E L S E V I E R Inorganica Chimica Acta 283 (1998) 202-210

The formation of ternary complexes between VO(maltolate)2 and small bioligands

Tamas Kiss a,., Erzs~bet Kiss b, Giovanni Micera c, Daniele Sanna c a Department oflnorganic andAnalytical Chemistry, Attila JJzsef University, PO Box 440, H-6701 Szeged, Hungary

b Department of Inorganic and Analytical Chemistry, Lajos Kossuth University, H-4010 Debrecen, Hungary c Dipartimento di Chimica, Universitt~ di Sassari, Via Vienna 2, 1-071100 Sassari, Italy

Received 8 October 1997; received in revised form 1 December 1997; accepted 28 January 1998

This paper is dedicated to Professor Osamu Yamauchi

Abstract

In order to assess the solution state of VO (IV) in organisms when administered in the form of the bis (maltolato) complex (VO(malt)2), ternary systems of VO(IV)-maltol with various bioligands (potential metal ion binders of biofluids and tissues), such as inorganic phosphates, adenosine nucleotides, catechol derivatives, citrate and oxalate, were studied. The speciation of VO (IV) in these ternary systems was studied by pH-potentiometry, while the binding modes of the complexes were determined by spectral (UV-Vis and electron paramagnetic resonance) methods. The results support strongly the assumption that VO(malt)2 undergoes transformation into mixed ligand complexes formed with the various VO(IV) binders of biological fluids and tissues. © 1998 Elsevier Science S.A. All rights reserved.

Keywords: Oxovanadium complexes; Maltolate complexes; Ternary complexes

1. Introduction

Since the first report of the insulin mimetic activities of vanadium compounds in vitro in 1979 [ 1] and in vivo in 1985 [2], great efforts have been made to prepare vana- dium ( 1V ) and vanadium (V) complexes of high activity and low toxicity, that are readily absorbed. One of the most prom- ising complexes is bis(maltolato)oxovanadium(IV) or, as it is abbreviated, VO(malt)2 [3].

Maltol forms mono and bis complexes with the VO(IV) ion via the formation of five-membered (O-,--O)-chelates. The bis complex VO(malt)2 is the predominant species in the pH range 5-7, and can be easily isolated from aqueous solution [3]. As has been proved by electron paramagnetic resonance (EPR), it forms two geometric isomers depending on the position of the solvent molecule in the VO(IV) coor- dination sphere: the water molecule can be either cis or trans

to the oxo group of VO(IV). In aqueous solution at room temperature and at neutral pH, the cis form predominates [4].

* Corresponding author. Tel.: +36-62-312 505; fax: + 36-62-312 505; e-mail: tkiss @chem.u-szeged.hu

When VO(malt)2 is absorbed, it may meet many other potential VO(VI)-binding molecules (such as nucleotides, inorganic and organic phosphates, lactate, citrate, catechol- amines, etc.) present in extraceUular or intracellular biolog- ical fluids. These molecules may displace maltol, partly, via the formation of mixed ligand complexes, or completely, which results in the formation of the corresponding binary complexes. Accordingly, ternary complexes cannot be ignored in a description of the speciation of VO(malt)2 in biological fluids. Such complexes might be of importance in the absorption and transport processes of VO(malt)2, and even in the physiological activity.

The VO(iv)-binding capabilities of the potential low molecular mass VO(IV) binders of biological fluids (such as saliva, gastric juice, intestinal fluid, blood serum, etc.) have been studied extensively [ 5-14].

Adenosinephosphates were found to coordinate to VO(IV) through the phosphate moiety in the weakly acidic and neutral pH range, and through the ribose residue in basic solutions [ 5 ]. The involvement of the purine ring in VO (IV) binding via N7 was detected only with AMP [6].

Inorganic phosphate forms protonated 1:1 and 1:2 com- plexes with VO(IV) in the acidic pH range [7,8]; it then precipitates from pH~4.5, and redissolves at pH~8.5,

0020-1693/98/$ - see front matter © 1998 Elsevier Science S.A. All fights reserved. PII S 0 0 2 0 - 1 6 9 3 ( 9 8 ) 0 0 2 2 9 - 1

T. Kiss et al. / lnorganica Chimica Acta 283 (1998) 202-210 203

forming mononuclear mixed hydroxo species in addition to various poorly characterized oligonuclear hydroxo-bridged complexes [ 8 ]. Diphosphate and triphosphate, being capable of chelate formation, are much stronger VO (IV) binders [ 9 ] ; they form mostly mononuclear species. In the pH range 3-6, diphosphate acts as a chelating and bridging ligand through the four unshared oxygen atoms, forming a well characterized trinuclear complex (VO) 3A3 .

Citrate also displays a high tendency to form oligonuclear complexes: in addition to some mononuclear 1:1 complexes, VOAH and VOA, in the pH range 2-3, dinuclear species predominate up to pH ~ 8. In these complexes, citrate behaves as a bridging ligand through the alcoholate group [ 10].

Catechol derivatives are very strong VO(IV) binders; in addition to the normal mono- and bis-chelated VO (IV) com- plexes, a tris-catecholato species containing a 'bare' non- oxovanadium(1V) centre is also formed [ 11 ]. Desferri- oxamine B (DFB), a naturally occurring trihydroxamic ligand, forms extremely stable complexes with vana- dium(IV), and is also able to displace the oxo group ofVO 2 + in the acidic pH range through complex formation [ 12].

