Tuning the interactions of decavanadate with thaumatin, lysozyme, proteinase K and human serum proteins by its coordination to a pentaaquacobalt(II) complex cation

Abstract

The decavanadate anion, H_xV₁₀O₂₈^(6−x)− (V₁₀), is one of the most studied vanadium polyoxometalate species. In recent decades several works have pointed to its biological relevance coming mainly from its ability to bind to proteins (such as actin, myosin or ion pumps). On the other hand, non-functional binding was observed in several protein crystal structures, where V₁₀ was incorporated “accidentally” resulting from the presence of Na₃VO₄ as a phosphatase inhibitor. In this work we broaden the potential biological applications of V₁₀ by presenting the synthesis and characterization of two decavanadate species where the anion acts as a ligand: (2-hepH)(NH₄)[{Cu(H₂O)₂(2-hep)}₂V₁₀O₂₈]·4H₂O (V₁₀Cu) and (2-hepH)₂[{Co(H₂O)₅}₂V₁₀O₂₈]·4H₂O (V₁₀Co) (2-hep = 2-hydroxyethylpyridine). Unlike free decavanadate, the complex anions stay intact in model buffer solutions (0.1 M 2-(N-morpholino)ethanesulfonic acid, 0.5 M NaCl, pH = 5.8 and 8.0). It has been shown that V₁₀Co is stable also in the presence of proteins and for the first time it was possible to study the interaction of decavanadate with proteins without the interference of lower vanadate oligomers. This allowed comparison of interactions of V₁₀ and V₁₀Co with the model proteins thaumatin, lysozyme, proteinase K, human serum albumin and transferrin under conditions close to biological ones (0.1 M 2-(N-morpholino)ethanesulfonic acid, 0.5 M NaCl, pH = 5.8). The linewidths of the signals at half-height in ⁵¹V NMR spectra reflect the strength of interaction of a vanadium species with a protein, and thus it was shown that V₁₀ and V₁₀Co both bind strongly to thaumatin, V₁₀ binds to lysozyme and V₁₀Co binds to proteinase K. V₁₀ interacts with both human serum albumin and transferrin, but surprisingly V₁₀Co exhibits high affinity to transferrin but does not interact with albumin.

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1 Introduction

Polyoxometalates (POMs) are an important group of metal oxide clusters¹ exhibiting diverse archetypal structures of, particularly, vanadates, molybdates and tungstates.^2–4 The tremendous variability of POMs has given rise to their application in distinct areas of materials science,^5,6 catalysis,^7–10 electrochemistry and redox processes,^11,12 photochemistry^13–15 and magnetism.^16–18 In recent years, the roles of POMs in biological systems have been intensively investigated. Such studies may be divided into two groups: functional binding and interaction of POMs in biological systems widening the borders of medicinal chemistry;^19–24 and non-functional interaction with biomolecules, such as proteins, enhancing the current possibilities in macromolecular crystallography by promoting the crystallization or being useful in obtaining the initial phases while solving the structures of proteins.^25–28

Decavanadate, H_xV₁₀O₂₈^(6−x)− (V₁₀), is the predominant species formed in vanadate solutions at vanadium(V) concentrations above 1 mM in the pH range of ≈2–6.^29,30 The structure of V₁₀ consists of ten face-sharing octahedra (Scheme 1). The symmetrically non-equivalent vanadium atoms V_A, V_B and V_C give rise to three different signals in ⁵¹V NMR spectra depending on the conditions: while the low-field signal of V_A atoms stays in a narrow region around −425 ± 3 ppm, the other two peaks representing V_B and V_C atoms are more sensitive to changes in acidity and exhibit signals at approximately −505 ± 10 ppm and −525 ± 10 ppm, respectively. The oxygen atoms O_B and O_C are potential sites for protonation, and the atoms O_C, O_F, O_G and O_D are the most potential sites for ligation to transition metal ions.^31,32 As such, changes in ⁵¹V NMR parameters reflect the versatility of structural modifications of V₁₀. While protonation and coordination of V₁₀ manifest mostly in peaks’ movement, the interaction and binding to proteins result in significant peak broadening defined by linewidths at half-height of the signals, W_1/2. This is caused by the ligand bulkiness and decreased symmetry of the V₁₀ species upon interaction.³²

Scheme 1 The structure of [V₁₀O₂₈]⁶⁻ showing different types of vanadium and oxygen atoms.

