Borate complexes of benzohydroxamic acid and some of its derivatives

Ultraviolet spectrophotometric measurements in dilute aqueous solution give pK(a) values of 8.78, 8.27, 8.96 and 8.68, respectively, for benzohydroxamic, N-phenylbenzohydroxamic, p-methoxybenzohydroxamic and N-methyl-p-methoxybenzohydroxamic acids. The acids carrying no substituent on nitrogen form 1:1 complexes with boric acid according to the general equation RCONHOH + H(3)BO(3) --> (1:1 complex)(-) + H(+). Equilibrium constants (log K) were found to be -5.70 for benzohydroxamic acid and -5.8 for p-methoxybenzohydroxamic acid. The complexes behave as very weak monoprotic acids and decompose at high pH to yield borate ions and the corresponding hydroxamate ions. The N-substituted hydroxamic acids showed no reaction with boric acid under the same conditions.     

Nanophase iron phosphate, iron arsenate, iron vanadate, and iron molybdate minerals synthesized within the protein cage of ferritin

Nanoparticles of iron phosphate, iron arsenate, iron molybdate, and iron vanadate were synthesized within the 8 nm interior of ferritin. The synthesis involved reacting Fe(II) with ferritin in a buffered solution at pH 7.4 in the presence of phosphate, arsenate, vanadate, or molybdate. O2 was used as the oxidant to deposit the Fe(III) mineral inside ferritin. The rate of iron incorporation into ferritin was stimulated when oxo-anions were present. The simultaneous deposition of both iron and the oxo-anion was confirmed by elemental analysis and energy-dispersive X-ray analysis. The ferritin samples containing iron and one of the oxo-anions possessed different UV/vis spectra depending on the anion used during mineral formation. TEM analysis showed mineral cores with approximately 8 nm mineral particles consistent with the formation of mineral phases inside ferritin.     

Catechol releases iron(III) from ferritin by direct chelation without iron(II) production

It has been traditionally considered that catechols release iron from ferritin by reduction to iron(II), which diffuses through the ferritin channels into the intracellular milieu where it participates in the Fenton reaction, producing highly toxic hydroxyl radicals. However, in the present work we have proved that the mechanism of the release of iron from ferritin by catechol does not take place by iron(II) reduction but by direct iron(III) chelation and therefore without iron(II) production. A possible extension of these findings to other catechols is discussed on the basis of the stability with respect to the internal redox reaction of the iron(III)-catechol complexes.     

A novel dizinc bridged hydroxamate model for hydroxamate inhibited zinc hydrolases

Reaction of Zn(OAc)(2).2H2O with tmen leads to the formation of [Zn(tmen)(OAc)2] (I) which reacts with benzohydroxamic acid to form Zn(BA)2.H2O (II) and the novel dizinc hydroxamate bridged complex [Zn2(mu-OAc)2(OAc)(mu-BA)(tmen)] (III), which may also be prepared by self-assembly and whose structure closely mimics that of the native hydroxamate inhibited Aeromonas proteolytica aminopeptidase.     

Iron release from ferritin induced by light and ionizing radiation

The reductive release of iron from ferritin by UV light or ionizing radiation has been investigated in separate experiments. When ferritin is exposed to light, the mineral core is the main photoreceptor for the Fe(III) reduction. In radiolytic studies, we determined that, in the absence of oxygen, the hydrated electron (e_aq⁻) is the reducing agent triggering redox reactions associated with iron mobilization from ferritin. In an aerobic system, the superoxide radical anion (O₂^•−) is also involved in the iron release process. We found that, in photochemical and radiolytical studies, Fe(II) mobilization from ferritin required an iron chelator. Without a chelator, ferritin is an electron-storage molecule for a long period, on the order of at least several hours. The reductant or chelator entry into the ferritin core is not necessary for iron release. The ferrozine is a convenient chelating agent to monitor Fe(II) mobilization, due to a high extinction coefficient of \textFe ( \textferrozine )₃^{4 -} {\text{Fe}}\,\left( {\text{ferrozine}} \right)_{3}^{4 - } and a high rate constant of complexation process (2.65 × 10⁴ dm³ mol⁻¹ s⁻¹).     

