Complexes Formed in Solution Between Vanadium(IV)/(V) and the Cyclic Dihydroxamic Acid Putrebactin or Linear Suberodihydroxamic Acid

Abstract

An aerobic solution prepared from V(IV) and the cyclic dihydroxamic acid putrebactin (pbH₂) in 1:1 H₂O/CH₃OH at pH = 2 turned from blue to orange and gave a signal in the positive ion electrospray ionization mass spectrometry (ESI-MS) at m/z_obs 437.0 attributed to the monooxoV(V) species [V^VO(pb)]⁺ ([C₁₆H₂₆N₄O₇V]⁺, m/z_calc 437.3). A solution prepared as above gave a signal in the ⁵¹V NMR spectrum at δ_V = −443.3 ppm (VOCl₃, δ_V = 0 ppm) and was electron paramagnetic resonance silent, consistent with the presence of [V^VO(pb)]⁺. The formation of [V^VO(pb)]⁺ was invariant of [V(IV)]:[pbH₂] and of pH values over pH = 2–7. In contrast, an aerobic solution prepared from V(IV) and the linear dihydroxamic acid suberodihydroxamic acid (sbhaH₄) in 1:1 H₂O/CH₃OH at pH values of 2, 5, or 7 gave multiple signals in the positive and negative ion ESI-MS, which were assigned to monomeric or dimeric V(V)– or V(IV)–sbhaH₄ complexes or mixed-valence V(V)/(IV)–sbhaH₄ complexes. The complexity of the V-sbhaH₄ system has been attributed to dimerization (2[V^VO(sbhaH₂)]⁺ ↔ [(V^VO)₂(sbhaH₂)₂]²⁺), deprotonation ([V^VO(sbhaH₂)]⁺ – H⁺ ↔ [V^VO(sbhaH)]⁰), and oxidation ([V^IVO(sbhaH₂)]⁰ –e^– ↔ [V^VO(sbhaH₂)]⁺) phenomena and could be described as the sum of two pH-dependent vectors, the first comprising the deprotonation of hydroxamate (low pH) to hydroximate (high pH) and the second comprising the oxidation of V(IV) (low pH) to V(V) (high pH). Macrocyclic pbH₂ was preorganized to form [V^VO(pb)]⁺, which would provide an entropy-based increase in its thermodynamic stability compared to V(V)–sbhaH₄ complexes. The half-wave potentials from solutions of [V(IV)]:[pbH₂] (1:1) or [V(IV)]:[sbhaH₄] (1:2) at pH = 2 were E_1/2 −335 or −352 mV, respectively, which differed from the expected trend (E_1/2 [VO(pb)]^+/0 < V^V/IV–sbhaH₄). The complex solution speciation of the V(V)/(IV)–sbhaH₄ system prevented the determination of half-wave potentials for single species. The characterization of [V^VO(pb)]⁺ expands the small family of documented V–siderophore complexes relevant to understanding V transport and assimilation in the biosphere.

Short abstract

The monooxoV(V) complex [V^VO(pb)]⁺ was formed from solutions of VOSO₄ and the cyclic dihydroxamic acid putrebactin (pbH₂) over pH values of 2−7. The solution speciation of VOSO₄ and the linear dihydroxamic acid suberodihydroxamic acid (sbhaH₄) showed dimerization ([V^VO(sbhaH₂)]⁺ ↔ [(V^VO)₂(sbhaH₂)₂]²⁺), deprotonation ([V^VO(sbhaH₂)]⁺ − H⁺ ↔ [V^VO(sbhaH)]⁰), and oxidation ([V^IVO(sbhaH₂)]⁰ − e⁻ ↔ [V^VO(sbhaH₂)]⁺) phenomena. The characterization of [V^VO(pb)]⁺ expands the small family of V−siderophore complexes described and is relevant in understanding V transport and assimilation in the biosphere.

Introduction

The ability of microbial siderophores to coordinate metal ions, particularly Fe(III), continues to generate interest in the potential applications of these bioligands in the environment and medicine.¹⁻³ Siderophores produced by terrestrial and marine bacteria have evolved to sequester Fe(III) from insoluble Fe(III)-oxy/hydroxides under oxic and pH neutral environments.⁴⁻⁷ The formation of the Fe(III)–siderophore complex is the first step in siderophore-dependent iron uptake and is essential for the viable growth of almost all environmental and pathogenic bacteria.⁸⁻¹² Siderophores have diverse structures and have been classified according to the metal ion-binding functional group as catechol-, hydroxamic acid- or citric acid-based compounds.(13) Clinical applications of hydroxamic acid-based siderophores include the mesylate salt of desferrioxamine B for the treatment of iron-overload disease in patients undergoing frequent blood transfusions and suberoylanilide hydroxamic acid, which acts as an anticancer agent via the inhibition of Zn(II) containing histone deacetylases.¹⁴⁻¹⁸

While siderophores have evolved as high-affinity Fe(III) complexing agents, these ligands have a rich coordination chemistry with other transition-metal ions in the first and second row of the Periodic Table.(19) Siderophores excreted by marine bacteria compete for Fe(III) and other transition-metal ions present in greater abundance in the ocean, such as Mo(VI) and V(V).(20) Nitrogen fixing Azotobacter vinelandii produces a suite of siderophores that bind Fe(III) and V in order to meet its V requirement for the biosynthesis of V-containing nitrogenase.(21) Desferrioxamine B reactivated V(V)-inhibited enzymes by sequestering the V(V) ion at the active site(22) and the V(V)-hydroxamic acid complexes inhibited the β-lactamase of Enterobacter cloacae P99.(23) Complexes between V(IV) or V(V) and hydroxamic acids lowered blood glucose levels in rat adipocytes(24) and streptozotocin-induced diabetic mice(25) and are under increasing scrutiny as antidiabetic agents. Despite the importance of the coordination chemistry of V and siderophores, there have been few complexes characterized. The triscatecholate-based siderophore enterobactin formed an unusual desoxoV(IV) complex that has been characterized by X-ray crystallography,^26,27 and complexes between desferrioxamine B and V(V) have been characterized in solution.(28)

The cyclic dihydroxamic acid-based siderophore putrebactin (pbH₂, 1, Chart 1) is a 20-membered macrocycle produced by Shewanella putrefaciens.²⁹⁻³¹ The cyclic dihydroxamic acid-based class of siderophores features only two other members: alcaligin produced by pathogenic Bordetella pertussis and B. parapertussis and bisucaberin produced by Alteromonas haloplanktis and Aliivibrio salmonicida.³²⁻³⁵ It was shown by the group that first discovered and fully characterized pbH₂ that the Fe(III):pbH₂ stoichiometry changed from 1:1 at pH = 2 to 2:3 at pH = 6, with the 6 coordination sites of each of the 2 Fe(III) centers in the latter complex fully saturated by 3 tetradentate pb(2−) ligands.(29) This was similar to the 2:3 complex characterized in solution and in the solid state between Fe(III) and alcaligin^33,36 or between Fe(III) and the linear dihydroxamic acid rhodotorulic acid.^37,38 Due to the low yields produced in culture and the complex nature of the total syntheses of cyclic dihydroxamic acids,^39,40 the coordination chemistry of pbH₂ has not been studied beyond Fe(III). Since pbH₂ has four hydroxamic acid-derived oxygen donor atoms positioned within a constrained macrocycle, we predicted that pbH₂ would form stable mononuclear complexes with oxoV(IV) or oxoV(V) centers, with the oxo group in a five-coordinate species positioned above the metal–ligand plane. This posit was supported by the results from studies of the coordination chemistry of V(V) or Cr(V) and abiological hydroxamic acids.^41,42 With limited amounts of native pbH₂ available from culture, we have characterized species formed in solution between V(V)/(IV) and pbH₂ using electronic absorption spectroscopy, electrospray ionization mass spectrometry (ESI-MS), ⁵¹V NMR spectroscopy, electron paramagnetic resonance (EPR) spectroscopy and cyclic voltammetry. To gain insight into the differences in speciation between V(V)/(IV) and cyclic or linear dihydroxamic acids, experiments using the linear dihydroxamic acid suberodihydroxamic acid (sbhaH₄, 2,Chart 1) were conducted in parallel.

