JOURNAL OF

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Journal of Inorganic Biochemistry 100 (2006) 1660–1671

InorganicBiochemistry

New dimanganese(III) complexes of pentadentate (N2O3) Schiff baseligands with the [Mn2(l-OAc)(l-OR)2]3+ core: Synthesis,

characterization and mechanistic studies of H2O2 disproportionation

Hernan Biava a, Claudia Palopoli a, Sergiu Shova b,1, Monica De Gaudio a, Veronica Daier a,Manuel Gonzalez-Sierra c, Jean-Pierre Tuchagues b, Sandra Signorella a,*

a Departamento de Quımica, Facultad de Ciencias Bioquımicas y Farmaceuticas, UNR, Suipacha 531, 2000 Rosario, Argentinab Laboratoire de Chimie de Coordination du CNRS, 205 Route de Narbonne, 31077 Toulouse Cedex 04, France

c Instituto de Quımica Organica de Sıntesis (IQUIOS), CONICET – UNR, Suipacha 531, 2000 Rosario, Argentina

Received 2 March 2006; received in revised form 19 May 2006; accepted 29 May 2006Available online 13 June 2006

Abstract

Two new diMnIII complexes [MnIII2 L1ðl-AcOÞðl-MeOÞðmethanolÞ2�Br (1) and [MnIII

2 L2ðl-AcOÞðl-MeOÞðmethanolÞðClO4Þ� (2)(L1H3 = 1,5-bis(2-hydroxybenzophenylideneamino)pentan-3-ol; L2H3 = 1,5-bis(2-hydroxynaphtylideneamino)pentan-3-ol) were synthe-sized and structurally characterized. Structural studies evidence that these complexes have a bis(l-alkoxo)(l-carboxylato) triply bridgeddiMnIII core in the solid state and in solution, with two substitution-labile sites – one on each Mn ion – in cis-position. The two com-plexes show catalytic activity toward disproportionation of H2O2, with saturation kinetics on [H2O2], in methanol and dimethylformamide at 25 �C. Spectroscopic monitoring of the H2O2 disproportionation reaction suggests that (i) complexes 1 and 2 dismutateH2O2 by a mechanism involving redox cycling between MnIII

2 and MnIV2 , (ii) the complexes retain the dinuclearity during catalysis,

(iii) the active form of the catalyst contains bound acetate, and (iv) protons favors the formation of inactive MnII species. Comparisonto other dimanganese complexes of the same family shows that the rate of catalase reaction is not critically dependent on the redoxpotential of the catalyst, that substitution of phenolate by naphtolate in the Schiff base ligand favors formation of the catalyst-substrateadduct, and that, in the non-protic solvent, the bulkier substituent at the imine proton position hampers the binding to the substrate.� 2006 Elsevier Inc. All rights reserved.

Keywords: Manganese complexes; Schiff base ligands; Catalase models; Structure; Mechanism

1. Introduction

Catalases are enzymes that protect cells from deleteriouseffects caused by H2O2, a by-product of respiration, by dis-proportionating it into water and dioxygen. Besides hemetype catalases, there is a class of manganese catalases(MnCAT) that has been found in several bacterial organ-isms: Lactobacillus plantarum [1], Thermus thermophilus[2], Thermoleophilum album [3], Thermus sp. YS 8–13 [4]

0162-0134/$ - see front matter � 2006 Elsevier Inc. All rights reserved.

doi:10.1016/j.jinorgbio.2006.05.016

* Corresponding author. Tel./fax: +54 3414350214.E-mail address: signorel@infovia.com.ar (S. Signorella).

1 On leave from the Institute of Applied Physics, Academy of Sciences ofMoldova, Academiei strasse 3, 2028 Chisinau, Moldova.

and Pyrobaculum calidifontis VA1 [5]. Recent crystallo-graphic studies have evidenced that the active sites of theMnCaT from T. thermophilus [6,7] and L. plantarum [8]comprise two Mn ions triply bridged through a l1,3-carbox-ylate group of a glutamate residue and two solvent-derivedsingle atom bridges, the nature of which is yet to be ascer-tained. Although the structure of these enzymes has beendetermined, their catalytic mechanism is still poorly under-stood and the functional role of the bridges is yet unclear.Owing to their potential application as therapeutic agentsagainst oxidative stress and as catalysts for bleaching andorganic chemistry, a large number of dinuclear manganesecomplexes including various types of ligands have been syn-thesized and structurally characterized and many model

H. Biava et al. / Journal of Inorganic Biochemistry 100 (2006) 1660–1671 1661

complexes have been reported that show catalase activity[9–16]. An exhaustive and comprehensive review of therelevant research has recently been made [17]. However,the best reactivity of models is still three orders of magni-tude slower than that of MnCAT. The obtention of moreeffective mimics could find applications in catalysis whilealso elucidating the principles of catalysis of H2O2

disproportionation.In previous work, we have studied the catalase-like

activity of a series of diMnIII complexes of salpentOH(1,5-bis(salicylidenamino)pentan-3-ol) and its phenyl-ringsubstituted derivatives, and we have found that someminor changes in the Schiff base ligand have incidence onthe catalytic activity of the complexes [12,18,19]. Stericeffects determine the optimal approach of the substrate,but also electronic properties (electron-donor or withdraw-ing character) of the substituents in the aromatic ring of theligand induce variation of the kcat value. In this context, wedecided to explore the effect of replacing either the iminohydrogen of salpentOH by a phenyl group or the phenolateby a naphtolate group (where electrons are delocalized overtwo fused aromatic rings) on the catalytic activity of theresulting diMnIII complexes. Here, we report the synthesis,structure, properties and catalase-like activity of two newMnIII

2 complexes, [MnIII2 L1ðl-OAcÞðl-OMeÞðmethanolÞ2�Br

(1) and [MnIII2 L2ðl-OAcÞðl-OMeÞðmethanolÞClO4� (2),

where L1H3 = 1,5-bis(2-hydroxybenzophenylideneamino)-pentan-3-ol and L2H3 = 1,5-bis(2-hydroxynaphtylidena-mino)-pentan-3-ol (Scheme 1), and compare their activityto that of other complexes in the same family. Moreover,a detailed spectroscopic study of the intermediates formedduring catalysis is presented and a general reaction schemeis proposed in light of the spectroscopic and kineticsevidences.

OH NN

HOOH

OH N

HO

N

OH

L1H3

L2H3

Scheme 1. Ligands used in this work.

2. Experimental section

2.1. Materials

All reagents or AR chemicals were used as purchased.Solvents were purified by standard methods. The concen-tration of H2O2 stock solution was determined by iodomet-ric titration.

