Available online at www.sciencedirect.comJOURNAL OF

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Journal of Inorganic Biochemistry 102 (2008) 1589–1598

InorganicBiochemistry

Structural characterization and reactivity ofc-octamolybdate functionalized by proline

Els Cartuyvels, Kristof Van Hecke, Luc Van Meervelt, Christiane Gorller-Walrand,Tatjana N. Parac-Vogt *

Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, B-3001 Leuven, Belgium

Received 13 July 2007; received in revised form 12 February 2008; accepted 13 February 2008Available online 23 February 2008

Abstract

The reaction of molybdate and DL-proline at pH 3.4 results in the formation of a Na4[Mo8O26(proO)2] � 22H2O complex (pro = pro-line) in which two proline ligands are attached to molybdenum(VI) ions via monodentate coordination of the carboxylate groups. Thestructure of the complex was determined by single crystal X-ray diffraction and by combination of 1H, 13C and 95Mo NMR spectroscopytechniques in solution. The structure of the complex is strongly dependant on the pH. At native pH 3.4 the octamolybdate-type structureseems to be present in solution, but the increase of pH to 5.8 resulted in a rearrangement of the structure to a heptamolybdate-type struc-ture. At physiological pH, the polyoxometalate framework was completely dissociated into the monomeric MoO2�

4 unit. The reactivity ofthe Na4[Mo8O26(proO)2] � 22H2O towards the hydrolysis of ATP was tested at different pH values. While in solution at pH 3.4 the hydro-lysis proceeded to yield AMP (adenosine monophosphate) and ADP (adenosine diphosphate) in nearly equal amounts, reaction mixtureat pH 5.8 gave ADP as the only product of hydrolysis after 24 h of reaction. At neutral pH, the hydrolysis of ATP was slower, but itproceeded to yield 75% of ADP after 48 h of reaction.� 2008 Elsevier Inc. All rights reserved.

Keywords: Octamolybdate; Proline; ATP hydrolysis; 31P NMR; 95Mo NMR

1. Introduction

Polyoxometalates (POMs) are a large family of metal–oxygen clusters that contain early transition metals (mostcommonly V, Mo, and W) in their high oxidation states[1]. The diversity in structure and composition of POMsallows for a wide versatility in terms of shape, polarity,redox potentials, surface charge distribution, acidity,resulting in many possible applications in the fields ofcatalysis, electronics and magnetic materials, among others[2–4]. In recent years there has been a growing interest inthe biological activity of POMs and their relevance in med-icine, especially after it was shown that a range of POMsexhibits potent antiviral and anti-tumor activities [5–9].

0162-0134/$ - see front matter � 2008 Elsevier Inc. All rights reserved.

doi:10.1016/j.jinorgbio.2008.02.005

* Corresponding author. Tel.: +32 16 327612; fax: +32 16 327992.E-mail address: Tatjana.Vogt@chem.kuleuven.be (T.N. Parac-Vogt).

Various POMs have been reported to interact with viralsurface proteins and to inhibit the replication of severaltypes of viruses [10–14]. Recent studies have shown thatPOMs have superior ability to selectively precipitate prionproteins, a property which could be useful in the develop-ment of immonoassays capable of detecting extremelylow concentrations of infectious prions in blood and cere-brospinal fluid [15]. Selective interaction of POMs withbasic fibroplast growth factor, a globular single-chain hep-arin-binding polypeptide synthesized by different cell types,has been also demonstrated [16]. Compounds that selec-tively bind and recognize fibroplast growth factors mayoffer new platforms for the design and synthesis of noveltypes of inhibitors of tumor angiogenesis.

First reports describing the anti-tumoral activity ofPOMs revealed that anti-tumoral activity of heptamolyb-date, [Mo7O24]6�, on different type of tumor cells is poten-tially better than that of some commercial drugs [17–19].

