Gramicidin S: Structure, Activity, and New Synthetic Analogues
Gramicidin S (GS) is an important and powerful antibiotic, effective against a variety of Gram-positive and Gram-negative bacteria. The C2-symmetric cyclic decapeptide with primary structure cyclo(D-Phe-Pro-Val-Orn-Leu)2 forms an antiparallel β-sheet, with its polar Orn side-chains aligned on one side and its aliphatic Val and Leu side-chains directed oppositely (Figure 1). The amphiphilic nature of GS enables interaction with and disruption of bacterial lipid bilayers, responsible for its effectiveness as an antimicrobial agent.
Figure 1. Gramicidin S and Geyer’s Synthetic Analogues
Unfortunately, though a potent antibiotic, GS is limited to topical applications due to its lack of hemolytic selectivity. Recently, however, the Geyer Laboratory at Philipps-Universität Marburg (Marburg, Germany) and the Ulrich Laboratory at Karlsruhe Institute of Technology (Karlsruhe, Germany) have demonstrated the ability to alter the amphiphilic nature and biological selectivity of Gramicidin S through the synthesis of cis-dihydroxyproline (Dyp) and D-hydroxythreonine═thiaproline (D-Hot═Tap) analogues (Figure 1).1
Modification of Gramicidin S: Dyp and D-Hot═Tap Building Block Synthesis
To access Dyp and D-Hot═Tap analogues (15–19) of Gramicidin S, Geyer et al. first synthesized Fmoc-Dyp-acetonide and Fmoc-D-Hot═Tap-ketal building blocks. Fmoc-Dyp-acetonide 9 was synthesized from commercially available Hyp in a 30% yield over nine steps (Scheme 1). Upon hydroxyl group activation and elimination, resulting alkene 5 underwent dihydroxylation and a series of reprotection steps to yield 9, which was used directly in solid-phase peptide synthesis (SPPS) of Dyp analogue 15.
Scheme 1. Synthesis of cis-Dihydroxyproline (Dyp) Building Block.
Fmoc-D-Hot═Tap-ketals 14a–14d were synthesized from diol 11 over three steps (Scheme 2). Upon ketalization and saponification of 11, resulting N3-D-Hot═Tap-ketals 12a–12d underwent azide reduction and acylation to yield 14a–14d, which were used directly in respective SPPS of D-Hot═Tap analogues 16–19. Notably, though ketals typically undergo ready hydrolysis, in this tricyclic ring system the moiety remains resilient, even under acidic conditions.
Scheme 2. Synthesis of D-Hydroxythreonine═Thiaproline (D-Hot═Tap) Building Blocks.
Modification of Gramicidin S: Dyp and D-Hot═Tap Analogue Synthesis
Upon completing the synthesis of Fmoc-Dyp-acetonide 9 and Fmoc-D-Hot═Tap-ketals 14a–14d, Geyer et al. prepared Gramicidin S analogues 15–19. First, the linear precursors of the Dyp and D-Hot═Tap GS analogues were synthesized via SPPS on 2-CTC resin (Scheme 3). All GS analogue precursors were prepared with the Liberty Blue™ automated microwave peptide synthesizer, with the exception of 16, which was prepared manually.
Scheme 3. Synthesis of Dyp and D-Hot═Tap Analogues
Following linear precursor synthesis, the peptide was cleaved from the resin with 1% TFA in DCM, ensuring Boc-protected Orn remained intact. The linear peptide then underwent intramolecular cyclization, employing HATU/HOAt and DIPEA in DMF. Lastly, the Orn Boc-protecting groups were removed with TFA/DCM (1:6) and the crude cyclopeptide was purified by semipreparative RP-HPLC to yield analogues 15–19 (Scheme 3).
Modification of Gramicidin S: Dyp and D-Hot═Tap Analogue Bioactivity
To conclude the study, Geyer et al. tested GS analogues 15–19 against a number of Gram-positive and -negative bacteria for antimicrobial activity and hemolytic selectivity. Dyp analogue 15, demonstrated a decrease in antimicrobial activity, but with an increase in selectivity. Surprisingly, D-Hot═Tap-dimethylketal analogue 16 was inactive. D-Hot═Tap analogues 18 and 19, containing a C13- and two C7-alkyl chains respectively, experienced an increase in hemolytic activity, a decrease in activity against Gram-positive bacteria, and no activity against Gram-negative bacteria. C8-Alkyl chain containing analogue 17, however, displayed comparable antimicrobial activities to GS but with an increase in hemolytic selectivity