2.2. Physical measurements

Electrospray ionization (ESI)-mass spectra wererecorded on a PERKIN ELMER SCIEX 365 LCMSMSmass spectrometer. The electrospray solutions were pre-pared from dimethyl formamide (DMF) solutions of com-plexes 1–2 or reaction mixtures diluted with methanol to a�10�6 (1–2) or 10�5 M (reaction mixture) concentration.The diluted solutions were electrosprayed at a flow rateof 5 ll/min. IR spectra were recorded on a Perkin–ElmerSpectrum one FT-IR spectrophotometer. Samples wererun as KBr pellets. Electronic spectra were recorded on aJASCO V550 spectrophotometer with thermostated cellcompartments. The electrochemical experiments were per-formed with a computer-controlled Princeton AppliedResearch potentiostat, model VERSASTAT II, with model270/250 Research Electrochemistry Software. Studies werecarried out under Ar, in DMF or methanol solutions using0.1 M Bu4NPF6 as supporting electrolyte and �10�3 M ofthe complex. The working electrode was a Pt wire or aglassy carbon disk, the reference electrode was Ag/AgClwith Pt as the auxiliary electrode. Electrolyses were per-formed at controlled potential using a Pt gauze-workingelectrode. 1H NMR spectra were recorded on a BrukerAC 200 NMR spectrometer at ambient probe temperature(ca. 26 �C), with nominal operating frequencies of 200.1and 50.3 MHz. For operation conditions see Ref. [19]. Var-iable-temperature magnetic susceptibility data wereobtained with a Quantum Design MPMS SQUID suscep-tometer, under a magnetic field of 0.5 T in the temperaturerange 2–300 K. Diamagnetic corrections were applied byusing Pascal’s constants. A function-minimization programwas used for the least-squares computer fitting of the datato Eq. (1). Thermogravimetric measurements were per-formed on a Shimadzu TGA-50H (heating rate of20 �C min�1) equipped with a Nicolet model 550 IR gasanalyzer and a Fisons model Thermolab mass detector.The EPR (electron paramagnetic resonance) spectra wereobtained on a Bruker ESP 300 E spectrometer. The micro-wave frequency was generated with a Bruker ER 04 (�9–10 GHz). The microwave frequencies were measured witha Racal-Dana frequencymeter and the magnetic field wasmeasured with a Bruker NMR probe gaussmeter.

2.3. Synthesis of ligands

Bis(2-hydroxybenzophenylideneamino)pentan-3-ol (L1H3)and 1,5-bis(2-hydroxynaphtylidenamino)pentan-3-ol (L2H3)

1662 H. Biava et al. / Journal of Inorganic Biochemistry 100 (2006) 1660–1671

were prepared by Schiff-base condensation of 1,5-diamin-opentan-3-ol [20] with 2-hydroxybenzophenone and 2-hydroxy-1-naphtaldehyde, respectively, and were isolatedby precipitation from the reaction mixture (ethanol), aspure yellow solids.

L1H3. Anal. Calc. for C31H30N2O3: C, 77.79; H, 6.32; N,6.27. Found: C, 77.23; H, 6.36; N 6.02%. Significant IRbands (KBr, m cm�1): mOH 3430, mCH 3059, 2920, mC@N

1604. 1H NMR (CDCl3) d: 1.79 (m (multiplet), 4H,–CH2–), 3.42 (t (triplet), 4H, –CH2–N), 3.83 (m, 1H,–CH(OH)–), 6.77–7.46 (m, 18H, phenyl rings).

L2H3. Anal. Calc. for C27H26N2O3: C, 76.03; H, 6.14; N,6.57. Found: C, 75.61; H, 6.31; N 6.76%. Significant IRbands (KBr, m cm�1): mOH 3380, mCH 3046, 2928, mC@N

1636. 1H NMR (D6-dimethyl sulfoxide) d: 1.92 (m, 4H,–CH2–), 3.48 (m, 4H, –CH2–N), 3.87 (m, 1H, –CH(OH)–),6.7–8.2 (m, 12H, naphtyl ring), 9.19 (singlet, 2H,N@CH–).

3. Synthesis of complexes

Caution! Although we have experienced no difficultieswith the perchlorate salts they should nevertheless beregarded as hazardous and treated with care.

3.1. [Mn2L1(l-MeO) (l-AcO)(methanol)2]Br Æ 2H2O

(1 Æ 2H2O)

Mn(OAc)3 Æ 2H2O (1.43 g, 5.33 mmol) and 1,5-bis(2-hydroxybenzophenylideneamino)pentan-3-ol (1.2 g, 2.5mmol) were dissolved in methanol (20 mL) and stirredfor 20 min. The resulting brown solution was filtered, anda methanol solution (10 mL) of NaBr salt (1.03 g,10.0 mmol) was added to the filtrate. A green suspensionformed immediately. After stirring 20 h, the solid was col-lected by filtration, washed twice with cold methanol anddried under vacuum. Yield: 1.47 g (1.72 mmol, 69%). Anal.Calc. for Mn2C36H45N2O10Br Æ 2H2O: C, 50.50; H, 5.26; N,3.27; Br, 9.36; Mn, 12.9. Found: C, 50.79; H, 5.12; N,3.35; Br, 9.45; Mn 13.4%. Significant IR bands (KBr,m cm�1): mOH 3624, 3350, mC@N 1597, mCO�2

1536/1412.UV–vis (UV–visible) kmax nm (e M�1 cm�1) in methanol:350 (8300); in DMF: 370 (10300). These absorption bandsobey Beer’s law over the range of concentrations above10 lM used in this work. Single crystals of 1 Æ H2O suitablefor X-ray diffraction were formed upon standing of thereaction filtrate for about one week.

3.2. [Mn2L2(l-MeO)(l-AcO)(methanol)(ClO4)] Æ 1.5

H2O (2 Æ 1.5H2O)

The procedure used was similar to that for 1, exceptthat NaClO4 Æ H2O (1 mmol ClO�4 per mmol Mn3+) wasused instead of NaBr. Yield: 88%. Anal. Calc. forMn2C31H33N2O11Cl Æ 1.5H2O: C, 47.6; H, 4.60; N, 3.58;Cl, 4.54; Mn, 14.1. Found: C, 47.27; H, 4.50; N, 3.66; Cl,

3.97; Mn 13%. Significant IR bands (KBr, m cm�1): mOH

3660, 3418, mC@N 1618, mCO�21540/1414, mClO4 1128/1090/

1053/1030. UV–vis kmax nm (e M�1 cm�1) in methanol:301 (31,000), 326 (30,100), 400 (13,650), 414 (sh), 596(532); in DMF: 323 (23,100), 396 (8800). These absorptionbands obey Beer’s law over the range of concentrationsabove 10 lM used in this work. The content of 1.5 mole-cules of non-coordinated water was confirmed by thermo-gravimetric analysis of the complex which showed a 3.0%mass loss around 110 �C.

