A Fluorescent Teixobactin Analogue

Abstract

This report describes the first synthesis and application of a fluorescent teixobactin analogue that exhibits antibiotic activity and binds to the cell walls of Gram-positive bacteria. The teixobactin analogue, Lys(Rhod)₉,Arg₁₀-teixobactin, has a fluorescent tag at position 9 and an arginine in place of the natural allo-enduracididine residue at position 10. The fluorescent teixobactin analogue retains partial antibiotic activity, with minimum inhibitory concentrations of 4-8 μg/mL across a panel of Gram-positive bacteria, as compared to 1-4 μg/mL for the unlabeled Arg₁₀-teixobactin analogue. Lys(Rhod)₉,Arg₁₀-teixobactin is prepared by a regioselective labeling strategy that labels Lys₉ with an amine-reactive rhodamine fluorophore during solid-phase peptide synthesis, with the resulting conjugate tolerating subsequent solid-phase peptide synthesis reactions. Treatment of Gram-positive bacteria with Lys(Rhod)₉,Arg₁₀-teixobactin results in septal and lateral staining, which is consistent with an antibiotic targeting cell wall precursors. Concurrent treatment of Lys(Rhod)₉,Arg₁₀-teixobactin and BODIPY FL vancomycin results in septal colocalization, providing further evidence that Lys(Rhod)₉,Arg₁₀-teixobactin binds to cell wall precursors. Controls with either Gram-negative bacteria, or an inactive fluorescent homologue with Gram-positive bacteria, showed little or no staining in fluorescence micrographic studies. Lys(Rhod)₉,Arg₁₀-teixobactin can thus serve as a functional probe to study Gram-positive bacteria and their interactions with teixobactin.

Figure 1.

Structures of Arg₁₀-teixobactin, Lys(Rhod)₉,Arg₁₀-teixobactin, and seco-Lys(Rhod)₉,Arg₁₀-teixobactin.

Figure 2.

Fluorescence and differential interference contrast (DIC) micrographs of B. subtilis treated with 4 μg/mL of Lys(Rhod)₉,Arg₁₀-teixobactin in sodium phosphate buffer containing no polysorbate 80. Fluorescence micrographs were recorded with excitation at 561 nm. Scale bars of the top row images are 10 μm, while the scale bars of the bottom row images are 2 μm.

Figure 3.

Fluorescence and differential interference contrast (DIC) micrographs of (A) B. subtilis, (B) E. durans, (C) S. salivarius, (D) S. aureus, and (E) E. coli treated with 4 μg/mL of Lys(Rhod)₉,Arg₁₀-teixobactin in sodium phosphate buffer containing 0.05% polysorbate 80. Fluorescence micrographs were recorded with excitation at 561 nm. Scale bars are 2 μm.

Figure 4.

Fluorescence and differential interference contrast (DIC) micrographs of B. subtilis concurrently treated with 1 μg/mL of Lys(Rhod)₉,Arg₁₀-teixobactin and 1 μg/mL of Arg₁₀-teixobactin in sodium phosphate buffer containing 0.05% polysorbate 80. Fluorescence micrographs were recorded with excitation at 561 nm. Scale bars are 2 μm.

Figure 5.

Fluorescence and differential interference contrast (DIC) micrographs of (A) B. subtilis, (B) E. durans, (C) S. salivarius, (D) S. aureus concurrently treated with 4 μg/mL of Lys(Rhod)₉,Arg₁₀-teixobactin and 4 μg/mL of BODIPY FL vancomycin in sodium phosphate buffer containing 0.05% polysorbate 80. Fluorescence micrographs were recorded with excitation at 561 nm (left column panel) and 488 nm (right column panel). Scale bars are 2 μm.

Figure 6.

Fluorescence and differential interference contrast (DIC) micrographs of (A) B. subtilis, (B) E. durans, (C) S. salivarius, and (D) S. aureus treated with 4 μg/mL of seco-Lys(Rhod)₉,Arg₁₀-teixobactin in sodium phosphate buffer containing 0.05% polysorbate 80. Fluorescence micrographs were recorded with excitation at 561 nm. Scale bars are 2 μm.

Scheme 1.

Synthesis of Lys(Rhod)₉,Arg₁₀-teixobactin