compounds are well documented in the literature. 20 But there are only few reports on palladiumcatalyzed reactions involving squarates. 19d,21 Our initial studies involved the treatment of dibutyl squarate (1a) and diallylcarbonate 2a in the presence of Pd(PPh 3 ) 4 (5 mol%) in THF at room temperature. The reaction afforded 1,4-addition product 2,4-bis(allyloxy)-3,4-dibutoxycyclobut-2-enone (3aa) in 22% yield (Scheme 1). The structure of the 1,4-addition product was characterized by usual ...
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