Dicarboxylic acids, oxalic and malonic acids, owing to the low proton competition on the carboxylic groups, bind VO(IV) strongly in the acidic pH range, forming complexes VOA and VOA2 [13]. Above pH~6, OH- competition strengthens and mixed hydroxo complexes, presumably dimeric species, are formed [ 14].

To our knowledge, no data on the formation of ternary complexes of VO(IV) with these ligands have been reported so far. The aim of the present work was to study the possibility and importance of mixed ligand complex formation in the VO (iv)-maltol-l igand B (phosphate, diphosphate, triphos- phate, adenosine-5'-nucleotides AMP, ADP and ATP, oxalate, citrate, dopamine and L-dopa (3,4-dthydroxyphen- ylalanine)) systems, pH-potentiometry was used to deter- mine the stoichiometries and stability constants of the complexes formed, and spectral measurements (UV-Vis and EPR) were carried out to establish the binding modes of the ligands in the complexes. The data obtained are used to dis- cuss the possible fate of VO (malt)2 in vivo.

2.2. Potent iometric measurements

The stability constants of the proton and VO(IV) com- plexes of the ligands were determined by pH-potentiometric titration of 25.0 cm 3 samples. The concentration of the VO(IV) ion was 0.002 mol dm -3 and the molar ratios between metal ion, maltol and ligand B were 0:1:0, 0:0:1, 1:1:1, 1:1:2, 1:2:1 and 1:2:2 (plus 1:2:5 and 1:2:10 for the VO(IV)-maltol-phosphate system) in the investigation of the formation of mixed ligand complexes. For the VO( IV) - phosphate system, the concentration of phosphate was 0.004 mol dm-3 and the metal ion to phosphate molar ratios were 0:1, 1:1, 1:2, 1:4, 1:6, 1:8 and 1:10.

The pH was measured with a Radiometer priM 84 instru- ment equipped with a GK 2421 C combined glass electrode, calibrated for hydrogen ion concentration according to Irving et al. [ 17]. The pKw calculated from strong acid-strong base titrations was 13.76+0.01. The titrations of mixed ligand systems were performed in the pH range 2-11.5 with a car- bonate-free KOH solution of known concentration ( ~ 0.2 mol dm-3) under an argon atmosphere, in order to avoid oxidation of the metal ion and, in the case of VO(IV) - maltol-catecholic systems, to avoid oxidation of the ligands too. In the case of the VO(IV)-phosphate system, the titra- tions with KOH solution (normal titration) were performed in the pH range 2.0-4.0 because of precipitation, and in the pH range 11.5-5.0 with HC1 solution (back titration) of known concentrations ( ~ 0 . 2 mol dm -3) under an argon atmosphere.

Equilibria corresponding to the formation of the hydroxo complexes of VO(IV) were taken into account in the calcu- lation of the stability constants of the complexes. The fol- lowing species were assumed: [VO(OH)] + (log flH = -5 .94) , [(VO)2(OH)2] 2+ (log fl2.2 = -6 .95 calculated from the data published by Henry et al. [18], the Davis equation being used to take into account the different ionic s t r e n g t h s ) , [ W O ( O a ) 3 ] - (log/31-3 = - 18.0) and [ (VO)2- (OH)5] - (log/~2-5 = - 23.0) taken from Ref. [ 19].

The concentration stability c o n s t a n t s f l pqrs =

[MpAqB,}-I,] /[M]P[A]q[B]r[H]'~ were calculated with the PSEQUAD computer program [20].

2. Experimental 2.3. Spectroscopic measurements

2.1. Reagents

The ligands used were Aldrich, Sigma or Fluka products of puriss, quality. Their purities were checked and the exact concentrations of solutions were determined by the Gran method [15]. The VO(IV) stock solution was prepared as described in Ref. [ 16] and standardized for metal ion con- centration by permanganate titration and for hydrogen ion concentration by potentiometry, using the appropriate Gran function. The ionic strength of all solutions studied was adjusted to 0.20 mol dm-3 KC1. In all cases, the temperature was 25.0 + 0.1°C.

Anisotropic X-band EPR spectra (9.15 GHz) were recorded at 140 K in aqueous solutions, using a Varian E-9 spectrometer. As usual, the samples for low-temperature measurements contained a few drops of DMSO to ensure good glass formation in frozen solutions. Absorption spectra were recorded with a Hewlett Packard HP 8453 spectropho- tometer. All manipulations and titrations were done under an atmosphere of argon. Aqueous solutions with VO(IV)- maltol-ligand B ratios of 1:2:0, 1:5:0, 1:10:0, 1:0:1, 1:0:2, 1:0:5, 1:0:10, 1:1:1, 1:1:2, 1:1:5, 1:2:2, 1:2:5 and 1:2:10 and a VO(IV) concentration of 0.004 moldm -3 or 0.0004 mol dm- 3 were examined atpH 7-7.5 by spectrophotometry.

