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DaltonTransactions
PAPER
Cite this: Dalton Trans., 2015, 44,10392
Received 2nd April 2015,Accepted 30th April 2015
DOI: 10.1039/c5dt01279a
www.rsc.org/dalton
Oxaliplatin vs. cisplatin: competition experimentson their binding to lysozyme†
Daniela Marasco,a,b Luigi Messori,c Tiziano Marzoc and Antonello Merlino*b,d
The model protein hen egg white lysozyme was challenged with oxaliplatin and cisplatin. ESI mass
spectrometry, surface plasmon resonance and thermal shift analyses demonstrate the formation of a bis-
platinum adduct, though in very small amounts. Crystals of the bis-platinum adduct were obtained using two
different preparations and the X-ray structures were solved at 1.85 Å and 1.95 Å resolution. Overall, the
obtained data point out that, under the analyzed conditions, the two Pt drugs have similar affinities for the
protein, but bind on its surface at two non-overlapping sites. In other words, these two drugs manifest a sig-
nificantly different reactivity with this model protein and do not compete for the same protein binding sites.
Introduction
Platinum drugs such as cisplatin, carboplatin and oxaliplatin(Fig. 1) are routinely used for the medical treatment of varioustypes of cancers.1,2 The mechanism of action of these com-pounds is believed to rely mostly on Pt(II) covalent binding tothe N7 atoms of DNA guanine bases, leading to severe nucleicacid damage associated with inhibition of both replicationand transcription and eventually cancer cell apoptosis.3–5
Although these three Pt compounds have similar chemicalstructures and DNA binding properties, they manifest signifi-cant differences in their respective pharmacological profiles,resulting in different therapeutic uses and indications.6 In par-
ticular, the toxicity profile of oxaliplatin greatly differs fromthat of cisplatin by its moderate nephrotoxicity and from thatof carboplatin by its limited hematological toxicity. Owing toits atypical behaviour, oxaliplatin is used for the cure of color-ectal cancer, a type of tumor scarcely responsive to cisplatinand carboplatin.7–9
To better understand the pharmacological profiles of cispla-tin, carboplatin and oxaliplatin it is imperative to study themechanism by which these Pt drugs interact withproteins.10–13 During the last 15 years, protein platination bythese three Pt-based drugs has been investigated by varioustechniques.10,12,14,15 Electrospray mass spectrometry (ESI MS)measurements14,16–18 and crystallographic studies18–25 onsmall model proteins allowed a number of details on theprotein metalation process to be obtained. In this framework,hen egg white lysozyme (HEWL) has been extensively used as amodel protein.18–23 Notably, this well-behaved enzyme hasbeen studied in complexes with cisplatin,18–20 carboplatin,20
oxaliplatin,21 cis-PtI2(NH3)222 and trans-Pt derivatives.23
It was recently shown that HEWL is able to bind cisplatinand oxaliplatin in two distinct binding sites,21 whereas cispla-tin and carboplatin compete for the same residue side chain.20
In particular, upon reaction between cisplatin and HEWL, pla-tination occurs at the level of His15, with a cis-Pt(NH3)2
2+ frag-ment bound to the protein surface.18–20 On the other hand,the reaction between HEWL and oxaliplatin leads to the for-mation of a complex with the Pt(dach)2+ moiety bound toAsp119.21 These data suggest that, at least in principle, it ispossible for these two Pt-drugs to bind HEWL simultaneously,although it is unpredictable if the binding of the former drugcan alter the affinity of the protein for the latter. In fact, evensubtle structural alterations can affect the HEWL dynamics, asalready demonstrated in other studies,26 thus altering the drugaffinity for the protein.
Fig. 1 Structures of cisplatin, carboplatin and oxaliplatin (trans-l-oxalato-diaminocyclohexane platinum), a third-generation diammino-cyclohexane (dach) platinum compound.