Table 1Crystallographic data for the Na4[Mo8O26(proO)2] � 22H2O complex

Formula C10H62Mo8N2Na4O52

M (gmol�1) 1902.10Crystal dimensions (mm) 0.4 � 0.1 � 0.1Crystal system TriclinicSpace group P1a (A) 10.0027(9)b (A) 11.6214(10)c (A) 14.1881(12)a� 100.837(5)b� 109.377(7)c� 109.478(6)V/A3 1381.7(2)Z 1qcalc (lg/m3) 2.2862hmax (�) 142.80Radiation Cu-Kak (A) 1.54178F(000) 932T (K) 100(2)Measured reflections 12,609Unique reflections 5001Observed reflections (Io > 2r (Io)) 4382Parameters refined 343R1 0.0526xR2

a 0.1313R1 (all data) 0.0582xR2 (all data) 0.1346GOOF 0.990l (mm�1) 15.808CCDC-entry CCDC-660535

a Weighing scheme: x ¼ 1=½q2ðF 2oÞ þ ð0:0896PÞ2 þ 0:0000P �; P ¼ ½ðF 2

oþ2F 2

cÞ=3�.

1590 E. Cartuyvels et al. / Journal of Inorganic Biochemistry 102 (2008) 1589–1598

The most recent studies have shown that [Mo7O24]6� evenexhibits potent anti-tumor activity against human gastriccell lines as well as against pancreatic cancer cells thatare known to form a recalcitrant tumor which has a poorclinical outcome after surgery, chemotherapy, and radio-therapy [20,21].

Although the biological activity of POMs has been welldocumented, the molecular basis for their action remainslargely unexplored. In that respect, studies of interactionsbetween POMs and building blocks of proteins are of sub-stantial interest. Moreover, modification of POMs surfaceby amino acids and peptides may offer a way towards thetuning of the POMs biological properties and result innovel synergic effects. The natural amino acids with theirvariety of side chains represent a rich source for the synthe-sis of functionalized POMs. However, up to date only fewexamples of structurally characterized POMs with cova-lently bound amino acids have been described [22–25].Among these structures, c-type octamolybdates coordi-nated by alanine and methionine amino acids are of specialinterest since they have been recently reported to exhibitdifferential cell-growth inhibition on hepatocellular andbreast cancer cell lines in a dose-dependent manner [26].

In this study we report the synthesis and X-ray singlecrystal structure of a c-octamolybdate functionalized byDL-proline. Since a stability of functionalized POMs underphysiological conditions is of crucial importance for under-standing their biological activity, we have also examinedsolution chemistry of this novel compound by means ofmulti-nuclear NMR spectroscopy. The reactivity towardsadenosine triphosphate (ATP), which has been implicatedas a key in the anti-cancer activity of polyoxomolybdates[20,21,27], will be also discussed.

2. Experimental

2.1. Materials and measurements

ATP nucleotide was purchased from Applichem in theform of disodium salt. DL-proline was obtained as a pureproduct from Acros Organics. Sodium molybdate and allother reagents were of analytical grade and were used assuch. The pH of the sample solutions for the protonnuclear magnetic resonance (NMR) measurements wereadjusted with D2SO4 and NaOD.

Elemental analysis for carbon, hydrogen and nitrogenwas performed on a CE-Instruments EA-1110 elementalanalyzer. Fourier transform infra red (FTIR) spectra wererecorded on a Bruker IFS-66 spectrometer, using the KBrpellet method.