Encapsulation of a guest sodium cation by iron(III) tris-(hydroxamate)s

An unprecedented encapsulation of an exogenous sodium ion by iron(III) tris(hydroxamate)s was observed upon crystallization of an iron(III) complex with isonicotinylhydroxamic acid. The sodium cation is bound by bridging coordination of the amide oxygen atoms from two mononuclear iron(III) fac-tris(hydroxamate)s.     

Iron(III) coordination properties of a pyoverdin siderophore produced by Pseudomonas putida ATCC 33015

The iron complexation of a fluorescent green pyoverdin siderophore produced by the environmental bacterium Pseudomonas putida was characterized by solution thermodynamic methods. Pyoverdin binds iron through three bidentate chelate groups, a catecholate, a hydroxamate, and an alpha-hydroxycarboxylic acid. The deprotonation constants of the free pyoverdin and Fe(III)-pyoverdin complex were determined through a series of potentiometric and spectrophotometric experiments. The ferric complex of pyoverdin forms at very low pH (pH < 2), but full iron coordination does not occur until neutral pH. The calculated pM value of 25.13 is slightly lower than that for pyoverdin PaA (pM = 27), which coordinates iron by a catecholate and two hydroxamate groups. The redox potential of Fe-pyoverdin was found to be very pH sensitive. At high pH (approximately pH 9-11) where pyoverdin coordinates Fe in a hexadentate mode the redox potential is -0.480 V (NHE); however, at neutral pH where full Fe coordination is incomplete, the redox potential is more positive (E(1/2) = -0.395 V). The positive shift in the redox potential and the partial dissociation of the Fe-pyoverdin complex with pH decrease provides a path toward in vivo iron release.     

An iron reservoir model based on ferrichrome: iron(III)-binding and metal(III)-exchange properties of tripodal monotopic and ditopic hydroxamate ligands with an L-alanyl-L-alanyl-N-hydroxy-beta-alanyl sequence

To gain knowledge about biological iron mobilization, tripodal monotopic and ditopic hydroxamate ligands (1 and 2) are prepared, and their iron-chelating properties are investigated. Ligands 1 and 2 contain three Ala-Ala-beta-(HO)Ala units and three [Ala-Ala-beta-(HO)Ala](2) units connected with tris(alanylaminoethyl)amine, respectively, and form six-coordinate octahedral complexes with iron(III) in aqueous solution. Ligand 1 and 1 equiv of iron give Fe-1, and ligand 2 and 1 or 2 equiv of iron produce Fe(1)-2, or Fe(2)-2. These complexes exhibit absorptions at lambda(max) 425 nm of epsilon 2800-3000/Fe, characteristic of tris(hydroxamato)iron(III) complexes, and preferentially assume the Delta-cis configuration. Loading of Fe(III) on 1, 2, and M(III)-loaded ligands (M-1 and M(1)-2, M = Al, Ga, In) with ammonium ferric oxalate at pH 5.4 is performed, and the second-order rate constants of loading with respect to Fe(III) and the ligand or M(III)-loaded ligands are determined. The rates of loading of Fe(III) on M-1 increase in the order Al-1 < Ga-1 < In-1, and those on M(1)-2 in the order Al(1)-2 < Ga(1)-2 < Fe(1)-2 < In(1)-2, indicating that the dissociation tendency of M(III) ions from the hydroxamate ligand is an important factor. The iron complexes formed with 2 are subjected to an iron removal reaction with excess EDTA in aqueous pH 5.4 solution at 25.0 degrees C, and the collected data are analyzed by curve-fitting using appropriate first-order kinetic equations, providing the rate constants for the upper site and the lower site of 2. Similar analysis for FeM-2 affords removal rate constants for Fe(up)-2, M(up)-2, and Fe(low)-2, and the iron residence probability at each site. The protonation constants of the hydroxamate groups for 1 and 2 (pK(1,) pK(2), pK(3), and pK(1,) pK(2)., pK(6)) are determined, and the proton-independent stability constants for Fe-1, the upper site of Fe(2)-2, and the lower site of Fe(1)-2 are 10(28), 10(29), and 10(28.5), respectively.     