3.3. Kinetic measurements

Oxygen evolution studies were carried out with anYSI oxygen-monitoring system (Model 5300, YellowSprings Instruments Co., Inc.). The initial rate methodwas used to determine the rate constants (for experimen-tal details see Ref. [18]). Each rate constant reportedhere represents the mean value of multiple determinationsthat fall within ±5%. All experiments were carried out at25 �C.

3.4. Stoichiometric measurements

The stoichiometry of the reaction was measured bysimultaneous determination of O2 and H2O2 concentra-tions. The evolved dioxygen was measured volumetricallyas previously described [18], and H2O2 was determinedby titration with horseradish peroxidase [12]. Turnoversas high as 2000 have been measured for complexes 1–2 inDMF. However, successive additions of excess H2O2 tothe catalyst solution yielded the stoichiometric amount ofO2 but the initial rate of H2O2 dismutation graduallydecreased after each new addition. In methanol, complexes1 and 2 were able to disproportionate up to 200 and 450 eq.of H2O2, respectively, and were then inactivated.

3.5. X-ray crystal structure determination

Crystallographic measurements for 1 were carried outwith an Oxford-Diffraction XCALIBUR CCD diffractome-ter using graphite-monochromated Mo Ka radiation. Thecrystal was placed 60 mm from the CCD detector. Morethan one hemisphere of the reciprocal space was coveredby combination of four sets of exposures; each set had a dif-ferent u-angle (0�, 90�, 180�, 270�). Coverage of the uniqueset is 99.8% complete up to 2h = 50�. The unit cell determi-nation and data integration were carried out using theCrysAlis package of Oxford Diffraction [21]. Intensity datawere corrected for Lorentz and polarization effects. Thestructure was solved by direct methods using SHELXS-97[22] and refined by full-matrix least-squares on F 2

o withSHELXL-97 [23] with anisotropic displacement parametersfor non-hydrogen atoms. All H atoms attached to carbonwere introduced in calculations in idealized positions(dCH = 0.96 A) using the riding model with their isotropicdisplacement parameters fixed at 120% of the riding atom.

Fig. 1. Asymmetric unit of [Mn2L1(l-MeO)(l-AcO)(MeOH)2]Br Æ H2O(1 Æ H2O). The thermal ellipsoids are drawn at the 50% probability level.Hydrogen atoms have been omitted for clarity.

H. Biava et al. / Journal of Inorganic Biochemistry 100 (2006) 1660–1671 1663

Positional parameters of the H atom belonging to thehydroxyl group of the coordinated methanol was obtainedfrom difference Fourier syntheses and refined without anyconstraint. The H atoms of the solvent water molecule,which is disordered over two resolvable positions were notlocalized. Scattering factors were taken from Ref. [24].The molecular plot was obtained using the ZORTEP pro-gram [25]. The crystallographic data together with therefinement details are summarized in Table 1.

4. Results

4.1. Structure of [Mn2L1(l-OMe)(l- OAc)

(methanol)2] Br Æ H2O (1 Æ H2O)

The crystal structure of 1 Æ H2O consists of discretedimanganese complex cations, non-co-coordinated Br�

anions and solvate water molecules in a 1:1:1 molar ratio.As shown in Fig. 1, the two Mn atoms are six-coordinatewith NO5 donor sets, and are triply bridged by the centralalcoholate of L1(3�), and exogenous syn,syn-acetato andmethanolato ligands. The co-ordination sphere of eachMn is completed by the N-imine and the O-phenolato fromthe pentadentate ligand and the oxygen atom of an exoge-nous methanol molecule. Bond distances and angles listedin Table 2 indicate that the geometry around each Mnatom may be described as axially elongated octahedral,with the Mn(1)–O(3), Mn(2)–O(4), Mn(1)–O(6) and

Table 1Summary of crystal data for 1 Æ H2O

Empirical formula C36H43BrMn2N2O9

M 837.51Temperature (K) 180Wavelength (A) 0.71073Crystal system, space group Monoclinic, P21/na (A) 11.527(2)b (A) 23.778(5)c (A) 14.277(3)b (�) 105.76(3)V (A3) 3766.3(13)Z 4q(calcd) (Mg/m3) 1.477lMo (mm�1) 1.786F(000) 1720Crystal size (mm) 0.4 · 0.3 · 0.3h Range (�) 2.97–24.99Index ranges �13 6 h 6 9,

�28 6 k 6 28,�16 6 l 6 16

Number of reflections

Measured 23,649Unique 6602 [R(int) = 0.0790]Number of refined parameters 473Maximum and minimum transmission 0.805 and 0.979GOOF for F2 0.961aR [I > 2r(I)] 0.0554bwR 0.1302Dqmax and Dqmin (e A3) 1.095 and �0.667

a R ¼PkF oj � jF ck=

PjF oj.

b wR ¼ ½P

wðjF 2oj � jF 2

c jÞ2=P

wjF 2oj

2�1=2.

Mn(2)–O(7) bond distances (average 2.241 A) distinctlylonger than the equatorial Mn–O/N ones (average1.941 A), a fact attributable to the Jahn–Teller distortionof the high spin d4 electronic configuration of the MnIII

centres. The deviation from an ideal octahedral geometryis also evidenced by the range of angles observed aroundeach metal centre (from 79.94� to 96.03�). The Mn� � �Mnseparation of 2.942(1) A is in the range observed for otherdialkoxo-bridged MnIII complexes with axial elongationperpendicular to the bridging plane [26,27], and is 0.4–0.6 A shorter than observed for dialkoxo bridged MnIII

2

complexes with a Jahn–Teller elongation axis parallel tothe bridging core [28–32]. The Mn� � �Mn distance, theMn–O–Mn bond angles and the Mn–O/N bond lengthsof complex 1 Æ H2O are very close to those of the diMnIII

Table 2Selected bond lengths (A) and angles (�) for [Mn2L1(l-MeO)(l-AcO)(MeOH)2]Br Æ H2O (1 Æ H2O)

Bond lengths (A)

Mn(1)–O(1) 1.855(3) Mn(2)–O(2) 1.848(3)Mn(1)–O(5) 1.956(3) Mn(2)–O(5) 1.939(3)Mn(1)–O(8) 1.959(3) Mn(2)–O(8) 1.952(3)Mn(1)–O(3) 2.151(3) Mn(2)–O(4) 2.172(3)Mn(1)–O(6) 2.304(3) Mn(2)–O(7) 2.336(3)Mn(1)–N(1) 2.010(4) Mn(2)–N(2) 2.008(4)