Chart 1. Putrebactin (pbH₂, 1) and Suberodihydroxamic Acid (sbhaH₄, 2).

Mapping the species formed from solutions of V(V)/(IV) and a difficult-to-access cyclic dihydroxamic acid-based siderophore native to a bacterial species of Shewanella has implications for a better understanding of V(V)/(IV)-siderophore chemistry in marine environments.

Experimental Section

Chemicals and Bacterial Strain

All reagents were obtained from commercial sources and used without further purification. Suberodihydroxamic acid (sbhaH₄) and NaVO₃ were purchased from Sigma-Aldrich Co. VOSO₄·5H₂O was obtained from Merck. S. putrefaciens 8071^T was obtained from the American Type Culture Collection (ATCC). Methanol and acetonitrile were HPLC grade from LabScan. Milli-Q water (18.2 Ω) was used in all experiments. The pH values of the solutions were adjusted with 0.1 M NH₄OH, 0.1 M NaOH, or 0.1 M HCl, as stipulated. As measured for each solution at t = 15 min after preparation and at t = 1 h after analysis, the change in the pH value in all experiments was <0.1 pH unit.

Instrumentation

ESI-MS was carried out using a Finnigan LCQ mass spectrometer (San Jose, CA) in both positive and negative ion modes. The mobile phase was methanol, with a flow rate = 0.20 mL min^–1, cone voltage was 25 V, and the injection volume was 20 μL. Electronic absorption spectra were acquired with a Cary 1E UV–vis spectrophotometer. ¹H NMR spectra were recorded on a 300 MHz Bruker Avance300 NMR spectrometer at 300 K. ⁵¹V NMR spectra were recorded at 105 MHz on a 400 MHz Bruker DPX400 NMR spectrometer using a capillary VOCl₃ reference (0 ppm). EPR spectra were acquired using a Bruker Elexsys 500 spectrometer at 150 K under the following conditions: Center field, 3330 G; sweep width, 2000 G; resolution, 1024; modulation frequency, 100 kHz; modulation amplitude, 2 G; time constant, 1.28 ms; conversion time, 40.96 ms; microwave power, 6.241 mW; microwave frequency, 9.3470 GHz; and no. of scans, 20. Cyclic voltammetry experiments were carried out using a Bioanalytical Systems BAS 100B Electrochemical Analyzer (scan rate, 100 mV s^–1), 100% iR compensation) with a Pt working electrode, a Ag/AgCl reference electrode, and Pt wire as an auxiliary electrode. Samples were degassed with Ar for 10 min prior to data acquisition.

Purification of pbH₂ from S. putrefaciens 8071^T

The culture supernatant of two large-scale, Fe(III)-deficient cultures of S. putrefaciens 8071^T (30 and 50 L culture medium) were purified as previously described.^29,43 The pbH₂ obtained from the 30-L culture (26.6 mg) was dissolved in an aliquot (∼7 mL) 1:1 CH₃OH/H₂O at pH = 2 (∼10.2 mM, Batch 1), and the pbH₂ obtained from the 50-L culture (64.4 mg) was dissolved in an aliquot (17.2 mL) of 1:1 CH₃OH/H₂O at pH = 2 (∼10 mM, Batch 2). Based on RP-HPLC and ¹H NMR spectroscopic measurements, Batch 1 was ∼50% pure and Batch 2 was ∼40% pure. Some experiments used a solution of pbH₂ (∼11 μM in 40:60 CH₃OH/H₂O, pH = 2) that had not been purified by RP-HPLC (Batch 3).

Solution Preparation: Electronic Absorption Spectroscopy and ESI-MS

Solid VOSO₄·5H₂O (0.03 g, 0.12 mmol) was added to an aliquot of pbH₂ (2 mL, ∼20 μmol, Batch 1) to give a V(IV):pbH₂ solution in a ∼6:1 ratio at pH = 2. The resulting purple/red solution was stirred at room temperature. After standing overnight at room temperature, the solution was subjected to ESI-MS (positive ion mode) and an electronic absorption spectrum was obtained (300–900 nm). The solution was stored in a closed vial at room temperature for 15 months, and the positive ion ESI-MS was reacquired after this time. An aliquot of an aqueous solution of VOSO₄·5H₂O (200 μL, 1 mM) was added to an aliquot of pbH₂ (100 μL, Batch 3), and the solution was monitored by electronic absorption spectroscopy (300–900 nm) every 2 min over 90 min and again at 24 h. An aliquot of an aqueous solution of NaVO₃ (30 μL, 1 mM) was added to an aliquot of pbH₂ (750 μL of Batch 3) to give a V(V):pbH₂ solution in approximately a 3.5:1 ratio, and the pH value was adjusted to pH = 2. After stirring at room temperature overnight, the yellow/orange solution was analyzed by positive and negative ion ESI-MS. An aliquot of an aqueous solution of VOSO₄·5H₂O (200 μL, 1 mM) was added to an aliquot of pbH₂ (100 μL, Batch 3), the pH value of the solution was adjusted to pH = 2, and the solution was analyzed by negative ion ESI-MS. An aliquot of an aqueous solution of VOSO₄·5H₂O (4 mL, 10 mM) was added to an aliquot of pbH₂ (4 mL, Batch 2), and the orange solution was stirred at room temperature. After standing overnight at room temperature, the solution was lyophilized to yield a solid purple residue. The purple solid was dissolved in methanol (1 mL) to give an orange solution, which was applied to a Sephadex LH-20 column (16 × 1.2 cm i.d., CV = 18.1 cm³) which was eluted with CH₃OH (flow rate ∼0.5 mL min^–1). An orange band eluted at 10 mL, and a yellow band eluted at 25 mL. Both fractions were collected and analyzed by positive and negative ion ESI-MS. Solutions of VOSO₄·5H₂O (1 mM stock solution in 10 mM HCl) and pbH₂ were prepared in which [V(IV)]:[pbH₂] = 1:1 (pH = 2); 1:1.5 (pH = 2 or 7); 1:2 (pH = 7) and were analyzed by positive and negative ion ESI-MS and by electronic absorption spectroscopy.

An aliquot of an solution of VOSO₄·5H₂O in 0.1 M HCl (1 mL, 10 mM) was added to an aliquot of sbhaH₄ (2 mL, 10 mM) in 1:1 CH₃OH:H₂O and the pH value of the solution was adjusted to pH = 2 with 0.1 M HCl. Three additional solutions were prepared at pH = 5, 7, and 9. Each solution was analyzed by positive ion and negative ion ESI-MS and by electronic absorption spectroscopy.

