The development of potent adjuvants is an important step for improving the performance of subunit vaccines. CD1d agonists, such as the prototypical α‐galactosyl ceramide (α‐GalCer), are of special interest due to their ability to activate iNKT cells and trigger rapid dendritic cell maturation and B‐cell activation. Herein, we introduce a novel derivatization hotspot at the α‐GalCer skeleton, namely the N‐substituent at the amide bond. The multicomponent diversification of this previously unexplored glycolipid chemotype space permitted the introduction of a variety of extra functionalities that can either potentiate the adjuvant properties or serve as handles for further conjugation to antigens toward the development of self‐adjuvanting vaccines. This strategy led to the discovery of compounds eliciting enhanced antigen‐specific T cell stimulation and a higher antibody response when delivered by either the parenteral or the mucosal route, as compared to a known potent CD1d agonist. Notably, various functionalized α‐GalCer analogues showed a more potent adjuvant effect after intranasal immunization than a PEGylated α‐GalCer analogue previously optimized for this purpose. Ultimately, this work could open multiple avenues of opportunity for the use of mucosal vaccines against microbial infections.

Peptide-drug conjugates (PDCs) have recently gained significant attention for the targeted delivery of anticancer therapeutics, mainly due to their cost-effective and chemically defined production and lower antigenicity compared to ADCs, among other benefits. In this study, we designed and synthesized novel PDCs by conjugating new thiol-functionalized tubulysin analogs (tubugis) to bombesin, a peptide ligand with a relevant role in cancer research. Two tubulysin analogs bearing ready-for-conjugation thiol groups were prepared by an on-resin multicomponent peptide synthesis strategy and subsequently tested for their stand-alone in vitro anti-proliferative activity against human cancer cells, which resulted in IC50 values in the nanomolar range. In addition, various fluorescently labeled [K5]-bombesin(6–14) peptides, non-lipidated and lipidated with fatty acid chains of variable length, were also synthesized using the versatile multicomponent chemistry. These bombesin derivatives were tested for their gastrin-related peptide receptor (GRPR)-mediated internalization into cancer cells using flow cytometry, proving that the lipid tail (especially C14) enhances the cell internalization. Using the tubugi toxins and bombesin peptides, three different bombesin-tubugi conjugates were synthesized with different cleavage propensity and lipophilicity. Preliminary in vitro experiments revealed that, depending on the linker and the presence of a lipid tail, these novel PDCs possess good to potent anticancer activity and moderate selectivity for GRPR-overexpressing cancer cells.

Tubulysins are among the most recent antimitotic compounds to enter into antibody/peptide‐drug conjugate (ADC/PDC) development. Thus far, the design of the most promising tubulysin payloads relied on simplifying their structures, e.g., by using small tertiary amide N‐substituents (Me, Et, Pr) on tubuvaline residue. Cumbersome solution‐phase approaches are typically used for both syntheses and functionalization with cleavable linkers. p‐Aminobenzyl quaternary ammonium (PABQ) linkers were a remarkable advancement for targeted delivery, but the procedures to incorporate them into tubulysins are only of moderate efficiency. Here we describe a novel all‐on‐resin strategy permitting a loss‐free resin linkage and an improved access to super potent tubulysin analogs showing close resemblance to the natural compounds. For the first time, a protocol enables the integration of on‐resin tubulysin derivatization with, e.g., a maleimido‐Val‐Cit‐PABQ linker, which is a notable progress for the payload‐PABQ‐linker technology. The strategy also allows tubulysin diversification of the internal amide N‐substituent, thus enabling to screen a tubulysin library for the discovery of new potent analogs. This work provides ADC/PDC developers with new tools for both rapid access to new derivatives and easier linker‐attachment and functionalization.

In this study, the characteristic growth of Cladobotryum virescens on nine culture media was analyzed. The growing behavior of this fungus was dependent on the culture medium. In vitro analysis showed that oat agar was better than other media tested with the highest conidia production. The antifungal activity against Fusarium chlamydosporum and Alternaria brassicicola was evaluated by the Dual Culture method. C. virescens displayed high activity against both pathogens acting through antibiosis and mycoparasitism. This effect was increased by a higher competitiveness of the strain for the substrate. Furthermore, the crude ethyl acetate extract of the culture broth was tested in vitro against Botrytis cinerea and Septoria tritici, as well as the hemibiotrophic oomycete Phytophthora infestans using a microtiter plate assay at different concentrations. The extract showed excellent inhibition even below 5 ppm. According to these results, we concluded that C. virescens can be considered as a potential biological control agent in agriculture. To the best of our knowledge, this is the first study to investigate C. virescens as a biocontrol agent for different diseases caused by five relevant pathogens that affect cereals and vegetables.

Macrocyclization of peptides is typically used to fix specific bioactive conformations and improve their pharmacological properties. Recently, macrobicyclic peptides have received special attention owing to their capacity to mimic protein structures or be key components of peptide-drug conjugates. Here, we describe the development of novel synthetic strategies for two distinctive types of peptide macrobicycles. A multicomponent macrocyclo-dimerization approach is introduced for the production of interconnected β-turns, allowing two macrocyclic rings to be formed and dimerized in one pot. Also, an on-resin double stapling strategy is described for the assembly of lactam-bridged macrobicycles with stable tertiary folds.