204 T. Kiss et al. / Inorganica Chimica Acta 283 (1998) 202-210

Aqueous solutions with VO(IV)-maltol-ligand B ratios of 1:2:0, 1:0:1, 1:0:2, 1:1:1 and 1:2:2, and a VO(IV) concen- tration of 0.004 mol dm-3, were studied in the pH range 2-11.5 by EPR measurements, if not measured previously.

Table 1 Stability constants of proton (log K) and oxovanadium(IV) (log fl) com- plexes of maltol at 25°(2 and at 1= 0.2 mol dm-3 (KC1)

This work Ref. [3]

3. Results and discussion

The stability constants obtained for the proton and VO (IV) complexes of maltol are listed in Table 1. The recent stability data determined by Orvig and coworkers [ 3 ] at 0.1 mol dm- 3 KNO3 are included. The agreement between their stability constants and ours is reasonably good, especially concerning log/3VOA2. The somewhat poorer agreement between the stepwise constants may be explained by the fact that data from different pH ranges were used in the data evaluation; in order to avoid the slow hydroxo complex formation processes and to exclude any oxidation reaction between VO (IV) and maltol, Orvig and coworkers [3] limited their pH-metric study to the pH range 2-4 and evaluated their titration points assuming only the complexes VOA and VOA2. In our case, the pH-metric titrations were extended to higher pH values. A reversibility study of the pH-metric titrations performed with the rigorous exclusion of oxygen did not indicate a significant irreversible redox reaction up to pH 7.5-8, and 2-3 rain was always enough for pH equilibrium to be reached. Hence, we used a more complete range of formation of both the 1:1 and 1:2 complexes, which resulted in a somewhat different distribution of the cumulative constant log ~'VOA2 into log KvoA and log Kvog2. Although the reproducibility of the titration measurements was somewhat poorer at pH > 7.5-8, we attempted to fit the further base-consuming process occurring in this pH range by assuming another spe- cies, most obviously a mixed hydroxo species VOA2H_ i. This was supported strongly by the EPR spectra, which clearly indicated the presence of a new complex. Species distribution curves obtained with this speciation model are depicted in Fig. 1, which indicates that, in accordance with Orvig and coworkers [ 3 ], up to pH ~ 7 no hydroxo complex formation has to be taken into account, and almost exclusively the complex VOA2 is present in the physiological pH range.

Both the dominating 1:1 and 1:2 species can be detected and distinguished by spectral methods too. As discussed recently by Orvig and coworkers [3,4], the bis complex VO(malt)2 occurs in two geometrical isomeric forms depending on the position of the solvent molecule (the sixth coordinating): either cis (the water is cis to the oxo group) or trans. The EPR spectra indicate clearly that, similarly to oxalic acid, maltol prefers equatorial-axial cis coordination [4,14].

In equimolar solutions, a decrease in intensity and broad- ening of EPR signals suggested the probable formation of a dihydroxo-bridged dimer (VOAH_ ~)2 while in the case of an excess of ligand the occurrence of a new signal supported the formation of a mixed hydroxo complex VOA2H_ (=VOA2(OH)) . In accordance with the potentiometric

K(HA) 8.44( 1 ) 8.46 VOA 8.69(2) 8.80 VOA2 16.29(5) 16.31 (VOAH ~)2 9.88(6) VOA2H I 7.5(1) Fit a 0.0100 No. points 449 log K(VOA2) 7.60 7.51 log(K(VOA)/K(VOA2)) 1.09 1.29 VO 2+ + H A = V O A + H 0.25 0.34 VOA + HA = VOA2 + H - 0.83 - 0.95

The average difference between the calculated and experimental titration curves expressed in cm 3 of titrant.

1.0 VOA z

~ 0 . 6

~ 0 . 4

0.2 ).2A21:I.2 VOA2H. 1

o.o

3 4 5 6 7 8

pH

Fig. 1. Speciation curves for complexes formed in the VO(IV)-maltol system, Cvo = 0.002 mol dm 3 and Ctlg~nd = 0.004 mol d m - 3.

results, dimeric hydroxo complex formation did not affect the equilibria at ligand excess. The relatively low values for deprotonation of the maltolato complex VOA2 (pK~ 8.5- 8.8) can be explained by the fact that owing to the equatorial- axial cis arrangement of VOA2, this proton is released from a more acidic and equatorially coordinated water molecule, and not from a weakly bound apical water molecule, as would be the case for 'normal' trans complexes.

It is clear from Fig. 1 that the neutral complex VO(malt)2, which may have good membrane transport properties, exists in the wide pH range 5-7.5 which includes physiological pH. However, if the drug is administered orally, it will dissociate in the acidic milieu of the stomach and, for instance, at pH ~ 2 only 50% of the VO(IV) is bound to maltol in the uniposi- tively charged mono-complex [ VO (malt) ] +, the rest being the dipositively charged free aqua complex. Hence, this vana- dium will be ready to react with any other potential binders present. This should be an important factor in the formulation of the drug. With competitive ligands of the gastrointestinal tract, ternary complex function can likewise occur.

