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. 2023 Apr 14;9(4):739-748.
doi: 10.1021/acsinfecdis.3c00043. Epub 2023 Mar 31.

Total Synthesis and Structure Assignment of the Relacidine Lipopeptide Antibiotics and Preparation of Analogues with Enhanced Stability

Karol Al Ayed  1 , Denise Zamarbide Losada  1 , Nataliia V Machushynets  2 , Barbara Terlouw  3 , Somayah S Elsayed  2 , Julian Schill  4 , Vincent Trebosc  4 , Michel Pieren  4 , Marnix H Medema  3 , Gilles P van Wezel  2 , Nathaniel I Martin  1
Affiliations

Total Synthesis and Structure Assignment of the Relacidine Lipopeptide Antibiotics and Preparation of Analogues with Enhanced Stability

Karol Al Ayed et al. ACS Infect Dis. .

Abstract

The unabated rise of antibiotic resistance has raised the specter of a post-antibiotic era and underscored the importance of developing new classes of antibiotics. The relacidines are a recently discovered group of nonribosomal lipopeptide antibiotics that show promising activity against Gram-negative pathogens and share structural similarities with brevicidine and laterocidine. While the first reports of the relacidines indicated that they possess a C-terminal five-amino acid macrolactone, an N-terminal lipid tail, and an overall positive charge, no stereochemical configuration was assigned, thereby precluding a full structure determination. To address this issue, we here report a bioinformatics guided total synthesis of relacidine A and B and show that the authentic natural products match our predicted and synthesized structures. Following on this, we also synthesized an analogue of relacidine A wherein the ester linkage of the macrolactone was replaced by the corresponding amide. This analogue was found to possess enhanced hydrolytic stability while maintaining the antibacterial activity of the natural product in both in vitro and in vivo efficacy studies.

Keywords: AMR; NRPS; bioinformatics; lipopeptides; relacidine; total synthesis.

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Conflict of interest statement

The authors declare the following competing financial interest(s): J.S., V.T., and M.P. are employees of BioVersys AG. M.H.M. is a member of the Scientific Advisory Board of Hexagon Bio and cofounder of Design Pharmaceuticals.

Figures

Figure 1
Figure 1
Structures of brevicidine (1), laterocidine (2), and previously proposed structures for relacidine A (3) and B (4). For the structures of brevicidine and laterocidine, d-amino acids are labeled d. Amino acid numbering is indicated for relacidine A and B.
Figure 2
Figure 2
Comparison of the relacidine, laterocidine, and brevicidine biosynthetic gene clusters. (A) Architecture of the NRPS modules of the relacidine, laterocidine, and brevicidine BGCs. (B) Predicted chemical structures of relacidine A and B, including stereochemical assignments. (C = condensation domain; A = adenylation domain; PCP = peptidyl carrier protein domain; E = epimerization domain). d-amino acids are labeled d.
Figure 3
Figure 3
Comparison of the Gly-recognizing A-domains in the relacidine, laterocidine, and brevicidine biosynthetic gene clusters. (A) Comparison of the active sites of the Gly-recognizing A-domains; (B) Comparison of the 3D structures predicted for the active sites of A-domain 13 containing Trp (laterocidine BGC; dark gray) or Tyr (relacidine BGC; gray), which shows interactions of these residues with the Ala substrate.
Scheme 1
Scheme 1. (A) Representative Total SPPS of Relacidine A (3a) and (B) Structures of Various Diastereomers of Relacidine A and B Also Prepared
Figure 4
Figure 4
Extracted ion chromatograms (EICs) of (A) relacidine A with m/z 517.2776 and (B) relacidine B with m/z 521.9492 from the crude extracts of B. laterosporus (red), overlaid with traces obtained for relacidine A diastereomers 3a,b and relacidine B diastereomers 4a–d. Synthetically prepared 3a and 4a coelute with relacidine A and B from B. laterosporus MG64, respectively.
Scheme 2
Scheme 2. Synthesis of Relacidamide (5)
Figure 5
Figure 5
Percent survival of G. mellonella larvae after infection with colistin resistant A. baumannii isolate BV94 and subsequent treatment with test articles.
See this image and copyright information in PMC

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