Angles (�)

O(1)–Mn(1)–O(5) 94.85(12) O(2)–Mn(2)–O(5) 94.05(13)O(1)–Mn(1)–O(8) 174.45(13) O(8)–Mn(2)–O(8) 174.53(13)O(5)–Mn(1)–O(8) 79.94(12) O(5)–Mn(2)–O(8) 80.53(12)O(1)–Mn(1)–N(1) 89.83(14) O(2)–Mn(2)–N(2) 89.44(14)O(5)–Mn(1)–N(1) 172.35(14) O(5)–Mn(2)–N(2) 172.16(13)O(8)–Mn(1)–N(1) 95.16(14) O(8)–Mn(2)–N(2) 95.84(13)O(1)–Mn(1)–O(3) 91.72(13) O(2)–Mn(2)–O(4) 91.80(13)O(5)–Mn(1)–O(3) 91.26(13) O(5)–Mn(2)–O(4) 90.88(12)O(8)–Mn(1)–O(3) 90.30(12) O(8)–Mn(2)–O(4) 89.08(12)N(1)–Mn(1)–O(3) 94.66(14) N(2)–Mn(2)–O(4) 96.03(13)O(1)–Mn(1)–O(6) 91.24(13) O(2)–Mn(2)–O(7) 91.56(13)O(5)–Mn(1)–O(6) 89.53(12) O(5)–Mn(2)–O(7) 89.53(12)O(8)–Mn(1)–O(6) 86.85(11) O(8)–Mn(2)–O(7) 87.63(12)N(1)–Mn(1)–O(6) 84.31(14) N(2)–Mn(2)–O(7) 83.36(13)O(3)–Mn(1)–O(6) 176.86(12) O(4)–Mn(2)–O(7) 176.58(13)Mn(1)–O(5)–Mn(2) 98.12(13) Mn(1)–O(8)–Mn(2) 97.58(13)

1664 H. Biava et al. / Journal of Inorganic Biochemistry 100 (2006) 1660–1671

complex obtained with salpentOH [33]. This fact showsthat substitution of the imino H atom by a phenyl groupin the Schiff base ligand does not modify the geometricparameters of the bimetallic core of the resulting complex.

The anionic and cationic parts of the structure are asso-ciated through H-bonds as shown by two O–H� � �Br shortcontacts between coordinated molecules of methanol andBr� counter-anion with distances O(6)–H(1) 0.854(10) A,H(1)� � �Br(1) 2.361(12) A, O(6)� � �Br(1) 3.210(3) A, O(7)–H(2) 0.854(10) A, H(2)� � �Br(1) 2.351(13) A and O(7)� � �Br(1) 3.199(3) A, and angles O(6)HBr(1) 173(5)� andO(7)HBr(1) 172(4)�. The crystal packing (Fig. 2) probablyresults from Van der Waals interactions. The disorderedwater molecules O(1w) reside inside the cavities betweenanions and cations with only one O–H(1w)� � �Br(1) shortcontact, 3.204(3) A.

4.2. IR spectra

The carboxylato bands of 1 occur at 1536 cm�1 (anti-symmetrical) and 1412 cm�1 (symmetrical), which identifya syn–syn l-carboxylato. For the powdered complex 2,the two bands attributable to the bridging acetato anionappear at 1540 and 1414 cm�1, respectively, indicatingthe same mode of bidentate acetato coordination in thetwo complexes. The two complexes exhibit a strong bandattributable to mC@N at 1597 and 1618 cm�1, respectively.Complex 2 also shows split bands at 1128, 1090, 1053and 1030 cm�1 indicative of coordinated perchlorate,which is consistent with its molecular structure. Complexes1 and 2 display a band at 3624 and 3660 cm�1, respectively,assigned to the presence of non-coordinated water, inagreement with the crystal structure (1) and the loss ofH2O at about 110 �C upon thermogravimetric analysis of2. Comparison of the IR spectra of these two complexesconfirms their homologous structures.

Fig. 2. Crystal packing diagram for 1 Æ H2O.

4.3. Magnetic properties

The variable-temperature (2–300 K) magnetic suscepti-bility (vM) and effective magnetic moment (leff) of complex1 are illustrated in Fig. 3. The leff value of 5.70lB/dinuclearunit at 300 K decreases monotonically with decreasing tem-perature to reach a value of 0.16lB/dinuclear unit at 2 K.This behavior is characteristic of two antiferromagneticallycoupled high-spin (S = 2) MnIII ions resulting in an S = 0ground state. The magnetic data for 1 could be accountedfor by using the Van Vleck equation, based on the mag-netic exchange Heisenberg Hamiltonian H = �2JS1S2

(S1 = S2 = 2), modified to include a fraction (p) of para-magnetic impurity:

vM ¼ð1� pÞfNg2l2Bð2e2x þ 10e6x þ 28e12x þ 60e20xÞ

=kT ð1þ 3e2x þ 5e6x þ 7e12x þ 9e20xÞg þ pvc ð1Þ

where vM is the magnetic susceptibility, x = J/kT, and vc isthe Curie law magnetic susceptibility for the monomer. Thebest least-squares fit of the experimental data (solid line inFig. 3) was obtained with the following parameters: J =�13.1 cm�1, g = 1.90 and p = 0.2%. The calculated ex-change parameter J falls in the range observed for otherdialkoxo bridged diMnIII complexes with short Mn� � �Mndistances (2.98–3.00 A) [18,19,27,34–37] that allow directoverlap between the dxy orbitals.

5. Solution studies

5.1. EPR spectroscopy

Complexes 1 and 2 are EPR silent in frozen DMF ormethanol solution (T � 100 K), a fact consistent with thepresence of MnIII ions.

0 50 100 150 200 250 3000.000

0.005

0.010

0.015

0.020

0.025

0 50 100 150 200 250 3000

1

2

3

4

5

6

χ M (

cm3 m

ol-1)

T (K)

μ(B

M)

Fig. 3. Variable-temperature magnetic susceptibility data for 1 Æ H2O. Thesolid lines are theoretical curves based on Eq. (1) and the circles representthe experimental data.

