Peptide-Resorcinarene Conjugates Obtained via Click Chemistry: Synthesis and Antimicrobial Activity.

Antimicrobial resistance (AMR) is one of the top ten threats to public health, as reported by the World Health Organization (WHO). One of the causes of the growing AMR problem is the lack of new therapies and/or treatment agents; consequently, many infectious diseases could become uncontrollable. The need to discover new antimicrobial agents that are alternatives to the existing ones and that allow mitigating this problem has increased, due to the rapid and global expansion of AMR. Within this context, both antimicrobial peptides (AMPs) and cyclic macromolecules, such as resorcinarenes, have been proposed as alternatives to combat AMR. Resorcinarenes present multiple copies of antibacterial compounds in their structure. These conjugate molecules have exhibited antifungal and antibacterial properties and have also been used in anti-inflammatory, antineoplastic, and cardiovascular therapies, as well as being useful in drug and gene delivery systems. In this study, it was proposed to obtain conjugates that contain four copies of AMP sequences over a resorcinarene core. Specifically, obtaining (peptide)4-resorcinarene conjugates derived from LfcinB (20-25): RRWQWR and BF (32-34): RLLR was explored. First, the synthesis routes that allowed obtaining: (a) alkynyl-resorcinarenes and (b) peptides functionalized with the azide group were established. These precursors were used to generate (c) (peptide)4-resorcinarene conjugates by azide-alkyne cycloaddition CuAAC, a kind of click chemistry. Finally, the conjugates' biological activity was evaluated: antimicrobial activity against reference strains and clinical isolates of bacteria and fungi, and the cytotoxic activity over erythrocytes, fibroblast, MCF-7, and HeLa cell lines. Our results allowed establishing a new synthetic route, based on click chemistry, for obtaining macromolecules derived from resorcinarenes functionalized with peptides. Moreover, it was possible to identify promising antimicrobial chimeric molecules that may lead to advances in the development of new therapeutic agents.

Copper(I)-Catalyzed Alkyne-Azide Cycloaddition (CuAAC) "Click" Reaction: A Powerful Tool for Functionalizing Polyhydroxylated Platforms.

Click chemistry is currently one of the most used tools for the generation of complex organic molecules. The advantages of using click chemistry in organic synthesis are remarkable; in many cases, the reactions occur under mild conditions and are free of solvents, with high yields and short reaction times. This makes it an extraordinarily effective and viable alternative for obtaining complex/conjugated molecules. In this review, the use of click chemistry CuAAC is especially emphasized for polyhydroxylated platforms such as resorcinarenes or calixarenes, focusing mainly on aspects of synthesis, specifically conditions, reagents, and methodologies.

Efficient Separation of C-Tetramethylcalix[4]resorcinarene Conformers by Means of Reversed-Phase Solid-Phase Extraction.

A reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed to study the conformer formation generated during the reaction for obtaining C-tetramethylcalix[4]resorcinarene. The chromatographic method was used to design a strategy for purifying the reaction products, using solid-phase extraction columns (RP-SPE) and gradient elution. The chromatographic profiles of the cyclocondensation reaction between resorcinol and acetaldehyde show the presence of three products under the different reaction and precipitation conditions studied. Using RP-SPE, it was possible to enrich the products, which were later characterized by means of RP-HPLC and 1H nuclear magnetic resonance (NMR). This investigation explored and established a new method for RP-HPLC analysis and RP-SPE separation of conformational isomers obtained in the formation reaction of C-tetramethylcalix[4]resorcinarene.

In Vitro Antifungal Activity of Chimeric Peptides Derived from Bovine Lactoferricin and Buforin II against Cryptococcus neoformans var. grubii.

Cryptococcosis is associated with high rates of morbidity and mortality. The limited number of antifungal agents, their toxicity, and the difficulty of these molecules in crossing the blood-brain barrier have made the exploration of new therapeutic candidates against Cryptococcus neoformans a priority task. To optimize the antimicrobial functionality and improve the physicochemical properties of AMPs, chemical strategies include combinations of peptide fragments into one. This study aimed to evaluate the binding of the minimum activity motif of bovine lactoferricin (LfcinB) and buforin II (BFII) against C. neoformans var. grubii. The antifungal activity against these chimeras was evaluated against (i) the reference strain H99, (ii) three Colombian clinical strains, and (iii) eleven mutant strains, with the aim of evaluating the possible antifungal target. We found high activity against these strains, with a MIC between 6.25 and 12.5 µg/mL. Studies were carried out to evaluate the effect of the combination of fluconazole treatments, finding a synergistic effect. Finally, when fibroblast cells were treated with 12.5 µg/mL of the chimeras, a viability of more than 65% was found. The results obtained in this study identify these chimeras as potential antifungal molecules for future therapeutic applications against cryptococcosis.