†Electronic supplementary information (ESI) available. See DOI: 10.1039/c5dt01279a
aDepartment of Pharmacy, University of Naples Federico II, via Montesano 12, 80120
Napoli, ItalybInstitute of Biostructures and Bioimages, University of Naples Federico II, via
Mezzocannone 16, 80100 Napoli, ItalycDepartment of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto
Fiorentino (FI), ItalydDepartment of Chemical Sciences, University of Naples Federico II, Via Cintia,
I-80126 Napoli, Italy. E-mail: [email protected]; Fax: +39081674090;
Tel: +39081674276
10392 | Dalton Trans., 2015, 44, 10392–10398 This journal is © The Royal Society of Chemistry 2015
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These arguments prompted us to investigate the formationof the adducts that are obtained when oxaliplatin and cisplatinare incubated with HEWL. These results are of potential inter-est if one considers that cisplatin and oxaliplatin might beclinically used in combination and that studies of the cispla-tin/oxaliplatin combination on patients with advanced ovariancancer already revealed acceptable hematologic toxicity, andreversible cumulative neurosensory toxicity.27–29 Simultaneousinjection of cisplatin and oxaliplatin was reported to produce asynergistic antitumor activity against cisplatin-resistantmurine leukemia L1210.30,31 In addition, the feasibility andefficacy of a cisplatin/oxaliplatin combination were evaluatedin patients with testicular tumors and encouraging resultswere obtained.32
Along this line of reasoning, it should be also recalled thatphase I clinical studies of a carboplatin/oxaliplatin combi-nation revealed that the combination treatment seems feasibleand active, although it does not allow a significant increase inplatinum dose-intensity delivery.33
Results and discussionElectrospray mass spectrometry
First, we recorded ESI MS spectra of the HEWL sample in thepresence of a mixture of equimolar concentrations of cisplatinand oxaliplatin (Fig. 2). A number of HEWL–platinum adductsare clearly visible in this experiment whose assignment isreported in Table 1. Interestingly, a protein–metallodrugcomplex is detected corresponding to a bis-Pt adduct (bis-adduct) with lysozyme simultaneously bearing a Pt(NH3)
2+
fragment and an oxaliplatin molecule (peak l). The obser-vation of this bis-adduct, though in a quite small amount,
implies that the two drug binding sites on the HEWL surfaceare independent of each another.
Crystallography
Afterward, we tried to grow crystals of the bis-adduct followingdifferent strategies (see Methods for further details). Attemptsto obtain crystals of the bis-adduct by mixing the protein witha mixture of cisplatin + oxaliplatin or adding first cisplatin andthen oxaliplatin to the protein sample failed. In these cases weobtain crystals of the adduct between HEWL and cisplatinalone, i.e. structures of HEWL with the Pt centre only bound toHis15. On the other hand, tetragonal crystals of the bis-adductwere successfully grown using a protein sample that was firstincubated for 24 h with an excess of oxaliplatin and then incu-bated for 24 h with an excess of cisplatin (final protein tometal ratio 1 : 10) and a reservoir solution containing 0.6 MNaNO3, 0.1 M sodium acetate pH 4.4 and 20% ethylene glycol.Under the same experimental conditions, we also grew tetra-gonal crystals of a protein solution obtained when a HEWLsample with an excess of cisplatin (1 : 10 protein to metal ratio)is mixed with equal amounts of protein with an excess of oxali-platin (1 : 10 protein to metal ratio). The results of the struc-tural analyses carried out on crystals grown using these lasttwo strategies show that the bis-adduct is formed (Fig. 3). The
Fig. 2 ESI MS spectrum recorded to investigate the possible compe-tition between oxaliplatin and cisplatin for the same binding site. Thespectrum has been collected after 24 h incubation of equimolar con-centrations of the two drugs (cisplatin and oxaliplatin) with HEWL in20 mM ammonium acetate buffer pH = 6.8 at 37 °C (see Methods forfurther details). For the peak assignment see Table 1.
Table 1 Assignment of peaks in the ESI MS spectrum (Fig. 2)
Label Bound fragment Mass (Da)
a Pt + Na+ 14 517.76b [Pt(NH3)2]
2+ 14 530.77c [Pt(NH3)Cl]
+ 14 550.50d [Pt(NH3)2Cl]
+ 14 567.76e [Pt(NH3)2Cl]
+ + Na+ 14 590.80f [Pt(dach)]2+ 14 610.82g [Pt(NH3)2Cl2] + Na+ 14 627.78h [Pt(NH3)2Cl2] + 2Na+ 14 648.82i Oxaliplatin 14 701.84l Oxaliplatin + [Pt(NH3)]
2+ 14 915.82j 2 Oxaliplatin 15 098.89
Fig. 3 Overall structure of the bis-Pt HEWL adduct. Coordinates andstructure factors were deposited in the Protein Data Bank (PDB code4Z46).
