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Radical mediated indirect oxidation of a PEG-coupled polycyclic aromatic hydrocarbon (PAH) model compound by fungal laccase
Biochimica et Biophysica Acta 1474 (2000) 157^162
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Radical mediated indirect oxidation of a PEG-coupled polycyclic aromatic hydrocarbon (PAH) model compound by fungal laccase Andrzej Majcherczyk *, Christian Johannes Institut fu«r Forstbotanik, Georg-August-Universita«t Go«ttingen, Bu«sgenweg 2, 37077 Go«ttingen, Germany Received 14 September 1999; received in revised form 20 January 2000; accepted 28 January 2000
Abstract A high molecular model compound of polycyclic aromatic hydrocarbon was synthesised by coupling pyrene to PEG5000 . The pyrene-PEG was used for the study of a laccase-mediator-system. To prevent direct contact between the substrate and the enzyme the two were kept in their own compartments separated by a membrane. The low molecular mediators, 1-hydroxybenzotriazole and 2,2P-azino-bis-(3ethylbenzothiazoline-6-sulphonic acid), which were oxidised by laccase to the corresponding radicals or cations permeated the membrane and reacted with the pyrene-PEG model compound. Oxidation of the model compound resulted in an K-oxidation of the alkyl-chain leading to two main oxidation products. The same oxidation products were obtained in the reaction system without a membrane. ß 2000 Elsevier Science B.V. All rights reserved. Keywords : Polycyclic aromatic hydrocarbon; Laccase-mediator-system ; Laccase; Pyrene
1. Introduction The successful application of fungal laccases together with low molecular mediator compounds, so called mediators, in the bio-bleaching of wood pulp (e.g. [1,2]) and organic synthesis (e.g. [3,4]) has increased the interest in these enzymes during the last few years. Recently, the degradation of environmentally relevant chemicals by the laccase-mediator-system (LMS) was demonstrated through the oxidation of polycyclic aromatic hydrocarbons (PAH) [5^7]. The mechanism of these reaction systems has been discussed and the applied mediators were speculated : (1) to modify the enzyme reaction centre and thus extend the substrate spectrum, (2) to in£uence the oxidation mechanism and allow a two electron transfer from the substrate or (3) to mediate the oxidation as an electron transfer agent. The formation of radicals (or radical cations) from the mediator compound by laccases and the ability of these species to perform the oxidations of LMS were recently demonstrated [8] making the latter hypothesis the most probable explanation of the LMS reaction mechanism. This supposition is conform with a report on the depolymerisation of synthetic lignin by laccase from Pyc-
* Corresponding author. Fax: +49-551-39-27-05; E-mail : [email protected]
noporus cinnabarinus using 1-hydroxyanthranilic acid as a mediator compound [9]. The mechanism of the reaction and its general applicability, however, are still unknown and veri¢cation of this indirect mechanism by the oxidation of non-phenolic compounds in comparable experiments is still lacking. In the present study a high molecular PAH model compound was synthesised and applied in a compartment system with a membrane physically separating the enzyme from the substrate and only allowing the low molecular mediator compounds to reach both compartments. 2. Materials and methods 2.1. Chemicals 1-Hydroxybenzotriazole (HBT) was purchased from Sigma (Deisenhofen, Germany). All other reagents and substrates were provided by Aldrich (Steinheim, Germany) and Fluka (Neu-Ulm, Germany). 2.2. Model compounds K-(2-Bromoethyl)-g-monomethoxy-poly(oxyethylene) (Br^PEG, 2, see Scheme 1) was prepared according to Johansson et al. [10] modi¢ed by Bu«ckmann and Morr
0304-4165 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 0 0 ) 0 0 0 1 2 - X
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Scheme 1.
