pubs.acs.org/IC Article ESI-MS Study of the Interaction of Potential Oxidovanadium(IV) Drugs and Amavadin with Model Proteins Valeria Ugone, Daniele Sanna, Giuseppe Sciortino, Debbie C. Crans, and Eugenio Garribba* Cite This: Inorg. Chem. 2020, 59, 9739−9755 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: In this study, the binding to lysozyme (Lyz) of four important VIV compounds with antidiabetic and/or anticancer activity, [VIVO(pic)2(H2O)], [VIVO(ma)2], [VIVO(dhp)2], and [VIVO(acac)2], where pic−, ma−, dhp−, and acac− are picolinate, maltolate, 1,2-dimethyl-3-hydroxy-4(1H)-pyridinonate, and acety- lacetonate anions, and of the vanadium-containing natural product amavadin ([VIV(hidpa)2]2−, with hidpa3− N-hydroxyimino-2,2′- diisopropionate) was investigated by ElectroSpray Ionization-Mass Spectrometry (ESI-MS). Moreover, the interaction of [VIVO- (pic)2(H2O)], chosen as a representative VIVO2+ complex, was examined with two additional proteins, myoglobin (Mb) and ubiquitin (Ub), to compare the data. The examined vanadium concentration was in the range 15−150 μM, i.e., very close to that found under physiological conditions. With pic−, dhp−, and hidpa3−, the formation of adducts n[VIVOL2]−Lyz or n[VIVL2]−Lyz is favored, while with ma− and acac− the species n[VIVOL]−Lyz are detected, with n dependent on the experimental VIV/protein ratio. The behavior of the systems with [VIVO(pic)2(H2O)] and Mb or Ub is very similar to that of Lyz. The results suggested that under physiological conditions, the moiety cis-VIVOL2 (L = pic−, dhp−) is bound by only one accessible side-chain protein residue that can be Asp, Glu, or His, while VIVOL+ (L = ma−, acac−) can interact with the two equatorial and axial sites. If the VIV complex is thermodynamically stable and does not have available coordination positions, such as amavadin, the protein cannot interact with it through the formation of coordination bonds and, in such cases, noncovalent interactions are predicted. The formation of the adducts is dependent on the thermodynamic stability and geometry in aqueous solution of the VIVO2+ complex and affects the transport, uptake, and mechanism of action of potential V drugs. ■ INTRODUCTION Vanadium compounds (VCs) show a wide variety of such as voltammetry and polarography, HPLC-ICP-MS, size- exclusion chromatography, gel-electrophoresis, and MALDI- pharmacological actions, among which are antiviral, antipar- TOF were used in combination with the other techniques.3o,5 asitic, and antituberculosis effects, even though the most All these tools present some limitations: for example, NMR studied and promising application in medicine could be the can be used mainly with VV, and EPRthe spectroscopy most treatment of diabetes and some types of cancer.1 The study of frequently used for paramagnetic vanadium(IV) com- the biospeciation of potential vanadium drugs is of pounds6provides information on the type of residues fundamental importance to the prediction of (i) the form in involved in the metal binding but less on the stoichiometry which they are transported to the target organs, (ii) which is of the formed adducts. Moreover, the relatively high vanadium the active species in the organism, and (iii) the mechanism of concentration necessary to ensure a good signal-to-noise ratio action and binding to the biological receptors. In this context, in NMR and EPR measurements exceeds by several orders of the interaction of antidiabetic and anticarcinogenic VCs with magnitude that found in healthy humans (0.2−15 nM1e,7) and the main components of the blood, including proteins, has in the patients treated with V drugs (1−10 μM for inorganic been widely investigated.2,3 In particular, proteins play a salts1b,3a,8 and 40−60 μM when complexes such as bis- central role in the biospeciation and biotransformation of a VC in the organism, for both their high affinity toward V and high concentration in biological fluids. Received: April 1, 2020 In addition to X-ray diffraction, alternative techniques to Published: June 25, 2020 obtain information on the metal−protein adducts are desirable; among them, NMR, EPR, ESEEM, ENDOR, UV− vis, and CD spectroscopy have been applied to systems containing VIII, VIV, or VV.4 More recently, other methods, © 2020 American Chemical Society https://dx.doi.org/10.1021/acs.inorgchem.0c00969 9739 Inorg. Chem. 2020, 59, 9739−9755 Inorganic Chemistry pubs.acs.org/IC Article Scheme 1. Structure in Aqueous Solution of the VCs Studied in This Work: cis-[VIVO(pic)2(H2O)], [VIVO(acac)2], [VIVO(dhp)2] ⇄ cis-[VIVO(dhp)2(H2O)], cis-[VIVO(ma)2(H2O)], and [VIV(hidpa)2]2− (amavadin)a a The charges of the ligands and VO ion (2+) are omitted for clarity. (maltolato)oxidovanadium(IV) (BMOV) or bis(ethyl- structural features which make them ideal compounds for MS maltolato)oxidovanadium(IV) (BEOV) are administered to experiments: a small or moderate size (with molecular masses rats3m,9). Therefore, alternative techniques that are more ranging from 5500 to 33000 Da) and high stability in solution sensitive at the physiological concentrations are needed. under physiological-like conditions, commercial availability Over the past decade, the potentiality of mass spectrometry with high purity, water-solubility, and easy ionizability. Further (MS) to study the behavior of metallodrugs in biological ESI-MS studies have been carried out with potential molecular samples and characterize at the molecular level their targets, such as metallothioneins, glutathione-S-transferases, interaction with biomolecules and potential targets, such as cytochrome c, and calmodulin.11 Model studies with the above- proteins, has been discussed.10,11 Among the instrumental mentioned proteins can be very useful, since the proteins techniques based on MS, ESI (ElectroSpray Ionization) and involved in the transport and mechanism of action of a MALDI (Matrix-Assisted Laser Desorption/Ionization) are metallodrug (albumin and transferrin, for example) are not very powerful methods to ascertain metallodrug−protein available in a purified form and have often high molecular binding. While MALDI generates mainly singly charged masses.10c pseudomolecular ions, ESI gives singly or multiply charged Until now, the ESI technique was applied to inert metal metal−protein adducts (upon deprotonation or association complexes of the second and third transition series in which with protons and alkali ions).10c,11 For example, ESI-MS has proteins bind with a coordination bond.11 However, it has been used to explore the interaction of cisplatin and its been pointed out that such a technique can also be used to derivatives to low molecular mass bioligands, such as DNA study metal species which form labile coordinative bonds with nucleic bases, amino acids, oligonucleotides, and peptides, and biomolecules (such as vanadium, copper, and other metals of high molecular mass biomolecules, such as proteins;10a,c,12 the first transition row) or to systems where noncovalent moreover, the study of the reactivity of organometallic binding occurs (i.e., coordinatively saturated, such as PtIV complexes of RuII and AuIII with potential antitumoral complexes).11,15 Despite the high number of articles published applications is another example of its potentiality.10b,13,14 An on Pt, Ru, and Au complexes with antitumoral activity,11 very important advantage over other instrumental techniques is that few papers have been devoted to potential V drugs: beyond the the metal concentrations for ESI-MS experiments are in the “classical” applications of MS in the determination of the range 1−100 μM, therefore very close to those found under molecular mass