2.2. Synthesis and characterization of

Na4[Mo8O26(proO)2] � 22H2O

DL-Proline (0.23 g, 2 mmol) was added to an aqueoussolution of Na2MoO4 (0.5 g, 2 mmol) and the solutionwas acidified by addition of HNO3 to pH 3.4. After solu-

tion was left for several days at room temperature colorlesscrystals were obtained. Calculated for Na4[Mo8O26-(proO)2] � 22H2O (Na4Mo8O52C10N2H62, Mw = 1902.06):C, 6.31; H, 3.28; N, 1.47. Found: C, 6.42; H, 3.33; N,1.49. IR (KBr pellet, cm�1): 699 multiplet (m), 846 singlet(s), 926m, 1384s and 1632s. 1H NMR d (D2O): 1.85–2.22(m, 3H, c-CH2, b-CH); 2.33 (m, 1H, b-CH); 3.37 (m, 2H,d-CH2); 4.13 (m, 1H, a-CH). 13C NMR d (D2O): 25.10(Cc); 30.48 (Cb); 47.52 (Cd); 62.46 (Ca); 176.32 (COOH).

2.3. Crystal structure determination

Intensity data were collected at low temperature (100 K)on a SMART 6000 diffractometer equipped with a CCDdetector using Cu-Ka radiation (k = 1.54178 A). Theimages were interpreted and integrated with the programSAINT from Bruker. The structure was solved by directmethods and refined by full-matrix least-squares on F2

using the SHELXTL program package [28,29]. Non-hydrogen atoms were refined anisotropically and thehydrogen atoms in the riding mode with isotropic temper-ature factors fixed at 1.2-times U(eq) of the parent atoms.

Crystal parameters, data collection and refinement sta-tistics are shown in Table 1. Selected bond lengths are listedin Table 2.

Table 2Bond lengths (A) in the coordination sphere around molybdenum atomsin the Na4[Mo8O26(proO)2] � 22H2O complex

Atom 1 Atom 2 Bond length

Mo1 O2 2.119(4)Mo1 O3 1.732(4)Mo1 O4 1.708(4)Mo1 O5 2.274(4)Mo1 O6 2.102(4)Mo1 O13 1.879(5)Mo2 O6 1.924(4)Mo2 O7 1.724(4)Mo2 O8 1.916(5)Mo2 O9 2.229(4)Mo2 O11 1.711(5)Mo2 O12 2.365(4)Mo3 O5 1.897(4)Mo3 O6 2.141(4)Mo3 O9 1.927(4)Mo3 O10 1.717(4)Mo3 O9 1.927(4)Mo3 O12[2�x, 1�y, 1�z] 1.739(4)Mo4 O5 2.282(4)Mo4 O13 1.982(4)Mo4 O14 1.709(5)Mo4 O15 1.739(5)Mo4 O8[2�x, 1�y, 1�z] 1.930(4)Mo4 O9[2�x, 1�y, 1�z] 2.254(4)

Fig. 1. Molecular structure of the Na4[Mo8O26(proO)2] � 22H2O complexwith labeling scheme of the asymmetric unit. Sodium cations and watermolecules are omitted for clarity.

E. Cartuyvels et al. / Journal of Inorganic Biochemistry 102 (2008) 1589–1598 1591

2.4. Nuclear magnetic resonance (NMR) measurements

1H, 13C and 31P NMR spectra were recorded in deuter-ated water solutions on a Bruker Avance 400 spectrometer.95Mo NMR spectra were recorded at Bruker Avance spec-trometer operating at 600 MHz. In the case of 1H and 13CNMR measurements chemical shifts were referenced toDSS (sodium 2,2-dimethyl-2-silapentane-5-sulfonate). For31P NMR measurements 85% H3PO4 solution was usedas a 0 ppm reference. In a typical hydrolysis experiment,500 lL of 99.8% D2O solution containing 30 mM ofNa4[Mo8O26(proO)2] and 10 mM of ATP were kept atroom temperature. The solutions were analyzed by meansof 31P NMR spectroscopy 5 min, 6 h and 24 h after mixing.

