Lecture Notes:

Lecture Notes: Siderophores and Iron Metabolism Structures, Functions, Role in Infection and Potential as a Novel Class of Antibiotics

Prof. Dr. Berthold F. Matzanke Isotopes Laboratory TNF University of Lübeck Ratzeburger Allee 160 D-23538 Lübeck Germany Tel. +49-(0)451-5004140 Fax: +49-(0)451-5004139 e-mail: [email protected]

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1.Introduction 2. Structures of Siderophores 2.1. Hydroxamates 2.2. Catecholates 2.3 Carboxylates 2.4. Mixed ligands

3. Coordination Geometry of Siderophores 3.1Geometrical and optical isomers 3.2UV/Vis and CD spectra 3.3Band assignment of chromic siderophores 3.4 Ligand denticity

4. Equilibrium Thermodynamics of Siderophore Iron Binding 4.1 Ligand protonation and complex formation constants 4.2 Iron exchange kinetics 4.3 Redox chemistry of siderophores

5. Siderophore Transport in Microorganisms 5.1. Siderophore receptors 5.2 Specificity and stereospecificity of siderophore recognition 5.3 Transport mechanisms 5.4Reductive removal from iron siderophores in vivo 5.4a. Bacterial iron metabolism analyzed by in situ Mössbauer spectroscopy 5.5 Siderophore uptake regulation 5.6 Siderophore secretion

6. Siderophores in Medicine 6.1 Iron overload diseases, ß-thalassemia 6.2 Siderophores and infection 6.3 Siderophore-based antibiotics 6.4 Siderophores and MRI 6.5 Iron chelators and cancer

7. Additional functions of siderophores 7.1 Siderophores as iron storage compounds 7.2 Other applications of siderophores 8. References

3 4 5 7 8 9 10 11 12 13 13 14 14 17 17 19 20 22 24 26 27 29 32 32 32 32 33 37 37 38 38 38 39

Abbreviations AL= alcaligin; BC= bisucaberin; DHBS = dihydroxybenzoylserine; DMB = N,N-dimethyl-2,3dihydroxybenzamide; Feent = ferric enterobactin; LICAMS = N,N′,N″-tris(2,3-dihydroxy-5sulfobenzoyl)-1,5,10-triazadecane; MECAM = N,N′,N″-tris(2,3-dihydroxy-5-benzoyl)-1,3,5tris(aminomethyl)benzene; MECAMS = N,N′,N″-tris(2,3-dihydroxy-5-sulfobenzoyl)-1,3,5tris(aminomethyl)benzene; men = N-methyl-l-menthoxyacet-hydroxamato; OMR = outer membrane receptor; RA = rhodotorulic acid; Sid = siderophore; TREN = tris(2-aminomethyl)amine; TRIMCAM = N,N′,N″-tris(2,3-dihydroxy-5-benzoyl)-1,3,5-tricarbamoylbenzene; TRIMCAMS = N,N′,N″-tris(2,3-dihydroxy-5-sulfobenzoyl)-1,3,5-tricarbamoylbenzene. Page 2 of 47

