Fluorescently-Labeled Ferrichrome Analogs as Probes for Receptor-Mediated, Microbial Iron Uptake

Abstract

Biomimetic analogs 1 of the microbial siderophore (iron carrier) ferrichrome were labeled with a fluorescent marker at a site which does not interfere with iron binding or receptor recognition to provide iron(III) carriers 5 and 10 (Figure ). These carriers were built from a tetrahedral carbon as an anchor which was symmetrically extended by three converging iron-binding chains and a single, exogenous anthracenyl residue. Carriers 5 varied in the nature of the amino acids (G = glycyl, A = alanyl and L = leucyl) linking the anchor with the iron-binding hydroxamate groups, while the alanyl derivative 10A differed from 5A in the spacer between the anthracenyl label and the anchor. Examination of these binders by 1H NMR confirmed that their conformations were analogous to those of the nonlabeled parent compounds. Titration experiments using UV/vis and fluorescence spectroscopy demonstrated the quenching of these compounds' fluorescence upon iron(III) loading and its recovery upon iron(III)'s release to a competing chelator. The quenching process fits the Perrin model for static quenching and was more efficient in derivatives 5, where the label could approach the iron-binding domain, than in derivative 10A, where the label's approach was prohibited. These data are in compliance with an intramolecular quenching process. In vivo examination of the labeled derivatives 5 with Pseudomonas putida as the indicator organism established that their behavior parallels that of the nonlabeled analogs 1, with the added benefit of signaling microbial activity by fluorescence emission. Thus incubation of P. putida with iron(III)-loaded (and therefore nonfluorescent) 5G caused buildup of the label's fluorescence in the culture medium. These observations provide direct evidence for a shuttle-mechanism of iron delivery where the fluorescent, iron-unloaded carrier is released to the medium. Inhibition of both phenomena by natural ferrichrome or NaN3 demonstrates the involvement of the microbial ferrichrome receptor and transport systems, respectively. On the other hand, 5A induced only modest iron(III) uptake by P. putida and failed to generate fluorescence in the culture medium, concurring with its action as an inhibitor. The fact that two strains of different Pseudomonas species did not respond to the ferrichrome analog 5G illustrates the specificity of these compounds. The performance of these carriers as structural and functional probes, paired with their high species specificity, encourage their consideration as diagnostic tools for the detection and identification of pathogenic bacteria and fungi.

Siderophores for molecular imaging applications

“…Table 1 summarizes applications of siderophores as imaging agents. They can be prepared either by the introduction of appropriate radiometal to the natural (iron-)siderophore complex via the exchange of iron or artificially by the modification of natural siderophore with a chromophore suitable for optical imaging [31][32][33][34]. Already in the 1970s and 1980s, first investigations of radiolabelled siderophores, including desferrioxamine (DFO), were already reported with gamma-emitting radionuclides- 67 Ga and 111 In [35][36][37][38].…”

“…Several groups have developed strategies of synthesizing siderophore-chromophore conjugates for optical imaging [31][32][33][55][56][57][58]. Siderophores (e.g.…”

“…Pseudomonas spp.) [31,32,58] and fungi (Ustilago maydis, Saccharomyces cerevisiae, Candida albicans and Rhizzopus arrhizus) [33,55,57]. The microbial activity was not altered by the attachment of various functionalities and fluorescent siderophore analogues became invaluable tools in the investigation of molecular mechanisms involved in microbial iron transport and acquisition.…”

A New Ferric Ion-Selective Fluorescent Chemosensor with a Wide Dynamic Range

“…Since iron ion is paramagnetic, many iron sensors gave rise to the fluorescence-quenching process, 4,5 although the measurement of fluorescence enhancement upon binding with an analyte is more efficient.…”

Iron uptake in Ustilago maydis: studies with fluorescent ferrichrome analogues

“…We have recently synthesized siderophore analogues that possess fluorescent markers at a site remote from the iron (111)-binding domain and receptor-recognition sites (Weizman et al, 1996). These derivatives act as structural probes, by tracing the structural requirements for recognition by the fungal iron-uptake system, and as dynamic probes for iron-exchange processes, by converting from a non-fluorescent to a fluorescent state upon iron (111) release.…”