3. Results and discussion

3.1. Structure of Na4[Mo8O26(proO)2] in the solid state

The crystal structure of Na4[Mo8O26(proO)2] consists ofc-octamolybdate [Mo8O26]4� anions, DL-proline ligands,sodium cations and water molecules. The DL-proline iscoordinated to molybdenum atoms via monodentate car-boxylate-oxygen atoms (Fig. 1). The space group is the cen-trosymmetric P1, hence both enantiomers D- and L-prolineare present in the structure and the asymmetric unit con-tains half of the total molecule. The c-octamolybdate[Mo8O26]4� anions are built up by eight edge-sharingMoO6 octahedra, but with two additional terminal posi-tions, thereby satisfying the octahedral coordination ofall molybdenum atoms. This arrangement is similar as in

the previously reported Na4[Mo8O26(alaO)2] andNa2[Mo8O26(lysO)2] complexes where ala and lys representalanine and lysine amino acids [22,26]. Since the com-pounds were prepared at pH 3.4, the proline a-aminogroup is protonated (pKa � 10.6). Therefore, the prolineligands are in their zwitterionic form, which was confirmedby a difference Fourier electron density map.

An intramolecular hydrogen bond is formed betweenthe proline amino hydrogen atom H1B(N1) and O10 ofthe same octamolybdate complex (N1-O10 distance of2.888(7) A) and an intermolecular hydrogen bond isformed between the amino H1A(N1) and O5[1 � x,1 � y,1 � z] of another complex (N1-O5 distanceof 2.719(7) A). The free carboxyl oxygen atom O1 of theproline ligand is contacting O18 [x,y, 1 + z] (2.870(6) A)and O20 [1 � x, 2 � y, 1 � z] of a sodium–water chain(2.921(7) A). Furthermore, a contact with water moleculeO26 [1 � x, 2 � y, 1 � z] is observed (2.986(7) A) (Fig. 2).

The IR spectrum (KBr pellet, cm�1) of Na4[Mo8O26-(proO)2] exhibited strong absorption maxima at 699m,846s, 926m which confirm the presence of Mo–Oterminal

and Mo–Obridging. The results are in good agreement withother structures containing octamolybdate skeleton [26].

Fig. 2. Packing diagram of the Na4[Mo8O26(proO)2] structure along the b-axis, showing layers of c-octamolybdate complexes, along the (002) plane,alternated with layers containing sodium–water chains along the (001) plane. Sodium and molybdenum cations are colored purple and green, respectively.

1592 E. Cartuyvels et al. / Journal of Inorganic Biochemistry 102 (2008) 1589–1598

Strong absorption maxima at 1384 and 1632 cm�1 arecharacteristic of COOas and COOsym stretching frequen-cies of proline ligand. Shifts of ca. 10 cm�1 to lower energywere observed for these stretching frequencies upon com-plexation, indicating carbonyl oxygen coordination to themolybdenum(VI) ion.

It is noteworthy mentioning that all our attempts to iso-late crystals from the reaction mixtures prepared under dif-ferent conditions, such as pH and temperature, wereunfortunately not successful. Similarly, efforts to obtainsingle crystals of c-octamolybdate functionalized by otheramino acids, except than those already reported in litera-ture [22,26], failed. This may be due to inefficient packingof the amino-acid side chains in the crystal lattice of thecomplex.

3.2. 1H, 13C and 95Mo NMR solution studies of

Na4[Mo8O26(proO)2]

Dissolving the Na4[Mo8O26(proO)2] � 22H2O in D2Oresulted in a solution with the pH value of 3.4. ProtonNMR spectrum of the dissolved complex exhibited peaksthat are characteristic of the proline ligand (Fig. 3a). Com-parison of 1H NMR spectra of Na4[Mo8O26(proO)2] �22H2O and free proline ligand indicated that they werenearly identical, which may be due to the weak bindingconstant and/or fast ligand exchange on the NMR timescale. In addition, due to the diamagnetic character ofMo(VI), the complexation shifts are expected to be rathersmall. However, the 13C NMR spectrum of Na4[Mo8O26-(proO)2] � 22H2O revealed that the carboxylate resonancein this compound is slightly shifted by 0.48 ppm upfieldas compared to the free proline ligand (Fig. 3b), consistent

with the coordination of the oxygen atom tomolybdenum(VI).