1 Introduction For all plants and animals, and for virtually all microbes, with the exception of some Lactobacilli and a Borrelia species1, life without iron is impossible. A multitude of essential enzymes bind iron in their active centers. Therefore, up to 105 Fe-ions are typically required in key metabolic processes of a single bacterial cell. Why iron has gained such an eminent role in the course of biological evolution remains open to speculation.11 Though iron is the fourth most abundant element in the Earth's crust, it is present under aerobic conditions at nearly neutral pH in the form of extremely insoluble minerals like hematite, goethite, and pyrite or as polymeric oxidehydrates, carbonates, and silicates which severely restrict the bioavailability of this metal. In response to this, microorganisms secrete high-affinity iron-binding compounds called siderophores (Greek: σιδηρος=iron, φορέας=carrier).3-10 In any natural environment of microbial activity, siderophores are present. Significant amounts of siderophores can be extracted from soil and from fresh or salt water. Even some foods contain siderophores.8 Biosynthesis of siderophores is executed on a cellular level by a set of enzymes specific for the respective siderophore. The corresponding genes are located on the chromosome or on a plasmid.12 Expression of these genes is controlled by the amount of cellularly available iron12-15. Therefore, microbial metabolic products (mainly secondary metabolites) can be classified as siderophores, if (i) they exhibit iron chelating capability, (ii) they participate in active transport across the cell membrane(s) and (iii) their biosynthesis is regulated by the intracellular iron level. During the last decade we witnessed an exceptional progress in the field of iron-transport research3-6. More than 500 naturally occurring siderophores have been isolated and characterized (Section 2)3,4, and the discovery of new siderophores is continuing at a good rate. The structural features of siderophores are diverse. The ligating groups contain oxygen atoms of hydroxamate, catecholate, α-hydroxy carboxylic and salicylic acids, or oxazoline and thiazoline nitrogen. Siderophores display a selectivity for iron which is taken into account in the corresponding complex stability constants that are higher with Fe3+ than with Al3+, and with bivalent cations like Ca2+, Cu2+ or Zn2+. The physical properties of siderophores yield important information on biological mechanisms involving siderophore iron complexes. Fundamental data on solution thermodynamics (Section 4.1), electrochemistry (Section 4.3), and kinetic studies (Section 4.2) have been used in the search for likely intracellular iron release mechanisms (Section 5). Moreover, sufficient thermodynamic and kinetic data facilitate an estimation of the advantages of certain siderophores over others in their competition for iron. Siderophore uptake in microorganisms is, in general, a receptor-dependent process (Section 5.1). A diversity of high-affinity receptors has evolved either to recover iron loaded endogenous siderophores that have been excreted to scavenge iron from the environment, or to utilize xenosiderophores or iron sequestering agents of a host. Crystal structures have been solved of a variety of siderophore receptors and of other components of the high-affinity, energy-dependent siderophore-transport systems as well (Section 5.1). The permeation of cell walls or bacterial membranes by siderophores is in most microbes a highly specific process requiring an array of up to eight proteins. The advent of modern molecular biology delivered a cornucopia of methods enabling high-yield production of specific gene products relevant to siderophore-synthesis and -transport, analyses of structure-function relationships (employing site directed mutagenesis), and detailed insights into the regulation of the corresponding processes. Nevertheless, siderophores may exhibit both optical and geometrical isomers (Section 3). Synthesis of Cr3+ or Rh3+ siderophores enables isolation and characterization of these isomers (Section 3.3). Stereochemically wellcharacterized isomers are an indispensable prerequisite for studying the specificity of siderophoremediated iron uptake in microorganisms (Section 5.2). A variety of cellular iron-release mechanisms Page 3 of 47

from siderophores have been characterized, the majority of which involve reduction of ferric iron either on the membrane level or in the cytoplasm(Section 5.4). Reduction potentials of ferric siderophore complexes vary between -700-and -150mV . In particular at the low potential end below -450mV special biological strategies of reductive iron removal are required because these potentials are too negative for typical cellular reductases. The molecular nature of the overall transport process is complex (Section 5.3) and strictly regulated (section 5.5). In vivo Mössbauer spectroscopic investigations on the time course of siderophore mediated iron assimilation revealed, that (i) iron removal from siderophores is a fast and in most cases a reductive process (ii) few main metabolites of iron metabolism are found (iii) a ferrous iron complex, ferrochelatin, is present in all microorganisms analyzed (iv) siderophores function as iron storage compounds in various fungi (Section 6.1). One siderophore, ferrioxamine B, serves as a detoxifier in iron overload diseases and in the treatment of ß-thalassemia. This drug is also employed clinically for removal of aluminum from the body (Section 6.2) Siderophores may serve as MRI imaging agents and as templates for novel classes of antibiotics. Invading microorganisms exposed to circulating blood produce siderophores to compete for iron with the human transport protein transferrin, thus constituting one aspect of virulence and pathogenicity (Section 6.3). Siderophores and siderophore analogs play also a role as basic models for actinide chelators in order to remove these metals from the environment or from a contaminated body .