H. Biava et al. / Journal of Inorganic Biochemistry 100 (2006) 1660–1671 1665

5.2. 1H NMR spectroscopy

We used NMR spectroscopy as a way to establishwhether the solid-state structure was retained upon dissolu-tion. The paramagnetic 1H NMR spectra of 1 and 2 in D4-methanol (Fig. 4(a) and (d)) revealed a simple pattern forthese complexes, containing two or three proton reso-nances that lie outside the diamagnetic region (d = 0–14).The resonances span a wide range of �60 ppm as a resultof the weak antiferromagnetic coupling between the MnIII

ions in the MnIII2 species. One (2) or two (1) resonances are

observed up field between d�13 and �27 ppm, which canbe assigned to the H4 and H5 protons of 1, and to theH4 of 2, in accordance with previous studies [38–41]. Theprotons ortho to the donor groups of the Schiff base ligand

60 40 20 -10 -20 -30

40 20 -20 -40

b

c

δ / ppm

a

d

δ / ppm

Mn

O

O

N

O

N

Mn

OOO

YY

X

Z Z

X

CH3

45

Fig. 4. 1H NMR spectra of 1 (15 mM), before (a) and after addition of (b)1.0, (c) 2.0 equiv. of NaOAc; and (d) 2 (14 mM) in D4-methanol Æ 1:Y = Ph, X = Z = H; 2: Y = H, XZ = C4H4.

(H3 and H6 of 1 and H3 of 2) are not observed, and this isconsistent with previous finding for related MnIII com-plexes [38–41].

The two intense resonances observed at 7.20 and8.30 ppm for complex 1, and at 9.40 and 10.5 ppm for com-plex 2, correspond to the aromatic protons of the phenyland the external fused ring of naphtyl moieties, respec-tively. These signals are in the diamagnetic range but arebroader than in the free ligand.

The broad resonance at 33–38 ppm may be assigned tothe methyl group of the bridging acetato of 1–2 on the basisof comparison with the spectra of other MnIII

2 -l-OAc com-plexes previously studied [39,40]. This assignment was con-firmed by addition of excess acetate to the solution of 1 inD4-methanol, which resulted in an up-field shift of the ace-tate protons resonance (Fig. 4(b–c)). As the NaOAc con-centration increased, the signal shifted to lower d valuesand sharpened, indicating the dynamic averaging resultingfrom a rapid exchange mechanism between bridging andfree acetate.

The spectrum of complex 2 was also registered in D7-DMF. In this solvent, the H4 resonance appears at�24.5 ppm and the bridging acetate protons appear at37 ppm.

A strong linear correlation between the isotropic shift ofthe bridging acetate protons and the magnetic coupling con-stant (J) has been found for a number of dinuclear MnIII

2

complexes [42]. Based on this correlation, the observedisotropic shift (38 ppm) for the acetate protons of complex1 is as expected for acetate bridged MnIII

2 complexes withJ � �15 cm�1. This result strongly suggests that the solid-state structure of this complex is retained in solution. Thesimilar isotropic shift for the acetate protons of 2 and 1 sug-gests homologous geometries for the diMnIII centre of thetwo complexes in solution.

5.3. Electrospray ionization-mass spectrometry (ESI-MS)

The ESI-mass spectra of 1–2 in DMF confirmed that din-uclearity of the complexes is retained in solution. The massspectrum of 1 is dominated by the peak at m/z = 675 (100%)corresponding to the monocation [Mn2L1(OAc)(OMe)]+

which results from the loss of two coordinated methanolmolecules from the complex in the solid state. Otherobserved peaks correspond to the exchange of OMe byOH ([Mn2L1(OAc)(OH)]+, m/z = 661, 14%), the replace-ment of acetate by methanolate originating from the solventused in the spray experiments ([Mn2L1(OMe)2]+, m/z =647, 32%), and the loss of acetate and methanolate([Mn2L1]+, m/z = 585, 36%). Complex 2 afforded a similarmass spectrum with the following major peaks: m/z = 623([Mn2L2(OAc)(OMe)]+, 100%), m/z = 609 ([Mn2L2(OAc)-(OH)]+, 21%), m/z = 595 ([Mn2L2(OMe)2]+, 45%), and m/z = 533 ([Mn2L2]+, 33%). One additional peak which mightcorrespond to the Mn2Lþ2 mixed valence dimer alsoappeared in the ESI-mass spectra of both complexes. Thesespecies are not observed by EPR spectroscopy and are most

-800 -400 0 400

a

b

E (mV) vs Ag/AgCl

Fig. 5. Cyclic voltammograms of (a) 1 and (b) 2, in methanol. Conditions:Pt/Pt/Ag–AgCl; conc. = 1 mM; scan rate = 100 mV/s; supportingelectrolyte = Bu4NPF6.

-600 -300 0 300 600

-200 0 200 400 600 800

b

E (mV)

a

E (mV)

Fig. 6. Cyclic voltammograms (solid lines) of (a) 1 and (b) 2, in DMF atscan rate = 100 mV/s. Square-wave voltammogram (dashed line) of 1 inDMF, scan frequency = 5 Hz. Conditions: Pt/Pt/Ag–AgCl; conc. =1 mM; supporting electrolyte = Bu4NPF6.

1666 H. Biava et al. / Journal of Inorganic Biochemistry 100 (2006) 1660–1671

likely to be generated within the spectrometer. Tandem MS/MS experiments showed that the collision induced dissocia-tion of the ion at m/z = 1060 (Mn2L1þ

2 ) produced the ion atm/z = 530. Thus, the [MnL1]+ (m/z = 530, 28%) and[MnL2]+ (m/z = 478, 25%) species observed in the massspectra of 1 and 2 can result from fragmentation of therespective Mn2Lþ2 dimers.

5.4. Stability of complexes

The stability of complexes 1–2 was checked by UV–visspectroscopy. Spectra of the complexes registered at differ-ent time-lengths (hours to days) after the preparation of Arsaturated solutions in anhydrous DMF and methanol, orwhen water was added (up to 5%), showed identical kmax

and molar absorbance coefficients.

5.5. Electrochemistry

The electrochemical properties of complexes 1–2 wereinvestigated by cyclic voltammetry (CV) in methanol andDMF solutions, containing 0.1 M Bu4NPF6, using a Ag/AgCl reference electrode. In methanol, the two complexesexhibit a quasi-reversible reduction wave at E1/2 = 98 mV(DEp = 140 mV, Ipa/Ipc = 0.93) and 28 mV (DEp =126 mV, Ipa/Ipc = 0.95), respectively, followed by a non-reversible reduction at around to �530 mV (Fig. 5). Plotsof Ipc and Ipa vs. v1/2 for the quasi-reversible reductionare linear in the 25–1000 mV/s range of scan rates (v), indi-cating a diffusion-controlled process. Exhaustive electroly-sis of 2 at a potential of �200 mV required 0.96 electron/molecule of complex, indicating that the quasi-reversiblereduction corresponds to the MnIII

2 =MnIIMnIII couple.The second irreversible reduction wave can be attributedto the MnIIIMnII=MnII

2 couple, as suggested for previouslyreported complexes with similar ligand environment[12,18,19]. In the case of complex 2, an additional re-oxida-tion wave at �141 mV appears associated to the secondreduction wave. This extra peak should correspond to theoxidation of the chemically transformed MnII

2 form of thecomplex.