Solution Preparation: ⁵¹Vanadium NMR Spectroscopy

An aliquot of an aqueous solution of VOSO₄·5H₂O (0.4 mL, 10 μM) was added to an aliquot of pbH₂ (0.4 mL, 10 μM, Batch 2), and the orange solution (pH = 2) was left to stand overnight at room temperature. The solution was lyophilized to dryness, and the residue was dissolved in methanol (∼1 mL) before passing the solution through a Sephadex LH-20 column, as described above. The orange band was collected, the solution was lyophilized, and the residue was dissolved in CD₃OD (0.5 mL) for analysis by ⁵¹V NMR spectroscopy. An aliquot of a D₂O solution of VOSO₄·5H₂O (0.167 mL, 2 mM) was added to an aliquot (0.333 mL, 2 mM) of sbhaH₄ in D₂O, and the pH value of the solution was adjusted to pH = 2 using 0.1 M HCl (5 μL of 10.17 M HCl in 5 mL D₂O). Two additional V(IV):sbhaH₄ = 1:2 solutions were prepared at pH = 5 or 7, with the pH values of the solutions adjusted using 0.1 M NaOH (20 mg NaOH in 5 mL D₂O).

Solution Preparation: EPR Spectroscopy

Methanol stock solutions of: VOSO₄·5H₂O at 10 mM (0.063 g in 25 mL) or 1 mM (0.0063 g in 25 mL) or sbhaH₄ at 10 mM (0.0511 g in 25 mL) were prepared. An aliquot of a methanol solution of VOSO₄·5H₂O (5 μL, 1 mM) was added to an aliquot of pbH₂ (900 μL, ∼11 μM, Batch 3) to give an approximately V:pbH₂ = 1:2 solution. The solution was purified by Sephadex LH-20 column chromatography, as described above, and the orange band was subject to EPR analysis at 150 K. An aliquot of a methanol solution of VOSO₄·5H₂O (1 mL, 10 mM) was added to a methanol solution of sbhaH₄ (2 mL, 10 mM), and the pH value of the solution was adjusted with 0.1 M HCl to pH = 2. The solution was left at room temperature for 15 min prior to EPR analysis at 150 K. A second V(IV):sbhaH₄ = 1:2 solution was prepared as above, and the pH value was adjusted to pH = 5 with 0.1 M NH₄OH.

Solution Preparation: Electrochemistry

An aliquot (1 mL) of an aqueous solution of VOSO₄ (10 mM) was added to ∼25 μmol pbH₂, and the orange solution was passed through an LH-20 column as described above, and was eluted with methanol. The orange band was collected, and the methanol was removed to yield 10.5 mg of purple solid which was dissolved in 5 mL of 0.1 M NaCl. The pH value of the solution was adjusted to pH = 2. An aliquot (2 mL) of sbhaH₄ (10 mM in 0.1 M NaCl) was added to an aliquot (1 mL) of VOSO₄ (10 mM in 0.1 M NaCl). The volume was made to 6 mL with 0.1 M NaCl solution to give a final solution of [V(IV)]:[sbhaH₂] = 1.67 mM:3.33 mM, and the pH value of the solution was adjusted to pH = 2.

Results and Discussion

Rationale

In this study, the coordination chemistry of V(IV) and cyclic pbH₂ or linear sbhaH₄ has been explored. Due to the limited yields of pbH₂ obtained from iron-limited cultures of S. putrefaciens, the coordination chemistry of pbH₂ has not been studied beyond Fe(III).(29) The configuration of the oxygen donor atoms of pbH₂ suggested that this ligand would form stable complexes with oxoV(IV) or oxoV(V) which is of interest in the context of siderophores in the marine environment, particularly given that many species of Shewanella are marine bacteria(44) and that in seawater V is present at 20–35 pM and is second to Mo as the most abundant transition metal.^20,45 This work has mapped solution species formed between V(IV) and pbH₂ or sbhaH₄ using electronic absorption spectroscopy, positive and negative ion mode ESI-MS, ⁵¹V NMR spectroscopy, EPR spectroscopy, and cyclic voltammetry.

Electronic Absorption Spectroscopy

The solution formed from mixing V(IV) with pbH₂ (1:1 CH₃OH/H₂O at pH = 2) in an approximate 6:1 ratio was initially blue in color. After stirring for 1 h, the solution became dark-purple/red in color and gave an electronic absorption spectrum with λ_max at 452 and 760 nm. Hydroxamic acids are known to form highly colored complexes with V, which has been used for the analytical detection of submillimolar amounts of V.^46,47 A time-course study with [V(IV)] > [pbH₂] showed that the distribution of species formed between V and pbH₂ reached equilibrium at t = 90 min, with the spectrum acquired at 24 h similar to that at t = 90 min (Figure 1). There were two isosbestic points at 354 and 630 nm, indicating the presence of at least two species in solution. One of these species was VOSO₄, as indicated by the peak at 760 nm. The peak at 452 nm compared well with the electronic absorption spectra of previously characterized V(V)–hydroxamato complexes in CH₃OH/H₂O solutions(41) and was similar to the spectrum from a red solution prepared from V(IV) and sbhaH₄ (λ_max = 444 nm, Figure 1 inset, dashed line) in which the V(IV) had been oxidized to V(V). The electronic spectrum from a solution prepared from V(IV) and pbH₂, where [pbH₂] > [V(IV)] (Figure 1 inset, solid line) was similar to that of the V(V)-sbhaH₄ spectrum, which suggested that a solution of V(IV) and pbH₂ yielded V(V)-pbH₂ species.

Figure 1.

Electronic absorption spectra from an aqueous solution of VOSO₄·5H₂O (1 mM) and pbH₂ (<1 mM) acquired over 2 min intervals for 90 min. The inset shows the electronic absorption spectra from an aqueous 1:2 solution of V(IV) and shbaH₄ (pH = 5, dashed line) or an aqueous 1:1 solution of V(IV) and pbH₂ (pH = 2, solid line).

ESI-MS from Solutions of V(IV) or V(V) and pbH₂

The ESI-MS (positive ion) spectrum from the ∼6:1 V(IV):pbH₂ solution at pH = 2 gave a major signal at m/z 437.0 (100%, Figure 2a), which formulated as the V(V) species [V^VO(pb)]⁺ ([C₁₆H₂₆N₄O₇V]⁺, m/z_calc 437.3) in which pb(2−) coordinates in a tetradentate fashion to a monooxoV(V) center to give a five-coordinate species (Scheme 1, 3). A model of [V^VO(pb)]⁺ built using HyperChem 7.5 from X-ray crystal structure data of Fe₂(alcaligin)₃(36) and bis(thiomaltolato)oxoV(IV)(48) shows pb(2−) forming a bucket-type structure below the V(V) ion, which, similar to Fe(III)-alcaligin, lifts the metal ion out of the plane (Figure 3). This structure would be expected to prevent other ligands coordinating trans to the oxo group.(41) An additional methanol, hydroxyl or water ligand would give a six-coordinate complex, such as [V^VO(OH)(pb)], with the ancillary ligand cis to the oxo group.

Figure 2.