Novel unimolecular bivalent glycoconjugates were assembled combining several functionalized capsular polysaccharides of Streptococcus pneumoniae and Neisseria meningitidis to a carrier protein by using an effective strategy based on the Ugi 4-component reaction. The development of multivalent glycoconjugates opens new opportunities in the field of vaccine design, but their high structural complexity involves new analytical challenges. Nuclear Magnetic Resonance has found wide applications in the characterization and impurity profiling of carbohydrate-based vaccines. Eight bivalent conjugates were studied by quantitative NMR analyzing the structural identity, the content of each capsular polysaccharide, the ratios between polysaccharides, the polysaccharide to protein ratios and undesirable contaminants. The qNMR technique involves experiments with several modified parameters for obtaining spectra with quantifiable signals. In addition, the achieved NMR results were combined with the results of colorimetric assay and Size Exclusion HPLC for assessing the protein content and free protein percentage, respectively. The application of quantitative NMR showed to be efficient to clear up the new structural complexities while allowing the quantitative assessment of the components.

Background: Human infectious diseases caused by bacteria are a worldwide health problem due to the increased resistance of these microorganisms to conventional antibiotics. For this reason, the identification of novel molecular targets and the discovery of new antibacterial compounds is urgently required. Metalo-aminopeptidases are promising targets in bacterial infections. They participate in crucial processes for bacterial growth and pathogenesis, such as protein and peptide degradation to supply amino acids, protein processing, access to host tissues, cysteine supply for redox control, transcriptional regulation, site-specific DNA recombination, and hydrogen sulfide production. Although several of these enzymes are not essential, they are required for virulence and maximal growth in conditions of nutrient limitation and high temperatures.Objective: In this review, we describe the structural, functional and kinetic properties of some examples of bacterial metalo-aminopeptidases, in the context of their use as antibacterial targets. In addition, we present some inhibitors reported for these enzymes.Conclusion: It is necessary a meticulous work to validate these peptidases as good/bad targets and to identify inhibitors with a potential therapeutic use.

The replacement of the disulfide bridge by other types of side chain linkages has been a continuous endeavor in the development of cyclic peptide drugs with improved metabolic stability. Octreotide is a potent and selective somatostatin analog that has been used as an anticancer agent, in radiolabeled conjugates for the localization of tumors and as targeting moiety in peptide-drug conjugates. Here, we describe an onresin methodology based on a multicomponent macrocyclization that enables the substitution of the disulfide bond by a tertiary lactam bridge functionalized with a variety of exocyclic moieties, including lipids, fluorophores, and charged groups. Conformational analysis in comparison with octreotide provides key information on the type of functionalization permitting the conformational mimicry of the bioactive peptide.

Multicomponent reactions (MCRs) are recently expanding the plethora of solid-phase protocols for the synthesis and derivatization of peptides. Herein, we describe a solid-phase-compatible strategy based on MCRs as a powerful strategy for peptide cyclization and ligation . We illustrate, using Gramicidin S as a model peptide, how the execution of on-resin Ugi reactions enables the simultaneous backbone N-functionalization and cyclization, which are important types of derivatizations in peptide-based drug development or for incorporation of conjugation handles, or labels.

Solid-phase synthesis represents the methodological showcase for technological advances such as split-and-pool combinatorial chemistry and the automated synthesis of peptides, nucleic acids and polysaccharides. These strategies involve iterative coupling cycles that do not generate functional diversity besides that incorporated by the amino acids, nucleosides and monosaccharide building blocks. In sharp contrast, multicomponent reactions (MCRs) are traditionally used to generate both skeletal and appendage diversity in short, batchwise procedures. On-resin MCRs have traditionally been employed for the construction of heterocycle and peptidomimetic libraries, but that scenario has changed recently, and today the focus is more on the solid-phase derivatization of peptides and oligonucleotides. This review presents relevant experimental details and addresses the synthetic scope of such on-resin multicomponent protocols employed to accomplish specific biopolymer covalent modifications that are practically inviable by traditional solution-phase methodologies. Recommendations are provided to facilitate the implementation of solid-supported protocols and avoid possible pitfalls associated with the selection of the polymeric resin, the solvent and the order and amount of the reagents employed. We describe procedures comprising the multicomponent lipidation, biotinylation and labeling of both termini and the side chains, as well as the use of MCRs in the traceless on-resin synthesis of ligated and cyclic peptides. Solid-phase protocols for the assembly of α-helical and parallel β-sheet peptides as well as hybrid peptide–peptoid and peptide–peptide nucleic acid architectures are described. Finally, the solid-supported multicomponent derivatization of DNA oligonucleotides is illustrated as part of the DNA-encoded library technology relying on MCR-derived heterocyclic compounds.