title: Synthesis of a polymyxin derivative for photolabeling studies in the gram-negative bacterium Escherichia coli.
abstract: The antimicrobial activity of polymyxins against Gram-negative bacteria has been known for several decades, but the mechanism of action leading to cell death has not been fully explored. A key step after binding of the antibiotic to lipopolysaccharide (LPS) exposed at the cell surface is 'self-promoted uptake' across the outer membrane (OM), in which the antibiotic traverses the asymmetric LPS-phospholipid bilayer before reaching the periplasm and finally targeting and disrupting the bacterial phospholipid inner membrane. The work described here was prompted by the hypothesis that polymyxins might interact with proteins in the OM, as part of their self-promoted uptake and permeabilizing effects. One way to test this is through photolabeling experiments. We describe the design and synthesis of a photoprobe based upon polymyxin B, containing photoleucine and an N-acyl group with a terminal alkyne suitable for coupling to a biotin tag using click chemistry. The resulting photoprobe retains potent antimicrobial activity, and in initial photolabeling experiments with Escherichia coli ATCC25922 is shown to photolabel several OM proteins. This photoprobe might be a valuable tool in more detailed studies on the mechanism of action of this family of antibiotics.
authors: Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland.
authors: Benjamin
authors: B
authors: van der Meijden
authors: John A
authors: JA
authors: Robinson
mesh_terms: D000480:Alkynes / Q000737:chemistry; D000900:Anti-Bacterial Agents / Q000138:chemical synthesis* / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects*; D057930:Click Chemistry; D004926:Escherichia coli / Q000737:chemistry / Q000187:drug effects* / Q000502:physiology; D008027:Light; D008070:Lipopolysaccharides / Q000737:chemistry; D008826:Microbial Sensitivity Tests; D015336:Molecular Probe Techniques; D015335:Molecular Probes / Q000138:chemical synthesis* / Q000494:pharmacology; D010743:Phospholipids / Q000737:chemistry; D055668:Photochemical Processes; D011112:Polymyxin B / Q000031:analogs & derivatives / Q000138:chemical synthesis* / Q000494:pharmacology; D060327:Solid-Phase Synthesis Techniques / Q000379:methods; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D000480:Alkynes; D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D008070:Lipopolysaccharides; D015335:Molecular Probes; D010743:Phospholipids; D001710:Biotin; D011112:Polymyxin B
keywords: antibiotic; click reaction; mechanism of action; membrane protein; peptide; polymyxin