[11]. 70 mmol of poly(oxyethylene)monomethylether (PEG; Mw 2000) were dissolved in 1.6 l toluene and then 450 ml toluene were distilled o¡ to remove traces of moisture; an additional 100 ml were distilled o¡ separately for dilution of thionylbromide. The solution of PEG was chilled to 35³C and 28 ml of triethylamine (203 mmol) were added. Approximately 160 mmol thionylbromide (12 ml) were diluted with 100 ml dry toluene and added dropwise to the PEG solution during 1 h with continuous stirring under a dry nitrogen atmosphere. After the addition was completed, the reaction mixture was stirred with gentle re£uxing for another 1 h. Triethylammonium bromide was removed by passing the hot solution through a sintered glass funnel with a 2 cm thick layer of diatomeous earth. After 4 h incubation at room temperature, the solution was warmed to 50³C, treated with 40 g charcoal, and ¢ltered again over a new layer of diatomeous earth; the ¢ltercake was washed with a small amount of hot toluene. The combined solutions were left overnight at 4³C to crystallise the product and toluene was removed by ¢ltration. Crude Br^PEG was dissolved in 1.0 l of absolute ethanol at 60³C and treated again with 20 g charcoal. After ¢ltration through diatomeous earth the solution was kept at 4³C overnight to re-crystallise the Br^PEG. The solid material was separated by ¢ltration, washed with cold ethanol and dried in a vacuum over P2 O5 . The pale yellow product (282 g, yield 80%) had a substitution grade of 75% as determined by 13 C-NMR. K-(2-Mesyl-ethyl)-g-monomethoxy-poly(oxyethylene) (Mesyl^PEG, 3) was synthesised from the corresponding PEG (Mw 5000) by a modi¢ed method of Harris et al. [12]. 50 mmol of PEG were dissolved in 500 ml dichloromethane containing 10 g triethanolamine and chilled on ice. A solution of methanesulfonyl chloride (28 g) in 500 ml dichloromethane was added slowly and the reaction mixture stirred for 3 h at 0³C. The solution was reduced to about 500 ml using a rotatory evaporator and the product was precipitated from a viscous solution by the addition of 1000 ml distilled diethylether followed by 500 ml petroleum ether (boiling point 40^60³C). The precipitate was separated by ¢ltration, dissolved in a small amount of dichloromethane and then washed successively with 100 ml 0.1 N HCl, 100 ml saturated sodium hydrogencarbonate, and water until neutral. The product was precipitated with diethyl ether, dissolved in a small amount of dichloromethane and precipitated again. After drying in a vacuum Mesyl^PEG was obtained as a white powder (218 g, yield
86%). The substitution grade was determined after hydrolysis of 1 g sample with 10 ml 0.1 N NaOH (16 h at 90³C) followed by titration with 0.1 N HCl to be s 95%. K-(1-Pyrene-butyl)-g-monomethoxy-poly(oxyethylene) (pyrene^PEG, 4) was obtained from the Mesyl^PEG (or Br^PEG) and 1-pyrenebutanol. 10 mmol 3 were dissolved in dry tetrahydrofuran to obtain a 10% solution. 1-Pyrenebutanol (1), in a small excess amount with respect to the functionalised part of PEG, was dissolved in dry tetrahydrofuran (ca. 20 ml per 1 g PAH) and an excess of potassium-tert-butylate (ca. 5 g per 1 g of PAH) was added. The solution was stirred for 30 min at room temperature under argon and slowly added to a solution of (3). The reaction mixture was stirred at 50³C for 70 h under a re£ux condenser with a moisture trap. Progress of the reaction was monitored by thin layer chromatography (TLC) using Silicagel-F254 plates (Merck) with chloroform/methanol (9:1) as the eluent and inspected under UV ; PEG was detected with iodine vapour. Finally, an excess of acetic acid was added and after addition of 1 g charcoal, the mixture was stirred for 15 min at 50³C. The suspension was ¢ltered through 1 cm layer of diatomeous earth and the ¢ltercake washed with dichloromethane. The ¢ltrate was concentrated using a rotatory evaporator and the product precipitated twice with an excess of diethyl ether. Pyrene-PEG (4) was dissolved in dichloromethane, passed through a small silica gel column (3U10 cm) and eluted with 10% methanol in dichloromethane. The eluate was concentrated and the pyrene^PEG precipitated again with diethyl ether, dissolved in chloroform and puri¢ed on a silica gel column (3U40 cm, Type 60, Merck). 4 was eluted with chloroform, 10% and 50% methanol in chloroform successively (450 ml each) while being inspected with a UV-detector. Fractions containing 4 (inspected by TLC) were pooled and chloroform was evaporated in vacuum. After dissolving in water the product was washed in an Amicon cell with Y3 membrane and the concentrated solution was freeze-dried resulting in a 60% yield. The structure of the pyrene^PEG was con¢rmed by UV-vis- and 13 C-NMR spectroscopy and compared to the data obtained from pyrene and PEG alone. The amount of bonded PAH was measured by absorption in 20% methanol. This indicated an 18% substitution degree. The purity of the product was checked with TLC and HPLC, indicating, in addition to 4, only free PEG and a small amount of oxidation products; 1-pyrenebutanol was not detected.