Vanadium is naturally omnipresent in biological matrices^33,34 in a few enzymes such as vanadium dependent haloperoxidases and nitrogenases³⁵ or acts as a crucial component in the energetic metabolism of Ascidians.³⁶ Some artificial vanadium compounds, on the other hand, exhibit insulin mimetic properties, antitumor activity, antibacterial activity or anti-HIV activity.^35–39

Specifically decavanadate itself has also been studied with respect to many biological aspects,^40–43 and it was shown that V₁₀ binds to several proteins such as actin,⁴⁴ myosin,⁴⁵ ion pump Ca²⁺–ATPase,⁴⁶ bovine serum albumin and gelatine,⁴⁷ and microtubule-associated proteins.⁴⁸ Protein crystallography revealed the presence of V₁₀ in the crystal structures of acid phosphatase A (F. tularensis),⁴⁹ human activated receptor tyrosine kinase,⁵⁰ NTPDase1 (L. pneumophila),⁵¹ NTPDase1 (R. norvegicus),⁵² and human TRPM4 channel.⁵³ In all cases, V₁₀ was formed from the initially employed Na₃VO₄ (used as a phosphatase inhibitor) and its role may be explained as stabilization and rigidification of the protein structure.²⁵

In this work we compare the interaction of both free and ligated decavanadate with commercially available model proteins thaumatin, lysozyme, proteinase K, as well as human serum albumin and transferrin. We utilize ⁵¹V NMR spectroscopy as a powerful tool to investigate the stability of two decavanadates coordinated to metal centres Cu(II) and Co(II) under conditions usually used for protein crystallization.

2 Experimental

2.1 Materials and methods

All chemicals were of analytical grade and used as received without further purification. All proteins were supplied as lyophilized powders (supplier, reference code): thaumatin from Thaumatococcus danielii (Sigma, T7638; a mixture of thaumatin I and thaumatin II with traces of other sweet proteins), proteinase K from Tritirachium album (Sigma, P6556), lysozyme from chicken eggwhite (Carl Roth GmbH & Co. KG, 8259.3), albumin from human serum (Sigma, A1653) and apo-transferrin from human serum (Sigma, T1147). The determination of C/H/N was carried out by using an ‘EA 1108 CHNS-O’ elemental analyzer by Carlo Erba Instruments at the Mikroanalytisches Laboratorium, University of Vienna. Metal elements’ analyses were performed in aqueous solutions containing 2% HNO₃ using inductively coupled plasma mass spectrometry (PerkinElmer Elan 6000 ICP MS) for Mo and V, and atomic absorption spectroscopy (PerkinElmer 1100 Flame AAS) for Cu and Co. Standards were prepared from single-element standard solutions of concentration 1000 mg L⁻¹ (Merck, Ultra Scientific and Analytika Prague). FT-IR spectroscopy was performed on a Bruker Vertex 70 IR Spectrometer equipped with a single reflection diamond-ATR (attenuated total reflectance) unit in the range of 4000–100 cm⁻¹.

2.2 ⁵¹V NMR spectroscopy

⁵¹V nuclear magnetic resonance spectroscopy measurements of aqueous solutions were performed on a Bruker Avance II 500 MHz instrument operating at 131.60 MHz for ⁵¹V nucleus (2000 scans, accumulation time 0.05 s, relaxation delay 0.01 s). Chemical shift values are given with reference to VOCl₃ (δ = 0 ppm) as a standard. The solutions were in general prepared by dissolving 0.01 mmol of the given decavanadate in 700 μL of the buffer solution (0.11 M MES, 0.55 M NaCl, pH = 5.8; MES = 2-(N-morpholino)ethanesulfonic acid) and addition of 100 μL of D₂O used for locking. Next, the solution was either made up to 1000s μL with the buffer (Section 3.3.1) or a solution of a protein in the same buffer was slowly added. The protein solutions were prepared by dissolution of the given amount of solid protein in 200 μL of the buffer solution. The solution of proteinase K was prepared as 3.5 M solution in 0.1 M TRIS (tris(hydroxymethyl)aminomethane) buffer (pH = 7.0), and subsequently 1 μL of this solution was diluted in 200 μL of the MES buffer solution. The spectra were collected at RT one hour after preparation of the solutions.