3.1. VO(IV)-phosphate system

Because of the controversies relating to earlier results [7,8,21-23] and the different experimental conditions,

T. Kiss et al. / Inorganica Chimica Acta 283 (1998) 202-210 205

Table 2 Proton (logK) and oxovanadium(W) (log 13) stability constants for the complexes of phosphate at 25°C and 1= 0.20 tool dm-3 (KCI)

Complex log K/log/3

K(HA) 11.46( 1 ) K(HzA) 6.63(1) K(H3A) 1.83(1) VOAH2 20.3(3) VOAH 16.8(2) VOA 10.8(5) VOAH_2 -3 .0(3) (VO)2A2H_2 13.35(10) Fit a 0.011 No. points 230

"The average difference between the calculated and experimental titration curves expressed in cm 3 of titrant.

detailed potentiometric measurements were carried out in order to describe the species distribution in the VO(IV)- phosphate system in a wide pH range.

The protonation constants (log K(HnA)) measured for phosphate are 11.46, 6.63 and 1.83. These data are in reason- ably good agreement with those measured earlier, when the differences in experimental conditions (ionic strength and temperature) are taken into account. Literature values reported earlier are as follows: 11.39, 6.61 and 1.96 at I = 0.15 moldm -3 at 37°C [21]; 11.80, 6.51 and 1.20 at 1=0.15 moldm -3 at 25°C [22]; 11.54, 6.68 and 2.00 at 1=0.15 tool dm -3 at 37°C [23].

Potentiometric speciation results are presented in Table 2 where log/3 values for the complexes are listed. Phosphate forms a protonated species VOAH2 at pH ~ 2. This complex loses a proton with pK 3.49 and VOAH, which is the domi- nant species at pH ~ 4, is formed. Monodentate coordination of the ligands is very likely in these complexes. Accurate measurements could not be performed in the pH range 4.0- 8.5 because of precipitation. IR analysis of the precipitates obtained at different pH values showed that all contained both VO(IV) and phosphate [ 8]. In order to obtain reliable pH-metric data over as wide a pH range as possible, back- titrations with 0.2 mol din- 3 HCI were also carried out start-

ing from pH 11.5. In this way, precipitation could be avoided down to pH 5.0 at a metal ion-ligand ratio of 1:8, probably because of the formation of oversaturated solutions. These measurements indicate the formation of a mononuclear com- plex VOA and a dimeric species (VO)2A2H_2, rather than its monomeric form VOAH_ t, in the pH range 5-8. The fitting of the pH-metric titration curves improved a little when the model included the dimer (VO)zA2H_2 instead of VOAH_ ~. Additionally, the EPR measurements strongly supported the formation of an EPR-silent dinuclear species. There might be either monodentate or bidentate coordination of the phosphate in VOA. Earlier EPR studies indicated bidentate coordination [8]. (VO)2A2H_2 is probably a hydroxo-bridged dimer [8]. In a more basic solution, the mononuclear complex VOAH_2 is formed (in addition to the hydroxo complexes of the metal ion), in which one phos- phate ligand and two hydroxo groups are coordinated to the VO(/V) ion. Mononuclear bis-phosphato complexes were rejected by the computer program.

The complex-forming properties of the other ligands B with VO (IV) have been studied and stability constants under similar experimental conditions have been reported previ- ously [5,6,9-12].

3.2. VO(IV)-maltol-ligand B systems

The speciation data on ternary complex formation of VO(W)-maltol with several bioligands, potential VO(W) binders ofbioflnids, are listed in Table 3. Spectroscopic meas- urements were used to detect mixed ligand complex forma- tion and the characteristic spectral changes are discussed in the text.

3.3. VO(IV)-maltol-inorganic phosphates

Ternary complex formation with orthophosphate may have a significant impact on the absorption efficiency of VO(malt)2, as phosphate (beside lactate) seems to be the most important VO(IV) binder in the gastrointestinal tract [211.

Table 3 Stability constants of mixed ligand vanadium(W) complexes of maltol (A) with several ligands B at 25 :i: 0. I°C and I = 0.2 mol dm- 3 (KC1)

Ligand B VOABHz VOABH VOAB VOABH_ ~ VOABH_ 2

Phosphate 28.69(3) 25.00(2) 18.81 (2) Diphosphate 17.41 ( 4 ) 7.4( 1 ) Triphosphate 16.31 (5) AMP 12.5(2) ADP 13.97(5) - 2.8(I) ATP 13.72(8) -3 .1(3) Dopanline 34.69(2) 25.32(6) 15.03(7) L-dopa 34.15(4) 25.08(9) 15.26(5) Tiron 23.01(5) Oxalate 13.92 ( 3 ) Citrate 18.85(6) 15.41(3) 7.54(3)

206 T. Kiss et al. / Inorganica Chimica Acta 283 (1998) 202-210

Although phosphate alone in its protonated forms, H2A and HA, is not a strong VO(IV) binder, and above p H > 4 it forms a precipitate, in the presence of a two-fold excess of maltol (with respect to VO(IV)) there is no precipitation and pH-metric titrarions suggest the formation of ternary complexes throughout the whole pH range studied. Without the assumption of ternary species, the fitting parameter was 0.0209 cm3; it improved to less than half of this when for- marion of the mixed ligand complexes listed in Table 2 was assumed. Surprisingly enough, ternary complex formation proved to be highly favoured. The usual factors, such as charge neutralization, steric factors, w-bond formation and intramolecular ligand-ligand interactions, which may influ- ence ternary complex formation, cannot be so important in the VO(IV)-maltol-orthophosphate system as to explain such an enhanced mixed ligand complex formation.