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2.3. HPLC analysis Pyrene^PEG was analysed by HPLC (HP 1090 with a DAD detector, Hewlett-Packard) using a LiChrospher 100 RP-18 5 Wm column (4U125 mm). The separation was obtained in a gradient mode from 10% acetonitrile/10% methanol (solvent A) to 50% acetonitrile/50% methanol (solvent B) with a £ow rate of 1 ml/min. After an isocratic run for 1 min in solvent A, the mobile phase was changed by a linear gradient over 15 min to solvent B and the elution was continued for an additional 2 min. Absorption spectra within a range of 200^600 nm were recorded. 2.4. Laccase and enzyme assay Laccase from Trametes versicolor was puri¢ed as previously described [7,13]. The enzyme activity was determined by the oxidation of 2,2P-azino-bis-(3-ethylbenzothiazoline6-sulphonic acid) (ABTS) [6]. 2.5. Oxidation of pyrene^PEG by laccase-mediator-systems All experiments were performed under sterile conditions using sterile-¢ltered solutions and autoclaved equipment. 2 ml of the 0.2 mM pyrene^PEG solution placed in a dialysis tube (cut-o¡ 3000; Serva) were immersed in 100 ml £asks with 20 ml of 0.1 M phosphate bu¡er (pH 4.5) containing 0.2 mM PEG5000 . Laccase (4 U/ml) and mediator compounds (2 mM ABTS or 1 mM HBT ¢nal concentrations) were added to the outer bu¡er and the system was incubated for 48 h on a rotary shaker (40 rpm). Control samples were performed as above without the addition of mediators or the enzyme. Pyrene^PEG and 1-pyrenebutanol were also incubated in the same system without a separating membrane. The samples from the outer bu¡er and from the dialysis tube were analysed by HPLC. 2.6. Oxidations by ABTS2 + and HBT ABTS dication was prepared by the oxidation of ABTS with potassium peroxodisulphate [14] as previously described [8]. HBT radical was obtained by the oxidation of HBT (saturated solution, approximately 1.5 mM) by PbO2 in water- and ethanol-free chloroform [15]. 3. Results The HPLC method made a separation of the pyrene^ PEG and 1-pyrenebutanol possible and revealed the model compound to be free of the latter. The absorption spectrum of the model compound was identical to that of pyrene or 1-pyrenebutanol (210^600 nm). The water soluble PAH-model compound, pyrene^PEG, was completely oxidised by LMS utilising ABTS or HBT. No signi¢cant degradation by laccase without a mediator
Fig. 1. Chromatogram of pyrene^PEG model compound after oxidation by LMS using ABTS (upper panel) and HBT (lower panel) as the mediator compounds in a membrane-separated system. Inserts: absorption spectra (HPLC-on-line) of pyrene^PEG, compounds I and II.