of synthetic V complexes, the number of other physiological conditions. Among others, lysozyme, ubiquitin, studies is rather limited. One example is the ICP-MS myoglobin, superoxido dismutases, and insulin have been investigation of the biospeciation of some antidiabetic extensively studied by ESI-MS as model proteins;11 they have VIVO2+ compounds in the blood serum,16 in which the binding 9740 https://dx.doi.org/10.1021/acs.inorgchem.0c00969 Inorg. Chem. 2020, 59, 9739−9755 Inorganic Chemistry pubs.acs.org/IC Article Figure 1. ESI-MS spectrum of lysozyme (concentration 5 μM). Figure 2. Deconvoluted ESI-MS spectrum of lysozyme (concentration 5 μM). of VCs to serum proteins, in particular transferrin, was literature.18 [VIVO(acac)2] was purchased from Sigma-Aldrich. The suggested. calcium salt of amavadin, [Ca(H2O)5][VIV(hidpa)2]·H2O, was In this study, the interaction of VIV compounds with prepared according to the reported synthesis.17 pharmacological and biological relevance with some model Preparation of the Solutions and ESI-MS Measurements. The solutions were prepared dissolving in ultrapure water (LC-MS proteins has been investigated by ESI-MS spectrometry. In grade, Sigma-Aldrich) a weighted amount of the VC to obtain a metal particular, the binding to lysozyme (Lyz) with four important ion concentration of (1.0−2.0) × 10−3 M. They were subsequently VCs with potential antidiabetic or anticancer application,1e,3m diluted in ultrapure water and mixed with aliquots of a stock protein [VIVO(pic)2(H2O)], [VIVO(ma)2], [VIVO(dhp)2], and [VIVO- solution (500 μM) in order to have a metal-to-protein molar ratio of (acac)2 ], where pic −, ma −, dhp −, and acac− are the 3:1, 5:1, or 10:1 with a final protein concentration of 5 or 50 μM. In abbreviations for picolinate, maltolate, 1,2-dimethyl-3-hy- all the solutions, pH was in the range 5.0−6.0. Argon was bubbled droxy-4(1H)-pyridinonate, and acetylacetonate, and with through the solutions to ensure the absence of oxygen and avoid the [V(hidpa)2]2− (amavadin, with hipda3− N-hydroxyimino-2,2′- oxidation of the VIVO2+ ion. ESI-MS spectra were recorded diisopropionate anion4,17) was investigated (Scheme 1). immediately after the solution preparation. Mass spectra in positive- and negative-ion mode were obtained on [VIVO(pic)2(H2O)], [VIVO(ma)2], and [VIVO(dhp)2] exist a Q Exactive Plus Hybrid Quadrupole-Orbitrap (Thermo Fisher in aqueous solution, at least in part, as cis-octahedral species Scientific) mass spectrometer. The solutions were infused at a flow (with cis referring to the position of the water molecule with rate of 5.00 μL/min into the ESI chamber. The spectra were recorded respect to the VO bond, Scheme 1) with an accessible site in in the m/z range 50−750 for the binary and 300−4500 for the ternary the equatorial position where an amino acid side-chain donor systems at a resolution of 140 000 and accumulated for at least 5 min can replace the water ligand. In contrast, [VIVO(acac)2] is to increase the signal-to-noise ratio. Generally the experiments were square pyramidal and amavadin is an unusual nonoxido VIV run more than once; when measurements were repeated, indis- species with no available coordination sites. Subsequently, the tinguishable results were obtained. Only one set of the reproducible interaction of [VIVO(pic)2(H2O)], chosen as a representative data is reported here. VIVO2+ species, was examined with myoglobin (Mb) and The experimental settings for the measurements of positive-ion mode spectra were: spray voltage, 2300 V; capillary temperature, 250 ubiquitin (Ub), to compare the behavior of different proteins. °C; sheath gas, 10 (arbitrary units); auxiliary gas, 3 (arbitrary units); The main goal of this work is to ascertain if the ESI-MS could sweep gas, 0 (arbitrary units); probe heater temperature, 50 °C. The be considered as a valid technique to infer information on the settings used for negative-ion mode spectra were: spray voltage, VCs−protein interactions, trying to highlight the pros and −1900 V; capillary temperature, 250 °C; sheath gas, 20 (arbitrary cons, and to understand its possible applications in the design units); auxiliary gas, 5 (arbitrary units); sweep gas, 0 (arbitrary units); and development of new potential V drugs. probe heater temperature, 14 °C. ESI-MS spectra were analyzed with ■ Thermo Xcalibur 3.0.63 software (Thermo Fisher Scientific), and the EXPERIMENTAL SECTION average deconvoluted monoisotopic masses were obtained through the Xtract tool integrated in the software. ■ Chemicals. The chemicals oxidovanadium(IV) sulfate trihydrate (VIVOSO4·3H2O), pyridine-2-carboxylic acid (picolinic acid, Hpic), 3-hydroxy-2-methyl-4H-pyran-4-one (maltol, Hma), 1,2-dimethyl-3- RESULTS AND DISCUSSION hydroxy-4(1H)-pyridinone (deferiprone, Hdhp), N-hydroxyimino- Interaction of [VIVO(pic)2(H2O)] with Lysozyme. 2,2′-diisopropionic acid (H3hidpa), lysozyme from chicken egg Lysozyme represents one of the most frequently used models white (abbreviated with Lyz; code 62970), myoglobin from equine heart (abbreviated with Mb; M1882), and ubiquitin from bovine to study the metallodrug−protein interactions by mass erythrocytes (abbreviated with Ub; U6253), are all Sigma-Aldrich spectrometry techniques. Lyz is a relatively small protein products and were used without further purification. (with mass of ca. 14300 Da) formed of 129 amino acids with The complexes [VIVO(dhp)2], [VIVO(pic)2(H2O)], and [VIVO- an enzymatic activity and a significant relevance in a number of (ma)2] were synthesized following the procedure established in the host defense processes.19 The specific structure of this enzyme 9741 https://dx.doi.org/10.1021/acs.inorgchem.0c00969 Inorg. Chem. 2020, 59, 9739−9755 Inorganic Chemistry pubs.acs.org/IC Article Figure 3. ESI-MS spectra recorded on the system containing [VIVO(pic)2(H2O)] and lysozyme (5 μM): molar ratios 3:1 (top) and 5:1 (bottom). Figure 4. Deconvoluted ESI-MS spectra recorded on the system containing [VIVO(pic)2(H2O)] and lysozyme (5 μM): molar ratios 3:1 (top) and 5:1 (bottom). L indicates the picolinato ligand. relies on its stable three-dimensional conformation associated V exists in aqueous solution as a mixture of [VIVO- with the presence of four disulfide bonds between Cys residues (pic)2(H2O)] and [VIVO(pic)2(OH)]−, as demonstrated by alongside the peptide chain. It has only one histidine in its the combined characterization by EPR and pH-potentiometric structure (His15), which could represent a general binding site experiments.22 The positive-ion mode ESI-MS spectrum for some transition metal complexes.11 recorded in this study, after dissolution of the solid complex The positive-ion mode ESI-MS spectrum of lysozyme shows [VIVO(pic)2(H2O)] in ultrapure water at spontaneous pH a series of well-resolved peaks with a charge distribution from z (5.15), is reported in Figure S1 of the Supporting Information. = 7 to z = 12, which fall in the m/z range of 1100−2100 The spectrum shows the presence of two intense peaks at m/z (Figure 1).20 To determine the exact mass of the protein, the = 124.04 and m/z = 146.02 attributed to the protonated spectrum was deconvoluted with the Xtract software, which ligand, [Hpic+H]+, and its sodium adduct, [Hpic+Na]+. It is allows an estimation of the mass of macromolecules from the also possible to observe the signals of the H+ and Na+ adducts mass-to-charge spectra consisting of several peaks for multiply of [VIVO(pic)2], at m/z 311.99 and 333.98, respectively, charged ions.21 The result is reported in Figure 2, where a confirming the presence in solution, under these experimental central intense peak at 14305 Da can be