Fe(III) coordination properties of a new saccharide-based exocyclic trihydroxamate analogue of ferrichrome

The coordination chemistry of a saccharide-based ferrichrome analogue, 1-O-methyl-2,3,4-tris-O-[4-(N-hydroxy-N-methylcarbamoyl)-n-butyrate]-alpha-d-glucopyranoside (H(3)L), is reported, along with its pK(a) values, Fe(III) and Fe(II) chelation constants, and aqueous-solution speciation as determined by spectrophotometric and potentiometric titration techniques. The use of a saccharide platform to synthesize a hexadentate trihydroxamic acid chelator provides some advantages over other approaches to ferrichrome models, including significant water solubility and hydrogen-bonding capability of the backbone that can potentially provide favorable receptor recognition and biological activity. The pK(a) values for the hydroxamate moieties were found to be similar to those of other trihydroxamates. Proton-dependent Fe(III)-H(3)L and Fe(II)-H(3)L equilibrium constants were determined using a model involving the sequential protonation of the iron(III)- and iron(II)-ligand complexes. These results were used to calculate the formation constants, log beta(110) = 31.86 for Fe(III)L and 12.1 for Fe(II)L(-). The calculated pFe value of 27.1 indicates that H(3)L possesses an Fe(III) affinity comparable to or greater than those of ferrichrome and other ferrichrome analogues and is thermodynamically capable of removing Fe(III) from transferrin. E(1/2) for the Fe(III)L/Fe(II)L(-) couple was determined to be -436 mV from quasi-reversible cyclic voltammograms at pH = 9, and the pH-dependent E(1/2) profile was used to determine the Fe(II)L(-) protonation constants.     

FTIR spectroscopic analyses of the temperature and pH influences on stratum corneum lipid phase behaviors and interactions

A thermotropic investigation of different lipid dispersions containing ceramide 3 (CER3) or sphingomyelin (SPM), perdeuterated palmitic acid (PA-d(31)) and cholesterol (CH) or cholesterol sulfate (CS) at pH 5.2 and 7.4 used as model membranes, has been carried out by Fourier transform infrared (FTIR) spectroscopy in order to estimate the importance of these lipids and of the temperature and pH for the maintenance of the structural organization of the stratum corneum (SC) lipid lamellae. The results obtained for the CER3 and SPM mixtures at pH 5.2 and 7.4 indicated that the little size of the polar headgroup of CER3 compared with that of SPM could permit a more closely packing in the CER3 acyl chains. Moreover, the CH and CS induced an increase of the order in the CER3 acyl chains over the physiological temperatures while a disordering was seen above 60 degrees C. In addition, the thermal phase behaviors observed for the CER3/PA-d(31) dispersion at pH 5.2 and 7.4, suggested a phase separation between the CER3 and PA-d(31) molecules in this mixture. Nevertheless, the miscibility between the CER3 and PA-d(31) was raised in the presence of CH or CS at pH 5.2. In particular, the incorporation of these sterols into the CER3/PA-d(31) dispersion at pH 5.2 appeared to result in an increase of the order in the acyl chains of CER3 and PA-d(31) at about 37 degrees C. In contrast, a phase separation was observed between the CER3 and PA-d(31) in the CER3/PAd(31)/CH and CER3/PA-d(31)/CS dispersions at pH 7.4. Interestingly, the pH change from 5.2 to 7.4 in these tertiary dispersions was also accompanied by a substantial deprotonation of the PA-d(31) molecules which seemed more pronounced in the presence of CH as compared with CS. Altogether, the results suggested that the ceramides, fatty acids and sterols could play an important structural role in the SC cohesion.     