At first sight, the spectroscopic measurements do not seem to fully support the pH-metric speciation results. The pres- ence of phosphate has a minor effect on the electronic absorp- tion spectra of VO(malt)2 at physiological pH. For instance, as seen in Fig. 2, the molar absorptivity values exhibit a significant decrease, although the hma x values remain almost unchanged (the Area x (,~ (tool-] cm- 1 dm 3) ) values for the VO(iv)-maltol-phosphate system are as follows: at 1:2:0, 882 nm (40), 622 nm (19), 439 nm (94); at 1:2:2, 885 nm (35), 620 nm (20), 433 nm (91); at 1:2:10, 878 nm (29), 620 nm(15) , 432 nm (81 ) ). The EPR spectra hardly change even at ten-fold phosphate excess (see Fig. 3). However, the broad resonances attributable to the hydroxo-bridged oligo- nuclear species formed in the binary VO (IV)-phosphate sys- tem are lacking, which indicates the importance of maltol in the metal binding. More significantly, some splitting of the -s]V hyperfine resonances could indicate the coexistence of different complexed species with very similar EPR parame- ters. If one takes into account that the spectral features of VO(IV) are mainly sensitive to the equatorial donor set, and that V O A 2 involves a cis geometry, only minor changes in the EPR parameters and electronic absorption maxima are expected on changing three maltolate O with two maltolate

0 .4 1

~ 0 .3 2 1

0 .2 <

o.I

0 .0 i I i I i

400 500 600 700 800 900

W a v e l e n g t h (rim)

Fig. 2. Visible spectra of ( 1 ) VO(IV)-maltol 1:2 and (2) VO(1V)-maltol- phosphate 1:2:10 systems at pH 7.3.

dpph

~fferenee

Fig. 3. X-band EPR spectra of (1) VO(1V)-maltol 1:2 and (2) VO(IV) - maltol-phosphate 1:2:10 systems at pH 7.3 recorded at 140 K.

O and a phosphate O in the equatorial plane. Most likely, the same reasons may be taken into account to interpret the par- ameters of VOABH_ ~ which are almost coincident with those of VOA2H_ 1. In the light of these contradictory results, it can be concluded that phosphate, as the most potent 'endog- enous' VO (IV) binder of the gastrointestinal tract, may have some effect on the speciation of the VO (IV)-maltol system, but this will affect the absorption behavior of VO(malt)2 only slightly.

Diphosphate and triphosphate, as chelating agents, are much stronger VO(IV) binders than orthophosphate, and form unambiguously mixed complexes with VO(IV)-mal- tol. The pH-metric titration data on the ternary systems could be fitted well on assuming a mixed ligand complex VOAB in addition to the binary species (see Table 3). Predominance curves (the sums of VO(IV) fractions bound in the binary complexes with ligand A or B and those bound in the ternary complexes with ligand A and B) for the VO(IV)-maltol- triphosphate system are depicted in Fig. 4.

In complete accordance with the pH-metric speciation results, the spectral data show that in both systems the phos- phate chain is the primary VO(IV) binder in the weakly

1.0

0.8 VO(IV)-B VO(IV)-A-B

~ 0 . 6

~ 0.4 VO(ZV)-A

~- 0 .2

0.0 j

3 4 5 6 7 8

pit

Fig. 4. Predominance diagram of the VO ( IV)-maltol( A ) -triphosphate (B) system at 1:2:2 metal ion to ligand ratio, Cvo = 0.002 tool d m - ~.

T. Kiss et al./ lnorganica Chimica Acta 283 (1998) 202-210 207

10 rnT L

O)

Fig. 5. High field parallel region of frozen solution EPR spectra (X-band) recorded at 140 K: (1) VO(IV)-maltol 1:2, (2) VO(IV)-maltol-tdphos- phate 1:1:1 and (3) VO(IV)-triphosphate 1:2 systems at pH~7.5, Cvo = 0.004 mol dm- 3.

acidic pH range 2-5; the ternary species VOAB then becomes the predominant complex (its spectral parameters are gll = 1.934, AI1=174×10 -4 cm -~ and Amax ( e (mol -~ cm -1 dm3)) 852 nm (26) , 655 nm (14) ) up to p H ~ 9 , although both cis-VOA2 (gll = 1.939, All = 171 × 10 -4 cm - I ) and VOB2 (gll = 1.937, All = 176× 10 -4 cm -1) can be clearly seen by EPR as minor species. As an illustration, the EPR spectra of the VO(IV)-mal to l , VO(IV)- t r iphosphate and VO(IV)-mal to l - t r iphosphate systems in the physiological pH range are shown in Fig. 5. Above pH ~ 9, the signal intensity decreases as a consequence of the formation of polynuclear hydrolytic species. The main monomeric com- plex exhibits the spectral features of the cis-VOA2H_ j spe- cies of maltol (gll = 1.942, All = 167 × 10-4 c m - ~ ).

As a conclusion, it may be said that diphosphate and tri- phosphate are strong competitors of maltol and can displace one of the maltol molecules of VO (malt)2 in the physiolog- ical pH range, where the ternary complex VOAB is the pre- dominant species.