Chimeric Peptides Derived from Bovine Lactoferricin and Buforin II: Antifungal Activity against Reference Strains and Clinical Isolates of Candida spp.

Antimicrobial peptides (AMPs) are considered to be a valuable source for the identification and/or design of promising candidates for the development of antifungal treatments, since they have advantages such as lower tendency to induce resistance, ease of production, and high purity and safety. Bovine lactoferricin (LfcinB) and buforin II (BFII) are AMPs to which great antimicrobial potential has been attributed. The minimum motives with antimicrobial activity derived from LfcinB and BFII are RRWQWR and RLLR, respectively. Nine chimeras containing the minimum motives of both peptides were synthesized and their antifungal activity against fluconazole (FLC)-sensitive and resistant C. albicans, C. glabrata, and C. auris strains was evaluated. The results showed that peptides C9: (RRWQWR)2K-Ahx-RLLRRRLLR and C6: KKWQWK-Ahx-RLLRRLLR exhibited the greatest antifungal activity against two strains of C. albicans, a FLC-sensitive reference strain and a FLC-resistant clinical isolate; no medically significant results were observed with the other chimeras evaluated (MIC ~200 µg/mL). The chimera C6 was also active against sensitive and resistant strains of C. glabrata and C. auris. The combination of branched polyvalent chimeras together with FLC showed a synergistic effect against C. albicans. In addition to exhibiting antifungal activity against reference strains and clinical isolates of Candida spp., they also showed antibacterial activity against both Gram-positive and Gram-negative bacteria, suggesting that these chimeras exhibit a broad antimicrobial spectrum and can be considered to be promising molecules for therapeutic applications.

Designing Chimeric Peptides: A Powerful Tool for Enhancing Antibacterial Activity.

Chimeric peptides containing short sequences derived from bovine Lactoferricin (LfcinB) and Buforin II (BFII) were synthetized using solid-phase peptide synthesis (SPPS) and characterized via reversed-phase liquid chromatography and mass spectrometry. The chimeras were obtained with high purity, demonstrating their synthetic viability. The chimeras' antibacterial activity against Gram-positive and Gram-negative strains was evaluated. Our results showed that all the chimeras exhibited greater antibacterial activity against the evaluated strains than the individual sequences, suggesting that chemical binding of short sequences derived from AMPs significantly increased the antibacterial activity. For each strain, the chimera with the best antibacterial activity exerted a bacteriostatic and/or bactericidal effect, which was dependent on the concentration. It was found that: (i) the antibacterial activity of a chimera is mainly influenced by the linked sequences, the palindromic motif RLLRRLLR being the most relevant one; (ii) the inclusion of a spacer between the short sequences did not significantly affect the chimera's synthesis process; however, it enhanced its antibacterial activity against Gram-negative and Gram-positive strains; on the other hand, (iii) the replacement of Arg with Lys in the LfcinB or BFII sequences improved the chimeras' synthesis process without significantly affecting their antibacterial activity. These results illustrate the great importance of the synthesis of chimeric peptides for the generation of promising antibacterial peptides.

Short peptides conjugated to non-peptidic motifs exhibit antibacterial activity.

Short peptides derived from buforin and lactoferricin B were conjugated with other antimicrobial molecules of different chemical natures. The sequences RLLR, RLLRLLR, RWQWRWQWR, and RRWQWR were conjugated at their N-terminal end with non-peptidic molecules such as 6-aminohexanoic acid, ferrocene, caffeic acid, ferulic acid, and oxolinic acid. Peptide conjugates and unmodified peptides were synthesized by means of solid-phase peptide synthesis using the Fmoc/tBu strategy (SPPS-Fmoc/tBu), purified via RP-SPE, and characterized via RP-HPLC and MS. The peptides' antibacterial activity against bacterial strains E. coli ATCC 25922 and S. aureus ATCC 25923 was evaluated, and the results showed that the peptide conjugates exhibited higher antibacterial activity than the original unconjugated peptides. Conjugation of AMPs is a promising strategy for designing and identifying new drugs for treating bacterial infections.

Synthetic Peptide Purification via Solid-Phase Extraction with Gradient Elution: A Simple, Economical, Fast, and Efficient Methodology.

A methodology was implemented for purifying peptides in one chromatographic run via solid-phase extraction (SPE), reverse phase mode (RP), and gradient elution, obtaining high-purity products with good yields. Crude peptides were analyzed by reverse phase high performance liquid chromatography and a new mathematical model based on its retention time was developed in order to predict the percentage of organic modifier in which the peptide will elute in RP-SPE. This information was used for designing the elution program of each molecule. It was possible to purify peptides with different physicochemical properties, showing that this method is versatile and requires low solvent consumption, making it the least polluting one. Reverse phase-SPE can easily be routinely implemented. It is an alternative to enrich and purified synthetic or natural molecules.