Electrochemistry of unfolded cytochrome c in neutral and acidic urea solutions

The present investigation reports the first experimental measurements of the reorganization energy of unfolded metalloprotein in urea solution. Horse heart cytochrome c (cyt c) has been found to undergo reversible one-electron transfer reactions at pH 2 in the presence of 9 M urea. In contrast, the protein is electrochemically inactive at pH 2 under low-ionic strength conditions in the absence of urea. Urea is shown to induce ligation changes at the heme iron and lead to practically complete loss of the alpha-helical content of the protein. Despite being unfolded, the electron-transfer (ET) kinetics of cyt c on a 2-mercaptoethanol-modified Ag(111) electrode remain unusually fast and diffusion controlled. Acid titration of ferric cyt c in 9 M urea down to pH 2 is accompanied by protonation of one of the axial ligands, water binding to the heme iron (pK(a) = 5.2), and a sudden protein collapse (pH < 4). The formal redox potential of the urea-unfolded six-coordinate His18-Fe(III)-H(2)O/five-coordinate His18-Fe(II) couple at pH 2 is estimated to be -0.083 V vs NHE, about 130 mV more positive than seen for bis-His-ligated urea-denatured cyt c at pH 7. The unusually fast ET kinetics are assigned to low reorganization energy of acid/urea-unfolded cyt c at pH 2 (0.41 +/- 0.01 eV), which is actually lower than that of the native cyt c at pH 7 (0.6 +/- 0.02 eV), but closer to that of native bis-His-ligated cyt b(5) (0.44 +/- 0.02 eV). The roles of electronic coupling and heme-flattening on the rate of heterogeneous ET reactions are discussed.     

MALDI mass spectrometric determination of dendritic iron chelation stoichiometries and conditional affinity constants

The iron chelation stoichiometries of a dendritic iron(III) chelator with N(1), N(3), N(5)-trimethylbenzene-1,3,5-tricarboxamide at its core, and containing 3 identical hexadentate tris-hydroxypyridinone branches D was studied by MALDI mass spectrometry. At pH 7.2, the speciation of the system included FeD, Fe(2)D and Fe(3)D species with the respective conditional stability constants of 26.74, 26.03 and 25.36. The differences in the stepwise affinity constants arise from the statistical distribution of iron(III), and there was no evidence for cooperativity between the iron-binding sites.     

Ionically crosslinked alginate/carboxymethyl chitin beads for oral delivery of protein drugs

Complex beads composed of alginate and carboxymethyl chitin (CMCT) were prepared by dropping aqueous alginate-CMCT into an iron(III) solution. The structure and morphology of the beads were characterized by IR spectroscopy and scanning electron microscopy (SEM). IR confirmed electrostatic interactions between iron(III) and the carboxyl groups of alginate as well as CMCT, and the binding model was suggested as a three-dimensional structure. SEM revealed that CMCT had a porous morphology while alginate and their complex beads had a core-layer structure. The swelling behavior, encapsulation efficiency, and release behavior of bovine serum albumin (BSA) from the beads at different pHs were investigated. The BSA encapsulation efficiency was fairly high (>90%). It was found that CMCT disintegrated at pH 1.2 and alginate eroded at pH 7.4 while the complex beads could effectively retain BSA in acid (>85%) and reduce the BSA release at pH 7.4. The results suggested that the iron(III)-alginate-CMCT bead could be a suitable polymeric carrier for site-specific protein drug delivery in the intestine.     

Iron chelation equilibria, redox, and siderophore activity of a saccharide platform ferrichrome analogue