3.4. VO(IV)-maltol-adenosine-5' phosphates

Adenosinenucleotides are widespread in cellular fluids, hence their interactions with 'exogenous' compounds of the organism are very likely.

On the basis of the VO (IV) -binding ability of nucleotides in their binary complexes at weakly acidic and neutral pH, coordination of the terminal phosphate donor(s) can be assumed in the ternary complexes too. In the highly basic pH range, as the proton competition on the alcoholate donors decreases, the ribose moiety becomes a more efficient binding site and its participation in metal binding can also be assumed. The potentiometric data could be fitted with the assumption of two mixed ligand complexes, VOAB and VOABH_ 2 (see Table 3). The predominance diagram for the V O ( I V ) - m a l - to l -ATP system depicted in Fig. 6 shows that formation of

1.0

.~ 0.8

~0 .6

.~0.4 /~VO(IV)

~" 0.2

0.0 ; , 3 4 5 6 7 8

pl-I Fig. 6. Predominance diagram of the VO ( IV ) -maltol ( A ) -ATP ( B ) system at 1:2:2 metal ion to ligand ratio, Cvo = 0.002 mol dm- 3.

the ternary complex VOAB between maltol and nucleotides is a little favored in the weakly acidic pH range.

In accordance with the pH-metric speciation results (see Fig. 6), in the V O ( I V ) - m a l t o l - A T P or V O ( I V ) - m a l t o l - ADP systems, both EPR and electronic absorption spectros- copy reveal unambiguously a new species (gll = 1.937, All = 175 × 10-4cm-l ,Amax ( 8 ( m o l - I c m - L din3)) 838nm (25), 605 nm (22) and 425 nm (51) ) in the pH range 4.5-6, which is presumably the mixed ligand complex VOAB. The EPR spectra of the VO(IV) -mal to l , V O ( I V ) - ATP and V O ( I V ) - m a l t o l - A T P systems are given in Fig. 7. The EPR results indicate that the complex VOAB is formed in maximum concentration at a somewhat lower pH ( ~ 4.5) than that obtained from pH-metry; this difference can be explained by the shifts in the formation equilibria due to the different temperatures. VOA2 predominates in more basic solution (pH 7-9) . At around pH ~ 9, VOA2H_ t is detected, then hydrolysis occurs (the intensity of the spectra weakens and the bands broaden). In very basic solutions (pH > 10),

10 mT

(I)

Fig. 7. High field parallel region of frozen solution EPR spectra (X-band) recorded at 140 K: (1) VO(IV)-maltol 1:2, pH 5.5; (2) VO(IV)-maltol- ATP 1:1:1, pH 4.5; (3) VO(IV)-maltoI-ATP l:l:l,pH 5.5; (4) VO(IV)- ATP 1:2, pH 5.5; Cvo =0.004 mol dm -3.

208 T. Kiss et aL / lnorganica Chimica Acta 283 (1998) 202-210

the metal ion is transferred to the ribose sites and the binary ATP complex VOBEH_4 can be detected (g1=1.957, All = 151 × 10 -4 cm- ~).

At pH > 9, pH-metry suggests the formation of another ternary complex, in which the nucleotide coordinates through the ribose alcoholates in positions 2 and 3, and thus the stoi- chiometric composition is VOABH_ 2. This species, how- ever, can be characterized only with a large uncertainty, because the species distribution for the VO(IV)-maltol binary system at pH > 9 is rather uncertain owing to redox reactions [3,4] and/or slow hydroxo complex formation processes [ 14].

The above results confirm that ADP and ATP (like diphos- phate and triphosphate) are strong VO (IV) binders, although not so potent as the latter ligands. Accordingly, they readily form ternary complexes VOAB with VO(IV)-maltol in the weakly acidic pH range while at physiological pH, VO(IV) is bound mostly in VO(malt)2. Furthermore, formation of the ternary species is less favored with AMP because of the lack of chelate formation at the phosphate moiety. Owing to the low concentration of VOAB, the EPR spectra of the ter- nary system mostly detect the binary species of maltol, except in basic solutions (pH> 10), where the hydroxyl sites of ribose prevail over those of maltol and the species VOB2H_ 4 is formed.

3.5. VO(IV)-maltol-catechol derivatives

Catechol derivatives were found to have high affinity to VO(IV). They can even displace the oxo group of VO(IV) and form non-oxo 3 × ( O - , O - ) coordinated tris complexes. The high affinity of catecholates for VO(IV), however, is hindered in the acidic pH range because of the high proton competition on the phenolate functions. At the same time, maltol, which is also a strong VO(IV) binder, has a high affinity for VO(IV) in the acidic pH range, owing to the weaker proton competition on the chelating ( O - , = O ) func- tion. The complex-forming abilities of the two ligands are reflected in the species distributions of the ternary systems. Fig. 8 depicts the predominance diagrams for the VO(IV)- maltol-dopamine system. The stability constants obtained from an evaluation of the pH-metric titration data for the VO(IV)-dopamine, VO(IV)-L-dopa and VO(1V)-tiron systems are listed in Table 3.

