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.

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.

Calix[4]resorcinarenes are polyhydroxylated macrocyclic compounds with four units of resorcinol. These compounds can be derivatized through modifications at the upper rim, allowing reactivity with secondary amines to produce Mannich base derivatives via Mannich-type aminomethylation reactions. In this paper, we report the reaction of C-tetra(propyl)calix[4]resorcinarene with piperidine in acetonitrile. The aminomethylated compound C-2,8,14,20-tetra(propyl)-5,11,17,23-tetrakis(N–(piperidine)methyl)calix[4]resorcinarene was obtained with a 52% yield, with an exact mass of 1044.6994 u and a mass error of 7.6 ppm. The reaction progress and product formation were monitored by RP-HPLC, and the compound was characterized using LC ESI-TOF/MS, one- and two-dimensional ¹H and ¹³C NMR, and FTIR spectroscopy. Chromatographic and spectroscopy data are presented and discussed.