In DMF, the cyclic voltammogram of complex 1

(Fig. 6(a)) displays two oxidation waves besides that ofthe Br� anion at 828 mV. The first oxidation at E1/2 215(DE = 102 mV) is quasi-reversible at scan rates of 100–500 mV s�1. The DE values in CV and w1/2 values insquare-wave voltammetry (SWV) experiments indicate thatthis oxidation corresponds to a one-electron process, andmay be attributed to the MnIII

2 =MnIIIMnIV redox couple.The second one-electron oxidation wave at Ep = 369 mVis non-reversible, and the SWV experiments show that thiswave is much less intense than that of the first oxidation,although its relative intensity increases with increasing scanfrequency. These facts suggest the possible oxidation of 1via an alternative pathway involving ligand-centered oxida-tion to generate a MnIII-phenoxyl radical [43]. In thenegative scan, only a reductive non-reversible peak is

observed at �200 mV (not shown), which may be assignedto the MnIII

2 =MnII2 redox couple based on bulk coulometry

studies.

Table 3Kinetic parameters based on fits of the Michaelis–Menten equation to therate data at 25 �C

Complex kcat (s�1) KM (mM) kcat/KM (M�1 s�1) Solvent

1 3.3(1) 78(6) 42 DMF2 1.18(5) 16(2) 74 DMF1 2.18(7) 11(1) 198 Methanol2 0.79(4) 3.5(7) 226 Methanol

H. Biava et al. / Journal of Inorganic Biochemistry 100 (2006) 1660–1671 1667

The cyclic voltammogram of complex 2 in DMF showstwo quasi-reversible waves at 188 mV (DE = 96 mV) and320 mV (DE = 80 mV), and one irreversible two-electronreduction wave at Ep = �136 mV (Fig. 6(b)).

6. Catalytic activity

6.1. Kinetics

Addition of H2O2 to DMF or methanol solutions ofcomplexes 1–2 causes the immediate vigorous evolutionof dioxygen coupled to color changes. The initial rate ofdisproportionation of H2O2 was measured as a functionof the complex and substrate concentrations in methanoland DMF. At constant [H2O2]0, the initial rate of H2O2

disproportionation varies linearly with the [catalyst], mean-ing that the reaction is first-order on catalyst. At constant[catalyst], the initial rate of H2O2 dismutation exhibits sat-uration kinetics with [H2O2]0 (Fig. 7) and the experimentaldata could be fitted to the Michaelis–Menten equationfrom which the catalytic turnover number (kcat) and theMichaelis constant (KM) were determined. Values of kcat

and KM obtained for complexes 1 and 2, at 25 �C in meth-anol and DMF, are listed in Table 3.

6.2. Spectroscopic monitoring of the catalase-like reaction

In order to get an insight into the mechanism of theH2O2 disproportionation catalyzed by complexes 1–2, ineither methanol or DMF, the reaction was monitored byusing a combination of electronic and EPR spectroscopiesand ESI-MS.

6.2.1. Reaction studies in methanol

In methanol, complex 1 exhibits an intense ligand-to-metal charge transfer (LMCT) band at 350 nm. Afteraddition of an excess amount of aqueous H2O2 to the

0 50 100 150 200 250 300

0 10 20 30 40 500.0

0.5

1.0

1.5

2.0

2.5

3.0

2, MeOH

2, DMF

[H2O

2] / mM

1, MeOH

1, DMF

r i, [H

2O2][

cata

lyst

]−1 t−1

/ s

−1

[H2O

2] / mM

Fig. 7. Effect of [H2O2] on the initial rate of H2O2 disproportionation at25 �C, in DMF and methanol.

methanolic solution of 1, the intensity of this banddecreased concurrently with the appearance of the twobands of the ligand at 320 and 396 nm. When a 250-foldexcess of H2O2 over 1 was used (complex 1 can dispropor-tionate up to 200 eq. of H2O2 in methanol), the absorptionband at 350 nm completely disappeared. In this solvent,complex 2 behaves similarly. For H2O2 to catalyst ratios<500:1, the naphtolate-to-Mn charge transfer band at400 nm decreased during the reaction time. When 500:1 orlarger excess of H2O2 over the catalyst was employed (com-plex 2 can disproportionate up to 450 eq. of H2O2 in meth-anol), the MnIII spectral pattern was lost at the end of theO2 evolution. EPR spectra of the reaction mixture withexcess H2O2 were registered at various time-lags in frozenmethanol. The EPR spectra showed a 6-line signal (hyper-fine splitting of �90 G) centred at g � 2 which grew-inand persisted once the reaction was completed. The signalshowed weak doublets (Dm = 1 nuclear-spin-forbiddentransitions) [44] inserted between the six absorptions char-acteristic of the hyperfine structure of an uncoupled MnII

ion. This EPR signal persisted and intensified upon succes-sive additions of H2O2, indicating formation of an irrevers-ible final product, not of an intermediate species.

6.2.2. Reaction studies in DMF

The change in the electronic spectrum of 1 upon reactionwith H2O2 in DMF, is shown in Fig. 8(a). The UV–visiblespectrum of the DMF solution of complex 1 is characterizedby an intense absorption band at 370 nm assigned to thephenolate-to-metal charge-transfer. After addition ofH2O2, the spectrum exhibited a new absorption band at505 nm. This band grew in and persisted after complete con-sumption of H2O2 concurrently with the decrease of theabsorbance at 370 nm. In a 200:1 mixture of [H2O2]/[1],the band at 505 nm showed its maximal intensity when95% of the H2O2 had been consumed, while in a 2000:1[H2O2]/[1] mixture the maximal intensity was reached afterconsumption of 70% H2O2. The Abs505 vs. time profilesshowed that the maximal absorption was always the sameindependently of the [H2O2]/[1] ratio, although it wasreached at shorter time as the [H2O2] increased. A monitor-ing of the reaction by ESI-MS gave valuable indication onthe nuclearity of the catalyst during the catalytic cycle.The positive ESI-mass spectra of reaction mixtures takenwithin short times (few minutes) were dominated by thepeak of [Mn2L1(OMe)(OAc)]+ at m/z = 675 – together withthe other peaks observed in the starting solution of 1 –, andprovided a clear indication that the MnIII

2 complex persisted

0.0

0.2

400 500 600 700

400 500 600 700

0.0

0.2

0.4

0.6

400 500 600 7000.0

0.5

1.0

aAbs

b

c

λ (nm)

λ (nm)

λ (nm)

Fig. 8. Time-evolution of the UV–vis spectrum of a mixture of (a) 2000:1[H2O2]:[1]; (b) 200:1 [H2O2]:[2]; and (c) 120:1:2:20 [H2O2]:[L2H3]:[Mn2+]:[Bu4NOH], in DMF.

