Total chemical synthesis of glycocin F and analogues: S-glycosylation confers improved antimicrobial activity

Abstract

Glycocin F (GccF) is a unique diglycosylated bacteriocin peptide that possesses potent and reversible bacteriostatic activity against a range of Gram-positive bacteria. GccF is a rare example of a 'glycoactive' bacteriocin, with both the O-linked N-acetylglucosamine (GlcNAc) and the unusual S-linked GlcNAc moiety important for antibacterial activity. In this report, glycocin F was successfully prepared using a native chemical ligation strategy and folded into its native structure. The chemically synthesised glycocin appeared to be slightly more active than the recombinant material produced from Lactobacillus plantarum. A second-generation synthetic strategy was used to prepare 2 site selective 'glyco-mutants' containing either two S-linked or two O-linked GlcNAc moieties; these mutants were used to probe the contribution of each type of glycosidic linkage to bacteriostatic activity. Replacing the S-linked GlcNAc at residue 43 with an O-linked GlcNAc decreased the antibacterial activity, while replacing O-linked GlcNAc at position 18 with an S-linked GlcNAc increased the bioactivity suggesting that the S-glycosidic linkage may offer a biologically-inspired route towards more active bacteriocins.

Scheme 1. Synthesis of GccF (1) and [²⁷d-His]-GccF, with the problematic His highlighted in red. Reagents and conditions: (i) 6 M Gn·HCl, 0.2 M Na₂HPO₄, 100 mM MPAA, 20 mM TCEP, pH 6.8, r.t, 2 h; (ii) 6 M Gn·HCl, 0.2 M Na₂HPO₄, methoxylamine·HCl, pH 4, r.t, 16 h; (iii) 6 M Gn·HCl, 0.2 M Na₂HPO₄, 100 mM MPAA, 20 mM TCEP, pH 6.8, r.t, 4 h; (iv) 6 M Gn·HCl, 1 M HEPES, hydrazine, 2-mercaptoethanol, 0 °C, 30 min; (v) 1.5 M Gn·HCl, 50 mM Na₂HPO₄, 2 mM cysteine, 0.25 mM cystine, 0.025 mM EDTA, 0.25 mM peptide, pH 8.2, 4 °C, 16 h.

Scheme 2. Optimised synthesis of GccF fragment 9, which was then employed for the synthesis of glycol-mutants 10 and 11. Reagents and conditions: (i) Fmoc-l-Cys(β-GlcNAc(OAc)₃)-OH for 9 and 13 or Fmoc-l-Ser(β-GlcNAc(OAc)₃)-OH for 12, iPr₂EtN, CH₂Cl₂, r.t, 1 h; (ii) Fmoc-SPPS (Fmoc deprotection: 20% piperidine in DMF (v/v)), r.t, (2 × 5 min); coupling: Fmoc-amino acid, HATU, iPr₂EtN, DMF, r.t, 40 min except Fmoc-¹⁸Ser(β-GlcNAc(OAc)₃)-OH, HATU, HOAt, TMP, DMF, r.t, overnight and Fmoc-²¹Cys(Trt)-OH, HATU, HOAt, TMP, CH₂Cl₂: DMF (1 : 1, v/v), r.t, 2 × 1 h; (iii) 94% TFA, 2.5% EDT, 2.5% H₂O, 1% iPr₃SiH (v/v/v/v), r.t, 2 h; (iv)–(vi) for 14 and 15, (iv) 6 M Gn·HCl, 0.2 M Na₂HPO₄, 100 mM MPAA, 20 mM TCEP, pH 6.8, r.t, 4 h; (v) 6 M Gn·HCl, 1 M HEPES, hydrazine, 2-mercaptoethanol, 0 °C, 30 min; (vi) 1.5 M Gn·HCl, 50 mM Na₂HPO₄, 2 mM cysteine, 0.25 mM cystine, 0.025 mM EDTA, 0.25 mM peptide, pH 8.2, 4 °C, 16 h.