Ferrioxamine B Analogues: Targeting the FoxA Uptake System in the Pathogenic Yersinia enterocolitica Hagit Kornreich-Leshem, † Carmit Ziv, ‡ Elzbieta Gumienna-Kontecka, § Rina Arad-Yellin, † Yona Chen, ‡ Mourad Elhabiri, § Anne-Marie Albrecht-Gary,* ,§ Yitzhak Hadar,* ,‡ and Abraham Shanzer* ,† Contribution from the Department of Organic Chemistry, Weizmann Institute of Science, RehoVot, Israel, Faculty of Agricultural, Food and EnVironmental Quality Sciences, The Hebrew UniVersity of Jerusalem, RehoVot, Israel, and Laboratoire de Physico-Chimie Bioinorganique, ULP, UMR 7509 CNRS, Strasbourg, France Received March 16, 2003; Revised Manuscript Received November 5, 2004; E-mail: Abraham.Shanzer@weizmann.ac.il Abstract: A series of ferrioxamine B analogues that target the bacterium Yersinia enterocolitica were prepared. These iron carriers are composed of three hydroxamate-containing monomeric units. Two identical monomers consist of N-hydroxy-3-aminopropionic acid coupled with -alanine, and a third unit at the amino terminal is composed of N-hydroxy-3-aminopropionic acid and one of the following amino acids: -alanine (1a), phenylalanine (1b), cyclohexylalanine (1c), or glycine (1d). Thermodynamic results for representatives of the analogues have shown a strong destabilization (3-4 orders of magnitude) of the ferric complexes with respect to ferrioxamine B, probably due to shorter spacers and a more strained structure around the metal center. No significant effect of the variations at the N-terminal has been observed on the stability of the ferric complexes. By contrast, using in vivo radioactive uptake experiments, we have found that these modifications have a substantial effect on the mechanism of iron(III) uptake in the pathogenic bacteria Yersinia enterocolitica. Analogues 1a and 1d were utilized by the ferrioxamine B uptake system (FoxA), while 1b and 1c either used different uptake systems or were transported to the microbial cell nonspecifically by diffusion via the cell membrane. Transport via the FoxA system was also confirmed by uptake experiments with the FoxA deficient strain of Yersinia enterocolitica. A fluorescent marker, attached to 1a in a way that did not interfere with its biological activity, provided additional means to monitor the uptake mechanism by fluorescence techniques. Of particular interest is the observation that 1a was utilized by the uptake system of ferrioxamine B in Yersinia enterocolitica (FoxA) but failed to use the ferrioxamine uptake route in Pseudomonas putida. Here, we present a case in which biomimetic siderophore analogues deliberately designed for a particular bacterium can distinguish between related uptake systems of different microorgan- isms. Introduction Iron is an essential micronutrient for all living organisms and is involved in fundamental enzymatic reactions, such as oxygen metabolism, electron-transfer processes, and synthesis of DNA and RNA. To facilitate adequate iron(III) uptake, microorgan- isms have developed low molecular weight molecules, termed siderophores or iron carriers. Excreted into the environment, the siderophores bind ferric ions and deliver them to the microorganism via specific membrane receptors and transport proteins. The receptor-regulated process guarantees meticulous control of the intracellular iron concentration and operates against unfavorable concentration gradients. The properties and biological activity of the siderophores are dictated by their structure, chirality, and the extent by which their shape fits the binding sites of specific receptor proteins inside the membrane. 1 The importance of iron-acquisition processes has prompted us 2-4 and others 5-7 to consider artificial siderophores as structural probes for the study of microbial iron uptake processes. Investigation of analogues of the four natural siderophores, enterobactin, 8 ferrichrome, 9,10 coprogen, 11 and ferrioxamine, 11 has provided the methodology in which one can reproduce the † Weizmann Institute of Science. ‡ The Hebrew University of Jerusalem. § University Louis Pasteur of Strasbourg. (1) Winkelmann, G. Handbook of Microbial Iron Chelates; CRC Press: Boca Raton, FL, 1991. (2) Shanzer, A.; Libman, J. In Handbook of Microbial Iron Chelates; Winkelmann, G., Ed.; CRC: Boca Raton, FL, 1991; pp 309-338. (3) Shanzer, A.; Libman, J.; Yakirevitch, P.; Hadar, Y.; Chen, Y.; Jurkevitch, E. Chirality 1993, 5, 359-365. (4) Shanzer, A.; Libman, J. Met. Ions Biol. Syst. 1998, 35, 329-354. (5) Raymond, K. N.; Muller, G.; Matzanke, B. F. Top. Cur. Chem. 1984, 123, 49-102. (6) Telford, J. R.; Raymond, K. N. Compr. Supramol. Chem. 1996, 1, 245- 266. (7) Roosenberg, J. M.; Lin, Y. M.; Lu, Y.; Miller, M. J. Curr. Med. Chem. 2000, 7, 159-197. (8) Tor, Y.; Libman, J.; Shanzer, A.; Felder, C. E.; Lifson, S. J. Am. Chem. Soc. 1992, 114, 6661-6671. (9) Dayan, I.; Libman, J.; Agi, Y.; Shanzer, A. Inorg. Chem. 1993, 32, 1467- 1475. (10) Shanzer, A.; Libman, J.; Lazar, R.; Tor, Y.; Emery, T. Biochem. Biophys. Res. Commun. 1988, 157, 389-394. (11) Yakirevitch, P.; Rochel, N.; Albrecht-Gary, A.-M.; Libman, J.; Shanzer, A. Inorg. Chem. 1993, 32, 1779-1787. Published on Web 01/07/2005 10.1021/ja035182m CCC: $30.25 © 2005 American Chemical Society J. AM. CHEM. SOC. 2005, 127, 1137-1145 9 1137