Positive ion ESI-MS spectra from 1:1 H₂O:CH₃OH solutions at pH = 2 of: (a) V(IV):pbH₂ ∼6:1; (b) V(V):pbH₂ = ∼3.5:1; (c) V(IV):pbH₂ ∼1:1 after purification by Sephadex LH-20; and (d) V(IV):pbH₂ ∼6:1 after 15 months aging. Negative ion ESI-MS spectra from 1:1 H₂O:CH₃OH solutions at pH = 2 of: (e) V(IV):pbH₂ ∼1:1 and (f) V(V):pbH₂ ∼1:1.

Scheme 1. Structures of [V^VO(pb)]⁺ (3), [(V^VO)₂(pb)₂]²⁺ (4), and [V^VO(OH)(pb)] (5).

Figure 3.

Predicted structure of [V^VO(pb)]⁺ based upon the X-ray crystal structure of Fe₂(alcaligin)₃.(36) H atoms have been omitted for clarity.

The ESI-MS signal simulated well as [V^VO(pb)]⁺ (Figure 2a, inset). This species may be in equilibrium with the dimeric species [(V^VO)₂(pb)₂]²⁺ ([C₃₂H₅₂N₈O₁₄V₂]²⁺, m/z_calc 437.3) where two oxo groups bridge the two V(V) centers to form a hexacoordinate bis(μ-oxo) complex (4, Scheme 1). Compressed ESI-MS isotope patterns can be used to differentiate between mononuclear and dimeric coordination complexes with hydroxamic acid in cases where the metal ion, such as Fe(III) or Ga(III), is polyisotopic.(49) Since V is monoisotopic (natural abundance ⁵¹V, 99.75%), the nuclearity of complexes will not be reflected in compressed ESI-MS isotope patterns. Therefore, signals due to [V^VO(pb)]⁺ and [(V^VO)₂(pb)₂]²⁺ would be indistinguishable at the level of ESI-MS. Signals in the ESI-MS at m/z_obs 468.1 (7%) and 491.0 (21%) were attributed to [V^VO(pb)(CH₃OH)]⁺ (m/z_calc 469.4) and the Na⁺ adduct of [V^VO(pb)(OCH₃)] (m/z_calc 491.4), respectively. Signals corresponding to pbH₂ ([M + H]⁺m/z_calc 373.2) or its adducts were not observed. The presence of the intrinsically uncharged V(IV) complex [V^IVO(pb)] is discounted since the [M + H⁺]⁺ adduct of this species would be expected to give a signal in the positive ion ESI-MS centered at m/z_obs 438.4, which was not the case. The orange/red color of the solution, the electronic absorption spectrum and the ESI-MS data supported the formation of [V^VO(pb)]⁺ from solutions of V(IV) and pbH₂. Previous work has established the formation of V(V)–hydroxamic acid complexes from V(IV) and described the orange color of methanol solutions of these complexes and the violet color of methanol free residues.^41,47,50 This was similar to the physical characteristics of the [V^VO(pb)]⁺ system. If [V^VO(pb)]⁺ was being formed from a solution of V(IV) and pbH₂, then the ESI-MS from this solution would be expected to be similar to that from a solution of V(V) and pbH₂, where no redox chemistry was taking place. A dilute solution of NaVO₃ and pbH₂ gave a signal in the ESI-MS (positive ion) at m/z_obs 437.1 (Figure 2b) with other signals at m/z values similar to those observed for the solution prepared from V(IV) and pbH₂. The relative concentration of the signal at m/z_obs 437.0 in the ESI-MS ascribed to [V^VO(pb)]⁺ was largely invariant of pH value and of the [V(IV)]:[pbH₂] ratio (Table 1). This contrasts with the Fe(III)-pbH₂ or -alcaligin systems, which show a pH-dependent distribution of complexes of different stoichiometries.^29,33,36,38

Table 1. Positive and negative ion mode ESI-MS data from 1:2 solutions of V(IV) and pbH₂ or sbhaH₄ at pH = 2, 5 or 7.

			pH
			2	5	7a
species	no.	m/zcalcb	m/zobs (%)	m/zobs (%)	m/zobs (%)
[VVO(pb)]+c	3, 4	437.3	436.9 (100)	NCd	437.1 (100)
[VVO(OH)(pb)]e	5	455.3	454.7 (100)f
[sbhaH4]e	2	205.2	NDg	ND	204.7 (38)
[sbhaH3]−	2	203.2	ND	ND	203 (29)
[sbhaH3·sbhaH4]−	2	407.4	ND	ND	407 (10)
[VVO(sbhaH2)]+, [(VVO)2(sbhaH2)2]2+	6, 12	269.1	268.7 (7)	268.7 (4)	268.8 (3)
[VVO(sbhaH2)(CH3OH)]+, [(VVO)2(sbhaH2)2(CH3OH)2]2+	6·M, 12·M	301.1	300.4 (100)	300.5 (100)	300.5 (100)
[VVO(sbha)]−, [(VVO)2(sbha)2]2–	10, 24	267.1	266.6 (3)	266.9 (6)	267 (47)
[VVO(sbha)(CH3OH)]−, [(VVO)2(sbha)2(CH3OH)2]2–	10·M, 24·M	299.2	ND	ND	300.8 (20)
[(VIVO)(VVO)(sbhaH2)2]+	14	538.2	537.5 (6)	537.8 (5)	537.9 (5)
[(VVO)2(sbhaH2)(sbhaH)]+	15	537.3	ND	536.9 (6)	536.9 (12)
[(VVO)2(sbhaH2)(sbhaH)(CH3OH)]+	15·M	569.3	ND	568.8 (12)	568.8 (20)
[(VIVO)2(sbhaH2)(sbhaH)(CH3OH)]−	16·M	569.3	569.8 (47)	ND	ND
[VIVO(sbhaH)(CH3OH)]−, [(VIVO)2(sbhaH)2(CH3OH)2]2–	9·M, 19·M	300.1	300.6 (90)	ND	ND
[(VIVO)(VVO)(sbhaH)2(CH3OH)]−	20·M	569.3	568.7 (42)	ND	ND
[(VVO)2(sbhaH)(sbha)]−	21	535.3	ND	534.9 (6)	534.9 (42)
[(VVO)2(sbhaH)(sbha)(CH3OH)]−	21·M	568.3	ND	ND	568.9 (20)

^aSpectra acquired at pH = 9 were similar to those at pH = 7.

^bm/z_calc for M⁺ or M^– ion, unless otherwise specified.

^cThe metal:pbH₂ ratio was 1:1.5 at pH = 2.

^dNC is not acquired.

^em/z_calc for [M + H⁺]⁺.

^fAged solution.

^gND is not detected.

One orange and one yellow band were resolved using LH-20 size exclusion chromatography from a methanol solution prepared from V(IV) and pbH₂. The orange fraction analyzed in the positive ion ESI-MS as [V^VO(pb)]⁺ (m/z_obs 437.3) (Figure 2c). As a result of the methanol-based chromatographic procedure the intensity of the signal ascribed to [V^VO(pb)(CH₃OH)]⁺ in this solution (m/z_obs 468.7) was similar to the intensity of the signal for [V^VO(pb)]⁺. The complex signals between m/z_obs 356.1 to 358.1 could be ascribed to an pbH₂ adduct with one hydroxamic acid group reduced to an amine (C₁₆H₂₉N₄O₅, [M + H⁺]⁺m/z_calc 357.4). The positive ion ESI-MS from the yellow fraction contained no free pbH₂ and was likely to contain polyoxovanadates.