noticed with a series conditions, of the 1:2 VIVO−picolinato species; the equatorial of other signals due to the adducts containing Na+ ions (23 H2O is not revealed, in agreement with the literature data Da) and/or H2O molecules (18 Da). Each peak is also split in suggesting that a weak monodentate ligand can be removed several signals related to the isotopic distribution of the from the metal coordination sphere during the ionization revealed species. process.23,24 The comparison between the experimental and In order to interpret the spectra in the system containing calculated isotopic pattern for the peak of the [VIVO- [VIVO(pic)2(H2O)] and lysozyme, it must be considered that (pic)2+H]+ ion is shown as an example in Figure S2; in 9742 https://dx.doi.org/10.1021/acs.inorgchem.0c00969 Inorg. Chem. 2020, 59, 9739−9755 Inorganic Chemistry pubs.acs.org/IC Article Figure 5. Deconvoluted ESI-MS spectra recorded on the system containing [VIVO(pic)2(H2O)] and lysozyme (50 μM): molar ratios 3:1 (top) and 5:1 (bottom). L indicates the picolinato ligand. With the asterisks, the peaks of the adducts {[VIVOL]+ + n[VIVOL2]}−Lyz with n = 0−2 (ratio 3:1) and n = 0−4 (ratio 5:1) are denoted. particular, it must be observed the coincidence of the two (pic)2]−Lyz, in which one, two, or three cis-VIVO(pic)2 expected peaks due to the natural abundance of the 13C moieties (with mass of 311 Da) are bound to the protein. isotope (separated by m/z ∼ 1.00 for this adduct with charge If the spectra are recorded with a protein concentration of +1) at the third decimal figure. In the negative-ion mode ESI 50 μM (Figure 5), the outcome of the experiments significantly spectrum, the signal of the complex in which the water changes, and the signals of the species n[VIVO(pic)2]−Lyz, molecule is deprotonated, [VIVO(pic)2(OH)]−, was observed with n = 1−3 when the molar ratio is 3:1 and n = 1−5 when it together with the VV species [VVO2(pic)2]−, respectively at m/ is 5:1, are detected (Table 1). Therefore, an increase of the z 327.99 and 326.98 (Figure S3). The detection of [VVO2(pic)2]− is due to the partial oxidation of [VIVO- Table 1. Main Adducts Formed by Pharmacologically Active (pic)2(OH)]− in solution or in source.25 This oxidation VOL2 Complexes with Lysozyme (Lyz) Revealed by ESI-MS process is probably favored by the similarity of the structure of cis-VIVO(OH)− and cis-VVO2+ (Scheme S1). The species mass identified by ESI-MS spectrometry in the system with compound (Da) adducts picolinate are listed in Table S1. [VIVO(pic)2(H2O)] 14491.8 [VIVO(pic)]−Lyz The ESI-MS spectra on the system containing [VIVO- 14615.8 [VIVO(pic)2]−Lyz (pic)2(H2O)] and Lyz were recorded using two concentrations 14802.8 {[VIVO(pic)] + [VIVO(pic)2]}−Lyz of the protein (5 and 50 μM) and metal-to-protein ratios (3:1 14926.8 2[VIVO(pic)2]−Lyz and 5:1). The results indicate that these two variables 15114.8 {[VIVO(pic)] + 2[VIVO(pic)2]}−Lyz significantly influence the outcome of the experiments. In 15237.8 3[VIVO(pic)2]−Lyz Figure 3, the raw spectra obtained with a protein concentration 15425.8 {[VIVO(pic)] + 3[VIVO(pic)2]}−Lyz of 5 μM are represented. It can be noticed that, for each peak 15548.8 4[VIVO(pic)2]−Lyz of the protein, other signals are present with higher m/z values, 15736.8 {[VIVO(pic)] + 4[VIVO(pic)2]}−Lyz which correspond to the adducts n[VIVO(pic)2]−Lyz. Since 15859.8 5[VIVO(pic)2]−Lyz the arrangement of the two ligands is equatorial−equatorial [VIVO(ma)2] 14494.8 [VIVO(ma)]−Lyz and equatorial−axial, only one equatorial coordination site is 14685.8 2[VIVO(ma)]−Lyz 14812.8 {[VIVO(ma)] + [VIVO(ma)2]}−Lyz available for the protein. The comparison of the raw spectra [VIVO(dhp)2] 14527.3 [VIVO(dhp)(H2O)]−Lyz recorded in the system [VIVO(pic)2(H2O)]/Lyz with those 14649.4 [VIVO(dhp)2]−Lyz obtained in the system with lysozyme only (cfr. Figures 3 and 14870.5 {[VIVO(dhp)(H2O)] + 1) suggests that the VIVO(pic)2 binding does not alter the [VIVO(dhp)2]}−Lyz distribution of the protein charge states; this indicates that the 14992.6 2[VIVO(dhp)2]−Lyz conformation of lysozyme remains unchanged after the [VIVO(acac)2] 14470.0 [VIVO(acac)]−Lyz interaction with the metal moiety.26 To determine the mass 14571.2 [VIVO(acac)2]−Lyz of these adducts, the ESI-MS spectra were deconvoluted, and 14636.0 2[VIVO(acac)]−Lyz the results are reported in Figure 4. The spectra are similar, 14736.6 {[VIVO(acac)] + [VIVO(acac)2]}−Lyz and in addition to the peak of the free protein, the peaks at 14801.0 3[VIVO(acac)]−Lyz 14 616 and 14 926 Da (for the 3:1 ratio) and also at 15 238 Da 14837.1 2[VIVO(acac)2]−Lyz (for the 5:1 ratio) are revealed, which are attributed to the 14965.1 4[VIVO(acac)]−Lyz adducts [VIVO(pic)2]−Lyz, 2[VIVO(pic)2]−Lyz, and 3[VIVO- [V(hidpa)2]2− 14706.8 [VIV(hidpa)2]−Lyz 9743 https://dx.doi.org/10.1021/acs.inorgchem.0c00969 Inorg. Chem. 2020, 59, 9739−9755 Inorganic Chemistry pubs.acs.org/IC Article Figure 6. Deconvoluted ESI-MS spectra recorded on the system containing [VIVO(ma)2] and lysozyme (50 μM): molar ratios 3:1 (top) and 5:1 (bottom). L indicates the maltolato ligand. Figure 7. Deconvoluted ESI-MS spectra recorded on the system containing [VIVO(dhp)2] and lysozyme (5 μM): molar ratios 3:1 (top) and 5:1 (bottom). L indicates the 1,2-dimethyl-3-hydroxy-4(1H)-pyridinonato ligand. concentration seems to favor the interaction between the cis- were identified. In the negative-ion mode the signal of the VIVO(pic)2 moiety and lysozyme. Moreover, for each peak deprotonated ligand [ma]− (m/z = 125.02) and the VV n[VIVO(pic)2]−Lyz, the signal of the adducts {[VIVO(pic)] + complex [VVO2(ma)2]− (m/z = 332.98, simulation of the n[VIVO(pic)2]}−Lyz with n = 0−4 at the highest V/protein isotopic pattern in Figure S5) were detected. As previously ratio can be observed, where both VIVO(pic)+ (with mass of pointed out for the system with Hpic, the detection of these 189 Da) and a certain number of cis-VIVO(pic)2 fragments species can be related to the presence in solution of bind to lysozyme. It must be noticed that the intensity of the [VIVO(ma)2(H2O)] and [VIVO(ma)2(OH)]−, since a solvent signals due to the adducts with 1:1 species VIVO(pic)+ is lower molecule can be lost during the ionization process and a partial than that of the adducts formed by 1:2 fragment cis- oxidation of the VIVO2+ moiety can occur.23−25 The list of the VIVO(pic)2. species identified is reported in Table S2. Interaction of [VIVO(ma)2] with Lysozyme. In the solid In the ESI-MS spectra of the system [VIVO(ma)2]/Lyz state, maltolate forms [VIVO(ma)2] with a square pyramidal recorded with a protein concentration of 5 μM and molar geometry,27 but when the solid complex is dissolved in a ratios of 3:1 and 5:1, only the signal of [VIVO(ma)]−Lyz coordinating solvent such as water, it undergoes isomerization (mass of VIVO(ma)+ being 191 Da) was detected (spectra not to the cis-octahedral species [VIVO(ma)2(H2O)] (Scheme shown). However, when the lysozyme concentration is 10 1).28 times higher (i.e., 50 μM), the number of species increases, After dissolving [VIVO(ma)2] in ultrapure water, in the similarly to what was detected in the system with picolinate. In positive-ion mode ESI-MS spectrum, the species [Hma+H]+ particular, with molar ratios 3:1 and 5:1 [VIVO(ma)]−Lyz (m/z = 127.04) and [VIVO(ma)2+H]+ (m/z = 317.99, the (14495 Da) and 2[VIVO(ma)]−Lyz (14686 Da) were simulation of the isotopic pattern being shown in Figure S4) observed (Figure 6); moreover, a weak peak attributed to 9744 https://dx.doi.org/10.1021/acs.inorgchem.0c00969 Inorg. Chem. 2020, 59, 9739−9755 Inorganic Chemistry pubs.acs.org/IC Article Figure 8. Deconvoluted ESI-MS spectra