Separation of D-amino acid-containing peptide phenylseptin using 3,3'-phenyl-1,1'-binaphthyl-18-crown-6-ether columns.

Several D-amino acid-containing peptides (DAACPs) with antimicrobial, cardio-excitatory, or neuronal activities have been found in several species. Here, we demonstrated the chiral separation of the antimicrobial peptide diastereomers, D-phenylseptin and L-phenylseptin using (S) and (R) 3,3'-phenyl-1,1'-binaphthyl-18-crown-6-ether columns (CR-I (+) and CR-I (-), respectively) and also investigated the underlying mechanism. First, using D-amino acid-containing tripeptide Phe-Phe-Phe-OH, we found that CR-I (+) could be used to recognize diastereomeric tripeptides containing an L-amino acid as the first residue. On the contrary, CR-I (-) enabled separation of a series of diastereomers with D-amino acid as the first residue. Therefore, we achieved separation of the stereoisomers using the chiral columns depending on the position of the D- amino acid in the peptide and demonstrated the orthogonality of separations of the chiral columns. Then, using CR-I (+), we separated amphibian antimicrobial peptide diastereomers, L- and D-phenylseptin, which have the sequences, L-Phe-L-Phe-L-Phe and L-Phe-D-Phe-L-Phe at their N-termini, respectively. In order to understand the host-guest interactions, we performed molecular dynamics simulations for L-Phe-L-Phe-L-Phe tripeptide-CR-I molecule complex systems. Three hydrogen bonds between the N-terminal amine group -NH3+ and the crown ether oxygens were the dominant interactions. The hydrophobic interactions between phenyl-rings in the chiral selector unit of CR-I (+) and the side chains of 2nd and 3rd residues of the peptide also contributed to the affinity. Our results show that the CR-I (+)-column can be applied for the separation of endogenous DAACPs generated by the post-translational modification.

Self-assembly of tripeptides into γ-turn nanostructures.

Self-assembling phenylalanine-based peptides have garnered interest owing to their potential for creating new functional materials. Here, we designed four diastereomers, l-Phe-l-Phe-l-Phe (FFF), d-Phe-l-Phe-l-Phe (fFF), l-Phe-d-Phe-l-Phe (FfF) and l-Phe-l-Phe-d-Phe (FFf), to analyze the effect of the d-isomer on the self-assembly. Using SEM, TG, VCD, and solid-state NMR measurements, we found that only FFf forms a γ-turn conformation and self-assembles into a nanoplate with higher thermal stability. The supramolecular structure of FFf consists of intra- and intermolecular hydrogen bonds and π-π stackings. From our results, we have discovered that FFf forms a new type of self-assembling γ-turn conformation, clarifying the structural role of a d-amino acid residue in supramolecular formation.