Correlating the structure and reactivity of a contact allergen, DNCB, and its analogs to sensitization potential.

We report a study that seeks to find a correlation between the overall sensitization potential quantified by the expression of IL-8 by stimulated monocytes and the chemical structure of a model contact allergen, 2,4-dinitrochlorobenzene (DNCB). We show that structure and reactivity of the chemical compounds play an important role in activation of the monocytes and subsequent inflammation in tissue. However, we observed a non-linear correlation between the rate of reaction and biological activity indicating a required balance of stability and reactivity.

Protecting Triazabutadienes To Afford Acid Resistance.

Recent work on triazabutadienes has shown that they have the ability to release aryl diazonium ions under exceptionally mild acidic conditions. There are instances that require that this release be prevented or minimized. Accordingly, a base-labile protection strategy for the triazabutadiene is presented. It affords enhanced synthetic and practical utility of the triazabutadiene. The effects of steric and electronic factors in the rate of removal are discussed, and the triazabutadiene protection is shown to be compatible with the traditional acid-labile protection strategy used in solid phase peptide synthesis.

Light-Activated Triazabutadienes for the Modification of a Viral Surface.

Chemical crosslinking is a versatile tool for the examination of biochemical interactions, in particular host-pathogen interactions. We report the critical first step toward the goal of probing these interactions by the synthesis and use of a new heterobifunctional crosslinker containing a triazabutadiene scaffold. The triazabutadiene is stable to protein conjugation and liberates a reactive aryl diazonium species upon irradiation with 350 nm light. We highlight the use of this technology by modifying the surface of several proteins, including the dengue virus envelope protein.

A Photobasic Functional Group.

Controlling chemical reactivity using light is a longstanding practice within organic chemistry, yet little has been done to modulate the basicity of compounds. Reported herein is a triazabutadiene that is rendered basic upon photoisomerization. The pH of an aqueous solution containing the water-soluble triazabutadiene can be adjusted with 350 nm light. Upon synthesizing a triazabutadiene that is soluble in aprotic organic solvents, we noted a similar light-induced change in basicity. As a proof of concept we took this photobase and used it to catalyze a condensation reaction.

Water-soluble triazabutadienes that release diazonium species upon protonation under physiologically relevant conditions.

Triazabutadienes are an understudied structural motif that have remarkable reactivity once rendered water-soluble. It is shown that these molecules readily release diazonium species in a pH-dependent manner in a series of buffer solutions with pH ranges similar to those found in cells. Upon further development, we expect that this process will be well suited to cargo-release strategies and organelle-specific bioconjugation reactions. These compounds offer one of the mildest ways of generating diazonium species in aqueous solutions.

Improving the Adjuvanticity of Small Molecule Immune Potentiators Using Covalently Linked NF-κB Modulators.

Small molecule immune potentiators (SMIPs) such as imidazoquinolinone derivatives that activate Toll-like receptor (TLR) 7/8 have immense potential as vaccine adjuvants and as antitumor agents. However, these molecules have high bioavailability that results in unacceptable levels of systemic inflammation due to adjuvant toxicity, thereby greatly limiting their use. To address this challenge, here we report the design and synthesis of novel imidazoquinolinone-NF-κB immunomodulator dimers. Employing in vitro assays, we screened a select library of synthesized dimers and selected viable candidates for further in vivo experiments. With ovalbumin as a model antigen, we vaccinated mice and demonstrated that these dimers reduce the systemic toxicity associated with SMIPs to baseline levels while simultaneously maintaining the adjuvanticity in a vaccine formulation. Additionally, we showed that select dimers improved efficacy in a CT26 mouse colon carcinoma tumor model while eliciting minimal adjuvant toxicity.

Receptor-Ligand Kinetics Influence the Mechanism of Action of Covalently Linked TLR Ligands.

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.

Determining Whether Agonist Density or Agonist Number Is More Important for Immune Activation via Micoparticle Based Assay.

It is unknown if surface bound toll-like-receptor (TLR) agonists activate cells via density or total molecular number. To answer this question, we developed a TLR agonist surface conjugated polystyrene microparticle (MP) system. Using a library of MPs with varying TLR agonist density and number, we simultaneously observed innate immune cell MP uptake and TNFα expression using ImageStream flow cytometry on a cell by cell basis. The data shows that total TLR number and not density drives cellular activation with a threshold of approximately 105-106 TLR agonists. We believe that this information will be crucial for the design of particulate vaccine formulations.

Cu-Click Compatible Triazabutadienes To Expand the Scope of Aryl Diazonium Ion Chemistry.

Triazabutadienes can be used to readily generate reactive aryl diazonium ions under mild, physiologically relevant conditions. These conditions are compatible with a range of functionalities that do not tolerate traditional aryl diazonium ion generation. To increase the utility of this aryl diazonium ion releasing chemistry an alkyne-containing triazabutadiene was synthesized. The copper-catalyzed azide-alkyne cycloaddition ("Cu-click") reaction was utilized to modify the alkyne-containing triazabutadiene and shown to be compatible with the nitrogen-rich triazabutadiene. One of the triazole products was tethered to a fluorophore, thus enabling the direct fluorescent labeling of a model protein.