recorded on the system containing [VIVO(acac)2] and lysozyme with a molar ratio of 5:1 and a protein concentration of 5 μM (top) and 50 μM (bottom). L indicates the acetylacetonato ligand. {[VIVO(ma)] + [VIVO(ma)2]}−Lyz (14813 Da) was revealed. observed, while for maltolate the 1:1 moiety, VIVO(ma)+, It must be observed that the moieties [VIVO(ma)2] and binds preferentially to lysozyme. [VIVO(ma)]+ have one and three sites (two adjacent in the Interaction of [VIVO(acac)2] with Lysozyme. Spectro- equatorial plane plus the axial one) suitable for the protein scopic and potentiometric studies indicate that acetylacetonate binding. forms with VIVO2+ the species with 1:1 and 1:2 composition,30 Two major differences in comparison with the system with at which the stoichiometries [VIVO(acac)(H2O)2]+ and picolinate must be highlighted: (i) it is the moiety VIVOL+ [VIVO(acac)2], with square pyramidal geometry, are as- instead of cis-VIVOL2 which preferentially binds to the protein signed.31 and (ii) a smaller number of VIVO(ma)+ fragments compared The ESI-MS spectrum obtained after dissolution in ultrapure to cis-VIVO(pic)2 interact with lysozyme with varying both the water of the solid complex [VIVO(acac)2] was recorded in this ratio and concentration. study (Figure S7). At m/z 101.06 and m/z 123.04, the Interaction of [VIVO(dhp)2] with Lysozyme. In the protonated form of the ligand [Hacac+H]+ and the sodium binary system VIVO2+/Hdhp at a metal-to-ligand molar ratio of adduct [Hacac+Na]+ are observed. The spectrum shows also 1:2, VIVO2+ forms species with composition 1:1 and 1:2. The the peaks at m/z = 266.04 and m/z = 288.02, which were 1:2 complex exists, from pH 5 to 8, as a mixture of attributed to [VIVO(acac)2+H]+ and [VIVO(acac)2+Na]+, [VIVO(dhp)2(H2O)], cis-octahedral, and [VIVO(dhp)2], square respectively. The comparison between the experimental and pyramidal, in equilibrium between each other (Scheme calculated isotopic pattern for the peak of the [VIVO- (acac)2+Na]+ ion is shown as an example in Figure S8. In 1).18a,29 In the ESI-MS spectrum obtained after dissolving the negative-ion mode spectrum, the signal of the species [VIVO(dhp)2] in water, the signals at m/z 344.06 of [VIVO(acac)2−H]− at m/z 264.02 was observed with a low [VIVO(dhp)2+H]+ and at m/z 366.04 of [VIVO(dhp)2+Na]+ intensity. All the species detected are listed in Table S4. (Figure S6) are detected, confirming the existence of the 1:2 The ESI-MS spectrum recorded on the system [VIVO- complex in solution. The species revealed are listed in Table (acac)2]/Lyz with a ratio of 5:1 and protein concentration of 5 S3. μM shows the signal of the free protein at 14 305 Da and only In the ESI-MS spectra recorded on the system [VIVO- a weak signal attributable to the adduct [VIVO(acac)]−Lyz (dhp)2]/Lyz 3:1, the species observed were [VIVO(dhp)- (Figure 8, top). For this species the equatorial interaction with (H2O)]−Lyz (14526 Da) and [VIVO(dhp)2]−Lyz (14649 Da) one or two protein residues (plus a weak axial binding) is plus {[VIVO(dhp)(H2O)] + [VIVO(dhp)2]}−Lyz (14870 Da), possible. When a higher concentration is used (50 μM), the while when the ratio was 5:1 the peaks of 2[VIVO(dhp)2]−Lyz spectrum shows a series of peaks belonging to [VIVO(acac)]− (14993 Da) were also identified (Figure 7). With increasing Lyz (14 470 Da), 2[VIVO(acac)]−Lyz (14 636 Da), 3[VIVO- the VC/Lyz ratio, the relative amount of [VIVO(dhp)2]−Lyz (acac)]−Lyz (14 801 Da), and 4[VIVO(acac)]−Lyz (14 965 increases compared to [VIVO(dhp)(H2O)]−Lyz. It must be Da), while the weak signals at 14 571 and 14 837 Da can be noticed that in the moieties VIVO(dhp)2 and VIVO(dhp)- attributed to [VIVO(acac)2]−Lyz and 2[VIVO(acac)2]−Lyz (H2 O) + , one (equatorial) and two coordination sites (Figure 8, bottom). Therefore, the increase of the vanadium (equatorial and axial) are available for coordination to the concentration favors the interaction of the 1:2 complex protein. [VIVO(acac)2] with lysozyme, even thoughas discussed Comparing the results of Hdhp with those obtained with the above for maltolatethe binding of the 1:1 species VIVO- structurally similar ligand Hma, we found that in the first case (acac)+ is favored over [VIVO(acac)2] (see Table 1). adducts with the 1:2 fragment VIVO(dhp)2 are mainly Concerning the interaction between [VIVO(acac)2] with 9745 https://dx.doi.org/10.1021/acs.inorgchem.0c00969 Inorg. Chem. 2020, 59, 9739−9755 Inorganic Chemistry pubs.acs.org/IC Article Figure 9. Deconvoluted ESI-MS spectra recorded on the system containing [VIV(hidpa)2]2− and lysozyme with a molar ratio of 5:1 and a protein concentration of 5 μM (top) and 50 μM (bottom). L indicates the N-hydroxyimino-2,2′-diisopropionato ligand. Figure 10. Deconvoluted ESI-MS spectrum of myoglobin (concentration 5 μM). lysozyme, no equatorial sites are available, and so only two [V(hidpa)2]−Lyz was observed (14706.8 Da), whose intensity possibilities exist: a weak axial binding through an amino acid increases with the vanadium concentration. Considering that residue or a noncovalent interaction on the protein surface. several protonation states exist for the protein, it is not possible Interaction of Amavadin with Lysozyme. Amavadin, to determine if the oxidation state of vanadium in the adduct is isolated in 1972 by Kneifel and Bayer,32 is an anionic nonoxido +IV or +V. On the basis of the studies in the literature, one VIV compound accumulated in three species of mushrooms of would expect that it is the VIV complex, because the oxidation the genus Amanita: A. muscaria, A. regalis and A. velatipes. In A. to VV is not favored.17b muscaria the vanadium content exceeds 400 times the value Interaction of [VIVO(pic)2(H2O)] with Myoglobin. normally detected in other species of the same genus and is Myoglobin (Mb) is a small protein, found in skeletal muscles independent of the vanadium concentration in the soil.4,17 It is and in the heart, where it stores molecular oxygen. It formed by the ligand N-hydroxyimino-2,2′-diisopropionic acid, constitutes up to 5−10% of all the cytoplasmic proteins (S,S)-H3hidpa (Scheme 1), in its triply deprotonated form found in these muscle cells and consists of a polypeptide of 153 hidpa(3−). The XRD structure of the calcium salt of amino acid residues and a heme group. 33,34 Under amavadin, [Ca(H2O)5][VIV((S,S)-hidpa)2]·2H2O, shows an physiological conditions, 70% of the Mb backbone is folded eight-coordination around VIV, involving two η2-N,O− groups into eight alpha helices (named A−H), the folding leading to a and four monodentate carboxylates.17 tight globular structure with a cleft for the heme group at In this study, the negative-ion mode ESI-MS spectrum was helices C, E, and F.35 recorded dissolving the solid complex [Ca(H 2 O) 5 ]- The ESI-MS spectrum of myoglobin dissolved in ultrapure [VIV(hidpa)2]·2H2O in ultrapure water. It shows the presence water (Figure S10) shows a series of peaks which correspond of a peak at m/z = 399.02 attributed to the anionic species to different charge states (from +6 to +13). In the [VV(hidpa)2]−. The detection of the VV species is due to the deconvoluted spectrum, two series of peaks can be recognized presence in solution of [V IV (hidpa) 2 ] 2− and can be with an experimental mass of 16 951 and 17 566 Da, attributedas mentioned aboveto the oxidation of the respectively, and a predominance of the second one. The complex in source or in solution.25 The comparison between mass difference (615 Da) suggests that they belong to the apo the experimental and calculated isotopic pattern confirms the (without heme) and holo forms of the protein, with the latter presence of this species (Figure S9). maintaining