Biomimetic Catalysts for Oxidation of Veratryl Alcohol, a Lignin Model Compound

Kraft pulp has to be bleached to eliminate the chromophoric structures, which cause a darkening of the pulp. In Nature, an equivalent role is assumed by ligninolytic enzymes such as lignin peroxidases, manganese peroxidases and laccases. The development of low molecular weight manganese peroxidase mimics may achieve environmentally-safe bleaching catalysts for the industry. Herein we report the synthesis and characterization of six manganese(III) complexes 1–6, incorporating dianionic hexadentate Schiff base ligands (H 2 L 1-H 2 L 4) and different anions. Complex 4, Mn 2 L 2 2 (H 2 O) 2 (DCA) 2 was crystallographically characterized. Complexes 1–4 behave as more efficient mimics of peroxidase in contrast to 5–6. We have studied the use of these complexes as catalysts for the degradation of the lignin model compound veratryl alcohol. The biomimetic catalysts were used in conjunction with chlorine-free inexpensive co-oxidants as dioxygen or hydrogen peroxide. Yields up to 30% of veratryl alcohol conversion to veratraldehyde have been achieved at room temperature in presence of air flow using 0.5% of catalyst.


Introduction
The predominant chemical pulping process used nowadays is the Kraft process, which, at high temperatures and under forceful alkaline conditions, is able to remove large amounts of lignin from the cellulose fiber by reductive depolymerisation with sulfide [1].The formation of various chromophoric structures giving rise to absorption of visible light cannot be avoided in the Kraft process and will cause a darkening of the pulp [2].
The objective of pulping and bleaching of wood is the selective removal of lignin without degrading the polysaccharides, and the removal of colored structures, which are originally present in the wood pulp or have been formed during the pulping process.Since toxic chlorinated compounds have been detected in the waste water streams of mills that use molecular chlorine as bleaching agent, the paper and pulp industry has been sought to design alternative bleaching sequences which use other oxidizing agents [3].So far, several different bleaching processes have been developed using dioxygen, hydrogen peroxide and ozone as proposed oxidants [4].
In our search of biomimetic models for peroxidase, we have also reported active manganese complexes involving tetradentate ONNO Schiff bases, and the influence of the geometry around the manganese ion on peroxidase activity has also been studied by us [12][13][14].
The dimeric nature of the complexes is decisive on their peroxidase activity [15].Dimeric complexes can be achieved using the appropriate ligands, for instance polydentate Schiff bases with both inner and outer compartments.
Peroxidase-like activity of the complexes was followed by the oxidation of the diammonium salt of 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) at pH 6.8 in aqueous solution.The possibility of evaluating catalyst properties of 1-6 for pulp-bleaching purposes has been studied using veratryl alcohol (VA), which can be considered as a model compound for lignin substructures [16].

Synthesis and Characterization of the Complexes
Complexes 1-6 were prepared in high yield as detailed in Section 3. ESI (electrospray-ionization) mass spectra registered in methanol show peaks corresponding to the fragment [MnL] + for all the complexes, indicating the coordination of the Schiff base ligand to the metal centre.Other minor signals could be assigned to [Mn 2 L 2 (X)] + units (being X the anion), which could be attributed to the presence of dimeric species.
Values for the magnetic moments at room temperature are very close to the spin-only value of 4.9 B.M., as expected for a high-spin magnetically diluted d 4 manganese(III) ion.
Infrared spectroscopy (IR) verifies the coordination of the Schiff base ligand to the manganese ion, since in all cases we observe a negative shift (4-10 cm −1 ) of the ν(CN imine ) band and also a positive shift (10-30 cm −1 ) of the ν(CO phenol ) mode with respect to the free ligand.These data suggest the coordination of the Schiff base in its dianionic form through the inner phenol oxygen and the imine nitrogen atoms.Some other IR spectroscopical features allow understanding how the former manganese salt anions are bound to the central ion or even indicating their coordination behavior: a.The broad unsplit band at 1120 cm −1 in 1 and 5 is indicative of the presence of the uncoordinated perchlorate anion [12], its characteristic ν 4 stretching mode at ca. 635 cm −1 can also easily be identified; b.Two new bands at 1546 and 1438 cm −1 in 2 are assigned to the symmetric and asymmetric acetylacetonate bidentate modes suggesting its coordination to the manganese ion [17]; c.The appearance of a new strong and sharp band at 1384 cm −1 , together with bands at ca. 740 and 850 cm −1 are characteristic of the non-coordinated nitrate counterion in complexes 3 and 6 [18]; d.Two new bands at 2148 and 2266 cm −1 in 4 are assigned to the symmetric and asymmetric diacyanamide modes [19].