The solution of V-pbH₂ that had been stored at room temperature for 15 months, gave rise to a signal at m/z_obs 454.7 (100%) (Figure 2d), which was consistent with the six-coordinate species [V^VO(OH)(pb)] ([M + H⁺]⁺, m/z_calc 455.3) in which a hydroxide ligand has coordinated to [V^VO(pb)]⁺ (5, Scheme 1). The time period over which [V^VO(OH)(pb)] formed was not clear, since ESI-MS spectra were acquired from solutions of V(IV) and pbH₂ that were aged for <1 d and 15 months but not in between these times. Attempts to isolate the CF₃SO₂^– salt of [V^VO(pb)]⁺ and to bind [V^VO(pb)]⁺ to a cation-exchange resin from 1 to 2 day-old solutions of V(IV) and pbH₂ were unsuccessful, which indicates that the concentration of [V^VO(OH)(pb)] may be significant over time periods <15 months.

The presence of six-coordinate [V^VO(OH)(pb)] suggested that cis-dioxoV(V)-pbH₂ species, such as [V^VO₂(pb)]⁻, might be present. None of the signals in the negative ion ESI-MS from a solution of V(IV) and pbH₂ were ascribable to cis-dioxoV(V)-pbH₂ species (Figure 2e). The signal at m/z 485.1 in the negative ion ESI-MS from a solution of V(V) and pbH₂ could be ascribed to the methanol adduct [V^VO₂(pb)]⁻·CH₃OH (m/z_calc 485.4) (Figure 2f). The relative concentration of this species is low (7%). The remaining signals in the negative ion spectrum are attributed to background ions or impurities in pbH₂.

ESI-MS from Solutions of V(IV) and sbhaH₄: Opening Comments

Spectra were acquired from 1:2 solutions of V(IV):sbhaH₄ at pH = 2, 5, 7 (Figure 4), and 9 and from a 1:1 solution of V(IV):sbhaH₄ at pH = 2 (Table S1, Supporting Information). Compared to pbH₂, more signals were present in spectra from solutions of V(IV) and sbhaH₄, which suggested a more complex speciation profile. This increased complexity was made more acute by the fact that sbhaH₄ was pure and that signals were not attributable to ligand impurities. Based on previous work, it was reasonable to expect that dihydroxamate or dihydroximate complexes would predominate at low or high pH values, respectively.⁵¹⁻⁵⁶ The former complexes would more likely be detected by positive ion mode ESI-MS and the latter complexes by negative ion mode. Mixed hydroxamate/hydroximate complexes might form at intermediate pH values, some of which could be intrinsically uncharged and MS-inactive. Further, since the V(IV) ion is more acid stable than the V(V) ion, it was reasonable to expect that at pH = 2, [V(IV)-sbhaH₄] > [V(V)-sbhaH₄] and at pH = 7 [V(V)-sbhaH₄] > [V(IV)-sbhaH₄]. This was borne out from the results of the ⁵¹V NMR and EPR spectroscopy experiments (discussed further below).

Figure 4.

Positive ion ESI-MS spectra from 1:2 solutions of V(IV):sbhaH₄ in 1:1 H₂O:MeOH at: (a) pH = 2, (b) pH = 5, or (c) pH = 7. Negative ion ESI-MS spectra from 1:2 solutions of V(IV):sbhaH₄ in 1:1 H₂O:MeOH at: (d) pH = 2, (e) pH = 5, or (f) pH = 7. Simulated spectra of selected species are shown in the insets.

In the negative ion ESI-MS data there was a discontinuity in the signals observed from solutions at pH = 5, compared to pH = 2 and 7. The major signal at m/z_obs 300.6 present in the spectrum at pH = 2 and m/z_obs 300.8 at pH = 7 (Figure 4 panels d and f, respectively) was absent in the spectrum at pH = 5 (Figure 4, panel e). This suggested that MS-active species were present in relatively high concentrations at pH = 2 and 7 and that intrinsically uncharged MS-inactive species were present in relatively high concentrations at pH = 5. Further, this discontinuity indicated that although the m/z values of the major signals were similar, the signal at m/z_obs 300.6 at pH = 2 could not be ascribed to the same species that gave the signal at m/z_obs 300.8 at pH = 7. The most likely explanation for the observed discontinuity was that the two species giving rise to the signals with similar m/z values varied in the oxidation state of the V center(s) and/or the protonation state of the complex.

A theoretical map of the solution equilibria of V(V)/(IV)–sbhaH₄ complexes (Scheme 2) shows several phenomena, including dimerization (eqs 1 and 2, refer to dotted boxes in Scheme 2), deprotonation (eqs 3 and 4), and oxidation (eqs 5 and 6). At any given pH value, these processes would yield a mixture of complexes with different charges (refer to upper horizontal bars of Scheme 2). While ESI-MS data are not an accurate reflection of equilibrium conditions, this map served to aid assigning signals to species and in providing possible pathway(s) for the formation and transformation of species.

Scheme 2. Major Species Present in Solutions Analyzed by ESI-MS Prepared From V(IV) and sbhaH₄ As a Function of pH Value.

The m/z_calc for each species is given below the structure and the m/z_calc value of the methanol adduct is given in parentheses.

ESI-MS from Solutions of V(IV) and sbhaH₄: Positive Ion Mode

The major signal in the positive ion ESI-MS from a 1:2 V(IV):sbhaH₄ solution at pH = 2 (m/z_obs 300.4, 100%) was ascribed to the methanol adduct [V^VO(sbhaH₂)(CH₃OH)]⁺ (m/z_calc 301.1, 6·CH₃OH, Scheme 2) or its dimer [(V^VO)₂(sbhaH₂)₂(CH₃OH)₂]²⁺ (m/z_calc 301.1, 12·CH₃OH, Scheme 2), which would be indistinguishable from the monomer by ESI-MS. This signal was present at 100% relative intensity from the solutions at pH = 2, 5, and 7 (Figure 4, panels a–c). Signals ascribed to the methanol free adduct [V^VO(sbhaH₂)]⁺ (m/z_calc 269.1, 6) or its dimer [(V^VO)₂(sbhaH₂)₂]²⁺ (m/z_calc 269.1, 12) were observed at lower abundance at all pH values (m/z_obs 268.7, 3–7%).

Signals observed from the solution at pH = 5 (m/z_obs 536.9, 6%) and 7 (m/z_obs 536.9, 12%) were ascribed to [(V^VO)₂(sbhaH₂)(sbhaH)]⁺ (m/z_calc 537.3, 15), which according to eq 4 could result from the single deprotonation of [(V^VO)₂(sbhaH₂)₂]²⁺ (12) or other pathways (Scheme 2). Signals observed from the solution at pH = 2 (m/z_obs 568.7, 2%), 5 (m/z_obs 568.8, 12%), and 7 (m/z_obs 568.8, 20%) were ascribed to the methanol adduct of 15, [(V^VO)₂(sbhaH₂)(sbhaH)(CH₃OH)]⁺ (m/z_calc 569.3, 15·CH₃OH). The relative intensities of the signals ascribed to 15 and 15·CH₃OH would be expected to increase with increasing pH value, which was the case. The isotope patterns of the signal from the solution at pH = 5 and 7 centered at m/z_obs 536.9 were not able to be simulated as a single V-sbhaH₄ species (Figure 4, insets in panel b and c), suggesting that the signal comprised overlapping signals from 15 and one or more other V-sbhaH₄ species.