2 Structures of Siderophores As with secondary metabolites, various and complex chemical structures are typical within the siderophores, preventing their unequivocal and universal classification. Since the biosyntheses and structural features of siderophores are diverse, a classification scheme will be to some extent arbitrary. Criteria may include the producing organisms (bacteria, fungi, plants), the nature of the backbone (peptidic or non-peptidic, cyclic or open chain), or the nature of the chelating group. Despite the considerable structural variation found in the siderophores, their common feature is to form six-coordinate complexes with iron (III) of great thermodynamic stability. The ligating groups contain the oxygen atoms of hydroxamate, catecholate, α-hydroxy-carboxylic acids, and α-ketocarboxylic acids.

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In addition, siderophores with diverse FeIII ion binding groups were isolated, like salicylic acid, oxazoline and thiazoline nitrogen, and even negatively charged nitrogen (in the case of maduraferrin). Comprehensive reviews on the structural variety of siderophores are available3, 7-9, 16 Therefore we shall focus on salient structural features of siderophores. NMR studies of most siderophores have been part of the general chemical and structural characterization. The metal is generally removed from the complex prior to NMR spectroscopy, because FeIII causes severe line broadening of the NMR signals. To obtain spectra of the siderophore in the metal-chelated form, usually the diamagnetic Ga3+ or the Al3+ siderophore analogs are employed.16-19 1H and 13C NMR data of a variety of hydroxamate-type siderophores have been tabulated.20 Investigations on the biosynthesis of siderophores represent a major activity in the field.12 Considerable effort has been devoted to the chemical synthesis of natural siderophores, enantiomeric siderophores, and completely synthetic siderophore analogs. These topics will not be covered here and the reader is referred to the corresponding literature.8,10,21-24 Both, the pathways of siderophore biosyntheses as well as their chemical partial or total synthesis are central building blocks for the design of novel classes of antibiotics.

2.1 Hydroxamate-Type Siderophores Hydroxamate group-bearing siderophores are mainly synthesized by fungi and Gram-positive filament-forming bacteria (streptomycetes). In fungal systems the hydroxamic acid chelating group is commonly derived from acylated Nδ-acyl-Nδ-hydroxy-L-ornithine. The ferrichromes comprise one large family of hydroxamate siderophores (Figure 1a) and were isolated from low-iron cultures of many fungi.25 With few exceptions, ferrichromes possess cyclic hexapeptide backbones in which one tripeptide is linked to a second tripeptide of Nδ-acyl-Nδhydroxy-L-ornithine. Linear derivatives of the latter tripeptide form backbones of various antibiotics, termed albomycines. Crystal structures have been determined of several ferrichromes.26, 27 In all structures the iron coordination site is on one side of the molecule, the coordination of the metal is Λcis, and the conformation of the amino acids is L. A β(II) bend and a β(I) bend of the cyclic peptide skeleton is found. R1

R2 NH O

C

O C

C O

O

C

R3 NH

NH

NH

R3

NH

C

O

NH

C

C N

O

O

N

C

O

O

Fig. 1a Ferrichromes ferrichrome: R1=R2=H, R3=CH3 Ferricrocin: R1= R2=CH2=HH, R3=CH3

Fe O

O

N

C R3

O

Ferrioxamines, typical constituents of culture broths of Actinomycetes, occur as both linear and cyclic compounds containing 1-amino-5-hydroxyaminopentane (N-hydroxycadaverine) and succinic Page 5 of 47

acid as building blocks (Figure 1c). A cyclic trimer of succinyl-(N-hydroxycadaverine), is named ferrioxamine E. In some cases the pentane moiety is replaced by a butane carbon skeleton (putrescine). The most prominent representative of this siderophore family, desferrioxamine B (Figure1) , has become the drug of choice for the treatment of transfusional iron overload (Section 6.2).29 The crystal structure of ferric ferrioxamine B has been published recently.30 Certain derivatives of the ferrioxamines display antibiotic activity and therefore have been designated as ferrimycins.31