Crystal Structure of 4
Single crystals of Complex 4, suitable for X-ray diffraction studies, were obtained by slow evaporation of the methanolic solution at room temperature.Intensity data were collected on a Bruker X8 APEXII difractometer employing graphite-monochromated Mo-Kα radiation (λ = 0.71073 Å) at 100 K.
The geometry around the manganese ion can be described as distorted octahedral.Main bond distances and angles are collected in Table 1.An ORTEP view of 4 with the atomic numbering scheme is shown in Figure 2. The coordination sphere around each manganese center comprises the planar Schiff base ligand, tightly bound to the metal ion through the inner N 2 O 2 compartment by the N imine and O phenol atoms (Mn-N imine bond lengths of 1.98-1.99Å and Mn-O phenol of 1.88 Å which are typical of such complexes and corroborate the bisdeprotonation of the ligands), occupying the equatorial positions and giving rise to two six-membered chelate rings (which are nearly planar) and an additional five-membered chelate ring.The axial positions of the octahedron are occupied by a capping water molecule and a dicyanamide molecule.The Jahn-Teller elongation expected for d 4 high-spin manganese(III) appears outstanding in the axis orthogonal to the plane of the Schiff base ligand, with distances ranging from 2.26 to 2.30 Å, considerably longer than the equatorial Mn-O bond lengths quoted earlier [12][13][14][15].
The superstructure of 4 involves associations via a combination of π-aryl offset interactions [20] and hydrogen bonds between capping water molecules and both phenoxy and methoxy oxygen atoms of the neighboring Schiff base ligand, forming μ-aquo dimeric units.These hydrogen bonds are charge-assisted, that is, the hydrogen bond donor and/or acceptor carry positive and negative ionic charges, respectively, and hence are rather strong and short.As result of these supramolecular interactions, the Mn•••Mn distances of about 4.84 Å are short for monomeric compounds.

Electrochemical Studies
The electrochemical behavior of complexes 1, 3, 4 and 5 has been studied.Cyclic voltammograms of the complexes exhibit a quasi-reversible one-electron reduction-oxidation wave, which at slow scan rates of about 0.02 V s −1 is reversible (Figure 3, Table 2).
The shape of the cyclic voltammograms for all the complexes is quite similar, with a peak-to-peak separation from 0.080 to 0.108.The rate of reversibility of these compounds suggests that they could act as catalysts in different processes.The redox potentials of 1, 4 and 5 appear in the same range, the values of 5 being slightly more positive.The reduction wave of 3 is observed at more negative potentials (E red = −0.237V).Normal pulse voltammetry can be applied to such systems and provides an additional proof of the oxidation state of the manganese in the former complexes.Anodic and cathodic currents were observed when the initial potentials were more negative than the wage range.However, when the initial potentials were more positive than the wave voltage range, only cathodic currents were observed.These data indicate that in solution only the oxidized forms of the redox systems exist, i.e., manganese(III).

Peroxidase Studies
ABTS is colorless and it reacts readily with H 2 O 2 in the presence of a peroxidase catalyst to yield a stable green colored radical cation ABTS •+ [21], which presents characteristic absorption bands at 415, 650, 735 and 815 nm.The extent of the reaction can be measured quantitatively at λ = 650 nm since ε = 12000 M −1 cm −1 has been determined.The oxidation potential of ABTS to provide ABTS •+ is invariable over a wide range of pH.In the absence of the complex, a solution of ABTS and H 2 O 2 is stable for several hours without showing any formation of ABTS •+ .
The reaction of ABTS with H 2 O 2 in the presence of 1-4 generates ABTS •+ and the characteristic absorption bands of this species could be established (Figure 4).The rate of formation of ABTS• + of about 40-62% indicates a relevant peroxidase activity by Complexes 1-4.Complexes 5-6 do not show significant peroxidase-like activity since the UV absorbances corresponding to the ABTS •+ radical cation are negligible in the assays with these complexes.The extent of the conversion of ABTS increases to 70-76% using a larger amount of the oxidant (250 μL 10 M).However the higher turnovers numbers (TON; calculated as the number of moles of substrate that a mole of catalyst can convert before becoming inactivacted) of catalytic cycles is achieved by reducing the concentration of the catalyst in solution (Figure 5).This behavior can be explained by the reaction completion so that more turnovers are observed with lower catalyst load.The percentages of conversions in these conditions range from 53 to 61%.
The exact mechanism of oxidation process is not clear at present.High-valent intermediates such as Mn(IV) or Mn(V) have been proposed in different redox processes catalyzed by other synthetic manganese complexes [22][23][24][25].Accordingly, Complexes 1-4 could follow a pathway involving the interconversion between the Mn III  2 and [Mn IV =O] 2 forms.The resolution of the crystal structure of 4 reveals a short distance between the manganese ions.Both the short intermetallic separation and the occurrence of two labile coordination sites could facilitate the µ-bridging mode of peroxide leading to O-O cleavage and formation of the [Mn IV =O] 2 form of catalyst.
The way to achieve the dimeric nature of the complexes is decisive on their peroxidase activity since the oxidation of the hydroperoxide ion involves an intramolecular two-electron transfer reaction which is forbidden for a monomeric Mn(III) complex.In this sense, the self-assembly of the manganese complexes through hydrogen bonding arises as a key issue to enhance the peroxidase activity for this type of complex [13].The methoxy groups of the outer compartments of the Schiff bases H 2 L 1 -H 2 L 4 are essential to establish a supramolecular network that approximates the manganese ions to distances below 5 Å, which is considerably shorter than the 10 Å for monomeric compounds [26].