2.3 X-ray diffraction on single crystals

The X-ray diffraction data were collected on Bruker X8 APEXII (V₁₀Cu, Mo Kα) and Bruker D8 Venture (V₁₀Co, Cu Kα) instruments equipped with multilayer monochromators, Incoatec Microfocus sealed tubes, and Kryoflex and Oxford cooling devices. The structures were solved by direct methods and refined by full-matrix least-squares. Nonhydrogen atoms were refined with anisotropic displacement parameters. Hydrogen atoms were inserted at calculated positions and refined with riding coordinates except for solvent molecules and H atoms in NH₄⁺ in V₁₀Cu. This was not done because of disorder and partial occupancies of O and N atoms in question. The following software was used for the structure solving procedure: Bruker SAINT software package⁵⁴ (frame integration, cell refinement), SADABS⁵⁵ (absorption correction), SHELXS-2013⁵⁶ (structure solution), SHELXL-2013⁵⁷ (structure refinement), OLEX2⁵⁸ (user interface and publication material) and Diamond⁵⁹ (graphics). Experimental data and CCDC-codes can be found in Table 2.

2.4 Syntheses

(NH₄)₆[V₁₀O₂₈]·6H₂O (V₁₀): the compound was prepared as per the previously described procedure⁶⁰ and the composition was confirmed by FT-IR spectroscopy and elemental analysis. Analytical data for V₁₀O₃₄N₆H₃₆ in % (calc.): V 46.3 (43.4). IR: [V₁₀O₂₈]⁶⁻: 958vs, 816m, 727s, 588m, 516s, 452s, 392vs; NH₄⁺: 1405s, 3151sh, 3009sh, 2008sh.

(2-hepH)(NH₄)[{Cu(H₂O)₂(2-hep)}₂V₁₀O₂₈]·4H₂O (V₁₀Cu): NH₄VO₃ (0.468 g, 4 mmol) was dissolved in 30 mL of distilled water by heating. After cooling to RT, the pH of the solution was adjusted to 4.6 with 5 M HCl. Then, 2-hep (2-hep = 2-hydroxyethylpyridine) was added (0.45 mL, 4 mmol) and the pH was adjusted to 4.6 with 5 M HCl again. Finally, Cu(NO₃)₂·6H₂O was added (0.484 g, 2 mmol). After dissolution, the pH dropped to 4.0. The clear orange-green solution was left to crystallize at 18 °C. After three days, green crystals of V₁₀Cu were isolated. Yield 250 mg (39% based on V). Analytical data for V₁₀Cu₂O₃₉C₂₁H₄₈N₄ in % (calc.): V 30.7 (31.5), Cu 7.76 (7.86), C 15.36 (15.60), H 3.02 (3.00), N 3.41 (3.46). IR: [V₁₀O₂₈]⁶⁻: 983s, 959vs, 937s, 850s, 793s, 721s, 512s, 429s, 386s; 2-hep: 1609m, 1488m, 1313m, 1248w, 1109w, 1073w, 1051w, 1025w; NH₄⁺: 1439s, 3210sh.

(2-hepH)₂[{Co(H₂O)₅}₂V₁₀O₂₈]·4H₂O (V₁₀Co): a procedure similar to that in the previous synthesis was employed, except that upon addition of Co(NO₃)₂·6H₂O (0.596 g, 2 mmol) it was necessary to adjust the pH of the solution to 4.0 with 5 M HCl. The clear red solution was left to crystallize at RT. After three days, red crystals of V₁₀Co were isolated. Yield 400 mg (63% based on V). Analytical data for V₁₀Co₂O₄₄C₁₄H₄₈N₂ in % (calc.): V 31.7 (32.3), Co 7.17 (7.48), C 10.55 (10.67), H 3.11 (3.07), N 1.82 (1.78). IR: [V₁₀O₂₈]⁶⁻: 989s, 967vs, 921s, 811s, 724s, 596s, 554s, 505s, 446m, 398s; 2-hep: 1608m, 1772m, 1248w, 1024w. Crystals suitable for X-ray diffraction were grown by prolonged crystallization at 18 °C in a flask closed with perforated Parafilm.

3 Results and discussion

3.1 Syntheses

To the best of our knowledge, the work of Schwendt et al.⁶¹ presenting the synthesis and characterization of (2-hepH)₂[{Cu(H₂O)₂(2-hep)}₂V₁₀O₂₈]·6H₂O is the only one reporting a coordination compound of decavanadate that stays intact in aqueous solution. Inspired by this work, the synthetic protocol was modified in order to prepare new stable complexes of decavanadate. A reversed addition of the individual reaction components and stepwise acidification of the solution prevented formation of heavy precipitates that were described in the original procedure⁶¹ and such an approach resulted in successful isolation of V₁₀Cu containing the [{Cu(H₂O)₂(2-hep)}₂V₁₀O₂₈]²⁻ complex anion and a new compound V₁₀Co composed of the purely inorganic complex anion [{Co(H₂O)₅}₂V₁₀O₂₈]²⁻. In this case, 2-hep did not coordinate to the cobalt(II) center and acted only as a cation. Syntheses employing Ni^II, Zn^II, Mn^II, La^III and Ce^III nitrates were not successful. Despite the systematic study it was possible to isolate only two complexes of decavanadate, and even they differed in the coordination mode of decavanadate and in the structure of the coordinated transition metal. Table 1 summarizes 22 compounds where decavanadate coordinates to transition metal cations. There are ten Cu^II, four Zn^II, four Mn^II and two Ag^I complexes. Compound V₁₀Co is the first complex of decavanadate involving Co^II. There are about 100 salts of decavanadate and some of them contain a transition metal complex in the cationic part, including the [Co(H₂O)₆]²⁺ complex cation.^62a–c Therefore, in general it is still not clear what conditions are required for decavanadate to act as a ligand.