was observed. To test the assumption of the mediated oxidation of pyrene by LMS without contact to the enzyme, the pyrene^PEG model (Mw 5200 Da) was separated from the enzyme using a dialysis tube with a cuto¡ of 3000 Da. Laccase and the mediator compounds, ABTS or HBT, were added to the outer bu¡er also containing the same concentration of PEG (Mw 5000 Da) as the pyrene^PEG inside the tube. Under these conditions, only the mediator compounds or their oxidised species were able to pass through the membrane. This was also visible in the case of ABTS: the blue-green radical cation was observed both outside and inside the tube. After incubation with laccase and ABTS for 48 h only 6% þ 3% of the initial pyrene^PEG were detected in the inner solution. The model compound was completely metabolised by LMS using HBT (Table 1). Two main oxidation products were detected in the inner solution: compounds I and II (Fig. 1). The same compounds were detected in reaction systems without a membrane. No pyrene^PEG or reaction products were detected in the solution outside the dialysis tube. The relative amounts of the products were approximately two times higher in the case of ABTS than in the system utilising HBT as mediator. In the latter case, small amounts of other products (III and IV) were detected, indicating further metabolisation of pyrene^PEG. The absorption spectrum of compound I in the range 210^600 nm was identical to the spectrum of pyrene^PEG (or 1-pyrenebutanol and pyrene). Compound II revealed a
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Table 1 Oxidation of pyrene^PEG (0.2 mM, 18% pyrene content) model compound by laccase from T. versicolor (4 U/ml) in presence of ABTS (2 mM) or HBT (1 mM) Membrane separation
Pyrene^PEG (% of initial)
Laccase/ABTS Laccase ABTS
3 + +
97 þ 4 6þ7 0þ5
Laccase/ABTS Laccase/HBT Laccase HBT Laccase/HBT
+ 3 + + +
94 þ 3 100 þ 0 0þ0 0þ0 100 þ 0
4. Discussion
spectrum with broad absorption bands at 240, 278, 350 nm and a shoulder at 382 nm (see inserts in Fig. 1). A detailed characterisation of the pyrene^PEG oxidation products was not possible and therefore LMS was applied in the oxidation of the synthesis substrate, 1-pyrenebutanol. The oxidation of 1-pyrenebutanol using HBT resulted in 95% removal of this compound and the appearance of two reaction products in the HPLC (data not presented). A complete oxidation of this compound was also achieved using a chemically generated HBT in chloroform (Table 2). Comparable metabolisation of 1pyrenebutanol was obtained by precipitated ABTS2 , respectively by LMS using ABTS; in the latter case only the second product was detected. One product obtained by LMS oxidation of 1-pyrenebutanol revealed an absorption spectrum identical to that of the parent compound (or pyrene); the spectrum of the second displayed broad absorption bands at 240, 278, 350 nm and a shoulder at 382 nm and corresponded to the spectrum of compound II from the pyrene^PEG oxidation. The products were analysed by direct inlet MS (EI 70 eV, Finnigan MAT 95) and GC^MS (as TMS derivatives) and are assumed to correspond to the 1-pyrene-K-furan (m/z [relative intensity] : 42[9], 101[8], 202[36], 215[22], 229[49], 239[20], 272[100]) and 1-pyrene-K-keto-butanol (EI-MS : 100[8], 201[78], 244[18], 229[100], 270[39], 288[24]; GC^MS of its TMSethers: 73[6], 75[10], 201[47], 229[50], 244[100], 345[10], 360[19] ; di-TMS ethers - two cis-trans-isomers - of the enol compound: 73[100], 147[7], 201[12], 239[71], 255[26], 314[32], 329[84], 432[19]). To con¢rm the indirect oxidation of aromatics 13 PAH Table 2 Oxidation of pyrene (25 WM) and 1-pyrenebutanol (40 WM) by laccase from T. versicolor (4 U/ml) in presence of ABTS or HBT (1 mM) and by radical/cation ; 24 h incubation Laccase Laccase/ABTS ABTS2 Laccase/HBT HBT
were oxidised using chemically generated HBT . Most of the compounds were e¡ectively oxidised in this reaction (Table 3) and considering the partially di¡erent reaction conditions (e.g. a chloroform solution of HBT ) a very similar oxidation `pattern' and reaction yield of numerous PAH in comparison to the LMS reactions were obtained and thus strengthened the supposition of an indirect oxidation of PAH by LMS system.
