Biomimetic Enterobactin Analogue Mediates Iron-Uptake and Cargo Transport into E. coli and P. aeruginosa

The design, synthesis and biological evaluation of the artificial enterobactin analogue Ent_KL and several fluorophore-conjugates thereof are described. Ent_KL provides an attachment point for cargos such as fluorophores or antimicrobial payloads. Corresponding conjugates are recognized by outer membrane siderophore receptors of Gram-negative pathogens and retain the natural hydrolyzability of the tris-lactone backbone, known to be key for uptake into the cytosol. Initial density-functional theory (DFT) calculations of the free energies of solvation (ΔG(sol)) and relaxed Fe-O force constants of the corresponding [Fe-Ent_KL]^3- complexes indicated a similar iron binding constant compared to natural enterobactin (Ent). The synthesis of Ent_KL was achieved via an iterative assembly based on a 3-hydroxylysine building block over 14 steps with an overall yield of 3%. A series of growth recovery assays under iron-limiting conditions with Escherichia coli and Pseudomonas aeruginosa mutant strains that are defective in natural siderophore synthesis revealed a potent concentration-dependent growth promoting effect of Ent_KL similar to natural Ent. Additionally, four cargo-conjugates differing in molecular size were able to restore growth of E. coli indicating an uptake into the cytosol. P. aeruginosa displayed a stronger uptake promiscuity as six different cargo-conjugates were found to restore growth under iron-limiting conditions. Imaging studies utilizing BODIPY_FL-conjugates, demonstrated the ability of Ent_KL to overcome the Gram-negative outer membrane permeability barrier and thus deliver molecular cargos via the bacterial iron transport machinery of E. coli and P. aeruginosa.