Table 1 Coordination compounds of H_xV₁₀O₂₈^(6−x)−

Compound	Type of coordinated O atom^a	Ref.
a As in Scheme 1. Abbreviations: 2,2′-bipy – 2,2′-bipyridine, btx – 1,4-bis(triazol-1-ylmethyl)benzene, ppz – piperazine, pz – pyrazole, im – imidazole, mim – 1-methylimidazole, trz - 1,2,4-triazole, en – ethylenediamine, amp – 2-(aminomethyl)pyridine, 2-hep = 2-hydroxyethylpyridine, L – 5,5,7,12,12,14-hexamethyl-1,4,8,11-tetraazacyclotetradecane.
[Cu(2,2′-bipy)2]2[H2V10O28]·(2,2′-bipy)·H2O	OC	63
{[(CuL)0.5(H2L)1.5][H2V10O28]·6H2O}n	OF	64
(NH4)2[Cu2(NH3CH2CH2COO)4(V10O28)]·10H2O	OG	65
(Hpz)2[{Cu(pz)4}2V10O28]·2H2O	OG, OF	66
{Cu(pz)}4[{Cu(pz)3}2V10O28]	OG, OC	66
(2-hepH)2[{Cu(H2O)2(O,N-2-hep)}2(V10O28)]·6H2O	OC	61
(2-hepH)(NH4)[{Cu(H2O)2(2-hep)}2V10O28]·4H2O	OC	This work
[Cu(2-amp)2(H2O)]2H2V10O28·4H2O	OC	61
(H3O)2[{Cu(en)2(H2O)}2V10O28]·3H2O	OC, OD	67
[{Cu(en)2}3(V10O28)]·6H2O	OC, OD	67
[Zn2(H2O)14(V10O28)]·H2ppz	OD	68
[Zn(H2O)6][Zn2V10O28(H2O)10]·6H2O	OF	69
[Zn(en)2]3[V10O28]·5H2O	OC	70
[Zn(im)2(DMF)2]2[H2V10O28]·im·DMF	OC	71
{[Zn3(trz)3(H2O)4(DMF)]2[V10O28]·4H2O}n	OC	71
(2-hepH)2[{Mn(H2O)5}2V10O28]·4H2O	OF	72
{[Mn(mim)4]2[H2V10O28]}	OC, OF	73
(NMe4)2[{Mn(H2O)5}2V10O28]·5H2O	OF	76
[NH3C(CH2OH)3]2[{Mn(H2O)5}2V10O28]·2H2O	OF	76
[Ag(btx)}4H2V10O28]·2H2O	OC	74
[{Na3(H2O)8(μ2-H2O)6Ag2}HV10O28]·6H2O	OC, OG	75
(2-hepH)2[{Co(H2O)5}2V10O28]·4H2O	OF	This work

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3.2 Crystal structures

The structure solution and refinement details for compounds V₁₀Cu and V₁₀Co are summarized in Table 2. The crystal structure refinement of V₁₀Cu revealed the presence of the complex anion [{Cu(H₂O)₂(2-hep)}₂V₁₀O₂₈]²⁻ that has been already reported (Fig. 1).⁶¹ The decavanadate anion [V₁₀O₂₈]⁶⁻ is a bridging ligand between two {Cu(H₂O)₂(2-hep)}²⁺ fragments and binds to the Cu1 atom through oxygen atom O12 (O_C in Scheme 1) at 1.9612(8) Å. The organic ligand is chelating the Cu1 atom by the N atom of the pyridine ring [N1–Cu1 1.9802(11) Å] and the O atom of the hydroxyl group [O13–Cu1 1.9857(9) Å]. The tetragonal pseudoplane around the atom Cu1 is completed by a water molecule [O15–Cu1 1.9824(10) Å]; the water molecule in the axial position completes the pyramidal geometry [O14–Cu1 2.2860(10) Å].

