The design, synthesis and biological evaluation of the artificial enterobactin analogue EntKL and several fluorophore-conjugates thereof are described. EntKL provides an attachment point for cargos such as fluorophores or antimicrobial...

Iron is an essential growth factor for bacteria, but although highly abundant in nature, its bioavailability during infection in the human host or the environment is limited. Therefore, bacteria produce and secrete siderophores to ensure their supply with iron. The tris-catecholate siderophore enterobactin and its glycosylated derivatives, the salmochelins, play a crucial role for iron acquisition in several bacteria. As these compounds can serve as carrier molecules for the design of antimicrobial siderophore drug conjugates as well as siderophore-derived tool compounds for the detection of infections with bacteria, their synthesis and the design of artificial analogues is of interest. In this review, we give an overview on the synthesis of enterobactin, biomimetic as well as totally artificial analogues and related drug-conjugates covering up to 12/2021.

The design, synthesis and biological evaluation of the artificial enterobactin analogue <b>Ent_KL</b> and several fluorophore-conjugates thereof are described. <b>Ent_KL</b> 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 <i>tris</i>-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 <b>[Fe-Ent_KL]^3- </b>complexes<b> </b>indicated a similar iron binding constant compared to natural enterobactin (<b>Ent</b>). The synthesis of <b>Ent_KL</b> 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 <i>Escherichia coli</i> and <i>Pseudomonas aeruginosa</i> mutant strains that are defective in natural siderophore synthesis revealed a potent concentration-dependent growth promoting effect of <b>Ent_KL</b> similar to natural <b>Ent</b>. Additionally, four cargo-conjugates differing in molecular size were able to restore growth of <i>E. coli</i> indicating an uptake into the cytosol. <i>P. aeruginosa </i>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 <b>Ent_KL </b>to overcome the Gram-negative outer membrane permeability barrier and thus deliver molecular cargos via the bacterial iron transport machinery of <i>E. coli</i> and <i>P. aeruginosa</i>.

The design, synthesis and biological evaluation of the artificial enterobactin analogue <b>Ent_KL</b> and several fluorophore-conjugates thereof are described. <b>Ent_KL</b> 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 <i>tris</i>-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 <b>[Fe-Ent_KL]^3- </b>complexes<b> </b>indicated a similar iron binding constant compared to natural enterobactin (<b>Ent</b>). The synthesis of <b>Ent_KL</b> 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 <i>Escherichia coli</i> and <i>Pseudomonas aeruginosa</i> mutant strains that are defective in natural siderophore synthesis revealed a potent concentration-dependent growth promoting effect of <b>Ent_KL</b> similar to natural <b>Ent</b>. Additionally, four cargo-conjugates differing in molecular size were able to restore growth of <i>E. coli</i> indicating an uptake into the cytosol. <i>P. aeruginosa </i>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 <b>Ent_KL </b>to overcome the Gram-negative outer membrane permeability barrier and thus deliver molecular cargos via the bacterial iron transport machinery of <i>E. coli</i> and <i>P. aeruginosa</i>.

Overcoming increasing antibiotic resistance requires the development of novel antibacterial agents that address new targets in bacterial cells. Naturally occurring nucleoside antibiotics (such as muraymycins) inhibit the bacterial membrane protein MraY, a clinically unexploited essential enzyme in peptidoglycan (cell wall) biosynthesis. Even though a range of synthetic muraymycin analogues has already been reported, they generally suffer from limited cellular uptake and a lack of activity against Gram‐negative bacteria. We herein report an approach to overcome these hurdles: a synthetic muraymycin analogue has been conjugated to a siderophore, i. e. the enterobactin derivative EntKL, to increase the cellular uptake into Gram‐negative bacteria. The resultant conjugate showed significantly improved antibacterial activity against an efflux‐deficient E. coli strain, thus providing a proof‐of‐concept of this novel approach and a starting point for the future optimisation of such conjugates towards potent agents against Gram‐negative pathogens.