The presence of the signal ascribed to 15 (m/z_obs 536.9) supported the existence of dimeric V-sbhaH₄ species. Mononuclear 6 could form dimeric 12, in which each sbhaH₄ ligand bridges between two oxoV(V) centers (intermetal coordination) and dimeric 12a, in which each sbhaH₄ ligand binds to discrete V(V) centers (intrametal coordination) which themselves are bound via a bis(μ-oxo) bridge. With six methylene groups separating the flanking hydroxamic acid groups, sbhaH₄ sits at the boundary of the preference of dihydroxamic acids with methylene chain lengths n ≤ 6 to form dimeric complexes (eg, 12) and ligands with methylene chain lengths n ≥ 6 to form mononuclear complexes (eg, 6), as established by previous work.^49,57−60

There was close to one m/z unit difference between a signal from the solutions at pH = 5 and 7 at m/z_obs 536.9 and from the solution at pH = 2 at m/z_obs 537.5. The difference was consistent with the presence of [(V^VO)(V^IVO)(sbhaH₂)₂]⁺ (m/z_calc 538.2, 14) at pH = 2 and the presence of its one-electron oxidized, single-deprotonated product 15 at pH = 5 and 7. Species 14 could arise from the dimerization of mononuclear 6 and 7 (eq 2) and/or from the one-electron oxidation of 13 (eq 6), itself a dimer of 7. Based upon the acid stability of V(IV), the concentration of 14 would be expected to be higher at low pH and to decrease at higher pH values. Support for the presence of mixed-valence V(V)/(IV)–sbhaH₄ complexes at acid conditions is obtained from the crystallographically characterized mixed-valence V(V)₂-V(IV)-quinic acid trimer, which was isolated from a water–ethanol solution of the V(V)-quinic acid dimer upon reducing the pH value from 7.0 to 3.3.(61) Several mixed-valence V(IV)/V(V) μ-oxo-bridged complexes have been characterized with O and N donor atoms.⁶²⁻⁶⁴ While the signal at m/z_obs 537.5 from the spectra at pH = 2 simulated reasonably well as [(V^VO)(V^IVO)(sbhaH₂)₂]⁺ (14), an improved fit was obtained from assuming the presence of 80% [(V^VO)(V^IVO)(sbhaH₂)₂]⁺ (14) and 20% [(V^VO)₂(sbhaH₂)(sbhaH)]⁺ (15) (Figure 4, panel a, inset). The signal at m/z_obs 536.9 from the spectra at pH = 5 simulated as comprising 65% [(V^VO)₂(sbhaH₂)(sbhaH)]⁺ (15) and 35% [(V^VO)(V^IVO)(sbhaH₂)₂]⁺ (14) and at pH = 7 as 80% [(V^VO)₂(sbhaH₂)(sbhaH)]⁺ (15) and 20% [(V^VO)(V^IVO)(sbhaH₂)₂]⁺ (14) (Figure 4). In summary, the simplest model consistent with the positive ion ESI-MS data involved the presence of monomeric or dimeric [V^VO(sbhaH₂)]⁺ (6, 12) and the methanol adducts (6·CH₃OH, 12·CH₃OH), [(V^VO)₂(sbhaH₂)(sbhaH)]⁺ (15) and the methanol adduct (15·CH₃OH) and [(V^VO)(V^IVO)(sbhaH₂)₂]⁺ (14).

ESI-MS from Solutions of V(IV) and sbhaH₄: Negative Ion Mode

The major signal in the negative ion ESI-MS from a 1:2 V(IV):sbhaH₄ solution at pH = 2 (m/z_obs 300.6, 90%) was ascribed to methanol adduct [V^IVO(sbhaH)(CH₃OH)]⁻ (m/z_calc 300.1) or its dimer [(V^IVO)₂(sbhaH)₂(CH₃OH)₂]^2– (m/z_calc 300.1) (9·CH₃OH and 19·CH₃OH, respectively, Scheme 2). This signal was absent from the solution at pH = 5 and reappeared from the solution at pH = 7, which indicated that the signals were due to different species, even though the m/z values were similar. The signal (m/z_obs 300.8, 20%) from the solution at pH = 7 was ascribed to the methanol adduct [V^VO(sbha)(CH₃OH)]⁻ (m/z_calc 299.1) or its dimer [(V^VO)₂(sbha)₂(CH₃OH)₂]^2– (m/z_calc 299.1) (10·CH₃OH and 24·CH₃OH, respectively, Scheme 2). The one-electron oxidation of [V^IVO(sbhaH)(CH₃OH)]⁻ (9·CH₃OH) would produce the MS-inactive [V^VO(sbhaH)(CH₃OH)]⁰ (8·CH₃OH) consistent with the absence of a signal from the solution at pH = 5, and the deprotonation of [V^VO(sbhaH)(CH₃OH)]⁰ (8·CH₃OH) would form the MS-active [V^VO(sbha)(CH₃OH)]⁻ (10·CH₃OH) at pH = 7. Signals ascribed to dimeric [(V^VO)₂(sbhaH)(sbha)]⁻ (m/z_calc 535.3, 21) were observed from the solution at pH = 5 (m/z_obs 534.9, 6%) and 7 (m/z_obs 534.9, 42%). The isotope pattern of the signal simulated well as 21 present as a single species (Figure 4, insets in panel e and f). The relative concentration of 10, 24, and 21 would be expected to increase with increasing pH value, which was the case. The signals observed from the solution at pH = 2 (m/z_obs 568.7, 42% and m/z_obs 569.8, 47%) were ascribed to the methanol adduct of the mixed-valence dimer [(V^VO)(V^IVO)(sbhaH)₂(CH₃OH)]⁻ (m/z_calc 568.3, 20·CH₃OH) and the methanol adduct [(V^IVO)₂(sbhaH₂)(sbhaH)(CH₃OH)]⁻ (m/z_calc 569.3, 16·CH₃OH), respectively. The isotope pattern in this region of the spectrum from the solution at pH = 2 indicates that 16·CH₃OH and 20·CH₃OH were present at about equal relative concentration.

ESI-MS from Solutions of V(IV) and sbhaH₄: Closing Comments

At any given pH value, solutions of V(IV) and shhaH₄ analyzed by ESI-MS showed signals that could be ascribed to monomeric or dimeric V(V)/(IV)-sbhaH₄ species that differed in the level of deprotonation, the oxidation state of the V center(s), and in the overall charge of the complex. In total, the presence of 11 MS-active V(V)/(IV)-sbhaH₄ species have been proposed (marked by circles around the species number in Scheme 2; note 6 is shown twice in eqs 1 and 2). Since three of these 11 species (12, 19, and 24) are dimers that cannot be distinguished from the respective monomers (6, 9, and 10), the minimal speciation model from ESI-MS data supported the presence of 8 species. At all pH values, the dominant species from solutions of V(IV) and sbhaH₄ detected in the positive ion ESI-MS was 6·CH₃OH and/or its dimer 12·CH₃OH (Figure 5). At pH = 2, a higher relative concentration of V(IV)-sbhaH₄ species (9·CH₃OH, 19·CH₃OH, 16·CH₃OH) and V(IV)/(V) species (14, 20·CH₃OH) was present, with these species not evident in significant concentrations at pH = 5 and above. The V(V)-sbhaH₄ species 15, 15·CH₃OH, 10, 10·CH₃OH, 24, 24·CH₃OH, 21, and 21·CH₃OH were not evident at pH = 2 but were detectable at low levels at pH = 5 and increased in relative concentration at pH = 7 (Figure 5). The V(V)/(IV)-sbhaH₄ speciation profile was internally consistent across the positive and negative ion ESI-MS data. For example, the four-step deprotonation of dimeric [(V^VO)₂(sbhaH₂)₂]²⁺ (m/z_calc 269.1; m/z_obs 268.7, 12) to [(V^VO)₂(sbhaH₂)(sbhaH)]⁺ (m/z_calc 537.3; m/z_obs 536.9, 15) to [(V^VO)₂(sbhaH)₂]⁰ (MS-inactive, 18) to [(V^VO)₂(sbhaH)(sbha)]⁻ (m/z_calc 535.3; m/z_obs 534.9, 21) to [(V^VO)₂(sbha)₂]^2– (m/z_calc 267.1; m/z_obs 266.9, 24) was consistent with the positive and negative ion ESI-MS data.