Table 2 Crystallographic and refinement data for compounds V₁₀Cu and V₁₀Co

	V10Cu	V10Co
CCDC No.	1909251	1909250
Empirical formula	V10Cu2O39C21H48N4	V10Co2O44C14H48N2
M r	1605.02	1575.80
Crystal system, space group	Monoclinic, C2/c	Triclinic, P1̄
Temperature (K)	100	100
a (Å)	27.0345(15)	9.4219(5)
b (Å)	9.9170(5)	11.2451(6)
c (Å)	20.5851(12)	11.9820(7)
α (°)		78.5909(17)
β (°)	119.341(5)	75.1180(17)
γ (°)		66.3817(17)
V (Å^3)	4810.9(5)	1117.66 (11)
Z	4	1
Radiation type	Mo Kα	Cu Kα
μ (mm^−1)	2.82	23.51
Absorption correction	Multi-scan	Multi-scan
T min, Tmax	0.505, 0.747	0.534, 0.754
F(000)	3160	782
Theta range	2.2–35.7°	5.7–72.6°
Crystal size in mm	0.45 × 0.25 × 0.22	0.11 × 0.11 × 0.04
Index ranges	h = −44 → 44	h = −11 → 11
	k = −16 → 16	k = −13 → 13
	l = −33 → 33	l = −12 → 14
No. of measured, independent and observed [I > 2s(I)] reflections	343 398, 11 216, 9986	16 530, 4079, 3882
R int	0.062	0.127
No. of reflections, parameters, restraints	11216, 421, 9	4079, 337, 20
Goodness-of-fit on F^2	1.10	1.16
R[F^2 > 2s(F^2)]	0.024	0.098
wR(F^2)	0.071	0.222
Largest diff. peak and hole	0.79 e Å^−3, −0.65 e Å^−3	1.79 e Å^−3, −1 e Å^−3

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Fig. 1 Molecular structures of the anions [{Cu(H₂O)₂(2-hep)}₂V₁₀O₂₈]²⁻ (V₁₀Cu) and [{Co(H₂O)₅}₂V₁₀O₂₈]²⁻ (V₁₀Co) as revealed by X-ray crystallography showing atom labelling. Colour code: orange octahedra {VO₆}, green Cu, light blue Co, blue N, red O, black C, gray H. Ellipsoids are displayed at a 30% probability level.

The closest contact in the trans position towards this water molecule is oxygen atom O8 of [V₁₀O₂₈]⁶⁻ (O_G in Scheme 1). The short distance O8–Cu1 2.889 Å indicates some weak attraction. The crystal structure of V₁₀Cu differs from the previously described one⁶¹ in the presence of the NH₄⁺ cation that was confirmed by elemental analysis and IR spectroscopy.

The asymmetric unit of V₁₀Co contains one half of the centrosymmetric anion [{Co(H₂O)₅}₂V₁₀O₂₈]²⁻ (Fig. 1), one molecule of (2-hepH)⁺ balancing its charge and two water molecules of crystallization. The decavanadate anion [V₁₀O₂₈]⁶⁻ is acting as a bridging ligand for two {Co(H₂O)₅}²⁺ fragments. The slightly irregular octahedral coordination sphere of the Co1 atom is completed by one oxygen atom O1 coming from the terminal V=O group of the decavanadate (atom O_F in Scheme 1). Such a coordination fashion is not unknown and was already reported for manganese(II)^72,76 and zinc(II) derivatives.^68,69 The bond length O1–Co1 2.089(3) Å indicates a relatively strong coordination bond of decavanadate to the metal centre. The bond lengths of the Co1 atom and oxygen atoms of the coordinated water molecules are in the range of 2.105 – 2.115 Å for the equatorial ligands and 2.057(4) Å for the Co1–O15 bond of the water molecule in the trans position towards the decavanadate ligand.

The supramolecular structure of V₁₀Co is stabilized by a rich network constructed from hydrogen bonds in which all the individual components are involved. Due to the presence of {Co(H₂O)₅}²⁺ fragments the most prominent hydrogen bonds are formed between the adjacent [{Co(H₂O)₅}₂V₁₀O₂₈]²⁻ anions. Three oxygen atoms of the decavanadate O5, O6 and O11 are connected with hydrogen bonds to three water molecules of {Co(H₂O)₅}²⁺ at contact distances O5⋯O17 2.636 Å, O6⋯O17 2.636 Å and O11⋯O15 2.744 Å.

