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

Abstract

The design, synthesis and biological evaluation of the artificial enterobactin analogue <b>Ent<sub>KL</sub></b> and several fluorophore-conjugates thereof are described. <b>Ent<sub>KL</sub></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<sub>KL</sub>]<sup>3-</sup> </b>complexes<b> </b>indicated a similar iron binding constant compared to natural enterobactin (<b>Ent</b>). The synthesis of <b>Ent<sub>KL</sub></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<sub>KL</sub></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<sub>FL</sub>-conjugates, demonstrated the ability of <b>Ent<sub>KL</sub> </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>.

Advances in the Synthesis of Enterobactin, Artificial Analogues, and Enterobactin-Derived Antimicrobial Drug Conjugates and Imaging Tools for Infection Diagnosis

“…Although, the artificial backbone might reduce enterobactin associated immunogenicity, specific back bones can lead to cytotoxic properties of the resulting siderophores as proven by us for a close derivative of Miller’s enterobactin analogue 438 . 152…”

“…201 Nolan and co-workers further speculated on a cargo size limit for the uptake through FepA as they were not observing convincing evidence for marked uptake of larger cargos. 201 However, considering the pore size of FepA (see Figure 2 , vide supra ), later results of Nolan and co-workers 202 and our own work in the field of enterobactin-derived cargo-conjugates with attachment point in the backbone of the siderophore, 152 it is more likely that the carboxylate handle introduced by Nolan and co-workers leads to interference of recognition and uptake of the siderophore in the case of larger and more rigid cargo payloads.…”

“…In our own studies for the development of novel artificial enterobactin analogues, we generated a closely related derivative of Miller’s siderophore 438 , which showed significant two-digit micromolar cytotoxic activity against human HepG2 hepatocytes. 152 In addition, the structurally related artificial siderophore CaCAM 451 (Figure 17 ) based on 5-amino-2,2-bis(3-aminopropyl)pentanoic acid as backbone, introduced in 2000 by Pierre and co-workers, 329 as well as further related artificial enterobactin analogues 452a – c 330 (Figure 17 ) showed similar hepatotoxic activity. 252…”

“…54 , 141 142 143 Inspired by these natural antetypes, researchers have developed different artificial siderophores and utilized them for the construction of artificial siderophore-drug conjugates and reporter conjugates in order to explore this fascinating approach of active translocation over the bacterial cell envelope for the development of treatment options against Gram-negative pathogens 19 123 124 127 , 144 145 146 147 148 149 150 and molecular tool compounds for infection diagnosis. 151 152 153 154 155 156 In this context, enterobactin and its derivatives have attracted great interest due to its key role in the securing of iron supply during infection in human host.…”

“…Recently, our group developed the biomimetic enterobactin analogue Ent KL 240 (Scheme 16 A) in which we retained the natural cleavability of the tris -lactone backbone present in the natural antetype while providing a handle for the attachment of payloads in the backbone of the siderophore and thus out of the receptor recognition site by incorporation of the γ-modified l -allothreonine derivative 228 . 152 However, as the tin-template mediated transesterification approach utilized earlier by Shanzer, 180 220 Guiterrez, 181 and Raymond 190 for the synthesis of the enterobactin tris-serine lactone backbone, leads exclusively to the formation of the thermodynamic, symmetric product and threonines have been shown not to react under these conditions, 181 it was not applicable for the synthesis of the envisaged asymmetric tris -lactone backbone. Therefore, we decided to follow an approach of iterative assembly of a linear trimer containing the l -allothreonine derivative 228 with subsequent macrolactonization as used in the early synthesis of enterobactin by Corey 177 and Rastetter 178 in order to generate the desired asymmetric backbone of Ent KL 240 .…”