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The yeast Rhodotorula pilimanae produces the dihydroxamate siderophore rhodotorulic acid (RA) in prodigious amounts when starved for iron. Synthetic dihydroxamate analogs of RA have been prepared in which the diketopiperazine ring of RA is replaced by a simple chain of n methylene groups. It is found that R. pilimanae is able to accumulate iron using these achiral complexes, as well as from simple monohydroxamate analogs, at rates comparable to those of RA. While the Fe2RA3 complex does not enter the cell, there is a receptor system whose geometric requirements for siderophore recognition have been probed using analogs. In contrast to mono- or dihydroxamate ligands, the trihydroxamate siderophores such as ferrioxamine B are completely ineffective at delivering iron to R. pilimanae. This is ascribed to the greater stability of these complexes, which blocks release of the Fe(III) in a ligand exchange process that is required for uptake. To explore whether this ligand exchange involves redox catalysis, Ga(III) was substituted for Fe(III). The gallium was taken up at rates near those of iron and were also energy-dependent, as determined by metabolic inhibition with KCN.

Although the ferrioxamines are an important and well-characterized class of siderophores produced by several species of Nocardia, Streptomyces, Micromonospora, Arthrobacter, Chromobacterium, and Pseudomonas, no studies of the mechanism of ferrioxamine-mediated iron uptake have been performed for an organism which produces the siderophore. This is the first report of metal transport in Streptomyces pilosus mediated by the native ferrioxamines B, D1, D2, and E. 55Fe accumulation in these ferrioxamines was dependent on metabolic energy and was a saturable process with increasing complex concentration. The apparent Km for [55Fe]ferrioxamine B uptake was approximately 0.2 microM. Both chromic desferriferrioxamine B and [67Ga]desferriFerrioxamine B were transported at rates similar to those of the 55Fe complexes: this implies that no decomplexation or reduction of the metal ion is required for transport, since the chromic complexes are kinetically inert and the gallium complexes have no stable divalent state as a possible reduction product. In addition, isomers of inert chromic desferriferrioxamine B complexes were used to probe the stereospecificity of the ferrioxamine uptake system. The chromic complexes were separated into three fractions by cationic exchange chromatography and assigned as two cis and a (mixture of) trans geometrical isomer(s) by their visible spectra. [55Fe]ferrioxamine B uptake was equally inhibited by each isomer, suggesting that no differentiation between cis and trans geometrical isomers occurs. In the presence of chromic desferriferrioxamine B isomers, the uptake rates for 55Fe-labeled ferrioxamines E, D1, and D2 were even more strongly reduced than was that for [55Fe]ferrioxamine B itself. From these results we conclude that all the ferrioxamines tested are transported into the cells by the same uptake system.

Synthetic analogs of enterochelin (enterobactin) were tested for their ability to support the growth of Escherichia coli K-12 under iron-limiting conditions. The cyclic compound MECAM [1,3,5-N.N'; N"-tris-(2,3-dihydroxybenzoyl)-triamino-methylbenzene] and its N-methyl derivative Me3MECAM promoted growth, whereas the 2,3-dihydroxy-5-sulfonyl derivatives MECAMS and Me3MECAMS were inactive. The same results were obtained with TRIMCAM [1,3,5-tris(2,3-dihydroxybenzoylcarbamido)-benzene] and TRIMCAMS (the 2,3-dihydroxy-5-sulfonyl derivative of TRIMCAM). However, the sulfonic acid-containing linear compound LICAMS [1,5,10-N,N', N"-tris(5-sulfo-2,3-dihydroxybenzoyl)-triaza-decane] supported growth. In contrast, LIMCAMC, in which the sulfonyl groups at the five position of LICAMS are replaced by carboxyl groups at the four position, was inactive. The uptake of the active analogs required the functions specified by the fepB, fesB, and tonB genes. Surprisingly, growth promotion of mutants lacking the enterochelin receptor protein in the outer membrane was observed. Only MECAM protected cells against colicin B (which kills cells after entering at the enterochelin uptake sites) and transported Fe3+ at about half the enterochelin rate.

Rhodotorulic acid (RA), a dihydroxamate siderophore produced by Rhodotorula pilimanae, forms 3:2 complexes with ferric and chromic ions (M2RA3) at pH 7. Kinetically inert chromic complexes of RA have been separated into geometrical isomers and for the first time partially resolved into optical isomers. The three isomers delta-cis, delta-trans, and lambda-trans were characterized by their visible and circular dichroism spectra. Inhibition by both delta-isomers of radiolabeled ferric RA uptake in R. pilimanae was equally effective. However the lambda-cis isomer was significantly less effective as an inhibitor. Concentration-dependent uptake kinetics were performed with ferric RA and the ferric complex of synthetic enantio-RA, which form predominantly delta and lambda complexes, respectively. The lambda-enantio-Fe2RA3 was 50% less effective in supplying iron to R. pilimanae than was Fe2RA3. An additional synthetic analog of RA, which lacks a carbonyl group at the diketopiperazine ring, exhibited the same uptake rates as ferric RA. We conclude that stereoselective recognition of optical isomers takes place during iron uptake mediated by RA and that this recognition primarily involves the right-handed delta coordination "propellor" of the metal center and its adjacent functionalities.