A

B

C Fig 1B: (A) fusarinines (n = 1, fusarinine; n = 3, R = acetyl, cyclic, triacetylfusarinine); (B) coprogens (R1 = H, R2 = COMe, R3 = R4 = isopentenol, coprogen; R1 = H, R2 = COMe, R3 = R4 = Me, neocoprogen II); (c) ferrioxamines (X = NH2, m = n = 5, R = Me, ferrioxamine B; X = NH (cyclic), m = n = 5, R = (CH2)2CO-[X], ferrioxamine E);; Typical tetradentate representatives of the hydroxamate-siderophore family include rhodotorulic acid,33 dimerum acid,20 bisucaberin,34 alcaligin35 and putrebactin36. Bisucaberin (Figure 1d), a cyclic dimer of succinyl-(N-hydroxycadaverine), which sensitizes tumor cells to macrophage-mediated cytolysis, was isolated from the supernatant of cultures of the marine bacterium Alteromonas haloplanktis.34. Putrebactin is a cyclic dimer of succinyl-(N-hydroxyputrescine) whereas rhodotorulic acid is a linear tetradentate chelator assembled of two Nδ-(res)-Nδ-hydroxyornithine units which are cyclized to form a diketopiparazine ring.33 The crystal structure of the Fe-alcaligin complex disclosed a monobridged topology and a Fe2L3 stoichiometry at near neutral pH.37

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O R

OH N

O

NH

NH

R

N OH

O

O

CH3

A:

OH

Fig. 35. (a) Rhodotorulic acid: R = CH3 (b) Dimerum acid: R = A

Bisucaberin

2.2 Siderophores with Catecholate Ligands Under conditions of iron deficiency, many bacteria excrete siderophores exhibiting phenolate or 2,3dihydroxybenzoate (DHB) iron binding groups. In 1970, enterobactin (also known as enterochelin), the first tricatechol siderophore, was isolated from culture fluids of E. coli, Aerobacter aerogenes, and Salmonella typhimurium (Figure 2a).38 Enterobactin is the cyclic triester of DHBS (2,3dihydroxybenzoylserine) exhibiting extraordinary features: (i) an extremely high complex formation constant, (ii) a redox potential too low for physiological reductants, (iii) a strong pH-dependence of (i) and (ii), and (iv) a trilactone backbone the cleavage of which lowers the redox potential. Due to these exceptional properties relevant to its physiological reactions, enterobactin is one of the most intensively analyzed siderophores. Recently, enterobactin was also isolated from Gram-positive bacteria39 as well as the enterobactin homolog corynebactin from Corynebacterium glutamicum and Bacillus subtilis40,41 All other known tris-catecholate siderophores exhibit a linear backbone based on spermidine or norspermidine. Parabactin, N4-(2,3-dihydroxybenzene-3-methyloxazoline-2carboxamidyl)-N1,N8-bis(2,3-dihydroxybenzoyl) spermidine, is produced by Paracoccus denitrificans (Figure 2b). Exposure to acid destroys the oxazoline ring, producing a threonyl moiety.42 Other members of the linear tris-bidentate catecholate-type siderophores include vibriobactin,7 vulnibactin,7 fluvibactin,7 protochelin7, and salmochelin-243. Salmochelin is unique, because the backbone of the DHBS-chelating units is made up by glucose( see chapter pathogenicity).

A

B

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C

D

Figure 2 Structures of representative catecholate and mixed-ligand siderophores: (a) enterobactin; (b) parabactin; (c) mycobactins (R1 = various alkyl chains, R2 = R3 = R5 = Me or H, R4 = alkyl chains or H;3 (d) fluorescent chromophore of pseudobactins and pyoverdins