the binding with the heme group. It is known that The ESI-MS spectrum of the system [VIV(hidpa)2]2−/Lyz in myoglobin heme is bound to the protein chain by the 5:1 was recorded (Figure 9) and, together with the signal of covalent binding with the proximal histidine (His93) and that the free protein, only a peak attributable to the adduct the stability of this coordinative bond may be heavily affected 9746 https://dx.doi.org/10.1021/acs.inorgchem.0c00969 Inorg. Chem. 2020, 59, 9739−9755 Inorganic Chemistry pubs.acs.org/IC Article by experimental and instrumental conditions.36 In particular, intensity lower than that of [VIVO(pic)2]−Mb (cfr. Figures 11 the heme group can dissociate at low pH (2−3)due to and S13). protonation of the histidine residuesand strong ionization Interaction of [VIVO(pic)2(H2O)] with Ubiquitin. Ubiq- conditions, while it is preserved at high pH and mild uitin is a small regulatory protein constituted of a polypeptide electrospray source conditions. The results obtained in this chain of 76 amino acid residues with a molecular weight of 8.5 study are consistent with those reported in the literature36 and kDa. It contains a limited number of potential binding sites for suggest that, under our experimental conditions, the prosthetic metals, among them the N-terminal methionine (Met1), one group remains mainly bound to the protein. Analyzing the histidine residue (His68), and some carboxylate O-donor signal of holo-Mb in Figure 10, it can be noticed that the most groups.10c Ub is involved in the post-transductional signal intense peak at 17 566 Da is revealed with a series of other called ubiquination, which has a regulatory role in the cellular signals due to the adducts with a certain number of Na+ ions processes. Besides its functions in biological systems, ubiquitin (23 Da). is used as a model protein in mass spectrometry since it is The presence of the free heme group was confirmed by the commercially available with high purity, well characterized, and detection of a signal at m/z 616.2 in the raw spectrum of the allows to obtain accurate data.10c The interaction with some protein. In Figure S11, a comparison between the experimental metal compounds with antitumoral activity (such as cisplatin and calculated isotopic patterns attributable to [FeIII heme]+ is and Ru-based complexes) was already studied by MS.12c,38 shown; in agreement with the results in the literature, the The ESI-MS spectrum of the free protein dissolved in oxidation state of iron in the heme group released from Mb is ultrapure water at a concentration of 5 μM (Figure S14) was +III.37 recorded in this study and shows a series of peaks The interaction of [VIVO(pic)2(H2O)] with myoglobin was corresponding to the different charge states of ubiquitin studied by ESI-MS with the same ratio and concentration used (from +5 to +10). The deconvoluted spectrum reported in for the previous measurements with lysozyme. In the raw Figure 12 shows that the mass of the protein is 8564.6 Da, in spectrum, the signals at m/z values higher than the peaks of accordance with the literature data.12c,38 In addition to the free protein can be attributed to [VIVO(pic)]−Mb and major peak, a series of other signals due to the adducts with [VIVO(pic)2]−Mb adducts (Figure S12). The deconvoluted ubiquitous ions, such as Na+ (23 Da) and K+ (39 Da), are spectra with a protein concentration of 5 μM are shown in present. Figure S13. The mass difference between the peaks of the With ubiquitin, the isotopic pattern of its charged states was adducts and those of free protein suggest that n[VIVO(pic)2]− simulated, allowing us to determine the exact formula. As an Mb (n = 1−3) and {[VIVO(pic)] + n[VIVO(pic)2]}−Mb (n = example, in Figure 13 the comparison of the experimental and 0−2) are formed (Table 2). This indicates that, under these calculated isotopic pattern for the peak at m/z 952.63 with z = experimental conditions, only two or three vanadium species 9 is reported. The empirical formula of the neutral protein is are bound to the protein. C378H630N105O118S, in agreement with the previous studies.39 The ESI-MS spectra were recorded on the system Table 2. Main Adducts Formed by VIVO(pic)2(H2O) with [VIVO(pic)2(H2O)]/Ub with different protein concentrations Myoglobin and Ubiquitin Observed by ESI-MS (5 and 50 μM) and metal-to-protein molar ratios (3:1, 5:1, and 10:1). When the signals in the deconvoluted spectra are protein mass (Da) adducts analyzed, it can be noticed that, in comparison with the free Mb 17864.0 [VIVO(pic)]−Mb protein (with peak centered at 8564.6 Da, see Figure 12), 17986.0 [VIVO(pic)2]−Mb signals at higher mass appear, suggesting the formation of 18174.0 {[VIVO(pic)] + [VIVO(pic)2]}−Mb adducts VC−ubiquitin (Figure 14). At ratio 10:1, the major 18298.0 2[VIVO(pic)2]−Mb adducts revealed are n[VIVO(pic)2]−Ub with up to four cis- 18485.0 {[VIVO(pic)] + 2[VIVO(pic)2]}−Mb VIVO(pic)2 fragments bound to the protein; the other peaks 18609.0 3[VIVO(pic)2]−Mb correspond to {[VIVO(pic)] + n[VIVO(pic)2]}−Ub (n = 0−3), 18799.0 {[VIVO(pic)] + 3[VIVO(pic)2]}−Mb similarly to what was observed with lysozyme and myoglobin 18920.0 4[VIVO(pic)2]−Mb (Tables 1 and 2). It must be noticed that the intensity of the 19109.0 {[VIVO(pic)] + 4[VIVO(pic)2]}−Mb signals attributed to {[VIVO(pic)] + n[VIVO(pic)2]}−Ub is 19231.0 5[VIVO(pic)2]−Mb lower than those of n[VIVO(pic)2]−Ub. Ub 8752.6 [VIVO(pic)]−Ub To confirm the formation of the adducts, the simulation of 8875.6 [VIVO(pic)2]−Ub the experimental isotopic pattern was carried out for the 9063.6 {[VIVO(pic)] + [VIVO(pic)2]}−Ub species formed by VIVO(pic)2 with Ub obtaining an excellent 9186.6 2[VIVO(pic)2]−Ub agreement with the experimental data (Figure S15); it was 9374.6 {[VIVO(pic)] + 2[VIVO(pic)2]}−Ub found th at the adduct with molecular formula 9497.6 3[VIVO(pic)2]−Ub C390H638N107O123SV (m/z = 1110.46, z = 8) corresponds to 9685.5 {[VIVO(pic)] + 3[VIVO(pic)2]}−Ub [VIVO(pic)2]−Ub. 9809.6 4[VIVO(pic)2]−Ub Finally, in the spectra recorded with an ubiquitin concentration of 50 μM, the maximum number of fragments If the spectra are recorded with a protein concentration of bound to the protein increases up to 2 with the ratio 3:1 and 50 μM (Figure 11), the number of VIVO(pic)2 fragments up to 3 with the ratio 5:1 (Figure S16). Moreover, at a ratio bound to the protein raises up to 4 when the 3:1 ratio is used 5:1 also the adduct {[VIVO(pic)] + [VIVO(pic)2]}−Ub, not and up to 5 with a 5:1 ratio, and a general increase of the observed with the same ratio when Ub concentration is 5 μM, relative abundance of the metal−protein adducts is revealed. It is detected (Figure S16). Therefore, according to the data in can be noticed that at 50 μM the peaks of {[VIVO(pic)] + the literature,36a it can be concluded that with increasing the n[VIVO(pic)2]}−Mb are also observed, even though with an concentration of the species, the VC−protein interactions 9747 https://dx.doi.org/10.1021/acs.inorgchem.0c00969 Inorg. Chem. 2020, 59, 9739−9755 Inorganic Chemistry pubs.acs.org/IC Article Figure 11. Deconvoluted ESI-MS spectra recorded on the system containing [VIVO(pic)2(H2O)] and myoglobin (50 μM): molar ratios 3:1 (top) and 5:1 (bottom). L indicates the picolinato ligand. With the asterisks, the peaks of the adducts {[VIVOL] + n[VIVOL2]}−Mb with n = 0−3 (ratio 3:1) and n = 0−4 (ratio 5:1) are denoted. Figure 12. Deconvoluted ESI-MS spectrum of ubiquitin (concentration 5 μM). Figure 13. Experimental (top) and calculated (bottom) isotopic pattern for the peak revealed in the ESI-MS spectrum of ubiquitin, C378H639N105O118S (i.e., [Ub+9H+]) at m/z 952.63 with z = 9. become more