Pyrene (% oxidised)
1-Pyrenebutanol (% oxidised)
8þ4 6þ5 7þ4 48 þ 4 43 þ 3
63þ3 95 þ 3 88 þ 3 95 þ 3 100 þ 2
The coupling of PAH to high molecular PEG was performed to obtain a compound that can be applied in LMS without direct contact to the enzyme. Di¡erent means of synthesis and various derivatives were tested. Mesylateactivated PEG remained stable during storage ; it reacts very e¤ciently and quickly in displacement reactions [12]. Still, the results obtained were comparable to the reactions using Br-activated PEG - both alternatives are therefore presented. Alternative synthesis of PAH^PEG compounds via e.g. Grignard compounds from the corresponding PAH-bromide and PEG-bromide, or a coupling of PAH to PEG as an ester via imidazolide using the corresponding PAH-alkyl-acid (e.g. 2-naphthaleneacetic acid) and native PEG were not e¡ective enough or resulted in unstable products. The analogous napthalene^PEG was also obtained through the above synthesis from the corresponding naphthalene^alkyl derivative. Up to now, the only application of LMS demonstrating an indirect, membrane-separated oxidation using 1-hydroxyanthranilic acid as the mediator compound has been reported for the depolymerisation of a synthetic lignin model [9]. However, 1-hydroxyanthranilic acid was not e¡ective as a mediator for the oxidation of PAH by laccase from T. versicolor [7]. The formation of radical species resulting from the mediator compounds by the action of laccases and also the ability of these compounds to Table 3 Oxidation of PAH by HBT and laccase in bu¡er and HBT in chloroform PAH Naphthalene Acenaphthene Acenaphthylene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Benzo[a]anthracene Chrysene Benzo[b]£uoranthene Benzo[k]£uoranthene Benzo[a]pyrene Perylene
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Oxidation of PAH (% of control) Laccase/HBT
HBT
0 þ 3.0 99.0 þ 0.6 100.0 þ 0.0 91.9 þ 0.7 5.5 þ 2.4 98.7 þ 0.1 3.0 þ 4.1 48.1 þ 1.7 52.9þ 1.9 0.0 þ 3.1 9.0 þ 2.5 12.3 þ 4.3 91.3 þ 0.4 96.2 þ 0.1
4.7 þ 1.7 77.2 þ 1.2 94.1 þ 1.3 47.2 þ 1.3 12.1 þ 4.8 97.7 þ 1.2 16.3 þ 2.8 42.4 þ 2.0 90.8 þ 3.3 25.3 þ 4.1 43.5 þ 6.0 45.2 þ 11.1 99.0 þ 2.1 97.3 þ 3.3
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oxidise non-phenolic compounds were assumed (e.g. [3,8,16^18]) but the mediated indirect oxidation was not experimentally demonstrated. The application of a membrane separating the laccase and pyrene^PEG allowed us to study the oxidation reactions under conditions in which only the mediator compounds - respectively the radicals produced by laccase were able to react with the model compound. As expected, the mediator compound and laccase alone did not oxidise the pyrene^PEG in this system. In the system consisting of laccase and ABTS or HBT the model compound was nearly completely oxidised. The oxidation yield and the products obtained corresponded to results found by a direct reaction without a separating membrane. The results clearly demonstrated that the reactions were performed by oxidised species of the mediator compound (HBT or ABTS2 ) and that the laccase was not directly involved in the reactions. The oxidation of 1-pyrenebutanol and pyrene^PEG was more e¡ective than the oxidation of pyrene by LMS (approximately 50%). These results can not simply be explained by the lower solubility of pyrene but are probably the result of the presence of the alkyl side chain. The main reaction products detected were still coupled to PEG and were not able to permeate the membrane; minor oxidation products have not been described. The main products were characterised by their UV absorption spectra indicating that compound I possessed an unchanged aromatic system and was probably modi¢ed on the side chain only. Analogous to the results obtained with 1-pyrene-butanol, compound I was expected to be K-(1-pyrene-K-hydroxy-butoxy)-g-monomethoxy-poly(oxyethylene). The spectra of compound II indicated changes in an aromatic system or the presence of an additional conjugated double bond (e.g. carbonyl group) on the alkyl site. The UV^vis data of this compound correspond to the data of the product obtained from the oxidation of 1-pyrenebutanol and was therefore assumed to be K-(1-pyrene-K-keto-butoxy)-g-monomethoxy-poly(oxyethylene). The oxidation of the alkyl part of the substituent-activated aromatic compounds was previously reported (e.g. [3,19]). Under the reaction conditions used here only a very low oxidation of ethyl^benzene was obtained indicating that the reactions performed by laccase and mediators (ABTS and HBT) probably not proceeded via abstraction of the benzylic hydrogen atom and the aromatic structure was involved in the process. The mechanism involving an abstraction of benzylic H-atom would be theoretically possible for the reaction involving the N-oxide radical of HBT but seems to be unlikely for the ABTS2 . PAH radical cations (PAH ) formed by an abstraction of one electron are generally less reactive than the corresponding carbon-centred radicals and carbo-cations; their reactivity is dominated by the degree of coupling between the charge and the radical centres [20]. The cation centre of PAH may undergo a nucleophilic attack by water (poor nucleophile) and the resulting radical easily under-
161
Scheme 2. Proposed pathway for the oxidation of pyrene^PEG by LMS.