2.3 Carboxylate-Type Siderophores A great variety of siderophores exhibit carboxylate and hydroxy donor groups. Many of these siderophores belong to the mixed ligand group. In the nineties of the last century, however, a completely novel class of siderophores was detected whose members neither possess hydroxamate nor phenolate ligands. Rather, FeIII iron binding is achieved exclusively by α-hydroxycarboxylates and carboxylates. The late discovery of these compounds is mainly based on the fact that they are colorless, therefore requiring novel methods for screening and isolation (chromazurole test and iron nutrition bioassays).44 These siderophores are found in the kingdom of bacteria as well as in the realm of fungi. A very hydrophilic complex, termed staphyloferrin A, was isolated from Staphylococcus hyicus (Figure 3b).45 The molecule is composed of two moles of citrate linked by ornithine. Besides Staphyloferrin A,45 also staphyloferrin B,44 vibrioferrin,45 and rhizoferrin48 contain citric acid building blocks. Rhizoferrin has been isolated from culture filtrates of Rhizopus and other members of the class of Zygomycetes (Figure 3c).48 In this compound, two citric acid residues are linked to diaminobutane, resulting in N1,N4-bis(1-oxo-3-hydroxy-3,4dicarboxybutyl)diaminobutane. Although citrate is an intracellular primary metabolite, it may be regarded - from an evolutionary view - as the simplest siderophore from which the above mentioned siderophores, as well as the mixed ligand compounds schizokinen, arthrobactin, and aerobactin (section 2.4) have evolved. Rhizobium meliloti, capable of fixing atmospheric nitrogen when symbiotically associated with certain legumes, excretes and utilizes rhizobactin DM4 (Figure 3a), N2-[2-{(1Phytosiderophores, carboxymethyl)amino}ethyl]-N6-(3-carboxy-3-hydroxy-1-oxopropyl)lysine.50 detected in root washings of gramineous plants, represent a separate subclass of the carboxylate-type of siderophores. For eaxample, the phytosiderophores nicotianamin, mugineic, avenic and distichonic acid are produced and utilized by cereals, like barley, wheat, rye oat etc.51,52

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Figure 3 Structures of carboxylate siderophores: (a) rhizobactin; (b) staphyloferrin; (c) rhizoferrin

2.4.Siderophores with mixed ligands and heterocyclic chelating groups Fiftyfive years ago the very first siderophore, mycobactin, was isolated by the crystallization of the aluminum complex.53 Mycobactins from Gram-positive Mycobacteria and the closely related nocobactins from Nocardia embody a series of lipid-soluble siderophores located in the lipid-rich boundary layers of these bacteria (Figure 2c).54 The X-ray structure revealed that iron binding in mycobactins is accomplished by two hydroxamates, a phenolate group, and oxazoline nitrogen. It is generally assumed that in oxazoline and thiazoline containing donor-deficient siderophores, the imine-N will participate in ferric ion complexation. These heterocycles result physiologically from an enzymatic cyclization of cysteinyl, seryl, or threonyl side chains. The imine-type of Fecoordination is a common feature of the siderophores pyochelin, yersiniabactin, anguibactin, and acinetobactin (Figure 4).3,7 A variety of fluorescent chromopeptide siderophores, termed pseudobactins and pyoverdins, are synthesized by Pseudomonas species.57 The chromophores, derived from 2,3-diamino-6,7-dihydroxyquinoline (Figure 2d), are linked to a peptide chain exhibiting either two hydroxamate groups or one hydroxamate and one α-hydroxycarboxylate group. O

OH

N

O

CH3

N

S

OH

B

NH

OH

OH COO

S

OH

C

A

N

N

CH3

N

_

S

N

CH3

+

OH

N H2

CH3 HO

O

S

HO

N

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NH

O HN O

N H O

O O

O N CH3 H

HN

Fe

N

NH

O

H

O

CH2OH N

Figure 27. Maduraferrin

Fig. 4 Structures of siderophores with heterocyclic ligands: acinetobactin (a), anguibactin (b), yersiniabactin (c) and maduraferrin (Note: iron binding nitrogen of hexahydropyridazine-3corboxylic acid is negatively charged)

3 Coordination Geometry of Siderophores The stereochemistry of siderophores is a very important aspect of their role in receptor mediated iron uptake, since it has been shown that very subtle discrimination by microbial iron transport systems takes place between siderophore isomers. In fact, uptake of siderophores by microorganisms shows – at least in part - stereospecific preferences. The geometry at the metal center of a trihydoxamate complex is shown in Fig. 5a Figure 5a The geometry and dimensions of the iron-coordination octahedron in a natural tris(hydroxamate) complex, triacetylfusarinine. (ACS from M. B. Hossain et al.66)