favored. All the adducts revealed in the system solutions in the four systems under the experimental containing [VIVO(pic)2(H2O)] and ubiquitin are listed in conditions used for recording the ESI-MS spectra (pH in the Table 2. range 5−6 and metal concentration between 15 and 150 μM). Rationalization of the ESI-MS Data. Analyzing the data The results are summarized in Table 3. As it can be noticed, at presented in the previous sections, it can be observed that 15 μM, the formation of VIVOL2 species is favored with Hpic depending on the molar ratio, vanadium concentration, and and Hdhp, while with maltolate and acetylacetonate, VIVOL+ is type of VIV complexthe number and identity of the adducts the predominant complex in solution. Therefore, in the first revealed change significantly. two systems, the adducts n[VIVOL2]−Lyz are the major species An important difference between the four systems and, in contrast, with Hma and Hacac n[VIVOL]−Lyz containing VIVO2+ complexes and lysozyme is that for Hpic predominate. Conducting experiments at 150 μM, the relative and Hdhp the formation of adducts with the 1:2 complex, amount of VIVOL2 increases compared to VIVOL+ species; as a n[VIVOL2]−Lyz, is favored, while for Hma and Hacac the consequence, with Hpic and Hdhp the adducts n[VIVOL2]− adducts n[VIVOL]−Lyz are revealed (see Figures 4−8). The Lyz continue to dominate the spectra, whereas with Hma and reason for this apparent anomaly can be found examining the Hacac those with composition {[VIVOL] + [VIVOL2]}−Lyz, in different distribution of the VIVO2+ species formed in aqueous addition to n[VIVOL]−Lyz, are detected. It can be observed 9748 https://dx.doi.org/10.1021/acs.inorgchem.0c00969 Inorg. Chem. 2020, 59, 9739−9755 Inorganic Chemistry pubs.acs.org/IC Article Figure 14. Deconvoluted ESI-MS spectra recorded on the system containing [VIVO(pic)2(H2O)] and ubiquitin (5 μM): molar ratios 3:1 (top), 5:1 (center), and 10:1 (bottom). L indicates the picolinato ligand. Table 3. Distribution (%) of the Most Important Species Formed in the Systems Containing VIVOL2 (L = pic−, ma−, dhp−, acac−) at pH 5.5 and with Vanadium Concentration of 50 and 150 μM Used for Recording the ESI-MS Spectraa ligand V conc. VIVO2+ [VIVOL]+ [VIVOL2] [VIVOL2H−1]− [(VIVO)2OH)5]− [(VIVO)(OH)3]− − pic 15 μM 1.8 39.2 53.2 1.8 0.3 0.1 150 μM 0.2 14.4 78.5 2.6 0.0 0.0 ma− 15 μM 7.3 51.9 30.2 0.0 4.9 0.2 150 μM 0.9 26.2 65.4 0.0 0.7 0.3 dhp− 15 μM 0.1 15.1 84.7 0.0 0.0 0.0 150 μM 0.0 5.1 94.8 0.0 0.0 0.0 acac− 15 μM 11.2 54.8 17.3 b 11.9 0.4 150 μM 1.8 38.9 55.6 b 2.9 0.1 a Calculated using the stability data of the species reported in refs.22b,29,30,41 bSpecies not formed in the system with acac. that with picolinate the intensity of the peaks of {[VIVO(pic)] two iron binding sites which can accommodate hard metal ions + n[VIVO(pic)2]}−protein is considerably lower than the like the oxidovanadium(IV) ion;2a,d,3b,e,j,m metal ion abstrac- corresponding n[VIVO(pic)2]−protein, which reflects the tion could alter the species distribution of VIVO2+ and L−. In relative amount of VIVO(pic)2 compared to VIVO(pic)+ in contrast, when strong sites are not available (as in the case of the vanadium concentration range 15−150 μM (Table 3). The the proteins examined in this work), the preferential ability of the ligand to form stable adducts influences the type interaction is with VIVOL+ or VIVOL2 moieties and, for this of species observed by ESI-MS. This is clear when comparing reason, the data obtained by ESI-MS can be used to evaluate the results obtained with two structurally similar ligands the interaction of the metal species with proteins and stability maltolate and 1,2-dimethyl-3-hydroxy-4(1H)-pyridinonate. of the adducts. Specifically, Hma, the weaker ligand, forms n[VIVOL]−Lyz The validity of the discussion can be tested analyzing the low adducts, and Hdhp, a stronger ligand (for the presence of the m/z regions of the raw ESI-MS spectra. This is done for the endocyclic N−CH3 group instead of the O atom in the six- system VIVO(ma)2/Lyz and is shown in Figure 15 (ratio 3:1 membered ring18a,29,40), gives mainly n[VIVOL2]−Lyz species. and vanadium concentration 5 μM). Whereas at a high m/z This agrees well with the distribution data of the VIVO2+/Hma the signals of the (metal species)−protein adducts are well and VIVO2+/Hdhp systems at the concentrations used to resolved, at m/z lower than 350 the peaks of the free record the ESI-MS spectra (Table 3). In the overall complexes are revealed. This proves that an equilibrium examination of the data, it must be taken into account that between the free complexes and metal adducts with the protein the metal ion can be sequestered (that is completely removed exists according to the reaction VO(ma)x + protein ⇄ from the ligand L−) when the protein affinity for VIVO2+ is very [VO(ma)x]−protein, with x = 1−2. In the region with m/z strong: a clear example is transferrin, which in the apo form has 180−350, besides [VIVO(ma)2+H]+ and [VIVO(ma)2+Na]+ 9749 https://dx.doi.org/10.1021/acs.inorgchem.0c00969 Inorg. Chem. 2020, 59, 9739−9755 Inorganic Chemistry pubs.acs.org/IC Article Figure 15. ESI-MS spectrum recorded on the system containing [VIVO(ma)2] (150 μM) and lysozyme (50 μM). The region in the m/z range 180−350 is presented in the inset. (m/z = 318.0 and 340.0, respectively), the signals of n[VIVOL2]−protein, the second class includes VCs with [VIVO(ma)(H2O)]+ and [VIVO(ma)(H2O)2]+ are detected intermediate strength ligands (such as maltolate and (m/z = 210.0 and 228.0). Even though the intensity of the MS acetylacetonate) that form adducts n[VIVOL]−protein. signal does not accurately report on the concentration of the In general, for Hpic and Hdhp adducts, the protein binds species in solution, the relative intensity indicates that the 1:2 with an accessible protein residue indicated with an X in complex is favored, according to the data of Table 3. In Scheme 2 in the fourth equatorial position. Specifically, the contrast, the concentration of the hydrolytic species structure of the adduct between cis-VIVO(pic)2 and Lyz has [(VIVO)2(OH)5]− and [(VIVO)(OH)3]− is so small that, been determined by X-ray crystallography and computational under these experimental conditions, they cannot be observed. methods, and the Asp52 residue of lysozyme was found to The simulations with the software Thermo Xcalibur of the replace the equatorial water ligand of VIVO(pic)2(H2O) isotopic patterns of [VIVO(ma)(H2O)]+, [VIVO(ma)(H2O)2]+, forming a distorted octahedral complex.3h,42 Glu and His are [VIVO(ma)2+H]+, and [VIVO(ma)2+Na]+ are shown in Figures other residues able to bind V in a monodentate manner. S17−S20. Particularly, accessible residues for the coordination to a cis- In the other systems, a similar situation is observed. Notably, VIVOL2 moiety are Glu35 with Lyz,42 Glu16, Glu18, Asp21, the ratio between the signal intensity of the 1:2 complexes to and His68 for Ub,43 while for Mb they are mainly histidines that of the 1:1 ones increases from acac− to pic− and to dhp−, (His81, His113, His116, His199).44 With Hdhp, an identical in line with what is expected on the basis of the behavior is expected with the formation of n[VIVO(dhp)2]− thermodynamic data (Table 3). The regions with a low m/z protein species with the equatorial binding of Asp, Glu, and for these systems are reported in Figure S21, while the His; moreover, the formation of a minor adduct with the simulations of the peaks for 1:1 and 1:2 complexes are in composition [VIVO(dhp)(H2O)]−protein was observed, with Figures S22−S33. Another intriguing hypothesisthat could the coordination of the X residue in the equatorial