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. 2024 Sep 6;89(17):12479-12484.
doi: 10.1021/acs.joc.4c01414. Epub 2024 Aug 23.

Structure-Activity Relationship Studies of the Peptide Antibiotic Clovibactin

Jackson E H Brunicardi  1 , James H Griffin  1 , Michael J Ferracane  2 , Adam G Kreutzer  1 , Jeramiah Small  1 , Ana-Teresa Mendoza  1 , Joseph W Ziller  1 , James S Nowick  1   3
Affiliations

Structure-Activity Relationship Studies of the Peptide Antibiotic Clovibactin

Jackson E H Brunicardi et al. J Org Chem. .

Erratum in

Abstract

Our laboratory reported the chemical synthesis and stereochemical assignment of the recently discovered peptide antibiotic clovibactin. The current paper reports an improved, gram-scale synthesis of the amino acid building block Fmoc-(2R,3R)-3-hydroxyasparagine-OH that enables structure-activity relationship studies of clovibactin. An alanine scan reveals that residues Phe1, d-Leu2, Ser4, Leu7, and Leu8 are important for antibiotic activity. The side-chain amide group of the rare d-Hyn5 residue is not essential to activity and can be replaced with a methyl group with a moderate loss of activity. An acyclic clovibactin analogue reveals that the macrolactone ring is essential to antibiotic activity. The enantiomer of clovibactin is active, albeit somewhat less so than clovibactin. A conformationally constrained clovibactin analogue retains moderate antibiotic activity, while a backbone N-methylated analogue is almost completely inactive. X-ray crystallography of these two analogues reveals that the macrolactone ring adopts a crown-like conformation that binds anions.

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Conflict of interest statement

The authors declare the following competing financial interest(s): Initial support for work on clovibactin was provided by a subaward from NovoBiotic Pharmaceuticals LLC.

Figures

Figure 1
Figure 1
Improved synthesis of Fmoc-(2R,3R)-3-hydroxyasparagine-OH.
Figure 2
Figure 2
Clovibactin and representative analogues used for SAR studies.
Figure 3
Figure 3
Effect of alanine (residues 1, 2, 4, 7, and 8) or threonine (residue 5) substitution of clovibactin residues on MIC values. Ranges represent fold changes in activity compared to natural clovibactin.
Figure 4
Figure 4
Crystallographically based molecular model of clovibactin and X-ray crystallographic structures of N-methylated and stapled clovibactin analogues. (A) Molecular model of clovibactin based on the X-ray crystallographic structure of an analogue (PDB 8CUG). (B) X-ray crystallographic structure of N-Me-d-Leu2,d-Thr5-clovibactin bound to a sulfate anion. Eight peptide molecules comprise the asymmetric unit; a representative molecule is shown. The N-Me carbon of N-Me-d-Leu2 is highlighted in cyan. Hydrogen bonds are shown as yellow dashed lines. (C) X-ray crystallographic structure of stapled d-Thr5-clovibactin bound to a chloride anion. Three peptide molecules comprise the asymmetric unit; a representative molecule is shown. The hydrocarbon staple is highlighted in cyan.
Figure 5
Figure 5
d-Thr5-Clovibactin analogues.
See this image and copyright information in PMC

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