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.

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.

Scheme 1. Copper-catalyzed azide-alkyne cycloaddition of Pra-RRWQWR (1) with Lys(N 3 )-RLLR (2) for obtaining the chimeric peptide (3)

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.

Short peptides derived from buforin and lactoferricin B were conjugated with other antimicrobial molecules of different chemical natures.