Synthetic peptides are commonly obtained by means of solid-phase peptide synthesis (SPPS), in which separation of the peptide from the solid support requires treatment with 92.5% v/v trifluoroacetic acid (TFA); therefore, peptides are obtained as trifluoroacetate salts. For promising anticancer/antibacterial peptides it is essential to exchange the counterion from trifluoroacetate to hydrochloride or acetate, since the former are more widely studied in biological activity assays. In this research, RP-SPE-based methodologies were designed, developed, and implemented for simultaneous counterion exchange and peptide purification. Critical process steps were identified and parameters such as mobile phase composition, elution, and program were optimized. Analysis of the counterion exchange reaction and characterization of the final products was performed using high-performance liquid chromatography, attenuated total reflectance, nuclear magnetic resonance, and mass spectrometry. Peptides with purities between 82–97% and a trifluoroacetate ion content less than 0.36% were obtained. This novel counterion exchange proved efficient for peptides with different characteristics such as length, polarity, polyvalency, and presence of non-natural amino acids or non-protein molecules, therefore showing a wide range of applications in the field of therapeutic peptides. The methods developed are fast, efficient, low-cost, and do not require robust instrumentation and can be routinely implemented in SPPS.

The separation and purification of molecular compounds and their functionalized derivatives is a common challenge in organic synthesis. In particular, calix[4]resorcinarenes present a high potential for chemical derivatization at their upper edge by aminomethylation reactions, and these compounds and their derivatives require appropriate analytical methodologies for their analysis, separation, and purification. In this study, C-tetra(propyl)calix[4]resorcinarene was synthesized and functionalized with maleimide groups by optimized aminomethylation reactions, obtaining a mixture of mono-, di-, tri-, and tetrasubstituted compounds. Initial separation by RP-HPLC with a core-shell column showed poorly resolved peaks, indicating a loss of separation efficiency. Therefore, a monolithic C18 column was used, which significantly improved the separation, thanks to its larger pore volume and continuous structure facilitating the diffusion of these bulky molecules, notably improving efficiency. Finally, the six compounds functionalized with maleimide groups were efficiently separated and enriched by RP-SPE by analytical method transfer, and the two peptides of six and the thirteen residues derived from LfcinB (20–25): RRWQWR were synthesized by SPPS-Fmoc/tBu and purified. These were modularly linked by the Michael thiol-maleimide addition reaction obtaining six (peptide)_n-resorcinarene conjugates. The analytical method by RP-HPLC with a monolithic C18 column, the separation and purification by RP-SPE were used transversally in all the steps to obtain compounds with adequate purities and quantities. Finally, the antibacterial activities of the six conjugates were evaluated against E. coli and E. faecalis strains, and it was determined that three aminomethylated compounds and one monosubstituted conjugate showed activity against E. faecalis. Our work established a new modular conjugation strategy between calix[4]resorcinarenes and peptides by thiol-maleimide click chemistry, and a methodology of separation, purification, and enrichment for these products by RP-HPLC and RP-SPE, which permitted us to obtain quantities with purities appropriate for their characterization by NMR, LC-MS and antibacterial activity assays.

Palindromic antimicrobial peptides (PAMs) constitute versatile scaffolds for the design and optimization of anticancer agents with applications in therapy, diagnosis, and/or monitoring. In the present study, fluorolabeled peptides derived from the palindromic sequence RWQWRWQWR containing fluorescent probes, such as 2-Aminobenzoyl, 5(6)-Carboxyfluorescein, and Rhodamine B, were obtained. RP-HPLC analysis revealed that the palindromic peptide conjugated to Rhodamine B (RhB-RWQWRWQWR) exhibited the presence of isomers, likely corresponding to the open-ring and spiro-lactam forms of the fluorescent probe. This equilibrium is dependent on the peptide sequence, as the RP-HPLC analysis of dimeric peptide (RhB-RRWQWR-hF-KKLG)₂K-Ahx did not reveal the presence of isomers. The antibacterial activity of the fluorescent peptides depends on the probe attached to the sequence and the bacterial strain tested. Notably, some fluorescent peptides showed activity against reference strains as well as sensitive, resistant, and multidrug-resistant clinical isolates of E. coli, S. aureus, and E. faecalis. Fluorolabeled peptides 1-Abz (MIC = 62 µM), RhB-1 (MIC = 62 µM), and Abz-1 (MIC = 31 µM) exhibited significant activity against clinical isolates of E. coli, S. aureus, and E. faecalis, respectively. The RhB-1 (IC₅₀ = 61 µM), Abz-1 (IC₅₀ = 87 µM), and RhB-2 (IC₅₀ = 35 µM) peptides exhibited a rapid, significant, and concentration-dependent cytotoxic effect on HeLa cells, accompanied by morphological changes characteristic of apoptosis. RhB-1 (IC₅₀ = 18 µM) peptide also exhibited significant cytotoxic activity against breast cancer cells MCF-7. These conjugates remain valuable for elucidating the possible mechanisms of action of these novel anticancer peptides. Rhodamine-labeled peptides displayed cytotoxicity comparable to that of their unlabeled analogues, suggesting that cellular internalization constitutes a critical early step in their mechanism of action. These findings suggest that cell death induced by both unlabeled and fluorolabeled peptides proceeds predominantly via apoptosis and is likely contingent upon peptide internalization. Functionalization at the N-terminal end of the palindromic sequence can be evaluated to develop systems for transporting non-protein molecules into cancer cells.