Enhancing the Immune Response of a Nicotine Vaccine with Synthetic Small “Non-Natural” Peptides

The addictive nature of nicotine is likely the most significant reason for the continued prevalence of tobacco smoking despite the widespread reports of its negative health effects. Nicotine vaccines are an alternative to the currently available smoking cessation treatments, which have limited efficacy. However, the nicotine hapten is non-immunogenic, and successful vaccine formulations to treat nicotine addiction require both effective adjuvants and delivery systems. The immunomodulatory properties of short, non-natural peptide sequences not found in human systems and their ability to improve vaccine efficacy continue to be reported. The aim of this study was to determine if small “non-natural peptides,” as part of a conjugate nicotine vaccine, could improve immune responses. Four peptides were synthesized via solid phase methodology, purified, and characterized. Ex vivo plasma stability studies using RP-HPLC confirmed that the peptides were not subject to proteolytic degradation. The peptides were formulated into conjugate nicotine vaccine candidates along with a bacterial derived adjuvant vaccine delivery system and chitosan as a stabilizing compound. Formulations were tested in vitro in a dendritic cell line to determine the combination that would elicit the greatest 1L-1β response using ELISAs. Three of the peptides were able to enhance the cytokine response above that induced by the adjuvant delivery system alone. In vivo vaccination studies in BALB/c mice demonstrated that the best immune response, as measured by nicotine-specific antibody levels, was elicited from the conjugate vaccine structure, which included the peptide, as well as the other components. Isotype analyses highlighted that the peptide was able to shift immune response toward being more humorally dominant. Overall, the results have implications for the use of non-natural peptides as adjuvants not only for the development of a nicotine vaccine but also for use with other addictive substances and conventional vaccination targets as well

Structurally Diverse Diazafluorene-Ligated Palladium(II) Complexes and Their Implications for Aerobic Oxidation Reactions

4,5-Diazafluoren-9-one (DAF) has been identified as a highly effective ligand in a number of Pd-catalyzed oxidation reactions, but the mechanistic basis for its utility has not been elucidated. Here, we present the complex coordination chemistry of DAF and palladium­(II) carboxylate salts. Multiple complexes among an equilibrating mixture of species have been characterized by ¹H and ¹⁵N NMR spectroscopy and X-ray crystallography. These complexes include monomeric and dimeric Pd^II species, with monodentate (κ¹), bidentate (κ²), and bridging (μ:κ¹:κ¹) DAF coordination modes. Titration studies of DAF and Pd­(OAc)₂ reveal the formation of two dimeric DAF/Pd­(OAc)₂ complexes at low [DAF] and four monomeric species at higher [DAF]. The dimeric complexes feature two bridging acetate ligands together with either a bridging or nonbridging (κ¹) DAF ligand coordinated to each Pd^II center. The monomeric structures consist of three isomeric Pd­(κ¹-DAF)₂(OAc)₂ complexes, together with Pd­(κ²-DAF)­(OAc)₂ in which the DAF exhibits a traditional bidentate coordination mode. Replacing DAF with the structurally related, but more-electron-rich derivative 9,9-dimethyl-4,5-diazafluorene (Me₂DAF) simplifies the equilibrium mixture to two complexes: a dimeric species in which the Me₂DAF bridges the two Pd centers and a monomeric species with a traditional κ²-Me₂DAF coordination mode. The use of DAF in combination with other carboxylate ligands (CF₃CO₂^– or <i>t</i>BuCO₂^–) also results in a simplified collection of equilibrating Pd^II–DAF complexes. Collectively, the results highlight the ability of DAF to equilibrate rapidly among multiple coordination modes, and provide valuable insights into the utility of DAF as a ligand in Pd-catalyzed oxidation reactions