Dalton Transactions Paper
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structures were refined to an R-factor of 0.155 and 0.159 andan R-free of 0.218 and 0.230 for the datasets collecting usingthe former and the latter procedure, respectively. The resultsobtained using the two datasets are almost identical, and forthis reason only one structure will be described. Both struc-tures were deposited in the Protein Data Bank (accession code4Z46 and 4ZEE). The overall structure of HEWL in the bis-adduct is not significantly affected by the binding of the twodrugs.
The CA root mean square deviation (rmsd) from the struc-ture of ligand-free HEWL (PDB code 193L) is as low as 0.26 Å.When the structure of the bis-adduct is compared with that ofHEWL solved at ultrahigh resolution (PDB code 2VB1) andcrystallized under similar experimental conditions, but in thetriclinic space group, rmsd is in the range 0.79–0.81 Å. Themost significant differences between the atomic resolutionstructure of HEWL and the bis-adduct (Fig. S1 in the ESI†) arelocated in loop regions, close to residues 44–51 and 69–73(Fig. S2A in the ESI†), at the end of the helix constituted byresidues 88–101 and in the region connecting this helix to thatencompassing residues 108–115 (Fig. S2B in the ESI†). Thesestructural differences seem to be due to an altered dispositionof nitrate ions on the molecular surface rather than thebinding of the drugs (Fig. S2A and B†).
The inspection of the electron density maps of both bis-adduct structures suggests that there are two Pt binding siteson the HEWL surface (Fig. 4A and B and Fig. S3 and S4 in theESI†). As expected on the basis of the comparison between thestructure of HEWL–cisplatin20 and that of HEWL–oxaliplatin,21
the former binding site, i.e. presumably that of an oxaliplatinmoiety, is close to Asp119 (Fig. 4A). The latter, i.e. that presum-ably due to the binding of a cisplatin moiety, is found close toHis15 (Fig. 4B). No other binding sites are found, althoughmultiple sites of cisplatin on HEWL had been found by ESI-MScombined with trypsin digestion.34
In both the Pt binding sites observed in the bis-adduct, thePt center has a low occupancy (Pt occupancy factor = 0.40,B-factor = 52.5–55.8 Å2 for the Asp119 binding site and Pt occu-pancy factor = 0.35, B-factor = 63.6–67.0 Å2 for the His15binding site, respectively). The location of the Pt centres isconfirmed by the inspection of anomalous difference maps,which clearly identify the positions of the Pt atoms and ofsulphur atoms of Cys6, Met12, Cys30, Cys64, Cys76, Cys80,Cys94, Met105, Cys115, and Cys127 (Fig. 5 and S5 in the ESI†).
Due to the low occupancies, details on the Pt coordinationsphere cannot be easily resolved. However, a Pt(dach)2+ frag-ment close to Asp119 and a [Pt(NH3)Cl]
+ fragment close toHis15 have been identified and included in the final model.
An independent confirmation of these assignments derivesfrom the finding that the electron density maps of the Ptbinding sites are almost identical in the two structures of thebis-adduct obtained using the two different preparations(Fig. S3 and S4 in the ESI†).
In the previous crystallographic study on the HEWL/carbo-platin + cisplatin structures, both Pt drugs bind to His15, butcisplatin seems to have an overall higher binding affinity for
the His15 side-chain compared with carboplatin.35 However,our structures cannot be directly compared with that obtainedfor HEWL/carboplatin + cisplatin,35 since the bis-adduct hasnot been obtained using the same procedure by Helliwell andTanley.
Surface plasmon resonance and circular dichroism analyses
To further investigate the nature of the HEWL/cisplatin + oxali-platin interaction, surface plasmon resonance (SPR) and circu-lar dichroism studies were also performed. First, the affinitiesof cisplatin and oxaliplatin toward HEWL were measured sep-arately by SPR. This technique has been already used to evalu-ate the affinity of Pt complexes for G-quadruplexes.36 HEWLwas efficiently immobilized on a CM5-chip and Pt complexeswere employed as analytes (concentration range 0.1–2.0 mM).