Considering the biological activity of related octamolyb-date complexes, an important question that needs to beaddressed is the solution structure and the stability of thistype of complexes under conditions applied under in vitro

screening studies. When the solution of the complex waskept at 37 �C for several days no observable changes in1H and 13C NMR spectra were detected. Similarly, NMRspectra recorded in solutions with pH 5.5 and pH 7.4 didnot provide any further information about the stability ofNa4[Mo8O26(proO)2] � 22H2O under these conditions.

Due to the limited information that can be obtainedfrom 1H and 13C NMR spectroscopy, which cannot dis-criminate between bound and free proline, we employed95Mo NMR spectroscopy as at tool for assessing the poly-oxomolybdate structure in solution.

The 95Mo NMR spectrum of Na4[Mo8O26-(proO)2] � 22H2O solution at pH 3.4 exhibited two promi-nent signals at 86 and 10 ppm. Similar 95Mo NMRspectrum, that has been attributed to b-[Mo8O26]4� hasbeen previously reported [30]. Although this may suggestthe presence of an octamolybdate framework in the solu-tion, the NMR data are not sufficient to prove that thestructure, established in the solid state, is also preservedin solution.

When the pH of the Na4[Mo8O26(proO)2] � 22H2O solu-tion was increased to 5.7, clear changes in the 95Mo NMRspectra were observed (Fig 4b). A sharp signal at 0 ppm isindicative of the MoO2�

4 ion, while the broader signal at34 ppm could be unambiguously attributed to [Mo7O24]6�.This indicates that [Mo8O26]4� structure was transformedinto a [Mo7O24]6� polyanion, which under this conditions(pH and concentration) exists in an equilibrium with the

4.0 3.5 3.0 2.5 2.0 4.0 3.5 3.0 2.5 2.0 1.5

150 100 50 150 100 50 0

[ppm]

[ppm]

A

B

Fig. 3. Proton (A) and 13C (B) NMR spectra of Na4[Mo8O26(proO)2] � 22H2O solution in D2O at pH 3.4.

E. Cartuyvels et al. / Journal of Inorganic Biochemistry 102 (2008) 1589–1598 1593

MoO2�4 . Further increase of the pH resulted in the com-

plete dissociation of the polyoxomolybdate framework intothe monomeric MoO2�

4 unit. The 95Mo NMR spectrumrecorded at the physiological pH 7.4 exhibited only a sharppeak at 0 ppm, characteristic of the MoO2�

4 ion (Fig. 4c).

3.3. Reactivity of Na4[Mo8O26(proO)2] � 22H2O towards

ATP

The ability of molybdates to inhibit ATP (adenosine tri-phosphate) generation has been implicated as a key fortheir anti-tumor activity. Several studies have shown that

[Mo7O24]6� interacts with nucleotides to form a pentamo-lybdodiphosphate-type structure that is comprised of fiveMoO6 octahedra forming a ring structure, which is cappedon either side by two phosphate moieties of the nucleotide[31,32]. Formation of the complex between [Mo7O24]6� andflavinmononucleotide (FMN), which is a prosthetic groupin flavoprotein, is proposed to inhibit ATP generationdue to the inability of FMN to transfer electrons that areneeded for the generation of ATP. On the other hand,the ability of [Mo7O24]6� to promote rapid ATP hydrolysismay also play a role in its anti-tumor activity [27]. Withthis in mind, and also considering recent reports about

120 100 80 60 40 20 0 120 100 80 60 40 20 0

a

b

c

[ppm]-20

Fig. 4. 95Mo NMR spectra of 25 mM solution containing Na4[Mo8O26(proO)2] � 22H2O at (a) pH 3.4; (b) pH 5.8; and (c) pH 7.4.