1668 H. Biava et al. / Journal of Inorganic Biochemistry 100 (2006) 1660–1671

during the catalytic cycle. When increasing the [H2O2]/[1]ratio over 1000:1, a new peak appeared at m/z = 385.5and grew against those corresponding to the startingcomplex.

Fig. 8(b) shows the evolution of the electronic spectra ofa DMF solution of 2 after addition of H2O2. As the reac-tion progressed, the LMCT band of the starting complexat 396 nm decreased and a new band at 460 nm appearedand persisted after complete consumption of H2O2. In a500:1 mixture of [H2O2]/[2], the band at 460 nm showedits maximal intensity when 35% of the H2O2 had been con-sumed. For [H2O2]:[2] >500:1, this band grew in and thendecayed and the final intensity showed a reverse relation-ship with [H2O2]0, indicating partial loss of the catalyst.The disproportionation of H2O2 catalyzed by 2 was alsofollowed by ESI-MS. The spectra taken at short reaction

times and [H2O2]/[2] = 50–1000:1, were dominated by thepeak at m/z = 623 (100% intensity), corresponding to thestarting complex [Mn2L2(OAc)(OMe)]+ (M+). Besidesthe peaks present in the starting solution of 2, three othernew peaks of high relative intensity could be observed atm/z 346, 756 and 782, whose intensities built up to 27%,24% and 74% when a 1000:1 [H2O2]/[2] ratio was used.The new peaks appearing at higher m/z could correspondto solvated forms of the catalyst. Spectra taken after theend of the reaction showed peaks at m/z 605 (M+ – 18,54%), 591 (M+ – 32, 100%) and 764 (85%).

When 120 equivalents of H2O2 were added to a mixtureof L2H3 and Mn(ClO4)2 in DMF, the yellow solutionimmediately changed to orange but no O2 evolution wasobserved. However, after addition of 10 equivalents ofBu4NOH to the reaction mixture, O2 evolved vigorouslyand all the added H2O2 disproportionated in a few min-utes. Upon each successive addition of 120 equivalents ofH2O2 to a 2:1:20 Mn(ClO4)2 + L2H3 + Bu4NOH mixture,the orange solution changed to pink during O2 evolutionand then turned back to orange when the reaction wasover. The electronic spectra of the reaction mixture regis-tered at different times (Fig. 8(c)) showed the appearanceof a band at 460 nm immediately after addition of H2O2,and, as the reaction progressed, a multiplet centred at528 nm with line separation of ca. 20 nm superimposedthe band at 460 nm. Similar multiplets had previously beenobserved as intermediate in the catalytic disproportion-ation of H2O2 by several dimanganese complexes, and havebeen assigned to an oxo-to-manganese LMCT coupled tothe mMn@O vibration [45–49]. Once O2 evolution hadceased, this multiplet, responsible for the pink color ofthe solution, declined to the band at 460 nm (e460 =6500 M�1 cm�1, based on [L2]). The spectral features ofthe final spectrum were very close to those of the spectraobtained at the end of the reaction of 2 + H2O2 in DMF,suggesting that a similar species is formed in both cases.The ESI-mass spectra of a mixture of L2 + MnII +Bu4NOH + H2O2 (1:2:40:120) (pink solution where themultiplet and the band at 460 nm coexist) showed peaksat m/z = 275 (100%), 290 (60%) and 537 (60%) – also pres-ent in the orange solution –, and two peaks at m/z = 565(27%) and 583 (50%). Only peaks corresponding to per-chlorate and L2H�2 anions were observed in the negativeESI-mass spectra of the L2H3 + Mn(ClO4)2 + H2O2 +Bu4NOH reaction mixtures in DMF, thus revealing thatMnO�4 was not responsible for the multiline band observedin the electronic spectra. Besides, the negative mass spec-trum of MnO�4 using the level of MnO�4 calculated fromabsorbance at 530 nm, shows peaks that are absent inthe mass spectra of the reaction mixture. Therefore, itseems reasonable to assign the peaks at m/z = 565 and583 to oxo-MnIV species, probably, the monocations[Mn2L2(O)2]+ and [Mn2L2(O)2 + H2O]+, respectively.

EPR measurements performed on the reaction mixturesin DMF do not support the formation of mixed valenceMn2 complexes during the catalytic cycle. In this solvent,

H. Biava et al. / Journal of Inorganic Biochemistry 100 (2006) 1660–1671 1669

the reaction mixtures were essentially EPR silent, exceptfor a very small six-line signal at g � 2, which slowlyincreased for long reaction times. The intensity of this 6-line signal rapidly increased upon addition of p-toluensul-phonic acid (PPTS) to the reaction mixture in DMF,concomitantly to the loss of catalytic activity of the com-plex. Therefore, protonation of the catalyst favors the for-mation of an inactive non-coupled MnII species, which isonly a minor species when the reaction is performed inthe absence of proton source.

7. Discussion

Ligands L1H3 and L2H3 yield complexes 1 and 2 witha bis(l-alkoxo)(l-carboxylato) triply bridged diMnIII

core that are structural mimics of the MnIII2 form of

MnCAT, with Mn� � �Mn separation 0.1–0.2 A shorter thanthe Mn� � �Mn distance of 3.03 and 3.14 A found in theMnIII

2 form of L. plantarum and T. thermophilus, respec-tively [6–8]. Electronic and EPR spectroscopies show thatthese complexes are stable in methanol and DMF, evenin the presence of water, and the 1H NMR and ESI-massspectra evidence that the bis(l-alkoxo)(l-carboxylato) tri-ply bridged diMnIII core is retained in solution.

The two complexes possess the same diMnIII motifexcept for the ClO�4 anion co-coordinated at the sixth octa-hedral position of one Mn atom of 2 (instead of a methanolmolecule in 1). Therefore, the presence of one weaklybound ligand on each Mn provides two labile cis-positionsable to react with H2O2.