Fig. 4 Binding sites of oxaliplatin (A) and cisplatin (B). 2Fo − Fc electrondensity maps are contoured at 3σ (red) and 1σ (grey) level.
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Dose–response sensorgrams (Fig. S6A and B in the ESI†)revealed that, in 20 mM sodium citrate buffer at pH 4.4, thetwo molecules bind HEWL with similar affinities, in the lowmillimolar range (KD = 1.83 ± 0.03 mM and 1.51 ± 0.02 mM,for cisplatin and oxaliplatin, respectively). Kinetic and thermo-dynamic parameters obtained by the SPR analysis are reportedin Table 2. For comparative purposes, the same experimentswere then carried out in HBS (HEPES buffered saline) at pH7.4 (Fig. S6C†). Cisplatin binding assays provided a similarvalue of KD (1.69 ± 0.05 mM), while oxaliplatin binding couldnot be evaluated due to the instability of the complex in thepresence of NaCl.37 In fact, EXAFS spectra of oxaliplatin inNaCl solution at a concentration between 0.08 M and 3 Mshow that a slight enlargement of the band occurs after a fewhours, indicating that Pt chlorination occurs.37
Then, an experiment performed by injecting first oxali-platin and then cisplatin (after reaching the saturation curveassociated with the binding of the first ligand) was carried out(Fig. 6).
This experiment, performed using a Pt complex concen-tration of 1 mM and 20 mM sodium citrate buffer at pH 4.4 asrunning buffer, shows an increase of the resonance unit (RU)signal after the injection of the second Pt complex. Thisfinding clearly indicates that the two metallodrugs binddifferent sites on the HEWL surface. To further corroboratethese results, thermal shift assays were carried out. In particu-lar, we employed circular dichroism to determine the melting
temperature of HEWL alone and in the presence of the two Ptcomplexes. In Fig. 7, the overlay of denaturation profiles of thewild type protein, of its adducts with cisplatin and oxaliplatinand of the bis-adduct formed using different strategies isreported. In particular, for the formation of the bis-adduct thetwo drugs are concomitantly added to HEWL (HEWL/cisplatin
Fig. 5 Anomalous difference map calculated from anomalous data col-lected for the bis-adduct crystal. The anomalous map is rendered inblue and contoured at 2.0σ. The map clearly identifies the positions ofPt and sulphur atoms of Cys6, Met12, Cys30, Cys64, Cys76, Cys80,Cys94, Met105, Cys115, and Cys127.
Table 2 Equilibrium dissociation constants (KD) and kinetic parametersfor the interaction of HEWL with cisplatin and oxaliplatin obtained usingthe SPR method
Ligand kon (M−1 s−1) koff (s−1) KD (mM)
Cisplatin 9.4 0.0173 1.83 ± 0.03Oxaliplatin 16.9 0.0256 1.51 ± 0.02
Fig. 6 SPR sensorgram for the sequential injection of oxaliplatin and(then) cisplatin, both at 1 mM, to the immobilized HEWL in the presenceof 20 mM sodium citrate buffer at pH 4.4 (see Methods for furtherdetails).
Fig. 7 Unfolding curves of HEWL and HEWL/Pt complexes as followedby monitoring the variation of the molar ellipticity at 222 nm as a func-tion of temperature. Experiments have been performed using 20 µMprotein or protein adduct in 10 mM sodium acetate buffer at pH 4.4.
Table 3 Denaturation temperatures determined by circular dichroismspectroscopy for the protein–Pt complex adducts formed upon theinteraction of HEWL with cisplatin and oxaliplatin in 10 mM sodiumacetate buffer at pH 4.4
Complex Tm (°C)
HEWL 77.2 ± 0.1HEWL/cisplatin 76.5 ± 0.5HEWL/oxaliplatin 73.0 ± 0.1Bis-adductsHEWL/cisplatin + oxaliplatin 74.3 ± 0.3HEWL/cisplatin then oxaliplatin 74.0 ± 0.1HEWL/oxaliplatin then cisplatin 73.8 ± 0.5
Dalton Transactions Paper
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+ oxaliplatin), or added to the protein at different times, i.e.first adding cisplatin and then oxaliplatin (HEWL/cisplatinthen oxaliplatin) or vice-versa (HEWL/oxaliplatin then cispla-tin). The comparison of Tm values points out that the cisplatinbinding does not significantly alter the HEWL thermal stabi-lity, whereas oxaliplatin binding slightly decreases the proteinmelting temperature. The formation of the bis-adduct, whichseems to be afforded following different strategies, can be wellevidenced by comparing melting temperatures of the differentprotein complexes examined (Table 3).