H2N

N

N

HO

P

O

-

O

P

O

O-

β

γ

1594 E. Cartuyvels et al. / Journal of Inorganic Biochemistry 102 (2008) 1589–1598

potent anti-tumor activity of amino-acid containing octa-molybdates [26], we set out to examine the reactivity ofNa4[Mo8O26(proO)2] � 22H2O toward ATP. Since the95Mo NMR measurements showed that the solution struc-ture of the initial complex strongly depends on the pH, theATP hydrolysis was examined by means of 31P NMR spec-troscopy under different pH values.

OHHO

ONN

PO

O

O-

O O

α

Scheme 1. Chemical structure of ATP.

3.3.1. Hydrolysis of ATP at pH 3.4

After mixing [Mo8O26(proO)2]4� with ATP and adjust-ing the pH of solution to 3.4, the ATP resonance at�10.3 ppm, which corresponds to Pa and Pc (Scheme 1),is shifted to �13.1 ppm, while the peak of Pb at�22.3 ppm remains unaffected (Fig. 4). After the reactionwas kept 6 h at room temperature, 31P NMR indicatedthe presence of several new peaks that were assigned basedon the literature data [27,33,34]. The signals at �3.3 ppmand �7.2 ppm correspond to [(O3POPO3)Mo6O18-(H2O)4]4�, while the signals at �1.1 ppm and 1.8 ppmcan be attributed to a-A[H6PMo9O34]3� and [(PO4)2Mo5-O15]6�, respectively. The peak at �5.7 ppm has beenpreviously assigned to an AMP–molybdate complex.Integration of 31P NMR resonances indicated that after6 h at room temperature nearly 50% of ATP was hydro-lyzed. After the reaction mixture was kept for 12 h fullhydrolysis of ATP was observed, yielding [(O3POPO3)-Mo6O18(H2O)4]4� and [(PO4)2Mo5O15]6� as the two mostprominent products of the hydrolytic reaction (Fig. 4).

The 31P NMR data indicate that the hydrolysis of ATPat pH 3.4 proceeds via two pathways, which implicates the

presence of two different ATP-molybdate intermediates.The first intermediate is most likely analogous to the[(PO4)2Mo5O15]6� pentamolybdate structure in which theterminal Pc phosphate group of the two ATP moleculescoordinates to molybdate [33,34]. Subsequent cleavage ofthe Pc–O–Pb bond results in liberation of the phosphateion and formation of a [(PO4)2Mo5O15]6� complex. Thesecond intermediate is likely to be analogous to the[(O3POPO3)Mo6O18(H2O)4]4� hexamolybdate structure inwhich Pc and Pb phosphate groups of ATP coordinateto molybdate [27]. In this case, the breakage of the Pa–O–Pb bond of ATP takes place leading to liberation ofthe P2O4�

7 ion and the formation of [(O3POPO3)-Mo6O18(H2O)4]4� product. Inspection of 31P NMR spectraduring the course of reaction reveled that initially hydroly-

E. Cartuyvels et al. / Journal of Inorganic Biochemistry 102 (2008) 1589–1598 1595

sis of ATP proceeds via a hexamolybdate-type intermedi-ate, since after 6 h the [(O3POPO3)Mo6O18(H2O)4]4� waspresent in 97%, while [(PO4)2Mo5O15]6� was present inonly 3%. However, at the end of hydrolytic reaction,[(O3POPO3)Mo6O18(H2O)4]4� and [(PO4)2Mo5O15]6� weredetected in nearly equal amount, indicating that both pen-tamolybdate and hexamolybdate-type intermediates arehydrolytically active. It is interesting to note that the[(PO4)2Mo5O15]6� has been also found to be active towardsthe ATP hydrolysis, so an additional pathway in which theATP is hydrolyzed through the [(PO4)2Mo5O15]6� productonce it is formed, is also plausible.