goes further oxidation to the hydroxy^PAH [21]. The latter compounds are also readily oxidised to the ¢nal quinones. Alternatively, disproportionation of cation radicals to the parent compound and dication lead to the same quinones but is only probable under water-free conditions. The reactivity of the radical centres of PAH and its intermediates can result in a polymerisation of the aromatic compounds [22] or a coupling with the mediator radical
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- formation of both in LMS was reported in our previous studies [6,7]. In addition to the above reaction mechanisms, the aromatic cation radicals resulting from alkyl^arenes (e.g. alkyl^PAH) can react on another pathway via proton abstraction - a very quick and irreversible process - by bases or basic nucleophiles (e.g. [23^25]). The substitution on an aromatic ring and the deprotonation of the side chain can be strongly in£uenced by the oxidant [26] and also di¡ers in oxidative biochemical systems depending on the enzymes involved [27]. Water accelerates the loss of proton and the concurrence between this pathway and the substitution on the aromatic ring is in£uenced by the pH-value of the solution (e.g. [28^30]). The deprotonation of the alkyl chain results in a radical - probably resonance stabilised - which may undergo further oxidation to the corresponding cation and ¢nally, after addition of water (or hydroxyl ion) to benzylic alcohols that can be further oxidised to the aldehydes. The above process and enhanced reactivity of substituent activated aromatics is well explained by the hypothesis of Parker [20] with regard to the decoupling of cation and radical centres. Accordingly, the deprotonation of the 4-methoxytoluene cation radical proceeds extremely fast in contrast to the relative stable methylbenzenes cation radicals. This explains the high yield of the corresponding aldehydes or K-ketones from the substituent activated alkyl^benzenes obtained by the LMS- or chemical oxidation using the same mediator compounds (e.g. [3,4,31]). According to the above mechanism, the oxidation of the alkyl^pyrene (1-pyrenebutanol or pyrene^PEG) results (via cation radical) in an alkyl cation. The subsequent addition of water (or hydroxyl ion) and further oxidations lead to the K-keto products (Scheme 2). Alternatively, an intramolecular addition of the hydroxyl-group of the 1pyrenebutanol can result in a cyclic ether: 1-pyrene-K-furan. The mechanism of the reactions may be very complex and also involve sandwich- or charge^transfer complexes of the aromatics and oxidants [32,33]. Since the LMS oxidation of pyrene^PEG proceeds through the abstraction of an electron from the aromatic site, this compound can be treated as a suitable high molecular model of PAH. It permitted an experimental demonstration of the mediated oxidation of aromatic compounds by LMS and could also be useful in intracellular/extracellular degradation studies in vivo. Based on the above results, the role of natural mediator compounds - plants and fungal metabolites - in the laccase-mediated oxidation of PAH by wood degrading fungi could be demonstrated (submitted for publication). Acknowledgements The authors thank Dr. G. Remberg for EI^MS measurements and Dr. R. Machinek for NMR measurements