Conclusion
In conclusion, unambiguous evidence is provided here thatcisplatin and oxaliplatin can be used in combination toproduce a bis-metalated HEWL adduct where a [Pt(NH3)]
2+
fragment is bound to His15 and an oxaliplatin/[Ptdach]2+ frag-ment is bound to Asp119. The results of our experimentssuggest that the two drugs manifest similar affinities for theprotein, at least under the investigated experimental con-ditions, and that thermal shift analysis can be used to revealthe formation of the protein–Pt complex adducts. Obviously,the conditions required for the crystallization and the modelprotein used do not allow conclusions that are immediatelyrelevant for clinical cancer treatments to be drawn. However,although the combination of drugs acting synergistically canoffer a means of overcoming drug resistance,38,39 the results ofour analysis suggest that in principle combination treatmentof Pt drugs may lead to a greater array of side effects than pre-viously imagined as different protein sites may be damaged.
MethodsElectrospray mass spectrometry experiments
Cisplatin and oxaliplatin (3.0 × 10−4 M) were incubated for24 h with lysozyme (10−4 M) in ammonium acetate buffer pH =6.8 at 37 °C. After a 20-fold dilution with water, ESI MS spectrawere recorded by direct introduction at 5 μl min−1 flow rate inan Orbitrap high-resolution mass spectrometer (Thermo, SanJose, CA, USA), equipped with a conventional ESI source. Theworking conditions were the following: spray voltage 3.1 kV,capillary voltage 45 V, capillary temperature 220 °C, tube lensvoltage 230 V. The sheath and the auxiliary gases were set,respectively, at 17 (arbitrary units) and 1 (arbitrary unit). Foracquisition, Xcalibur 2.0. software (Thermo) was used andmonoisotopic and average deconvoluted masses were obtainedby using the integrated Xtract tool. For spectrum acquisition, anominal resolution (at m/z 400) of 100 000 was used.
Sample preparation, crystallization and X-ray diffraction datacollection
Cisplatin, oxaliplatin and hen egg white lysozyme (HEWL)were purchased from Sigma chemicals and used withoutfurther purification. A stock solution of HEWL was obtained
dissolving the protein at a concentration of about 40 mg ×mL−1 in 5 mM sodium acetate buffer at pH 5 and stored at4 °C. Cisplatin and oxaliplatin solutions were freshly preparedfor each experiment.
Crystals of the bis-adduct have been obtained following twodifferent protocols. Firstly, a protein sample has been firstincubated for 24 h in the presence of an excess of oxaliplatin(protein to metal drug ratio 1 : 10) and then for 24 h in thepresence of an excess of cisplatin (protein to metal drug ratio1 : 10). Secondly, a protein sample incubated for 24 h in thepresence of an excess of oxaliplatin (protein to metal drugratio 1 : 10) has been mixed with a protein sample incubatedfor the same time in the presence of an excess of cisplatin(protein to metal drug ratio 1 : 10). Crystals of the bis-adductwere grown by the hanging-drop vapor diffusion technique at298 K by mixing this sample containing 15 mg × mL−1 ofHEWL incubated for a total of 48 h with the two metallodrugswith equal volumes of reservoir solution. The best crystalsgrow within 2–10 days under the following conditions: 0.6 MNaNO3, 0.1 M sodium acetate pH 4.4 and 20% ethylene glycol.Attempts to obtain crystals of the bis-adduct by mixing theprotein with a mixture of cisplatin + oxaliplatin or adding firstcisplatin and then oxaliplatin to the protein sample failed. Inthese cases we obtain crystals of the adduct between HEWLand cisplatin alone, i.e. structures of HEWL with the Pt centreonly bound to His15.