It is interesting to note that compared to [Mo8O26]4�

polyoxometalate, the [Mo8O26(proO)2]4� was less activetowards ATP hydrolysis. While in the reaction with[Mo8O26(proO)2]4�, significant amount of non-hydrolyzedADP was still present in solution after 24 h (Fig. 5c), inthe reaction promoted with [Mo8O26]4� under the sameconditions, full hydrolysis of ADP was observed, yieldingAMP, [(O3POPO3)Mo6O18(H2O)4]4, [(PO4)2Mo5O15]6�

and a-A[H6PMo9O34]3� as the final products of hydrolysis.This difference in reactivity points to the structural differ-ence between [Mo8O26(proO)2]4� and [Mo8O26]4� suggeststhat the solution structure of the [Mo8O26(proO)2]4� com-plex resembles the one found in the solid state in which twoproline ligands are coordinated to the octamolybdateframe. The coordination of proline ligands to the octamo-lybdate structure could make the formation of pentamo-

0 - 10 0 - 10

[ppm]

Fig. 5. 31P NMR spectra of the reaction mixture containing 10 mM of ATP aafter 6 h and (c) after 24 h at room temperature. The peak assignment[(O3POPO3)Mo6O18(H2O)4]4�; H a-A[H6PMo9O34]3�.

lybdate and hexamolybdate-type intermediates moredifficult, resulting in slower ATP hydrolysis.

3.3.2. Hydrolysis of ATP at pH 5.4

Immediately after adjusting the pH of the ATP andNa4[Mo8O26(proO)2] � 22H2O solution to 5.4 the 31PNMR spectra indicated that Pc phosphate resonance shiftsupfield by 1.3 ppm units to �8.3 ppm, while Pa and Pb

phosphate resonances remained unaffected (Fig. 6). Thereaction products analyzed after 24 h of reaction, couldbe identified on the basis of their 31P NMR shiftsas [(PO4)2Mo5O15]6� (2.1 ppm), [(HAMP)2Mo5O15]2�

(1.5 ppm) and an ADP–molybdate complex (at �8.1 ppmand �10.1 ppm). The large shift of Pc phosphate resonanceobserved upon mixing in combination with the detection of[(PO4)2Mo5O15]6� and ADP (adenosine diphosphate) asonly products of hydrolysis, suggest that ATP hydrolysisproceeds exclusively via formation of a pentamolybdatestructure in which the cleavage of the Pc–O–Pb bond resultsin the formation of a [(PO4)2Mo5O15]6� product.

Interestingly, exactly the same results were obtained inthe [Mo7O24]6� promoted hydrolysis of ATP [27]. Thisstrengthens our findings obtained by 95Mo NMR spectros-copy that upon increasing the pH to 5.4 the Na4[Mo8O26-(proO)2] � 22H2O complex converts into the heptamolyb-date-type structure. The heptamolybdate is known to reactwith phosphate moieties to form a pentamolybdate-typeintermediate structure, as described in several other reports

- 20 - 20 -30

a

b

c

nd 30 mM Na4[Mo8O26(proO)2] � 22H2O at pH 3.4: (a) upon mixing, (b)is as follows: d ATP; j ADP; N AMP; h [(PO4)2Mo5O15]6�; 4

0 - 5 - 10 - 15 - 20 -25

[ppm]

a

b

Fig. 6. 31P NMR spectra of the reaction mixture containing 10 mM of ATP and 30 mM Na4[Mo8O26(proO)2], adjusted to pH 5.4: (a) upon mixing, (b)after 6 h and at room temperature. The peak assignment is as follows: d ATP; j ADP; N H2PO�4 ; h [(PO4)2Mo5O15]6�.

1596 E. Cartuyvels et al. / Journal of Inorganic Biochemistry 102 (2008) 1589–1598

[27,33,34]. Recently it has been shown that interactions ofheptamolybdate with a DNA model substrate results inthe hydrolysis of the very stable phosphodiester bond [35].