Figure 5.

Distribution of species from 1:2 solutions of V(IV) and sbhaH₄ at pH = 2, 5, and 7, as measured by ESI-MS. V(V)-, V(IV)-, or mixed-valence V(V)/(IV)-sbhaH₄ species are represented by closed (black), open, or closed (gray) symbols, respectively, and by the species number, according to Scheme 2. The relative concentrations are presented as the sum of the relative intensity of the signals of a species (eg, 6) and its methanol adduct (eg, 6.M), according to Table 1.

At higher pH values, the dominant species present in a solution prepared from V(IV) and sbhaH₄ tended toward the bottom right-hand region of Scheme 2. This was consistent with the increased concentrations at higher pH values of the V(V) ion and the hydroximate form of sbhaH₄. The V(V)/(IV)-sbhaH₄ speciation profile can be broadly represented as a vector sum (Scheme 2, inset, dotted vector) of a pH-dependent vector that describes the oxidation of V(IV) to V(V) (low to high pH, respectively) (Scheme 2, inset, shorter solid vector) and a pH-dependent vector that describes the deprotonation of hydroxamate to hydroximate (low to high pH, respectively) (Scheme 2, inset, longer solid vector).

⁵¹V NMR Spectroscopy

A solution prepared from V(IV) and pbH₂ containing [V^VO(pb)]⁺ should be detectable by ⁵¹V NMR spectroscopy (V(V), d⁰). The ⁵¹V NMR spectrum from an approximately V(IV):pbH₂ = 1:1 solution at pH = 2 showed a signal at −443.3 ppm (Figure 6a), which was within the range for selected five- and six-coordinate V(V)–hydroxamato complexes at low pH values and also other V(V) complexes with O,O′ coordination, such as diols and α-hydroxycarboxylic acids.⁶⁵⁻⁶⁹ There were no signals attributable to polyoxovanadate species ([HVO₄]^3–, −557 ppm; [V₂O₇]^4–, −571 ppm; [V₄O₁₂]^4–,–577 ppm) in the spectrum(70) which suggested that the signal was attributable to [V^VO(pb)]⁺ or [V^VO(OH)(pb)].

Figure 6.

⁵¹V NMR spectra referenced to VOCl₃ (0 ppm) from a: (a) 1:1 solution of V(IV):pbH₂ in CD₃OD; 1:2 solution of V(IV):sbhaH₄ in D₂O at (b) pH = 2, (c) pH = 5, or (d) pH = 7 or a (e) 1:3 solution of V(IV):sbhaH₄ in D₂O at pH = 7. Polyoxovanadate species are denoted by *.

A signal in the ⁵¹V NMR spectrum at −432.2 ppm from a 1:2 solution of V(IV):sbhaH₄ at pH = 2 suggested the presence of a single V(V)-sbhaH₄ species (Figure 6b). This is consistent with the posit that there exists a mixture of V(IV)-sbhaH₄ and V(V)-sbhaH₄ species in a solution prepared from V(IV) and sbhaH₄ at pH = 2. Similar to the case with pbH₂, there were no signals due to polyoxovanadate species at this pH value. The signal at −432 ppm was absent from V(IV):sbhaH₄ = 1:2 solutions at pH = 5 (Figure 6c) and 7 (Figure 6d) with broad signals ascribed to V(V)–sbhaH₄ complexes appearing at −478 or −493 ppm, at the respective pH values. The relative intensity of the signal at −493 ppm increased at V(IV):sbhaH₄ = 1:3, pH = 7 (Figure 6e). The significant upfield shift of these signals from −432.2 ppm at pH = 2 indicated the formation of different V(V)-sbhaH₄ species, possibly arising from a mixture of hydroxamate- and hydroximate-coordination modes. At pH = 5 and 7, signals due to polyoxovanadate species ([V₁₀O₂₈]^6–, [H₂V₁₀O₂₈]^4–, [HVO₄]^2–, [V₂O₇]^4–, [V₄O₁₂]^4–) were evident (signals marked with * in Figure 6).

EPR Spectroscopy

If [V^VO(pb)]⁺ was the dominant species formed from solutions of V(IV) and pbH₂, then these solutions would be expected to be EPR silent. Compared to the EPR signal (150 K) from a methanol glassy solution of VOSO₄ (10 mM, Figure 7a), there was no signal in the EPR spectrum from a methanol glassy solution of V(IV) and pbH₂ at pH = 2 (Figure 7b), which supports the notion that these solutions contained [V^VO(pb)]⁺. The calculated EPR parameters from the VOSO₄ spectrum as simulated using QPOW(71) (g_∥ = 1.933, A_∥ = 181.2 × 10^–4 cm^–1; g_⊥ = 1.976, A_⊥ = 70.7 × 10^–4 cm^–1) agreed well with literature values for VOSO₄ (g_∥ = 1.933, A_∥ = 183 × 10^–4 cm^–1; g_⊥ = 1.978, A_⊥ = 71 × 10^–4 cm^–1).^72,73 A methanol glassy solution of V(IV) and sbhaH₄ at approximately pH = 2 gave an EPR signal (Figure 7c) that indicated the presence of more than one species, which was more clearly evident from the signal at the −5/2 resonance (inset). It was reasonable to assume that this solution contained a mixture of VOSO₄ and V(IV)-sbhaH₄ species. Therefore, while solutions of V(IV) and pbH₂ exclusively yielded V(V)-pbH₂ species, solutions of V(IV) and sbhaH₄ yielded a mixture of V(IV)-sbhaH₄ species, as evidenced from the EPR spectroscopic data, and V(V)-sbhaH₄ species, as evidenced from the ⁵¹V NMR spectroscopic and electronic absorption spectroscopic data. This was also consistent with the distribution of V(V)- and V(IV)-sbhaH₄ species detected by ESI-MS (Table 1, Figure 5). A second spectrum was acquired from a different solution prepared from VOSO₄ and sbhaH₄ with the pH value approximately at pH = 2 (Figure 7d) which indicated the presence of VOSO₄ and V(IV)-sbhaH₄ species in different relative concentrations to Figure 7c (refer to −5/2 resonance in inset). The solution from Figure 7d contained a higher relative concentration of V(IV)-sbhaH₄ species and enabled the calculation of the EPR parameters of the V(IV)-sbhaH₄ species as: g_∥ = 1.939, A_∥ = 170.1 × 10^–4 cm^–1; g_⊥ = 1.979, A_⊥ = 63.4 × 10^–4 cm^–1. The values of the line widths at half height for the V(IV)-sbhaH₄ species (W_⊥ = 8.0 × 10^–4 cm^–1; W_∥ = 6.0 × 10^–4 cm^–1) were almost twice the values for VOSO₄ (W_⊥ = 5.0 × 10^–4 cm^–1; W_∥ = 4.0 × 10^–4 cm^–1), which suggested that more than one V(IV)-sbhaH₄ species was present. The most likely species to be attributed to the V(IV)-sbhaH₄ EPR signal were 9·CH₃OH, 19·CH₃OH, or 16·CH₃OH, since these were present in relatively high concentration according to the ESI-MS data. The relative concentrations of VOSO₄ and V(IV)-sbhaH₄ species in the solution from Figure 7c were 70:30 and in the solution from Figure 7d were 10:90. The increased signal-to-noise of the spectra in Figure 7d compared to Figure 7c indicated that the total concentration of V(IV) species in the solution from Figure 7d was less than that in Figure 7c. An EPR spectrum from a solution of VOSO₄ and sbhaH₄ at pH = 5 was EPR silent (Figure 7e). Therefore, the pH value of the solution in Figure 7d was likely to have been above pH = 2. The higher relative concentration of EPR silent V(V)-sbhaH₄ species at pH values >5 was consistent with the V(V)/(IV)-sbhaH₄ speciation model from the ESI-MS data.