X-ray diffraction data were collected for single crystals at theCNR Institute of Biostructure and Bioimages, Naples, Italy,using a Saturn944 CCD detector with CuKα X-ray radiationfrom a Rigaku Micromax 007 HF generator. Crystals wereslowly dehydrated in air40 and flash-frozen at 100 K usingnitrogen gas produced by an Oxford Cryosystem (and main-tained at 100 K during the data collection) without using cryo-protectants, following the procedure used in other studies.41,42
The dataset was processed and scaled using the HKL2000package.43 Data collection statistics are reported in Table S1.†
Structure resolution and refinement
The structures were solved by the molecular replacementmethod, using the PDB file 4J1A,44 without water moleculesand ligands, as the starting model. The refinement was carriedout with Refmac5.7,45 and model building and map inspec-tions were performed using Wincoot.46 5% of the data wasused for calculation of the R-free value. After several rounds ofrefinement using the maximum likelihood option in Refmac,manual adjustments of side-chain atoms and addition ofwater molecules to the coordinates, the structures convergedto an R-factor of 0.155/0.159 and to an R-free of 0.218/0.230.Refinement statistics are reported in Table S1.† Structure vali-dations have been carried out using Procheck.47 Coordinatesand structure factors of the two structures were deposited inthe Protein Data Bank (PDB codes 4Z46 and 4ZEE).
Surface plasmon resonance (SPR) experiments
Real time binding assays were performed at 25 °C on a Biacore3000 Surface Plasmon Resonance (SPR) instrument (GE
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Healthcare). HEWL was immobilized at 940 RU, on a CM5Biacore sensor chip in 10 mM sodium acetate pH 5.5, by usingthe EDC/NHS chemistry, with a flow rate of 5 μL × min−1 andan injection time of 7 min. Binding assays were carried out byinjecting 90 µL of the analyte, at 20 µL × min−1, with 20 mMsodium citrate at pH 4.4 as running-buffers. Experiments havealso been carried out at pH 7.4 using HBS (10 mM Hepes,150 mM NaCl, 3 mM EDTA, pH 7.4) as running buffer. Theassociation phase (kon) was followed for 270 s, whereas the dis-sociation phase (koff ) was followed for 300 s. The referencechip sensorgrams were opportunely subtracted to sample sen-sorgrams. Kinetic parameters were estimated assuming a 1 : 1binding model and using version 4.1 Evaluation Software (GEHealthcare).
A sequential injection experiment (by injecting first 30 µLof oxaliplatin and then 30 µL of cisplatin at 1 mM) has alsobeen performed using exactly the same experimental con-ditions of the single injection experiments. The inverse experi-ment where oxaliplatin is injected after cisplatin shows that inthe short time of the experiment oxaliplatin does not bind thepreformed HEWL–cisplatin adduct (Fig. S7†).
Circular dichroism thermal denaturation experiments
Protein thermal denaturation experiments were performed byfollowing the circular dichroism (CD) signal at 222 nm as afunction of temperature for HEWL, HEWL/cisplatin, HEWL/oxaliplatin (the protein sample was pre-incubated in the pres-ence of the metallodrug for 24 h at room temperature) and forthe bis-adduct that forms when (a) HEWL is concomitantlyincubated for 24 h in the presence of equal amounts of cispla-tin and oxaliplatin (HEWL/cisplatin + oxaliplatin), (b) HEWL isfirst incubated for 24 h with cisplatin and then for 24 h withoxaliplatin (HEWL/cisplatin then oxaliplatin) and (c) viceversa, i.e. HEWL is first incubated for 24 h with oxaliplatin andthen for 24 h with cisplatin (HEWL/oxaliplatin then cisplatin).In all cases, the final protein : metal ratio is 1 : 10 for eachcomplex and the protein concentration is about 20 µM.
The experiments have been carried out at pH 4.4 (10 mMsodium acetate buffer). A Peltier temperature controller wasused to set the temperature of the sample, with a slope of20 °C/1 hour.
Acknowledgements
The authors thank G. Sorrentino and M. Amendola for techni-cal assistance and G. Ferraro for her help with the crystalliza-tion trials, L.M. and T.M. acknowledge Beneficentia Stiftung(Vaduz, Liechtenstein), AIRC (IG-12085) for generous financialsupport and Elena Michelucci, CISM (University of Florence)for recording ESI-MS spectra.
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