A complete characterization of the aqueous solution Fe(III) and Fe(II) coordination chemistry of a saccharide-based ferrichrome analogue, 1-O-methyl-2,3,6-tris-O-[4-(N-hydroxy-N-ethylcarbamoyl)-n-butyryl]-alpha-D-glucopyranoside (H3LN236), is reported including relevant thermodynamic parameters and growth promotion activity with respect to both Gram-negative and Gram-positive bacterial strains. The saccharide platform is an attractive backbone for the design and synthesis of ferrichrome analogues because of its improved water solubility and hydrogen-bonding capabilities, which can potentially provide favorable receptor recognition and biological activity. The ligand deprotonation constants (pKa values), iron complex (FeIII(LN236) and FeII(LN236)1-) protonation constants (KFeHxL-236-N), overall Fe(III) and Fe(II) chelation constants (beta110), and aqueous solution speciation were determined by spectrophotometric and potentiometric titrations, EDTA competition equilibria, and cyclic voltammetry. Log betaIII110 = 31.16 and pFe = 26.1 for FeIII(LN236) suggests a high affinity for Fe(III), which is comparable to or greater than ferrichrome and other ferrichrome analogues. The E1/2 for the FeIII(LN236)/FeII(LN236)1- couple was determined to be -454 mV (vs NHE) from quasi-reversible cyclic voltammograms at pH 9. Below pH 6.5, the E1/2 shifts to more positive values and the pH-dependent E1/2 profile was used to determine the FeII(LN236)1- protonation constants and overall stability constant log betaII110 = 11.1. A comparative analysis of similar data for an Fe(III) complex of a structural isomer of this exocyclic saccharide chelator (H3LR234), including strain energy calculations, allows us to analyze the relative effects of the pendant arm position and hydroxamate moiety orientation (normal vs retro) on overall complex stability. A correlation between siderophore activity and iron coordination chemistry of these saccharide-hydroxamate chelators is made.     

Spectrophotometric titration of bismuth with EDTA

A new spectrophotometnc method for the estimation of bismuth with EDTA, using iron-salicylate complex as the indicator, has been developed. The determinations were carried out by measuring the absorbance at 520 mmu. of solutions containing bismuth, iron(III), salicylic acid and various quantities of EDTA, at pH 0.5. It has been shown from the stability constants of the complexes present that before the end-point iron(III) will not react appreciably with the Bi-EDTA complex. The interference from iron(III) in the estimation of bismuth, which is a serious drawback in many other methods, is eliminated in the present method, as iron(III) acts as the indicator.     

Selective extraction of iron(III) from aqueous nitrate solution in the presence of cobalt(II), copper(II) and nickel(II) ions using bis(delta2-2-imidazolinyl)-5,5'-dioxime.

The feasibility of using bis(delta2-2-imidazolinyl)-5,5'-dioxime (H2L) for the selective extraction of iron(III) from aqueous solutions was investigated by employing an solvent-extraction technique. The extraction of iron(III) from an aqueous nitrate solution in the presence of metal ions, such as cobalt(II), copper(II) and nickel(II), was carried out using H2L in binary and multicomponent mixtures. Iron(III) extraction has been studied as a function of the pH, equilibrium time and extractant concentration. From the extracted complex species in the organic phase, iron(III) was stripped with 2 M HNO3, and later determined using atomic-absorption spectrometry. The extraction was found to significantly depend on the aqueous solution pH. The extraction of iron(III) with H2L increases with the pH value, reaching a maximum in the zone of pH 2.0, remaining constant between 2 and 3.5 and subsequently decreasing. The quantitative extraction of iron(III) with 5 x 10(-30 M H2L in toluene is observed at pH 2.0. H2L was found to react with iron(III) to form ligand complex having a composition of 1:2 (Fe:H2L).     

Mössbauer studies of iron incorporation into ferritins

Iron(III) monomers, dimers and clusters have been identified by Mössbauer spectroscopy during the initial stages of iron incorporation into ferritins, following Fe(II) oxidation. Iron(III) monomers seem to arise from dimer dissociation. Some of the monomers are transferred from iron poor to iron rich ferritin molecules, where they join the iron core clusters. Horse spleen ferritin, several variants of human H chain ferritin andEscherichia coli ferritin (Ec-FTN) can all accept the iron from human H chain ferritin. The small iron cores of Ec-FTN are different from those of mammalian ferritins, which indicates that the structure of the iron core depends on the protein shell.     

Lipid peroxidation of the erythrocyte membrane caused by stimulated polymorphonuclear leukocytes in the presence of ferritin

Lipid peroxidation of erythrocyte membrane was caused by phorbol myristate acetate (PMA)-stimulated polymorphonuclear leukocytes (PMN) in the presence of ferritin. PMN themselves were not peroxidized. A lag period was observed before the start of the peroxidation reaction. In contrast, ferritin iron was continuously released by PMA-stimulated PMN, suggesting that accumulation of free iron in the reaction system was important for proceeding of the peroxidation reaction. Superoxide dismutase, catalase, hydroxyl radical scavengers and an iron chelator, diethylenetriaminepenta-acetic acid, inhibited the lipid peroxidation, indicating that the lipid peroxidation is initiated by a hydroxyl radical generated from the interaction of H2O2 with ferrous iron released from ferritin.     

