Development of Therapeutic Gramicidin S Analogues Bearing Plastic β,γ‐Diamino Acids

Abstract

Gramicidin S (GS), one of the most widely investigated antimicrobial peptides (AMPs), is known for its robust antimicrobial activity. However, it is restricted to topical applications due to undesired hemolytic activity. With the aim to obtain non-toxic GS analogues, we describe herein a molecular approach where the native GS β-turn region is replaced by synthetic β,γ-diamino acids (β,γ-DiAAs). Four β,γ-DiAA diastereomers were employed to mimic β-turn structure to afford GS analogues GS3-6 that exhibit diminished hemolytic activity. A comparative structural study demonstrates that the (βR,γS)-DiAA displays the most stable β-turn mimic. To further improve the therapeutic index (e.g. high antibacterial activity and low hemolytic activity) and to extend the molecular diversity, GS5 and GS6 were used as structural scaffolds to introduce additional hydrophobic or hydrophilic groups. We show that GS6K, GS6F and GS display comparable antibacterial activity while GS6K and GS6F possess significantly decreased toxicity. Moreover, antibacterial mechanism studies suggest that GS6K kills bacteria mainly through the disruption of membrane. Results and DiscussionScope of Lead Peptide Molecular Design and Synthesis. Initially, four diastereomeric GS analogues (GS3-6, Figure 1) were synthesized to evaluate in both biological and conformational aspects. The β,γ-DiAAs were prepared using the synthetic strategy previously described by our group starting from α-amino acids. [42] With respect to the aromatic character in the β-turn region, [36,39] D-and L-phenylalanine were chosen as starting materials (Scheme 1). Cbz-protected intermediates 7, 8, 9 and 10 were prepared as previously described. [23,43] Since the configuration of an analogue of compound 9 has been previously determined by crystallographic structure, [43] the other compounds could thereby be differentiated by the comparison of their NMR spectrum with their enantiomers. Noteworthy, the separation of the diastereomers is rather difficult when directly applying the silica gel chromatography. Alternatively, the minor diastereomers (8 and 10) can be easily precipitated in the presence of cold diethyl ether, and it consequently allows the major diastereomers (7 and 9) being purified by chromatography. To allow the solid phase synthesis, Cbz protecting group was further transferred to Fmoc protecting group (Scheme 1).Previous studies have demonstrated that GS and its analogues can be readily synthesized by solution-phase cyclization of the corresponding linear precursor peptides with protected side chains. [44] To this end, the linear precursors were assembled by following an either conventional or microwave-assistant solid phase Fmoc/tBu strategy. All Fmoc-protected α-amino acids are commercially available. Each projected peptide was assembled stepwise on the solid support starting from Fmoc-Pro-2-Chlorotrityl Chloride (CTC) Resin. During the sequential coupling, HBTU/HOBt (conventional synthesis) or HBTU (microwave-assistant synthesis) was used for activation, DiPEA as base,...

Gramicidin‐S‐Inspired Cyclopeptidomimetics as Potent Membrane‐Active Bactericidal Agents with Therapeutic Potential

“…Such an aliphatic character is of prime importance to maintain the biological activity of GS analogues. [35,37,43] Peptide synthesis. The synthesis of β,γ-DiAAs was achieved according to previously reported procedures.…”

“…The synthesis of β,γ-DiAAs was achieved according to previously reported procedures. [42,43,45] All other αamino acids are commercially available. The synthesis of cyclopeptidomimetics begins by building the linear sequence on solid phase starting from Fmoc-Pro-2-Chlorotrityl Chloride (CTC)-Resin.…”

“…Structure of gramicidin S (GS) and side view of its X-ray crystallographic structure (left panel). Structures of previously reported GS-based cyclopeptidomimetics [42,43] (middle panel) and GS-inspired cyclopeptidomimetics of this work (right panel). reverse-phase high performance liquid chromatography (RP-HPLC).…”

“…We have reported several studies which established the possible involvement of the supplemental amino groups in extra hydrogen bonds [39,40] . More recently, we have demonstrated that the replacement of D Phe‐Pro in the β‐turn region by turn mimics β,γ‐diamino acids (β,γ‐DiAAs) affords GS analogues endowed with retained activity but no hemolytic effect [42,43] . In this work, four aromatic β,γ‐DiAAs differing in stereochemistry were used to reinforce the amphiphilic character of GS‐inspired cyclopeptidomimetics (Figure 1, left panel).…”

“…[39,40] More recently, we have demonstrated that the replacement of D Phe-Pro in the β-turn region by turn mimics β,γ-diamino acids (β,γ-DiAAs) affords GS analogues endowed with retained activity but no hemolytic effect. [42,43] In this work, four aromatic β,γ-DiAAs differing in stereochemistry were used to reinforce the amphiphilic character of GS-inspired cyclopeptidomimetics ( Figure 1, left panel). We showed that peptidomimetic CP-2 is as toxic as GS towards bacterial cells while being relatively less toxic to human cells.…”