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3.1 Geometrical and Optical Isomers Upon metal chelation, a tris complex with a bidentate ligand forms a more or less distorted coordination octahedron via three five-membered chelate rings. According to IUPAC rules,63 the planes of these five-membered rings may form a right-handed (∆) propellor or a left-handed (Λ) propellor (Figure 5). If the ligand is not optically active, one will find a racemic mixture of ∆ and Λ optical isomers. If the ligand is chiral (e.g. D), two complexes are again possible, DΛ and D∆. However, they are not formed in equal amounts because they have different standard free energies of formation. Exclusively, one optical isomer may be formed if there is a large thermodynamic advantage for this isomer (as, for instance, in the case of ferrichromes where LΛ is exclusively observed). Figure 5 : Λ and ∆ optical configuration at the metal center (top) and eight geometrical isomers of a FeIII∆-trichelate complex, involving three unsymmetrical bidentate ligands attached to an asymmetric backbone. Not shown is the set of eight Λ diastereomers. R, X correspond to unique functional groups,e.g. amino terminus of ferrioxamine B(R) or diketopiperazine ring of coprogen (X)26 Many siderophores are hexadentate ligands with three asymmetrical bidentate functional units attached to an asymmetrical backbone. These complexes reveal a rather complicated stereochemistry. The bidentates are not equivalent and there will be 23 = 8 cis and trans isomers. Each can have a Λ and ∆ metal environment and when the siderophore is made from chiral residues there are 16 diastereomers, none of which is enantiomeric to one another. A selection of such diastereomers is shown in Figure 5. Nomenclature for the geometrical isomers of hydroxamate tris-bidentates was outlined by Leong and Raymond.10, 64 No general rule can be applied for an absolute assignment of the chelate ring sequence. However, if the structure exhibits a unique functional group, this group can be utilized to define the chelate ring sequence. In the case of ferrioxamine B or D1, the unique functional group is the N-terminus (a). In the case of the coprogens, this group is a diketopiperazine ring placed between rings 1 and 2

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. (b) Looking down the C3 axis the sequence of the chelate rings 1, 2, and 3 corresponds to the rotation direction, i.e. clockwise for Λ-isomers and counter-clockwise for ∆-isomers. (c) If the ring 1 has the carbon atom of the hydroxamate group below the nitrogen, it is denoted ‘C’; if the reverse is true, it is called ‘N’. (d) For rings 2 and 3, each is called cis or trans depending upon whether it has the same or opposite relative orientation with respect to the coordination axis as does ring 1. The X-ray crystallographic analysis of all ferrichrome siderophores yielded Λ-C-cis,cis configurations of the coordination octahedron.65 Neocoprogen I adopts a ∆-C-trans,trans configuration in the crystal structure.64 N,N′,N″-Triacetylfusarinine crystallizes as either the ∆ or Λ isomer, depending on the solvent system used.66 Achiral siderophores such as ferrioxamine E and ferrioxamine D1 crystallize as racemic mixtures of ∆ and Λ isomers.67,68 Similarily, the crystal structure of Ferrioxamine B displays a racemic mixture of Λ-N-cis,cis and ∆-N-cis,cis,30 although various geometrical isomers of the inert Cr-complex are present in solution (see section 3.3) . A multitude of siderophores achieve iron chelation via catecholates. Unlike hydroxamate, catecholate is a symmetric, bidentate ligand. Thus there are no geometrical isomers of simple tris(catecholate) metal complexes. The chirality at the metal center of (bis(catecholato)-siderophores) was determined in amonobactins, where a slight ∆ preference in Fe2L3 stoichiometry was observed whereas the corresponding FeL(H2O)2–complexes in the low-pH region are achiral.69 Enterobactin displays a ∆-cis configuration, dictated by the asymmetric centers (L) of the trilactone ring.70 Synthetic enterobactin analogs composed of tris(2-aminomethyl)amine (TREN) as anchors and amino acids linking the anchor to catechol units also form Fe3+ complexes of preferential ∆-cis configuration when L-amino acids are used. These complexes are stabilized by intramolecular Hbonds from the catechoylamides CONH to the catecholate oxygens, as in enterobactin. When the possibility of this type of H-bond is eliminated by replacing the amide proton CONH by an amide Nmethyl (CONMe), the chiral preference of the complex is inverted from the ∆-cis to the Λ-cis configuration.71 Surprisingly, the recently detected trilactone siderophore corynebactin forms a Λferric complex. The addition of a glycin spacer and the methylation of the trilactone ring as compared with enterobactin are sufficient to favor the opposite chirality.72, 73