3.3 Solution studies

3.3.1 Stability in a buffer solution. For the solutions studies 1 mM decavanadate solutions of V₁₀, V₁₀Cu and V₁₀Co were firstly prepared in a medium containing 0.1 M MES buffer and 0.5 M NaCl at pH = 5.8. These conditions were used to simulate the environment of protein crystallization where usually higher buffer concentrations and high ionic strengths are necessary. The acidic regime is preferred to ensure that the protein is positively charged and may therefore more profoundly interact with negatively charged POMs. The ⁵¹V NMR spectra of the given solutions are shown in Fig. 2. In the spectrum of V₁₀ the expected distribution of vanadium into several species can be observed. Due to hydrolysis, the equilibrated species include not only the originally employed decavanadate (−422.5, −498.2 and −513.8 ppm), but also monovanadate H₂VO₄⁻ (V₁, −558.5 ppm), divanadate H₂V₂O₇²⁻ (V₂, −571.2 ppm), tetravanadate V₄O₁₂⁴⁻ (V₄, −575.0 ppm) and pentavanadate V₅O₁₅⁵⁻ (V₅, −583.0 ppm). However, due to the high ionic strength decavanadate is still the dominant species and consumes about 80% of V^V present in the solution.

Fig. 2 ⁵¹V NMR spectra of 1 mM aqueous solutions of V₁₀, V₁₀Cu and V₁₀Co in 0.1 M MES buffer, 0.5 M NaCl at pH = 5.8.

The spectra of V₁₀Cu and V₁₀Co exhibit only peaks that can be assigned to vanadium atoms arising from decavanadate: −422.3, −496.9 and −511.9 ppm for V₁₀Cu and −422.3 and −495.9 ppm (broad peak) for V₁₀Co. This is the first indication that the complex anions stay intact and do not dissociate off the Cu(II) and Co(II) centres. As a matter of fact, the presence of free decavanadate would evoke vanadate self-condensation reactions and the overall picture of the present species should be similar to that of V₁₀. Next, similar to Schwendt et al.,⁶¹ we observed significant movement of the high-field signal of the V_C atom in V₁₀Cu to −511.9 ppm compared to −513.8 ppm in V₁₀, and the signal of the V_B atom is shifted by 1.3 ppm. For V₁₀Co, the shift of the peak is much more obvious and instead of two independent signals for V_B and V_C atoms we observe only a broad peak with the maximum at −495.9 ppm having a high-field shoulder. Based on peak integration, the integral intensities of the two present signals are in the ratio 2 : 8. In addition, significant peak broadening of the individual signals was observed (Table 3). This can be explained by at least two factors. Firstly, vanadates usually provide narrower lines in comparison to common vanadium(V) complexes because of higher symmetry (i.e. D_2h for ideal [V₁₀O₂₈]⁶⁻). Thus, peak broadening originates in the decrease of symmetry of the coordinated decavanadates. It is also important to note that for V₁₀Co the low field signal is broadened by only 10% in comparison to free decavanadate, while the second signal is broader by more than 220% compared to the sum of individual signals of V_B and V_C in V₁₀. This difference is naturally caused by the fact that the V_C atoms are the closest ones to the Co(II) atom and the inner V_A atoms are less affected by the coordination. The compound V₁₀Cu formed a cloudy precipitate in the buffer solution which might have also influenced the peaks’ width, but despite this, similar peak broadening as for V₁₀Co was observed – 14% and 200%, respectively. This leads to the conclusion that the peak coalescence is a consequence of its coordination to the Cu(II) center and not the presence of a precipitate. However, we excluded V₁₀Cu from further examination to prevent misinterpretation of protein interaction experiments.

Table 3 The ⁵¹V NMR parameters of peaks in the spectra of solutions of V₁₀, V₁₀Cu and V₁₀Co in 0.1 M MES, 0.5 M NaCl at pH = 5.8. The chemical shifts are given in ppm (upper) and the linewidths at half-height in Hz (lower). The linewidths are not given for species with concentrations <5%

	HxV10O28^(6−x)−			H2VO4^−	H2V2O7^2−	V4O12^4−	V5O15^5−
	VA	VB	VC
a The linewidth was calculated from the half-height for the low-field and high-field components of the merged broad signal.
V10	−422.5	−498.2	−513.8	−558.5	−571.2	−575.0	−583.0
	636.22	483.86	309.16			176.77
V10Cu	−422.3	−496.9	−511.9	—	—	—	—
	727.51	775.17	806.75
V10Co	−422.3	−495.9		—	—	—	—
	702.38	1794.05^a

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On the other hand, peak broadening may also originate in the presence of paramagnetic centres Cu(II) (d⁹) and Co(II) (d⁷). The experience has shown, however, that if an extraneous paramagnetic species interferes in a ⁵¹V NMR experiment, this manifests itself also in a lower signal-to-noise ratio and uneven lines (this was not the case).