Hard and soft X-ray absorption spectroscopic investigation of aqueous Fe(III)-hydroxamate siderophore complexes

Microorganisms release organic macromolecules, such as siderophores, to obtain Fe(III) from natural systems. While the relative stabilities of Fe(III)-siderophore complexes are well-studied, the structural environments of Fe(III) and ligands in the complex are not well-understood. Using the X-ray absorption spectroscopy (XAS) at the Fe- and N-K absorption edges, we characterized the nature of Fe(III) interactions with a hydroxamate siderophore, desferrioxamine B (desB), and its small structural analogue, acetohydroxamic acid (aHa), as a function of pH (1.4-11.4). These experimental studies are complemented with DFT calculations. The Fe-XAS studies suggest that Fe(aHa)3 is the dominant species in aqueous solutions in the pH range of 2.8-10.1, consistent with thermochemical information. However, the N-XAS and resonance Raman studies show that the chemical state of the ligand in the Fe(aHa)3 complex changes significantly with pH, and these variations are correlated with further deprotonation of the Fe(aHa)3 complex. The N-XAS studies also indicate that the overlap of Fe 3d orbitals with the molecular orbitals of the hydroxamate group is significant. The Fe- and N-XAS studies of Fe(III)-desB complexes indicated that Fe(desB)+ is the dominant species between pH values of 1.4 and 11.4, consistent with predicted stability constants. This information is useful in understanding the role of iron in bacterial transport, siderosis treatment, and actinide sequestration at contaminated sites. This is the first N-XAS study of aqueous metal ligand complexes, which demonstrates the applications of soft-XAS in studying the electronic structure of metal complexes of organic macromolecules in aqueous solutions.     

Formation and structure of 1:1 complexes between aryl hydroxamic acids and vanadate at neutral pH

Although aryl hydroxamic acids are well-known to form coordination complexes with vanadate (V(V)), the nature of these complexes at neutral pH and submillimolar concentrations, the conditions under which such complexes inhibit various serine amidohydrolases, is not well established. A series of qualitative and quantitative experiments, involving UV/vis, (1)H NMR, and (51)V NMR spectroscopies, established that both 1:1 and 1:2 vanadate/hydroxamate complexes form at pH 7.5, with the former dominating at submillimolar concentrations. Formation constants for the complexes of several aryl and alkyl hydroxamic acids were determined; for example, for benzohydroxamic acid, the stepwise formation constants of the 1:1 and 1:2 complexes were 3000 and 400 M(-1), respectively. The (51)V chemical shift of the 1:1 4-nitrobenzohydroxamic acid complex was -497 ppm, and that of its unsubstituted analogue was -498 ppm. A (1)H-(15)N HSQC spectrum of the 4-nitrobenzo-(15)N-hydroxamic acid/vanadate complex indicated the presence of an N-H group with (15)N and (1)H chemical shifts of 115 and 5.83 ppm, respectively. A (13)C NMR spectrum of the complex of 4-nitrobenzo-(13)C-hydroxamic acid with vanadate displayed a resonance at 170.1 ppm and thus a coordination-induced shift (CIS) of +3.8 ppm. In contrast, the CIS value of an established 1:2 complex, thought to contain chelated hydroxamic acid ligands, was +11.9 ppm. These spectral data led to the following structural picture of 1:1 complexes of vanadate and aryl hydroxamic acids. They contain penta- or hexa-coordinated vanadium. The ligand is in the hydroxamate rather than hydroximate form. The ligand is presumably bound to vanadium through the hydroxamic hydroxyl oxygen, but the hydroxamic acid carbonyl oxygen interacts weakly with vanadium. These species are the most likely candidates for the inhibitors of serine amidohydrolases found in vanadate/hydroxamic acid mixtures.