The predominance curves for the VO(IV)-maltol-dopa- mine system (see Fig. 8) demonstrate that the ternary com- plex VOABH formed in the intermediate pH range 4-8 (with a maximum of ~ 40 VO%) overlaps considerably with the formation of the two parent complexes. In VOABH, the cat- echols coordinate via the ortho-phenolate chelating site and the side-chain amino group is protonated.

A detailed analysis of the anisotropic EPR spectra leads to the same conclusion. At low pH (2-3), the EPR parameters (gu = 1.939 andAii = 178 × l0 -4 cm- 1) are slightly different from those of the aqua ion and can be ascribed to VOA. As the pH is increased to pH ~ 5, the spectra of three species can

1.0 VOOV)-A vo(tv)-e

0.8

~ 0 . 6

..~ 0.4

~" 0.2

0.0 . . . . .

2 3 4 5 6 7 8

pH

Fig. 8. Predominance diagram of the VO(1V)-maltol(A)-dopamine(B) system at 1:2:2 metal ion to ligand ratio, Cvo = 0.002 tool d m - 3.

be observed with EPR parameters characteristic of VOA, VOBH (gll = 1.943, All = 169X 10 -4 cm -~) and, overlap- ping strongly with the others (gll = 1.937, All = 161 X 10 - 4

cm-~), presumably the ternary complex VOABH. As the pH is increased further from pH ~ 7.5, purely catecholato complexes (VOB2H2, VOB2H and VOB2) predominate (gll = 1.952,AII = 156 X 10 - 4 c m - 1 ) . These complexes differ only in the protonation state of the non-coordinating side- chain amino group and thus their EPR spectra are practically the same.

At a metal ion-ligand ratio of 1:1:1, when there is no catecholate excess which could displace maltol completely from the coordination sphere, the stepwise deprotonation of the ternary complex VOABH takes place with pK values of 9.1, 9.7 and 9.8, 10.3; these can be ascribed to the parallel deprotonation of the side-chain ammonium group and the remaining water molecule in the coordination sphere of VO(IV).

Neither pH-metry nor EPR detected the formation of non- oxo tris(catecholato) complexes: the experimental condi- tions did not favor the oxo group displacement reaction as the concentration and the excess of catecholic ligands were not high enough. However, UV-Vis spectrophotometry did indicate their formation, as the color of the solution was reddish-blue and its intensity was about one order larger than that of the normal VO(IV) complexes (while e is in the range 10-30 cm- J mol- J dm 3 for VO(IV) complexes, the value for non-oxo complexes is a few thousands [ 11 ] ).

It may be concluded that catecholamines, being more potent VO (IV) binders than maltol at physiological pH, will partly (via the formation of ternary complexes) or fully dis- place maltol from the complex VO (malt)2.

3.6. VO(IV)-maltol-oxalic acid and VO(1V)-maltol-citric acid

Both oxalate and citrate are constituents of blood plasma: the concentration of the former is ~0.01 mmol dm -3, and that of the latter ten times higher [21]. Citrate generally behaves as a tridentate ligand in its complexes, and is regarded as one of the most important low molecular mass

T. Kiss et al. / lnorganica Chimica Acta 283 (1998) 202-210 209

metal ion binders in plasma, especially of hard or borderline metal ions.

Evaluation of the pH-metric speciation measurements revealed that ternary complex formation is favored slightly for oxalic acid (see Table 3). Comparisons of the binding donors, the stability constants and the ratios of the stepwise formation constants of the binary complexes do not demon- strate any specific difference between the VO(IV) binding abilities of the two ligands; this result is therefore in accor- dance with expectations. Also, the spectral parameters of the VO (IV) complexes formed by the two ligands are practically the same. Therefore it is not an unexpected result that the ternary species VOAB exhibits the same EPR parameters (gll = 1.939, All = 172X 10 -4 cm -1) as VOA2 (gtl = 1.939, AII=171×10 -4 cm -1) and VOB2 (g11=1.939, All = 172× 10 - 4 c m - 1 ) and therefore cannot be observed dis- tinctly. Moreover, the generally good agreement between the distribution curves calculated from potentiometric measure- ments and the experimental EPR measurement supports the goodness of the potentiometric fitting. In fact, consistent with the distribution curves, EPR spectroscopy substantiates that, in the ternary system, the oxalate complexes VOB and VOBe predominate below pH 4 whereas the VOA2H_ j species of maltol is the main mononuclear species in basic media above pH 9.

Unlike oxalic acid, citric acid strongly prefers ternary com- plex formation (see the data in Table 3). As illustrated in Fig. 9, the ternary complexes VOABH, VOAB and VOABH_ t predominate in the wide pH range between 5 and 9. As maltol can coordinate to VO(IV) exclusively through the (O- ,=O) donor set, only the binding mode of citric acid can change in the mixed ligand complexes protonated to different extents.

The EPR spectra of the VO (IV)-maltol-citric acid system at a metal ion-ligand ratio 1:1:1 and at different pH values highlight a very interesting feature (see Fig. 10): the spectra in the pH range 2-10 are well resolved and no decrease in signal intensity can be observed. This suggests that the pres- ence of maltol suppresses considerably the formation of di- nuclear VO(IV) complexes of citrate, (VO)2B2H_I and (VO)2B2H_2, which are predominant species in the binary

1.0 T

0.8 ~ VO(IV)-A-B

0 6 / vo(Iv)-B

~ °"4t\/ j \

~" 0.2

VO 0.0 F "1-.-. ~ r - , - - " q I r

2 3 4 5 6 7 8

pH

Fig. 9. Predominance d ia~am of the VO(IV)-maltol(A)-ci trate(B) sys- tem at i:2:2 metal ion to ligand ratio, Cvo =0.002 mol dm -3.