At pH = 8.0 (0.1 M MES, 0.5 NaCl) profound decomposition of V₁₀ into lower vanadates was observed, V₁₀Cu dissociated off the Cu(II) complex cation, while V₁₀Co was still the only species present (see Fig. S1 and Table S1, ESI†).

3.3.2 Interaction of decavanadates with thaumatin, lysozyme and proteinase K. The interaction of V₁₀ and V₁₀Co (1 mM) with model proteins thaumatin (10 μM), lysozyme (10 μM) and proteinase K (3.5 μM) was inspected by ⁵¹V NMR spectroscopy in 0.1 MES buffer, 0.5 M NaCl at pH = 5.8 one hour after careful addition of the protein solution to the solution of POMs (Fig. 3 and 4). Table 4 summarizes the data on chemical shifts and linewidths at half-height for the observed peaks. During the experiments, a peak movement larger than ±1 ppm was not observed indicating that no changes in protonation occurred. Table 4 includes data only for decavanadates and [V₄O₁₂]⁴⁻ as peaks corresponding to other vanadates had integral intensities <5%. Importantly, no visible reduction of vanadium(V) was recognized after 1 week of standing solutions.

Fig. 3 ⁵¹V NMR spectra showing interactions of 1 mM aqueous solutions of V₁₀ with model proteins under given concentrations in 0.1 M MES buffer, 0.5 M NaCl at pH = 5.8. The black line represents the spectrum of the referent V₁₀Co and the coloured lines show the spectra of solutions of V₁₀Co in the presence of different proteins at given concentrations.

Fig. 4 ⁵¹V NMR spectra showing interactions of 1 mM aqueous solutions of V₁₀Co with model proteins under given concentrations in 0.1 M MES buffer, 0.5 M NaCl at pH = 5.8. The black line represents the spectrum of the referent V₁₀Co and the coloured lines show the spectra of solutions of V₁₀Co in the presence of different proteins at given concentrations.

Table 4 The ⁵¹V NMR parameters of peaks in the spectra of solutions of V₁₀ and V₁₀Co in the presence of model proteins (c_V = 10 mM, 0.1 M MES, 0.5 M NaCl, pH = 5.8). The chemical shifts are given in ppm (upper) and the linewidths at half-height in Hz (lower)

Protein	Decavanadate	HxV10O28^(6−x)−				V4O12^4−
		VA	VB	VC
a The linewidth was calculated from the half-height for the low-field and high-field components of the merged broad signal.
None	V10	−422.5	−498.2	−513.8		−575.0
		636.22	483.86	309.16		176.77
	V10Co	−422.3	−495.9			—
		702.38	1794.05^a
Thaumatin 10 μM	V10	−421.5	−498.5	−514.1		−575.2
		1144.08	1198.98	746.33		274.76
	V10Co	−421.8	−495.9			—
		1265.82	2254.31^a
Lysozyme 10 μM	V10	−422.5	−498.3	−4513.9		−575.0
		580.67	530.95	342.1		199.5
	V10Co	−422.4	−495.7			—
		732.6	1810.91^a
Proteinase K 3.5 μM	V10	−422.6	−498.4	−513.9		−575.1
		653.95	510.83	326.98		194.65
	V10Co	−422.3	−495.6			—
		977.23	2041.13^a
Albumin 1 μM	V10	−422.3	−498.2	−513.8		−575.0
		943.72	851.65	532.05		192.97
	V10Co	−422.5	−495.6			—
		709.7	1629.61^a
Transferrin 1 μM	V10	−422.6	−499.5		−513.8	−575.5
		967.87	—		983.55	230.59
	V10Co	−421.6	−495.6			—
		1268.63	2404.42^a

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Both V₁₀ and V₁₀Co bind strongly to thaumatin resulting in significant peak broadening. In the case of lysozyme, very weak binding to V₁₀ may be deduced from slightly broadened higher-field peaks, but no obvious interaction was observed for V₁₀Co. Finally, proteinase K seemed not to interact with free decavanadate, but binds strongly to V₁₀Co. In this case we used about 3 times lower protein concentration; therefore, after extrapolation of the data it can be assumed that the binding of V₁₀Co to proteinase K is comparatively stronger than to thaumatin.