ESI-MS shows that the major Mn species occurring dur-ing the H2O2 disproportionation reaction in DMF corre-spond to the diMnIII complex [Mn2L1(2)(OAc)(OMe)]+.Therefore, these experiments clearly show that the complexretains dinuclearity during catalysis and that the activeform of the catalyst contains bound acetate. These resultsare in line with the lack of any significant signal in theEPR spectra of the reaction mixtures – since complexes 1and 2 are EPR silent, no EPR signal is expected for the cat-alyst occurring in the MnIII

2 oxidation state during cycling –except for the small six-line signal at g � 2 which increasesat long reaction times or high H2O2:catalyst ratios. On theother hand, the rapid growth of the MnII EPR signal in aprotic solvent or when PPTS is added to the DMF reactionsolution, suggests that protonation of the bridging ligandsbreaks down the bridges between the Mn ions and causesinactivity of the complex by conversion into a non-coupledMnII species. The absence of peaks in the ESI-mass spectracorresponding to the free ligand suggests that dissociationof the ligand to yield free Mn2þ

aq as the final Mn species doesnot occur. Partial inactivation of the catalyst by conversioninto MnII

2 species having lost the protonated exogenousbridging ligands (AcOH and/or methanol), could explainthe decrease of the catalytic activity of 1 and 2 with succes-sive additions of H2O2 to the complex DMF solution.

On the basis of the EPR and ESI-MS results, the LMCTband at 505 nm (for 1) and 460 nm (for 2) observed in the

electronic spectra during and at the end of O2 evolutioncorresponds to a MnIII

2 complex. The red shift of thePhO!Mn LMCT upon reaction with H2O2 can be inter-preted taking into account the effect of the carboxylatebinding mode on the spectroscopic properties of these com-plexes. If upon reaction with H2O2, the bridging acetatoshifts to a monodentate coordinated acetato, it shouldfunction as a weaker donor and should reduce the energygap between the dp and PhOp orbitals thus causing a redshift of the signal [50–52]. Besides, the shift of the bridgingmethanolato ligand to a weakly bound terminal ligandshould also contribute to the red shift of the band. Assign-ment of this band to a MnIV

2 complex can be disregardednot only based on the ESI-MS results, but also becauseof the lower intensity of the red shifted PhO!Mn CTband (PhO!MnIV electronic transitions occur at lowerenergies and have higher extinction coefficients thanPhO!MnIII ones) [53–57].

The electronic spectra of the reaction of H2O2 +Mn2+ + L2H3 + Bu4NOH, in DMF, show a band at460 nm immediately after addition of H2O2 (orange solu-tion), and then, as the reaction progresses, a multiplet cen-tered at 528 nm superimposes the band at 460 nm (pinksolution). The ESI-mass spectra recorded on the pink solu-tion show peaks that are not present in the orange solutionand could correspond to MnIV species ([Mn2L2(O)2 +H2O]+ and [Mn2L2(O)2]+) giving rise to the multipletobserved in the electronic spectra. It has been reported thatexogenous bases are able to stabilize a [MnIV = O] interme-diate species [58]. Therefore, in the present case, the addedbase would delay the reduction of the oxidized form of thecatalyst to the diMnIII species, and the MnIV

2 form could beobserved by ESI-MS and UV–vis spectroscopy.

Based on the spectroscopic results we propose that themechanism for the H2O2 dismutation by complexes 1 and2 involves redox cycling between MnIII

2 and MnIV2 , and that

MnII2 complexes, once formed, are inactive final reaction

species (Scheme 2). The fact that only the MnIII2 species

are observed suggests that the measured kcat should corre-spond to the oxidation of the catalyst with concomitantreduction of H2O2 (kox). Reduction of the oxidized formof the catalyst (kred) should occur in a fast step withinthe catalytic cycle thus preventing its spectroscopic obser-vation, except for the reaction performed in basic mediumwhere the MnIV

2 form would be stabilized (kred = f([H+]))and could be observed by electronic spectroscopy andESI-MS.

Complexes 1 and 2 catalyze the H2O2 dismutation withsubstrate saturation kinetics, first order dependence of thereaction rate on catalyst and without an initial time-lag.The kinetic parameters listed in Table 3 show that complex1 can reach maximal rates higher than complex 2, in eitherDMF or methanol (Fig. 7). On the other hand, 1 has higherKM values than 2, indicating that 1 is less efficient at bind-ing H2O2. Thus, although 1 is more effective at reducingH2O2 it has more difficulty to bind the substrate so thatmaximal rates can only be achieved at [H2O2] higher than

MnIVMnIV

O O

OAc

MnIIIMnIII

OAc

kred, fastkox, slow

MnIIIMnIII

O OH2O2

MnIIMnII

H2O2

O2H2O2

H2O

H+

H2O

(inactive)

Scheme 2. Proposed mechanism for the H2O2 disproportionation cata-lyzed by complexes 1 and 2.

1670 H. Biava et al. / Journal of Inorganic Biochemistry 100 (2006) 1660–1671

for 2. This is especially evident in DMF where up to300 mM [H2O2] had to be used to reach saturation.Replacement of H–C@N–R (salpentOH) by Ph–C@N–R(1) accelerates the catalase reaction in methanol (kcat higherthan for [Mn2(salpentO)(l-OAc)(l-OMe)]+) [18], but doesnot affect either the redox potentials or KM [18]. In DMF,the bulkier substituent at the imine proton positiondecreases the affinity of the complex for the substrate –KM is twice the value found for [Mn2(salpentO)(l-OAc)-(l-OMe)]+ in the same solvent – and diminishes the redoxpotential of the complex with no direct effect on kcat

[12,18]. Complex 2 possesses the lowest KM value amongcomplexes of this family [12,18,19]. Thus, substitution ofphenolate by the naphtolate moiety in the Schiff baseligand favors formation of the catalyst-substrate adduct,in the two solvents. However, the fact that this complexis easier to oxidize than all other complexes in this series[12,18,19] does not improve kcat, indicating that the rateof the catalase reaction is not critically dependent on theredox potential of the catalyst [59]. Based on the kcat/KM

criterion, in methanol, complexes 1 and 2 are the most effi-cient catalysts of this series and are among the 12 most effi-cient biomimetic catalysts known up to date [17].

Acknowledgements

We thank the National University of Rosario and theNational Agency for Sciences Promotion for financial sup-port, and CONICET and CNRS for a bilateral agreement(Res. 2311/2004). H. Biava thanks the Embassy of Francefor a fellowship.

Appendix A. Supplementary data

Supplementary crystallographic data (CCDC-279793)for complex 1 can obtained at www.ccdc.cam.ac.uk/conts/retreiving.htlm. Figures of ESI-mass spectra of 1, 2

and the reaction mixtures are available at doi:10.1016/j.jinorgbio.2006.05.016.

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