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,...
We have developed a new concept for reversible peptide stapling that involves macrocyclization between two amino groups and decyclization promoted via dual 1,4-elimination. Depending on the trigger moiety, this strategy could be employed to selectively deliver peptides to either intracellular or extracellular targets. As a proof of concept, a peptide inhibitor targeting a lysinespecific demethylase 1 (LSD1) was temporarily cyclized to enhance its stability and ability to cross the cell membrane. Once inside the cells, the biologically active linear peptide was released under reducing environment. Moreover, we have developed reversibly stapled peptides using antimicrobial peptides (RStAMPs) whose bioactive helical conformation can be temporarily destabilized by stapling the peptide backbone. The resulting helix-distorted RStAMPs are nontoxic and highly resistant to protease hydrolysis, while at the infection site, RStAMPs can be rapidly activated by the overproduced H 2 O 2 through the dual 1,4-elimination. The latter restored the helical structure of the native peptide and its antimicrobial activity. This work illustrates a highly valuable macrocyclization strategy for the peptide community and should greatly benefit the field of peptide delivery.
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