plane and, be investigated in a future studyis that one of the species eventually, Z in the axial position. [VIVO(L)(H2O)]+ or [VIVO(L)(H2O)2]+ could be interpreted With ma− and acac−, the adducts formed have the general as the monohydroxide derivative [VIVO(L)(OH)+H]+ or formula n[VIVOL]−protein. A considerable number of adducts [VIVO(L)(OH)(H2O)+H]+; these latter, in their turn, would are possible and should be considered. Since there are two free be the precursors of the μ-hydroxide dimer cis equatorial positions (plus the axial site) in the VIVOL+ [(VIVO)2(L)2(OH)2], detected by potentiometry in the moiety, the protein binding mode could be mono-, bi-, or systems with pic−, ma−, and dhp− and neglectedin a first tridentate (X, Y, plus a further axial Z, Scheme 2). The approximationwith acac−.22b,29,30,41 bidentate or tridentate binding mode of the protein depends Pharmacological and Biological Implications. The on the protein conformation and on the eventual presence of data obtained allow us to advance some hypotheses on the two or three neighboring residues able to occupy contempora- metal−protein adducts formed by VIVO2+ complexes with neously three facial positions of the octahedral vanadium concentrations used in this study (between 15 and 150 μM, geometry. This eventuality is rare in small proteins such as which is close to that found under the physiological lysozyme but could be more common in larger proteins such as conditions3m). The compounds can be divided into two transferrin or albumin. The candidate residues able to classes: the first class comprises VCs formed by strong ligands coordinate the V center are Asp, Glu, and His, with the (for example, picolinate and 1,2-dimethyl-3-hydroxy-4(1H)- possible assistance of Ser, Thr, Tyr, Cys, or backbone-CO if pyridinonate) which give adducts with a composition of they are in the right position. With the acac− ligand, adducts 9750 https://dx.doi.org/10.1021/acs.inorgchem.0c00969 Inorg. Chem. 2020, 59, 9739−9755 Inorganic Chemistry pubs.acs.org/IC Article Scheme 2. Adducts Formed at the Physiological Vanadium Concentration by [VIVO(pic)2(H2O)], [VIVO(ma)2], [VIVO(dhp)2], [VIVO(acac)2], and [VIV(hidpa)2]2−a,b a When more than one adduct is formed in aqueous solution, it is indicated with minor or major species. In round parentheses, the less probable binding of an axial residue Z is denoted. bThe charges of the ligands and V=O ion (2+) are omitted for clarity. with formula n[VIVO(acac)2]−protein are also observed. Since the ESI-MS technique could be considered in the design of [VIVO(acac)2] is square pyramidal and no equatorial sites are new potential vanadium therapeutics. First of all, ESI-MS available, in such species the proteins could interact weakly allows the study of biospeciation at the metal concentrations with a residue Z in the axial position or, alternatively, with found in the patients treated with potential drugs. Second, the weak noncovalent interactions with residues on the protein redox reactions in the biological media containing proteins of surface (see Scheme 2 and ref 45). If the interaction is strong the three oxidation states, VIII, VIVO, and VVO2, both in the enough, these adducts can be revealed by an ESI-MS free form and in the generated adducts, can be followed with technique. the aim to determine the most stable state. Third, ESI-MS can If the VC is very stable, such as amavadin, and no suggest if the metal species exists in the free form or bound to coordination sites in the complex are available, only non- the protein, and which equilibriums in solution are established covalent interactions could occur (Scheme 2). The strength of and which adducts are formed (see Figure 15). Depending on these interactions depends on the number of polar groups in the molecular receptor(s), the features of the organic ligand L− the VC able to form hydrogen bonds, van der Waals, or could be modulated to favor formation at physiological hydrophobic contacts with the exposed polar groups on the concentrations of moieties VOL+, that can form stable species protein surface.45 with proteins through the binding of two amino acid donors, These studies convincingly show that, if a VC reaches the or VOL2, thatin contrastyields moderately stable adducts blood, a significant amount of metal−protein adducts exist and if it is in the cis arrangement or unstable if the interaction the type of interaction can influence its fate in the organism occurs in the axial position (Scheme 2). and favor the cellular uptake and binding to the cellular However, all these considerations assume that the VCs receptor(s). For example, the adducts with transferrin, with the remain intact after administration and do not address binding of VIVO2+ to the active sites left free by Fe3+ or to the absorption, distribution, metabolism, and excretion (ADME) accessible surface donors, can promote a conformational and, particularly, the events that occur before the drug enters change which would lead to recognition by the cellular the bloodstream. The possibility and likelihood that the receptors in the endocytosis.46 Moreover, the binding to the complex survives the biological media and the uptake cellular targets can lead to pharmacological activity, as in the mechanisms is a question that is not addressed in this study. inhibition of phosphatases, to which the vanadium antidiabetic If the VC is administered orally, it will need to travel through activity is attributed.47 In this context, the results obtained with the digestive tract to first reach the acidic pH of the stomach 9751 https://dx.doi.org/10.1021/acs.inorgchem.0c00969 Inorg. Chem. 2020, 59, 9739−9755 Inorganic Chemistry pubs.acs.org/IC Article before continuing traveling into the intestine and being fragment in a bi- or tridentate fashion, generating stable absorbed into the blood. If the VCs are delivered intra- adducts with the contemporaneous binding of two amino acid venously, then they reach the blood immediately, however the side-chains in two adjacent equatorial positions. Finally, when presence of other blood components and salts may decrease the VIV complex is very stable without available coordination their concentration and, hence, reduce the chance of adduct sites, as happens for amavadin, the proteins are not able to formation. Alternative modes of administration exist, and the interact covalently and only weak noncovalent contacts are question of how much compound reaches the blood intact is predicted. Since the formed adducts have different thermody- not known, particularly if the complexes will need to pass any namic stability, it is clear that, depending on the properties of membranes in the process. Unfortunately, studies done after the ligand L, a pharmacologically active VIVO2+ complex would administration of the VCs to animals rarely have investigated reach the target organs in different forms, each of them being the blood content of vanadium, and if this amount was characterized by a different cellular uptake and biological determined, no speciation studies have been carried out. We activity. Thus, in relation to the pharmacological activity, not are therefore left conducting studies on model systems similar only the solubility and hydrophobicity of the VCs are to those described in this work. important but also their ability to interact with proteins, ■ CONCLUSIONS In this study, the first systematic application of the ESI-MS which determines the adducts existing under physiological conditions. Overall, the results further support the fact that the biological properties of VCs must be related with their technique on the binding to model proteins, such