Total Synthesis of Bacicyclin Analog, cyclo-(D-Phe-Lys-Ile-Val-MeLeu-Gly), using a Combination of Solid- and Solution-Phase Method

Abstract

Bacicyclin [cyclo-(D-Phe-D-Ala-Ile-Val-Leu-Gly)] is a cyclic hexapeptide with antimicrobial activity against Enterococcus  faecalis and Staphylococcus aureus  with MIC values ​​of 8 and 12 μM, respectively. Studies on bacicyclin analog were conducted to investigate how the substitution of bacicyclin sequences affects its antibacterial activity. In the studies, two residues, D-Ala and Leu, were replaced by respective Lys and MeLeu residues. Bacicyclin analog [cyclo-(D-Phe-Lys-Ile-Val-MeLeu-Gly)], was successfully synthesized by constructing the linear precursor on 2-chlorotrityl chloride resin using Fmoc-based strategy. The HATU/HOAt reagent was applied in all peptide bond formation. The desired linear peptide was released from the resin using trifluoroethanol:acetic acid: dichloromethane (2:2:6) giving the product in 92% yield. Cyclization of the linear peptide was carried out using HATU as a cyclization agent and DIPEA  as base in 1mM solution. Deprotection of Boc group was then carried out under 95% trifluoroacetic acid in dichloromethane. The synthesized product was then purified using semi-preparative RP-HPLC to obtain bacicyclin analog (10.3% yield). The purity of cyclic product was analyzed by analytical RP-HPLC (t_R= 35.2). The purified product was characterized by HR-TOF-MS ES⁺ giving molecular ion peak at m/z  [M+H]⁺= 672.4456 and confirmed with ¹H-NMR (500 MHz CD₃OD-d₄) and ¹³C-NMR (125 MHz CD₃OD-d₄). The antibacterial activity of bacicyclin analog was tested and showing a lower antibacterial activity than bacicyclin. The result indicated that the amino acid sequence dictates the antibacterial activity. This finding gives additional information on the relationship between peptide sequence and antibacterial activity.