Figure 7.

EPR spectra (150 K) from a methanol glass of: (a) VOSO₄·5H₂O, (b) V(IV):pbH₂ = 1:1 (pH = 2), (c) V(IV):sbhaH₄ = 1:2 (pH = 2), (d) V(IV):sbhaH₄ = 1:2 (2 < pH < 5), and (e) V(IV):sbhaH₄ = 1:2 (pH = 5). Traces in black or gray are experimental or simulated spectra, respectively.

Cyclic Voltammetry

As supported by data from ESI-MS and ⁵¹V NMR spectroscopy, the major species present in a solution of V(IV) and pbH₂ were due to [V^VO(pb)]⁺ or its dimer. The absence of a signal in the EPR spectrum from these solutions suggested that the concentration of V(IV) species was negligible. Analogous studies with the linear dihydroxamic acid sbhaH₄ showed a distribution of monomeric and dimeric V(IV) and V(V) species and mixed-valence V(V)/(IV) species. These results indicated that the thermodynamic stability of a complex formed between V(V) and the macrocyclic dihydroxamic acid pbH₂ would be greater than that with the linear dihydroxamic acid, which suggested that the V(V)/(IV) redox potential of [V^VO(pb)]⁺ would be less than that of the V-sbhaH₄ system. Cyclic voltammetry from a 1:1 solution of V(IV):pbH₂ at pH = 2 (Figure 8) showed a reversible reduction with a half-wave potential E_1/2 = −335 mV (Figure 8, solid line). The half-wave potential of the cyclic voltammogram from a 1:2 solution of V(IV):sbhaH₄ at pH = 2 was E_1/2 = −352 mV (Figure 8, dashed line). This was the opposite of what was predicted based on the solution speciation results (E_1/2 [VO(pb)]^+/0 < V^V/IV-sbhaH₄). The difference of 17 mV in the half-wave potential is rather small. Second, while a 1:1 solution of V(IV):pbH₂ at pH = 2 yielded [V^VO(pb)]⁺, the 1:2 solution of V(IV):sbhaH₄ at pH = 2 contained at a minimum VOSO₄ and a mixture of V(V)-sbhaH₄, V(IV)-sbhaH₄, and V(V)/(IV)-sbhaH₄ species (Table 1), which would prevent the determination of the half-wave potential of a unique species.

Figure 8.

Cyclic voltammograms from solutions of [V(IV)]:[pbH₂] = 1:1 (solid line) or [V(IV)]:[sbhaH₄] = 1:2 (dashed line) in 0.1 M NaCl at pH = 2.

Concluding Remarks

Multiple spectroscopic methods have shown that the monooxoV(V) complex [V^VO(pb)]⁺ was the dominant species formed from solutions of V(IV) and the cyclic dihydroxamic acid pbH₂. As predicted, the cyclic dihydroxamic acid is suitably preorganized for oxoV(V) coordination. The characterization of [V^VO(pb)]⁺ expands the small family of V–siderophore complexes that have been described. Solutions of V(IV) and the linear dihydroxamic acid sbhaH₄ gave mixtures with higher relative concentrations of hydroxamate-based V(IV)-sbhaH₄, V(IV)/V(V)-sbhaH₄, and V(V)-sbhaH₄ species at pH = 2 and higher concentrations of hydroximate-based V(V)-sbhaH₄ species at pH = 7. The complexity of the V-sbhaH₄ solution equilibria arose from phenomena including dimerization (2[V^VO(sbhaH₂)]⁺ ↔ [(V^VO)₂(sbhaH₂)₂]²⁺), deprotonation ([V^VO(sbhaH₂)]⁺ – H⁺ ↔ [V^VO(sbhaH)]⁰), and oxidation ([V^IVO(sbhaH₂)]⁰ −e^– ↔ [V^VO(sbhaH₂)]⁺) and can be described as the sum of two pH-dependent vectors, the first comprising the deprotonation of hydroxamate to hydroximate (low to high pH, respectively) and the second comprising the oxidation of V(IV) to V(V) (low to high pH, respectively).

The restricted conformational freedom of pbH₂ compared to linear sbhaH₄ likely accounts for the entropic-based increase in the thermodynamic stability of [V^VO(pb)]⁺ compared to that of the V-sbhaH₄ species. The preorganization of metal binding functional groups in macrocyclic peptides manifests as greater selectivity and stronger binding compared to their linear analogues^74,75 and is reflected in the greater stability of Fe(III) complexes of cyclic hydroxamic acid-based siderophores, such as alcaligin (1/2log β₂₃₀ = 32.4) and desferrioxamine E (log β₁₁₀ = 32.5), compared to linear siderophores, such as desferrioxamine B (log β₁₁₀ = 30.5).^76,77 The stability of [V^VO(pb)]⁺ suggested to us that the oxoV(V) ion could be used to preorganize the assembly of two endohydroxamic acid amino carboxylic acid units prior to end-to-end peptide-based cyclization to furnish [V^VO(pb)]⁺. We will shortly publish our results of the V(V)-based templated synthesis of [V^VO(pb)]⁺ and new cyclic dihydroxamic acids.

Each of pbH₂ and the related bisucaberin are siderophores native to marine bacteria, including species of the genus Shewanella. The formation of [V^VO(pb)]⁺ at pH values 2–7 is relevant in the context of the competition between pbH₂ and Fe(III) or V(V) in surface oxic marine waters and provides knowledge of the ligands potentially involved in V acquisition and uptake, so-called ‘vanadophores’, in microorganisms and other forms of life.(20)

Supporting Information Available

Positive and negative ion mode ESI-MS data from 1:2 solutions of V(IV):sbhaH₄ at pH = 2, 5, 7, and 9 and from 1:1 solutions of V(IV):sbhaH₄ at pH = 2 (positive ion mode). This material is available free of charge via the Internet at http://pubs.acs.org.