(Institute for Organic Chemistry, University of Go«ttingen). References [1] R. Bourbonnais, M.G. Paice, Appl. Microbiol. Biotechnol. 36 (1992) 823^827. [2] H.-P. Call, I. Mu«cke, J. Biotechnol. 53 (1997) 163^202. [3] E. Fritz-Langhals, B. Kunath, Tetrahedron Lett. 39 (1998) 5955^ 5956. [4] A. Potthast, T. Rosenau, C.L. Chen, J.S. Gratzl, J. Org. Chem. 60 (1995) 4320^4321. [5] P.J. Collins, M.J. Kotterman, J.A. Field, A.D. Dobson, Appl. Environ. Microbiol. 62 (1996) 4563^4567. [6] C. Johannes, A. Majcherczyk, A. Hu«ttermann, Appl. Microbiol. Biotechnol. 46 (1996) 313^317. [7] A. Majcherczyk, C. Johannes, A. Hu«ttermann, Enzyme Microb. Technol. 22 (1998) 335^341. [8] A. Majcherczyk, C. Johannes, A. Hu«ttermann, Appl. Microbiol. Biotechnol. 51 (1999) 267^276. [9] C. Eggert, U. Temp, J.F. Dean, K.-E.L. Eriksson, FEBS Lett. 391 (1996) 144^148. [10] G. Johansson, A. Hartman, P.A. Albertsson, Eur. J. Biochem. 33 (1973) 379^386. [11] A. Bu«ckmann, M. Morr, Makromol. Chem. 182 (1981) 1379^1384. [12] J.M. Harris, E.C. Struck, M.G. Case, M.S. Paley, M. Yalpani, v. Alstine, D.E. Brooks, J. Polym. Sci. 22 (1984) 341^352. [13] C. Johannes, A. Majcherczyk, A. Hu«ttermann, J. Biotechnol. 61 (1998) 151^156. [14] P. Maruthamuthu, L. Venkatasubramanian, P. Dharmalingam, Bull. Chem. Soc. Japan 60 (1987) 1113^1117. [15] H.G. Aurich, G. Bach, K. Hahn, G. Ku«ttner, W. Weiss, J. Chem. Res. (M) (1977) 1544^1566. [16] R. Bourbonnais, D. Leech, M.G. Paice, Biochim. Biophys. Acta 1379 (1998) 381^390. [17] K.C. Li, R.F. Helm, K.E.L. Eriksson, Biotechnol. Appl. Biochem. 27 (1998) 239^243. [18] P.J. Collins, A.D.W. Dobson, J.A. Field, Appl. Environ. Microbiol. 64 (1998) 2026^2031. [19] A. Potthast, T. Rosenau, C.L. Chen, J.S. Gratzl, J. Mol. Catal. A: Chem. 108 (1996) 5^9. [20] V.D. Parker, Acta Chem. Scand. 52 (1998) 145^153. [21] C.V. Ristagno, H.J. Shine, J. Org. Chem. 36 (1971) 4050^4055. [22] A. Gold, K. Jayaraj, L.M. Ball, K. Brust, J. Mol. Catal. A: Chem. 125 (1997) 23^32. [23] V.D. Parker, L. Eberson, Acta Chem. Scand. 24 (1970) 3542^3552. [24] C.J. Schlesener, C. Amatore, J.K. Kochi, J. Am. Chem. Soc. 106 (1984) 3567^3577. [25] C.J. Schlesener, C. Amatore, J.K. Kochi, J. Am. Chem. Soc. 106 (1984) 7472^7482. [26] E. Baciocchi, C. Rol, G.V. Sebastiani, Gazz. Chim. Ital. 112 (1982) 513^517. [27] P. Anzenbacher, T. Niwa, L.M. Tolbert, S.R. Sirimanne, F.P. Guengerich, Biochemistry 35 (1996) 2512^2520. [28] K. Sehested, J. Holcman, J. Phys. Chem. 82 (1978) 651^653. [29] L.M. Tolbert, R.K. Khanna, J. Am. Chem. Soc. 109 (1987) 3477^ 3478. [30] L.M. Tolbert, R.K. Khanna, A.E. Popp, L. Gelbaum, L.A. Bottomley, J. Am. Chem. Soc. 112 (1990) 2373^2378. [31] C. Einhorn, J. Einhorn, C. Marcadal, J.-L. Pierre (1997) Chem. Commun. 447^448. [32] M.E. Sigman, P.F. Schuler, M.M. Ghosh, R.T. Dabestani, Environ. Sci. Technol. 32 (1998) 3980^3985. [33] U. Svanholm, V.D. Parker, J. Chem. Soc. Perkin Trans. II (1976) 1567^1574.