Macromolecules containing four sulfonated catecholy (2,3-dihydroxybenzoyl) groups are effective for decorporation of newly acquired Pu(IV). However, multiple injections in mice and single injections in dogs of 30 mumole/kg of 3,4,3-LICAM(S), the most effective sulfonated poly(catechoylamide) ligand, indicated that it would be toxic, so the ligand structure was modified. Each ligand was injected into mice (30 mumole/kg, intraperitoneally) 1 hr after an intravenous injection of 238Pu(IV) citrate, and mice were killed 24 hr after the Pu injection. Excreta and tissues were analyzed for Pu. (a) The number of catechoyl groups per molecule was reduced to suppress affinity for Fe(III). Net excretion (treated - control) of 55% of the injected Pu was promoted by tetrameric 3,4,3-LICAM(S), 51% by trimeric 3,4-LICAM(S), 22% by dimeric 2-LICAM(S), and 7.4% by the monomer, Tiron. (b) A mesitylene platform was substituted for the linear backbone. Net Pu excretion promoted by MECAM(S), a structurally less flexible trimer, was only 26%, and excretion was delayed. (c) A carboxyl substituent on the catechoyl groups reduced the acidity and hydrophilicity of the ligands. Tetrameric 3,4,3-LICAM(C) promoted 63% net Pu excretion, and one-third of that was fecal. The Pu contents of liver and skeleton were 33 and 44% of their respective 1-hr control values--compared to 51 and 44%, respectively, for CaNa3-DTPA. Mice given 30 mumole/kg of 3,4,3-LICAM(C) 20 times in 4 weeks showed no ill effects. (d) Large N-terminal alkane substituents added to 3,4,3-LICAM(C) increased ligand lipophilicity, hindered Pu chelation, and delayed excretion.

Rhodotorulic acid (RA), a dihydroxamate siderophore produced by Rhodotorula pilimanae, forms 3:2 complexes with ferric and chromic ions (M2RA3) at pH 7. Kinetically inert chromic complexes of RA have been separated into geometrical isomers and for the first time partially resolved into optical isomers. The three isomers delta-cis, delta-trans, and lambda-trans were characterized by their visible and circular dichroism spectra. Inhibition by both delta-isomers of radiolabeled ferric RA uptake in R. pilimanae was equally effective. However the lambda-cis isomer was significantly less effective as an inhibitor. Concentration-dependent uptake kinetics were performed with ferric RA and the ferric complex of synthetic enantio-RA, which form predominantly delta and lambda complexes, respectively. The lambda-enantio-Fe2RA3 was 50% less effective in supplying iron to R. pilimanae than was Fe2RA3. An additional synthetic analog of RA, which lacks a carbonyl group at the diketopiperazine ring, exhibited the same uptake rates as ferric RA. We conclude that stereoselective recognition of optical isomers takes place during iron uptake mediated by RA and that this recognition primarily involves the right-handed delta coordination "propellor" of the metal center and its adjacent functionalities.

The effect of the intravenous administration of the synthetic siderophore LI-CAM-C on the biodistribution of Ga-67 was investigated. The ligand was found capable of the in vivo complexing with Ga-67, which hastened the renal clearance of the nuclide. The gallium concentration was decreased in all organs, with the exception of the liver and spleen, where we suggest that hydroxide precipitates interfere with gallium sequestration by LICAM-C. The gallium in abscess tissue was only slightly affected, giving rise to an increase in the Ga-67 abscess-to-soft-tissue concentration ratio when LICAM-C is administered. Dosimetry calculations show that the siderophore decreases the radiation burden from Ga-67 citrate. The advantage of clinical application of LICAM-C are discussed.

Isopropyl N-substituted tricatecholamide analogs of enterobactin have been found to form gallium and indium complexes with very high stability constants and to exhibit in vivo characteristics significantly different from gallium- or indium-transferrin and EDTA. The 3,4-DiP-LICAMS and TiP-MECAMS complexes were found to clear primarily through the kidneys, whereas the less polar 3,4-DiP-LICAM complex was eliminated through the liver. The rationale for developing new metal-binding analogs with larger organic groups attached to the amide nitrogens is discussed.

The yeast Rhodotorula pilimanae produces the dihydroxamate siderophore rhodotorulic acid (RA) in prodigious amounts when starved for iron. Synthetic dihydroxamate analogs of RA have been prepared in which the diketopiperazine ring of RA is replaced by a simple chain of n methylene groups. It is found that R. pilimanae is able to accumulate iron using these achiral complexes, as well as from simple monohydroxamate analogs, at rates comparable to those of RA. While the Fe2RA3 complex does not enter the cell, there is a receptor system whose geometric requirements for siderophore recognition have been probed using analogs. In contrast to mono- or dihydroxamate ligands, the trihydroxamate siderophores such as ferrioxamine B are completely ineffective at delivering iron to R. pilimanae. This is ascribed to the greater stability of these complexes, which blocks release of the Fe(III) in a ligand exchange process that is required for uptake. To explore whether this ligand exchange involves redox catalysis, Ga(III) was substituted for Fe(III). The gallium was taken up at rates near those of iron and were also energy-dependent, as determined by metabolic inhibition with KCN.