3.3.3. Hydrolysis of ATP at pH 7.0

Mixing of ATP and Na4[Mo8O26(proO)2] � 22H2O at pH7.0 did not result in any changes in the 31P NMR spectrumof ATP, indicating a weak or absent interaction betweenATP and molybdate. The 31P NMR spectrum recordedafter 48 h of reaction at room temperature revealed thatca 75% of ATP was converted into ADP, indicating that

0 - 5 - 10 [ppm

Fig. 7. 31P NMR spectra of the reaction mixture containing 10 mM of ATP aafter 48 h at room temperature. The peak assignment is as follows: d ATP; j

similarly to the hydrolytic reaction at pH 5.4, the ATPhydrolysis proceeds exclusively via formation of a penta-molybdate structure in which the cleavage of Pc–O–Pb

occurs. The phosphate ion, which is the expected productof the hydrolytic reaction, is in the presence of molybdateat neutral pH found to be in equilibrium with the[(PO4)2Mo5O15]6� ion [34]. Indeed, the presence of freephosphate and [(PO4)2Mo5O15]6� was also confirmed inthe 31P NMR spectra (Fig. 7).

According to our 95Mo NMR measurements of theNa4[Mo8O26(proO)2] � 22H2O complex, and other studies

- 15 -20 ]

-25

a

b

nd 30 mM Na4[Mo8O26(proO)2], adjusted to pH 7.0 (a) upon mixing, (b)ADP; h [(PO4)2Mo5O15]6�; N H2PO�4 .

E. Cartuyvels et al. / Journal of Inorganic Biochemistry 102 (2008) 1589–1598 1597

of polyoxomolybdates reported in the literature [30,36], theamount of the [Mo7O24]6� type structure significantlydecreases when increasing the pH from mildly acidic toneutral values, however, even these small amounts of thepolyoxometalate ion present at neutral pH are apparentlysufficient for the hydrolytic reaction to occur. It is in thiscontest important to point out that ATP hydrolysis cannot be induced by the monomeric MoO2�

4 ion [27].

4. Conclusions

Reaction of molybdate and DL-proline at pH 3.4 resultsin the formation of a Na4[Mo8O26(proO)2] � 22H2O com-plex in which two proline ligands are attached to molybde-num(VI) ions via monodentate coordination of carboxylategroups. The NMR data indicate that an increase in pH to5.7 results in the structural rearrangement into a heptamo-lybdate-type complex. Under physiological conditions, thepolyoxometalate ion is fully dissociated into monomericmolybdenum(VI) species. Our data suggest that due tothe instability of the Na4[Mo8O26(proO)2] � 22H2O struc-ture, the biological tests performed with this type of com-plexes have to be critically considered. Despite theapparent instability of the Na4[Mo8O26(proO)2] � 22H2Ostructure the complete hydrolysis of ATP was observed inthe pH range at which the polyoxometalate structure waspresent in solution. The hydrolysis of ATP occurred evenin solutions at neutral pH, in which polyoxometalate struc-tures are expected to be present in very small amounts.However, since it has been found that the extracellularpH in some solid tumors can be as low as 5.7, with thepH of the tumor interstitial fluid as low as pH 6.5 [37],one could imagine that the anti-tumor activity of this typeof polyoxometalate complexes can be somehow related totheir reactivity towards the ATP.

Abbreviations

ala alanine

CCD charge coupled device

DSS sodium 2,2-dimethyl-2-silapentane-5-sulfonate

lys lysine

POM polyoxometalate

pro proline

Acknowledgments

T.N.P.V. thanks FWO-Flanders (Belgium) for the post-doctoral fellowship. Financial support has been providedby the K.U. Leuven (GOA 03/03).

Appendix A. Supplementary data

Tables of atomic coordinates, bond lengths, bond anglesand thermal parameters are available from the Cambridge

Crystal Data centre. The CCDC reference number is660535.

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