Self-Assembly of Homo- and Hetero-Chiral Cyclodipeptides into Supramolecular Polymers towards Antimicrobial Gels

Abstract

There is an increasing interest towards the development of new antimicrobial coatings, especially in light of the emergence of antimicrobial resistance (AMR) towards common antibiotics. Cyclodipeptides (CDPs) or diketopiperazines (DKPs) are attractive candidates for their ability to self-assemble into supramolecular polymers and yield gel coatings that do not persist in the environment. In this work, we compare the antimicrobial cyclo(Leu-Phe) with its heterochiral analogs cyclo(D-Leu-L-Phe) and cyclo(L-Leu-D-Phe), as well as cyclo(L-Phe-D-Phe), for their ability to gel. The compounds were synthesized, purified by HPLC, and characterized by ¹H-NMR, ¹³C-NMR, and ESI-MS. Single-crystal X-ray diffraction (XRD) revealed details of the intermolecular interactions within the supramolecular polymers. The DKPs were then tested for their cytocompatibility on fibroblast cells and for their antimicrobial activity on S. aureus. Overall, DKPs displayed good cytocompatibility and very mild antimicrobial activity, which requires improvement towards applications.

Keywords: D-amino acids; antimicrobial; chirality; cyclo(Leu-Phe); cyclo(Phe-Phe); cyclodipeptides; diketopiperazines; gels; peptides; self-assembly.

Figure 1

Four cyclodipeptides or DKPs used in this work.

Figure 2

(a) Superimposition of single-crystal XRD structures of homochiral cyclo(Tyr-Leu) (green, [49]) and cyclo(Phe-Phe) (cyan, [31]), highlighting the βCH-π interaction (dashed black line). (b) Superimposition of single-crystal XRD structures of heterochiral DKP2 (green CCDC 2209459) and DKP4 (cyan, CCDC 2209458) highlighting the αCH-π interaction (dashed black line).

Figure 3

NH∙∙∙OC H-bonded (dashed lines) ribbons in (a) DKP2 (CCDC 2209459) and (b) DKP4 (CCDC 2209458). Oxygen atoms are shown in red, nitrogen atoms in blue, carbon atoms in grey, and hydrogen atoms in white.

Figure 3

NH∙∙∙OC H-bonded (dashed lines) ribbons in (a) DKP2 (CCDC 2209459) and (b) DKP4 (CCDC 2209458). Oxygen atoms are shown in red, nitrogen atoms in blue, carbon atoms in grey, and hydrogen atoms in white.

Figure 4

Crystal packing of (a) DKP2 (CCDC 2209459) and (b) DKP4 (CCDC 2209458) as viewed along the a and b axis, respectively. The independent molecules in the asymmetric unit are depicted in green, blue, red, and yellow in DKP2 and green and blue in DKP4.

Figure 5

Photographs of inverted-tube tests to probe for the gelation ability of DKPs 1–4 in soybean oil. (a) DKP1; (b) DKP2 (or DKP3); (c) DKP4.

Figure 6

Oscillatory rheological analyses for DKP2 soybean gels. (a) Time sweep. (b) Frequency sweep. G′ is the elastic or storage modulus. G″ is the viscous or loss modulus.

Figure 7

MTT assay on fibroblast cells treated with the DKPs 1–4, or 1% SDS (− ctrl), or untreated (+ ctrl). Metabolic activity % is shown relative to the + ctrl. * denotes p < 0.01.

Figure 8

Minimum inhibitory concentration assay on S. aureus for DKPs 1–4 measured as OD₆₀₀ normalized to the untreated S. aureus (100% control, ctrl). * denotes p < 0.01.