Oxidation of the Lignin Analogue Veratryl Alcohol
Veratryl alcohol (3,4-dimethoxybenzyl alcohol) is a commonly used lignin model [27].This substrate is optimal for our experiments since Complexes 1-6 have no absorbance at 310 nm, whereas its oxidized product veratraldehyde absorbs strongly at 310 nm (ε = 9300 M −1 cm −1 ) [28].The oxidation studies have been done both in presence and absence of air flow bubbling in order to check for the effect of a saturated atmosphere of molecular oxygen in the process.Dioxygen is an attractive choice as a nonpoisonous and inexpensive oxidant for industrial processes.The processes that are based on molecular oxygen can also be considered to be ecologically benign processes, because water and hydrogen peroxide are the main side products derived from the oxidant [29].
Veratraldehyde product were isolated and purified following the procedure described in Section 3.6. 1 H nuclear magnetic resonance (NMR) spectroscopy confirms the absence of the methylene proton signal at 4.61 ppm from the original veratryl alcohol as well as the appearance of a new peak at 9.62 ppm corresponding to the aldehyde.A control reaction under the same conditions without catalyst did not give any fraction of veratraldehyde.
The results of the oxidation of veratryl alcohol to veratraldehyde are shown in Figure 6.The activity of the catalysts 1-6 decreases as the length of the alkyl bridge between the coordinating amines increases.Complexes 1, 2 and 4, behave as better catalysts in this process.An explanation arises from the short two-carbon chain between imine groups in these complexes; the short chain length constricts the chelate ring once nitrogens coordinate to the metal and it leads to a tetragonally elongated octahedral geometry.An axial water molecule in this class of distorted geometries constitutes a quite labile ligand, which would generate a vacant position in the coordination sphere to accommodate the substrate molecule.On the other hand, the flexible three-membered alkyl chain between the imine groups in the Mn compounds 5-6 favors a better stabilization of a high-symmetry octahedral geometry, which subsequently makes the generation of a vacancy difficult.The activity of 3, intermediate between these two groups of complexes, may be derived from its more negative potentials observed in electrochemical studies.
Conversions up to 30% were obtained at 22 °C with an air flow bubbling for 10 h, using the Catalyst 4 and three equivalents of H 2 O 2 respective to the substrate.These are mild conditions compared with the experimental setup of other authors [30,31], where high temperatures and pressures are needed to reach similar yields with different catalysts.Moreover, most of the oxygen-activating homogeneous transition-metal catalysts are investigated in organic solvents [32,33]; however, only a few are known to be active in water [34].
No conversion has been shown in absence of catalyst or in absence of hydrogen peroxide.Air flow providing oxygen as oxidant is crucial to increase the yield, but a degradation of about 7% of veratryl alcohol is observed when the experiment was done without air flow.However, presence of both catalyst and H 2 O 2 is necessary to find some degradation of veratryl alcohol.This behavior suggests that hydrogen peroxide could induce the oxidation of the catalyst to a more catalytically active species, such as [Mn IV =O] 2 dimers, which subsequently would degrade the veratryl alcohol using dioxygen as oxidant.

Materials
All the starting materials (Aldrich) and solvents (Probus) used for the synthesis were of commercially available reagent grade and were used without further purification.