as lysozyme, biospeciation in the organism and that a mixture of species myoglobin, and ubiquitin, of four VIVOL2 complexes[VIVO- (intact VC, adducts with proteins and/or low molecular mass (pic)2(H2O)], [VIVO(ma)2], [VIVO(dhp)2], and [VIVO- bioligand, inorganic and hydrolytic ions, this latter eventually (acac)2], which are reported to normalize elevated glucose generated by redox reactions) could contribute to the levels in diabetic animals and to be effective on several tumor pharmacological action,48 with the active species depending cell linesis presented. The interaction with lysozyme of the both on the thermodynamic stability and structural require- vanadium-containing natural product amavadin is also ment of the administered VC and, eventually, on its redox behavior. ■ investigated. The results indicate that ESI-MS can be successfully applied to these systems with reproducible results and gives information on the number and stoichiometry of the ASSOCIATED CONTENT species formed in the metal concentration range found in the * Supporting Information sı organisms treated with VCs. In particular, adducts with The Supporting Information is available free of charge at different compositions were found, n[VIVOL2]−Lyz or https://pubs.acs.org/doi/10.1021/acs.inorgchem.0c00969. n[VIVOL]−Lyz, where the value of n varies with changing L. Tables with identified species in the binary systems This experimental evidence allows for ascertaining the (Tables S1−S4); raw ESI-MS spectra recorded on the biospeciation of a pharmacologically active VIVOL2 complex systems containing [VO(pic)2(H2O)], [VO(acac)2], with vanadium concentrations in the range 10−100 μM. For Mb, [VO(pic)2(H2O)]/Mb, and Ub (Figures S1, S7, example, if VIVOL2 is stable, as found with picolinate and 1,2- S10, S12, and S14); experimental and simulated isotopic dihydroxy-3-methyl-4(1H)-pyridinonate, it survives in solution patterns of [VIVO(pic)2+H]+, [VIVO(pic)2(OH)]−/ even at micromolar concentrations and can bind to proteins as [V V O 2 (pic) 2 ] − , [V IV O(ma) 2 +H] + , [V V O 2 (ma) 2 ] − , 1:2 species. The comparison between the [VIVO(pic)2(H2O)] [VIVO(dhp)2+Na]+, [VIVO(acac)2+Na]+, binding to lysozyme, myoglobin, or ubiquitin indicates a [VV(hidpa)2]−, heme group in myoglobin, [VIVO- similar interaction, with the cis-VIVO(pic)2 moiety which (pic)2]−Ub (Figures S2−S6, S8, S9, S11 and S15); coordinates to proteins through the free equatorial site. In deconvoluted ESI-MS spectra recorded on the systems contrast, if the VC is less stable, as for maltolate and containing [VO(pic) 2 (H 2 O)]/Mb and [VO- acetylacetonate, it undergoes hydrolysis, and the 1:1 moiety (pic)2(H2O)]/Ub (Figures S13 and S16); simulations VIVOL+ interacts with the proteins. It is expected that with of the peaks of the free species detected in the systems weaker ligands, such as 6-methylpicolinate or kojate, the [VIVO(ma)2]/Lyz, [VIVO(pic)2(H2O)]/Lyz, [VIVO- formation of hydrolytic species like [(VIVO)2(OH)5]− and (dhp)2]/Lyz, [VIVO(acac)2]/Lyz (Figures S17−S20 [VIVO(OH))3]− may occur, with the possible further oxidation and S22−S33); low m/z region of the ESI-MS spectra to VV inorganic anions. Two important findings are: (i) the recorded on the systems [VIVO(pic)2(H2O)]/Lyz, number of metal fragments bound to proteins increases with [VIVO(dhp)2]/Lyz, [VIVO(acac)2]/Lyz (Figure S21); the VC concentration and (ii) the oxidation to VV is possible in and oxidation process of cis-VIVO(H2O)2+ to cis-VVO2+ the binary systems VIVO2+−L, but it is prevented or moiety (Scheme S1) (PDF) significantly slowed down after the formation of the adducts VC−protein. In fact, in the studied systems, there is no evidence of oxidation of VIV to VV. The results could allow the prediction of the fate of an ■ AUTHOR INFORMATION Corresponding Author administered VIVOL2 potential drug. If the complex is stable, Eugenio Garribba − Dipartimento di Chimica e Farmacia, the proteins can coordinate the VIVOL2 moiety only in a Università di Sassari, I-07100 Sassari, Italy; orcid.org/ monodentate manner, remembering that the interaction and its 0000-0002-7229-5966; Email: garribba@uniss.it strength are related to the geometry assumed in solution, cis- octahedral with a free equatorial position (moderately strong Authors binding) or square pyramidal with an available axial site (weak Valeria Ugone − Dipartimento di Chimica e Farmacia, coordinative or noncovalent interaction). If the complex has an Università di Sassari, I-07100 Sassari, Italy; orcid.org/ intermediate stability, the proteins can interact with the VOL+ 0000-0002-2830-3869 9752 https://dx.doi.org/10.1021/acs.inorgchem.0c00969 Inorg. Chem. 2020, 59, 9739−9755 Inorganic Chemistry pubs.acs.org/IC Article Daniele Sanna − Istituto CNR di Chimica Biomolecolare, I- Buglyo, P.; Micera, G.; Garribba, E. Biotransformation of BMOV in 07040 Sassari, Italy; orcid.org/0000-0001-9299-0141 the presence of blood serum proteins. Metallomics 2012, 4, 33−36. Giuseppe Sciortino − Dipartimento di Chimica e Farmacia, (g) Sanna, D.; Bíró, L.; Buglyó, P.; Micera, G.; Garribba, E. Transport Università di Sassari, I-07100 Sassari, Italy; Departament de of the anti-diabetic VO2+ complexes formed by pyrone derivatives in the blood serum. J. Inorg. Biochem. 2012, 115, 87−99. (h) Sanna, D.; Quı ́mica, Universitat Autònoma de Barcelona, 08193 Ugone, V.; Micera, G.; Garribba, E. Temperature and solvent Cerdanyola del Vallés, Barcelona, Spain; orcid.org/0000- structure dependence of VO2+ complexes of pyridine-N-oxide 0001-9657-1788 derivatives and their interaction with human serum transferrin. Dalton Debbie C. Crans − Department of Chemistry, Colorado State Trans. 2012, 41, 7304−7318. (i) Sanna, D.; Micera, G.; Garribba, E. University, Fort Collins, Colorado, United States; orcid.org/ Interaction of Insulin-Enhancing Vanadium Compounds with Human 0000-0001-7792-3450 Serum holo-Transferrin. Inorg. Chem. 2013, 52, 11975−11985. Complete contact information is available at: (j) Sanna, D.; Serra, M.; Micera, G.; Garribba, E. Interaction of Antidiabetic Vanadium Compounds with Hemoglobin and Red Blood https://pubs.acs.org/10.1021/acs.inorgchem.0c00969 Cells and Their Distribution between Plasma and Erythrocytes. Inorg. Chem. 2014, 53, 1449−1464. (k) Sanna, D.; Serra, M.; Micera, G.; Notes Garribba, E. Uptake of potential anti-diabetic VIVO compounds of The authors declare no competing financial interest. picolinate ligands by red blood cells. Inorg. Chim. Acta 2014, 420, 75− ■ ACKNOWLEDGMENTS This work was financed by Regione Autonoma della Sardegna 84. (l) Koleša-Dobravc, T.; Lodyga-Chruscinska, E.; Symonowicz, M.; Sanna, D.; Meden, A.; Perdih, F.; Garribba, E. Synthesis and characterization of VIVO complexes of picolinate and pyrazine derivatives. Behavior in the solid state and aqueous solution and (grant RASSR79857) and Università di Sassari (fondo di biotransformation in the presence of blood plasma proteins. Inorg. Ateneo per la ricerca 2019). D.C.C. thanks Colorado State Chem. 2014, 53, 7960−7976. (m) Sanna, D.; Ugone, V.; Pisano, L.; University for support. ■ Serra, M.; Micera, G.; Garribba, E. Behavior of the potential antitumor VIVO complexes formed by flavonoid ligands. 2. Character- REFERENCES ization of sulfonate derivatives of quercetin and morin, interaction (1) (a) Sakurai, H.; Yoshikawa, Y.; Yasui, H. Current state for the with the bioligands of the plasma and preliminary biotransformation development of metallopharmaceutics and anti-diabetic metal studies. J. Inorg. Biochem. 2015, 153, 167−177. (n) Sanna, D.; Ugone, complexes. Chem. Soc. Rev. 2008, 37, 2383−2392. 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