3.3.3 Interaction of decavanadates with human serum albumin and transferrin. Using the same experimental conditions, the interaction of V₁₀ and V₁₀Co with the main proteins present in human plasma, namely, human serum albumin (1 μM) and transferrin (1 μM) (Fig. 3, 4 and Table 4), was also examined. Interestingly, free decavanadate V₁₀ binds strongly to albumin, but V₁₀Co does not interact. It seems that the Co(II) centers coordinated to V=O groups of decavanadate occupy the binding sites for interaction of decavanadate with this protein and block the interaction. In fact, this was the only case where the interaction was observed only for the free decavanadate but not for the coordinated one. The interaction of V₄O₁₂⁴⁻ with human serum albumin is much weaker than that of decavanadate based on only about 10% increase in the peak width.

The interaction of V₁₀ with transferrin is even stronger, and a significant binding to V₄O₁₂⁴⁻ was also observed. The shift of the peak corresponding to the V_B atom of the decavanadate by 1 ppm made it impossible to determine its half-height width. As expected, the presence of Co(II) causes extremely strong binding of V₁₀Co to transferrin, resulting in peak broadening comparable to that observed in the case of thaumatin, but at 100× lower protein concentration.

The stability of V₁₀Co, V₁₀Cu and potentially other coordinated decavanadates in the examined medium at 10 mM total vanadium concentration, in line with the potential of functionalized decavanadates to interact with various proteins, open new possibilities for the investigation of decavanadate's effects in biological systems – for the first time without the side effects of the always present lower oligovanadates (decavanadate may be the only species present in multicomponent solvents).⁷⁷ On the other hand, at physiological concentrations (c_V = 1 μM and less), vanadate exists only as a monomeric species (VO₂⁺, H₂VO₄⁻, HVO₄⁻ depending on the pH).³² We therefore checked the stability of V₁₀Co at 1 μM concentration (10 μM total vanadium) by ⁵¹V NMR (Fig. S2, ESI†). The chemical shifts corresponding to the decavanadate species (−421.2, −497.9 and −513.1) represent an undisturbed anion indicating that the Co(II) centers are no longer involved in coordination.

4 Conclusions

In this work, we showed that the complex anions [{Cu(H₂O)₂(2-hep)}₂V₁₀O₂₈]²⁻ and [{Co(H₂O)₅}₂V₁₀O₂₈]²⁻ involving coordinated decavanadate are stable in aqueous solution and do not decompose in the buffer solution compatible with proteins (0.1 M MES, 0.5 NaCl) at pH = 5.8 making them promising candidates for biological studies. For the first time, an interaction between modified decavanadate and biomolecules without the participation of lower oligovanadates was performed. The pilot interaction studies with several proteins used in model protein crystallization research showed that V₁₀ and V₁₀Co bind to thaumatin, V₁₀ binds also to lysozyme and V₁₀Co binds to proteinase K. As expected, V₁₀ interacts with human serum albumin and transferrin, but surprisingly V₁₀Co exhibits high affinity to transferrin but does not interact with albumin. The isolation and structural characterization of the crystalline products is the ultimate goal necessary for more precise understanding of the interaction between POMs and proteins. It is expected that, in addition to electrostatic interaction of V₁₀ with proteins, the presence of heterometals may induce a complementary interaction – even a covalent bond – when the coordinated transition metal contains labile ligands such as water molecules (as in V₁₀Co and V₁₀Cu) or a vacant accessible coordination position (as in V₁₀Cu). Furthermore, both V₁₀Cu and V₁₀Co contain biogenic transition metals that are known to interact with biomolecules to a great extent. In conclusion, the high potential of ligated decavanadate in medicinal chemistry and protein crystallography necessitates the challenging development of synthetic methods leading reliably to stable complexes of decavanadate (i.e. decavanadato complexes).

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This research was funded by the Austrian Science Fund (FWF): M2200 (LK) and P27534 (AR) and the University of Vienna. The authors are grateful to Dr Marek Bujdoš (Comenius University in Bratislava, Faculty of Natural Sciences, Institute of Laboratory Research on Geomaterials, Mlynská dolina, Ilkovičova 6, Bratislava, 84215, Slovakia) for elemental analyses and Ao. Univ.-Prof. Dr Markus Galanski (Universität Wien, Fakultät für Chemie, Institut für Anorganische Chemie, Währinger Straße 42, 1090 Wien, Austria) for great support with ⁵¹V NMR measurements.

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Footnotes

† Electronic supplementary information (ESI) available. CCDC 1909250 and 1909251. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c9nj02495f

‡ Comenius University in Bratislava, Faculty of Natural Sciences, Department of Inorganic Chemistry, Ilkovičova 6, 842 15 Bratislava, Slovakia.

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