Physical Measurements
Elemental analyses were performed on a Carlo Erba Model 1108 CHNS-O elemental analyzer.The IR spectra were recorded as KBr pellets on a Bio-Rad FTS 135 spectrophotometer in the range of 4000-400 cm −1 . 1 H NMR spectra were recorded on a Bruker AC-300 spectrometer using CD 3 OD (296 K) as solvent and SiMe 4 as an internal reference.The electro-spray mass spectra of the compounds were obtained on a Hewlett-Packard model LC-MSD 1100 instrument (positive ion mode, 98:2 CH 3 OH-HCOOH as mobile phase, 30 to 100 V).Room-temperature magnetic susceptibilities were measured using a digital measurement system MSB-MKI, calibrated using mercury tetrakis(isothiocyanato)cobaltate(II) Hg[Co(NCS) 4 ] as a susceptibility standard.Conductivities of 10 −3 M solutions in DMF were measured on a Crison microCM 2200 conductivity meter.
Electrochemical experiments were performed using an EG&G PAR model 273 potentiostat, controlled by EG&G PAR model 270 software.A Metrohm model 6.1204.000graphite disc coupled to a Metrohm model 628-10 rotating electrolyte device was used as a working electrode.A saturated calomel electrode was used as a reference and a platinum wire used as an auxiliary electrode.All measurements were made with ca. 10 −3 mol dm −3 solutions of the complexes in dimethylformamide using 0.2 mol dm −3 NBu 4 PF 6 as a supporting electrode.Cyclic voltammetry measurements were performed with a static graphite electrode, whilst direct-current and pulse voltammograms were recorded with the graphite disc rotating at 2000 revolutions per minute.

Synthesis of the Complexes
Mn 2 L 1 2 (H 2 O) 4 (ClO 4 ) 2 (1), 1.99 mmol (1.00 g) of H 2 L 1 was dissolved in 100 mL of a 1:1 methanol-ethanol mixture and 1.99 mmol (0.72 g) of Mn(ClO 4 ) 2 .6H 2 O was added to the initial yellow solution which changed to green (CAUTION: Although no problems were encountered in this work, perchlorates are potentially explosive and should be handled in only small quantities and with care!).
After stirring for 10 min, 3.98 mmol (0.16 g) of NaOH, dissolved in a small quantity of water, was added, and the mixture turned dark.The progress of the reaction was followed by thin-layer chromatography (TLC) for three days and the mixture was then filtered.The complex was obtained from the filtrate as a brown solid after crystallization.It was isolated by filtration and washed with diethyl ether and dried in air.Anal

Peroxidase Probes
Oxidation of ABTS with H 2 O 2 at ca. pH 7 in the presence of the complexes was tested in the following manner.An aqueous solution of ABTS (50 μL; 0.009 M; 4.5 × 10 −7 mol) and a methanolic solution of the complex (10 μL; 10 −3 M; 10 −8 mol) were added to water (3 mL).The intensity of the UV absorption bands of ABTS started to increase immediately after addition of an aqueous solution of H 2 O 2 (50 μL; 10 M; 5 × 10 −4 mol).The disproportionation of hydrogen peroxide by the Complexes 1-6 (catalase activity) was not observed for the concentrations of this peroxidase assay.

Oxidation of Veratryl Alcohol
Veratryl alcohol (200 mmol, 0.29 mL), previously dissolved in 20 mL of dihydrogenphosphate/NaOH buffer pH 8, was mixed with a methanolic solution (15 mL) of the catalytic complex (0.5 mmol, ca.6 mg).The oxidant (600 mmol) was added in three portions at 20 min intervals.The reaction mixture was stirred for 10 hours at 22 °C with an air flow bubbling through the solution and then filtered through a short silica gel plug to remove the catalyst and excess oxidant.The reaction mixture was extracted in CH 2 Cl 2 in the presence of saturated aqueous NaCl solution.The organic layer was dried over MgSO 4 and evaporated under reduced pressure.The mixture was then purified by column chromatography using a 1:1 mixture of ethyl acetate:hexane as eluent.The veratraldehyde fraction was characterized by NMR spectrometry. 1

Conclusions
This work again further emphasizes the suitability of Schiff bases with inner and outer O-R (R = CH 3 in the present work) groups for establishing rich hydrogen-bonding networks.The X-ray structure of 4 shows the self-assembly of the Mn(III)-Schiff base complexes through μ-aquo bridges.This type of structure is crucial to obtain peroxidase-like activity for these complexes.1-4 behave as efficient peroxidase mimics achieving TON close to 300 catalytic cycles.The four complexes are also able to decompose veratryl alcohol, a lignin analogue, with relevant yields in very mild conditions.

Figure 2 .
Figure 2. ORTEP view of the environment around the manganese ion in 4 (left) and mercury drawing of the resulting µ-aquo dimer (right).

Figure 6 .
Figure 6.Conversions of veratryl alcohol (VA) to veratraldehyde catalyzed by 1-6: (a) reaction conditions as detailed in Section 3.6 but in absence of air flow; (b) reaction conditions as detailed in Section 3.6 but reducing reaction time to 3 h; (c) reaction conditions as detailed in Section 3.6.

Table 2 .
Electrochemical data for the complexes (values in V).