3.2 UV/Vis and CD Spectra of Ferric Siderophores The d5 electronic configuration of Fe3+ rules out any crystal field stabilization energy (CFSE) and makes the complexes relatively labile with respect to isomerization and ligand exchange in aqueous solution. Furthermore, high-spin Fe3+ has no spin-allowed d–d transitions. Therefore the UV/Vis and CD spectra of iron(III) microbial iron chelates arise from the coulombic interaction between the positively charged metal ion and the negatively charged oxygen atoms (charge-transfer transition), which is not as readily interpreted as are ligand-field (d–d) transitions. Although transitions in high-spin iron(III) complexes are extremely weak, the determination of the metal center chirality of Fe3+ complexes in solution is possible through comparison of the solution and solid-state CD spectra. However, the correlation of the rotary power with left-handed or right-handed helical stereochemistry requires an absolute assignment based on crystal structure data which is usually determined employing the Bijvoet method for anomalous dispersion of Cu-Kα radiation by the Fe3+ ion in the crystalline solids. This correlation could be established for triacetylfusarinine and neurosporin.74, 66 This correspondence extends into the other Fe3+ complexes and shows that the CD spectra of the iron(III) complexes in solution can be used for determinations of the metal center chirality. Fe3+ complexes will have the Λ configuration (at least predominantly) if the CD band in the region of the absorption maximum (400–500 nm for hydroxamates) has a positive Page 12 of 47

sign. The visible and CD spectral parameters of siderophores in aqueous solution are summarized in Table 1. Table 1 Visible and CD spectral characteristics of aqueous siderophores and of a model complex Siderophore λmax (nm) λmax (nm) Λ λmax (nm) ∆ (ε [M−1 cm−1]) (∆ε [M−1 cm−1]) (∆ε [M−1 cm−1]) Ferrichrome 425 (2895) 360 (−3.7) 465 (2.4) Ferricrocin 434 (2460) 290 (−3.78) 360 (−1.62) 450 (+2.47) Ferrioxamine B 428 (2800) Coprogen 434 (2820) 375 (+2.1) 474 (−1.26) 370 (+3.25) N,N′,N″Triacetylfusarinine 467 (−2.04) 425 (2700)b 372 (+2.73) Fe2RA3 464 (−1.41) 435 (4910) 350 (+2.3) Fe(benz)3a 350 (−2.8) 455 (+1.1) 452 (−1.5) Enterobactin 495 (5600)b 553(−2.2)∆-cis Corynebactin 545(+1.7) Agrobactin 505 (4100) Λ-cis Parabactin 512 (3300) Λ-cis Pseudobactin 400 (15 000) 400 (+2.0) 436 (−0.8) 502 (+0.3) Pseudobactin A 400 (2000) Neurosporin 360 (−4.8) 465 (+4.5) a In acetone solution. b At pH 7.

3.3 UV/Vis and CD Band Assignments of Chromic Siderophores An alternative method that enables the classification of geometrical and optical isomers is accomplished by substituting the Fe3+ ion with kinetically more inert d3 Cr3+ or d6 Rh3+ ions.75 Since the substituted ions have almost the same ionic radii and the same charge as Fe3+, their complexes show a high degree of structural similarity with the corresponding Fe3+ complexes, as demonstrated by the crystal structures of model compounds.76, 77 The d-electron configurations of Cr3+ and Rh3+ grant significant CFSE for kinetic inertness and provide well characterized d–d transitions with distinct UV/Vis and CD spectra. For details, please refer to the literature

3.4 Ligand denticity and metal center binding mode The structure of the metal center is also depending on the denticity of the ligand. Tetradentate ligands are of particular interest because they are not able to achieve the octahedral coordination geometry in a 1:1 stoichiometry. The dihydroxamate siderophores rhodotorulic acid (RA), Page 13 of 47

bisucaberin (BC) and alcaligin (AL) were analyzed in some detail. 88,89 The binding mode of these complexes is pH-dependent. At low pH(