10 mT | I pH 2?

3.2

~ p H 9.2

pH 11.0

Fig. 10. High field parallel region of frozen solution EPR spectra (X-band) recorded at 140 K of the VO (IV)-maltol--citrate 1:1:1 system at various pH values, Cvo = 0.004 mol d m - 3

system between pH 3 and pH 9 [ 10 ]. Apart from the complex formed at low pH (<3 .5 ) , which has EPR parameters gll = 1.938 and Aji = 176 x 10 - 4 cm-1 (probably a carboxy- late-bound VO(IV)--citrate species VOBH), there is a major complex between pH 3 and pH 9 which exhibits spectral parameters ofgll = 1.942,AII = 167 X 10 - 4 cm- t and ~tma x ( 8

(mol -~ cm -1 dm3)) 765 nm (23), 576 (21). In this pH range, two ternary species, VOABH and VOAB, are formed. According to the EPR results, they should have the same binding mode, presumably containing (COO- ,COO-) coordinated citrate, and the proton loss of VOABH with pKvoABn = 3.44 can be ascribed to the dissociation of the non-coordinated terminal carboxylic function. The deproton- ation of the alcoholic-OH and the rearrangement of citrate to ( C O O - , O - ) coordination takes place only at higher pH (pKvoAB ---- 7.97 ). This is observed distinctly through the res- onances at gll = 1.947 and All = 161 × 10 - 4 cm-i . This spe- cies exhibits a vanadium parallel coupling constant which is intermediate between those of monomeric (COO- ,O- )2 [10] and the trans-VOA2 complex of maltol (157 and 1 6 3 × 10 - 4 cm - 1, respectively), indicating a mixed bonding mode. Minor amounts of VOA2 can also be observed at pH > 7, as deduced from the broader resonances. At higher pH (10.95), the decrease in the signal intensity indicates the formation of oligonuclear hydroxo complexes.

These results clearly indicate that, if they meet, citrate will certainly react with VO(malt)2, and the drug may be trans- ported in the blood serum, presumably in the form of its ternary species with citrate, VOAB. A more detailed study of the speciation of VO(IV) in serum is in progress in our laboratories.

210 T. Kiss et al. / Inorganica Chimica Acta 283 (1998) 202-210

Table 4 Speciation of vanadium (in VO%) in the different VO(IV)-maltolate- ligand B (1:2:2) ternary systems at pH 7.4

Ligand B VO(malt) ~ VO(malt) B VO(B)

Monophosphate ~ 100 (?) Diphosphate 8 42 50 Triphosphate 20 75 5 AMP 95 5 ADP 78 20 2 ATP 80 15 5 Dopamine 2 19 79 L-Dopa 1 19 80 Tiron 1 2 97 Citrate 22 60 18 Oxalate 80 20

The formation of vanadium(V) is assumed to be connected strongly with its capability to exert an insulin mimetic effect [24]. The redox properties of V O ( I V ) complexes are again influenced significantly by the chemical environment of the redox centre.

Acknowledgements

The work was supported by the Hungarian Research Fund (Project No. OTKA T23776, and the Hungarian Ministry of Education (FKFP 0013).

4. Conclusions

The stability data and structural considerations discussed above confirm the assumption that VO(mal t )2 undergoes transformations in the organism when it meets and reacts with potential low molecular mass V O ( I V ) binders in the various biological fluids. Table 4 clearly illustrates this, showing spe- ciation data for vanadium(IV) , expressed as percentages of the V O ( I V ) found in the various chemical environments in the various V O ( I V ) - m a l t o l ( A ) - l i g a n d B systems at physi- ological pH. The model calculations were performed in all systems at a metal ion- l igand ratio of 1:2:2, although the relative excess of ligand B under in vivo conditions may vary significantly.

The data in Table 4 (although the results are somewhat contradictory) reveal that inorganic monophosphate is too weak a V O ( I V ) binder to be able to affect the speciation of VO(malt)2. The chelate-forming diphosphate and tfiphos- phate, however, can displace maltolate to rather a large extent from the bis complex, partly via the formation of a ternary complex VOAB, or via the full transformation into the bis complex of ligand B. Interestingly, compared with inorganic oligophosphates, nucleotides seem to be less potent compet- itors for the displacement of VO ( IV) from its maltolato com- plexes. At the same time, catechol derivatives are much stronger V O ( I V ) binders than maltolate; accordingly, mal- tolate is almost completely displaced, and V O ( I V ) is bound almost exclusively to catechols at the physiological pH. The potentially tridentate citrate readily forms a ternary complex of 1:1:1 composition; this takes place to a much lower extent with the bidentate oxalate, which is a much weaker compet- itor than citrate to maltolate.

Finally it should be mentioned that ligand displacement reactions with bioligands are not the only possible transfor- mation reactions of VO(mal t )2 in biological fluids: oxidation of the binary and ternary complexes of maltol can also occur.

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