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.

The effect on the cytotoxicity against breast cancer cell lines of the substitution of ²⁶Met residue in the sequence of the Bovine Lactoferricin-derived dimeric peptide LfcinB (20-30)₂: (²⁰RRWQWRMKKLG³⁰)₂-K-Ahx with amino acids of different polarity was evaluated. The process of the synthesis of the LfcinB (20-30)₂ analog peptides was similar to the original peptide. The cytotoxic assays showed that some analog peptides exhibited a significant cytotoxic effect against breast cancer cell lines HTB-132 and MCF-7, suggesting that the substitution of the Met with amino acids of a hydrophobic nature drastically enhances its cytotoxicity against HTB-132 and MCF-7 cells, reaching IC₅₀ values up to 6 µM. In addition, these peptides have a selective effect, since they exhibit a lower cytotoxic effect on the non-tumorigenic cell line MCF-12. Interestingly, the cytotoxic effect is fast (90 min) and is maintained for up to 48 h. Additionally, through flow cytometry, it was found that the obtained dimeric peptides generate cell death through the apoptosis pathway and do not compromise the integrity of the cytoplasmic membrane, and there are intrinsic apoptotic events involved. These results show that the obtained peptides are extremely promising molecules for the future development of drugs for use against breast cancer.

The reaction between L-cysteine (Cys) and 6-maleimidohexanoic acid (Mhx) in an aqueous medium at different levels of pH was analyzed via RP-HPLC, finding the presence of two reaction products throughout the evaluated pH range. By means of solid-phase extraction (SPE), it was possible to separate the products and obtain isolated profiles enriched up to 80%. The products were analyzed individually through mass spectrometry, DAD-HPLC, NMR ¹H, ¹³C, and two-dimensional evidence of isomerization between the hydrogen atoms of the α-amino and the thiol group present in the cysteine. Thus, it was concluded that the products obtained corresponded to a mixture of the isomer Cys-S-Mhx, where the adduct is formed by a thioether bond, and the isomer Cys-NH-Mhx, in which the union is driven by the amino group. We consider that the phenomenon of isomerization is an important finding, since it has not previously been reported for this reaction.

In order to obtain gold electrode surfaces modified with Human Papillomavirus L1 protein (HPV L1)-derived peptides, two sequences, SPINNTKPHEAR and YIK, were chosen. Both have been recognized by means of sera from patients infected with HPV. The molecules, Fc-Ahx-SPINNTKPHEAR, Ac–C–Ahx-(Fc)KSPINNTKPHEAR, Ac–C–Ahx-SPINNTKPHEAR(Fc)K, C–Ahx–SPINNTKPHEAR, and (YIK)₂–Ahx–C, were designed, synthesized, and characterized. Our results suggest that peptides derived from the SPINNTKPHEAR sequence, containing ferrocene and cysteine residues, are not stable and not adequate for electrode surface modification. The surface of polycrystalline gold electrodes was modified with the peptides C-Ahx-SPINNTKPHEAR or (YIK)₂-Ahx-C through self-assembly. The modified polycrystalline gold electrodes were characterized via infrared spectroscopy and electrochemical measurements. The thermodynamic parameters, surface coverage factor, and medium pH effect were determined for these surfaces. The results indicate that surface modification depends on the peptide sequence (length, amino acid composition, polyvalence, etc.). The influence of antipeptide antibodies on the voltammetric response of the modified electrode was evaluated by comparing results obtained with pre-immune and post-immune serum samples.

Over the last few years, short peptides have become a powerful tool in basic and applied research, with different uses like diagnostic, antimicrobial peptides, human health promoters or bioactive peptides, therapeutic treatments, templates for peptidomimetic design, and peptide-based vaccines. In this endeavor, different approaches and technologies have been explored, such as bioinformatics, large-scale peptide synthesis, omics sciences, structure-activity relationship studies, and a biophysical approach, among others, seeking to obtain the shortest sequence with the best activity. The advantage of short peptides lies in their stability, ease of production, safety, and low cost. There are many strategies for designing short peptides with biomedical and industrial applications (targeting the structure, length, charge, or polarity) or as a starting point for improving their properties (sequence data base, de novo sequences, templates, or organic scaffolds). In peptide design, it is necessary to keep in mind factors such as the application (peptidomimetic, immunogen, antimicrobial, bioactive, or protein-protein interaction inhibitor), the expected target (membrane cell, nucleus, receptor proteins, or immune system), and particular characteristics (shorter, conformationally constrained, cycled, charged, flexible, polymerized, or pseudopeptides). This review summarizes the different synthetic approaches and strategies used to design new peptide analogs, highlighting the achievements, constraints, and advantages of each.

Metabolites have great potential for the design of biomarkers, since their presence or absence provides valuable information about a biological system. In this context, polyclonal antibodies are important metabolites for diagnostic procedures, but in some pathologies, it has been found that these metabolites are present at low concentrations, so it could be difficult to detect them. In this investigation, an organic monolithic material of poly(GMA-co-EDMA) was functionalized with a peptide via Michael addition (thiol–maleimide) click chemistry. The peptide, covalently bound to the monolith, contains the SPINNTKPHEAR sequence derived from the human papilloma virus L1 protein. It was determined that the obtained monolithic support allows selectively isolating polyclonal antibodies against the SPINNTKPHEAR sequence, since they are retained on the chemical surface of the material by an immunoaffinity interaction. The monolithic material functionalization protocol reported here could be applied to incorporate any peptide with a terminal cysteine in order to recover a specific analyte. A new method was developed for isolating and pre-concentrating antibodies using monolithic materials, which could contribute to the improvement of disease detection strategies based on immunoaffinity interactions.