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Radical mediated indirect oxidation of a PEG-coupled polycyclic aromatic hydrocarbon (PAH) model compound by fungal laccase Andrzej Majcherczyk *, Christian Johannes Institut fu«r Forstbotanik, Georg-August-Universita«t Go«ttingen, Bu«sgenweg 2, 37077 Go«ttingen, Germany Received 14 September 1999; received in revised form 20 January 2000; accepted 28 January 2000
Abstract A high molecular model compound of polycyclic aromatic hydrocarbon was synthesised by coupling pyrene to PEG5000 . The pyrene-PEG was used for the study of a laccase-mediator-system. To prevent direct contact between the substrate and the enzyme the two were kept in their own compartments separated by a membrane. The low molecular mediators, 1-hydroxybenzotriazole and 2,2P-azino-bis-(3ethylbenzothiazoline-6-sulphonic acid), which were oxidised by laccase to the corresponding radicals or cations permeated the membrane and reacted with the pyrene-PEG model compound. Oxidation of the model compound resulted in an K-oxidation of the alkyl-chain leading to two main oxidation products. The same oxidation products were obtained in the reaction system without a membrane. ß 2000 Elsevier Science B.V. All rights reserved. Keywords : Polycyclic aromatic hydrocarbon; Laccase-mediator-system ; Laccase; Pyrene
1. Introduction The successful application of fungal laccases together with low molecular mediator compounds, so called mediators, in the bio-bleaching of wood pulp (e.g. [1,2]) and organic synthesis (e.g. [3,4]) has increased the interest in these enzymes during the last few years. Recently, the degradation of environmentally relevant chemicals by the laccase-mediator-system (LMS) was demonstrated through the oxidation of polycyclic aromatic hydrocarbons (PAH) [5^7]. The mechanism of these reaction systems has been discussed and the applied mediators were speculated : (1) to modify the enzyme reaction centre and thus extend the substrate spectrum, (2) to in£uence the oxidation mechanism and allow a two electron transfer from the substrate or (3) to mediate the oxidation as an electron transfer agent. The formation of radicals (or radical cations) from the mediator compound by laccases and the ability of these species to perform the oxidations of LMS were recently demonstrated [8] making the latter hypothesis the most probable explanation of the LMS reaction mechanism. This supposition is conform with a report on the depolymerisation of synthetic lignin by laccase from Pyc-
* Corresponding author. Fax: +49-551-39-27-05; E-mail : [email protected]
noporus cinnabarinus using 1-hydroxyanthranilic acid as a mediator compound [9]. The mechanism of the reaction and its general applicability, however, are still unknown and veri¢cation of this indirect mechanism by the oxidation of non-phenolic compounds in comparable experiments is still lacking. In the present study a high molecular PAH model compound was synthesised and applied in a compartment system with a membrane physically separating the enzyme from the substrate and only allowing the low molecular mediator compounds to reach both compartments. 2. Materials and methods 2.1. Chemicals 1-Hydroxybenzotriazole (HBT) was purchased from Sigma (Deisenhofen, Germany). All other reagents and substrates were provided by Aldrich (Steinheim, Germany) and Fluka (Neu-Ulm, Germany). 2.2. Model compounds K-(2-Bromoethyl)-g-monomethoxy-poly(oxyethylene) (Br^PEG, 2, see Scheme 1) was prepared according to Johansson et al. [10] modi¢ed by Bu«ckmann and Morr
0304-4165 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 0 0 ) 0 0 0 1 2 - X
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Scheme 1.
[11]. 70 mmol of poly(oxyethylene)monomethylether (PEG; Mw 2000) were dissolved in 1.6 l toluene and then 450 ml toluene were distilled o¡ to remove traces of moisture; an additional 100 ml were distilled o¡ separately for dilution of thionylbromide. The solution of PEG was chilled to 35³C and 28 ml of triethylamine (203 mmol) were added. Approximately 160 mmol thionylbromide (12 ml) were diluted with 100 ml dry toluene and added dropwise to the PEG solution during 1 h with continuous stirring under a dry nitrogen atmosphere. After the addition was completed, the reaction mixture was stirred with gentle re£uxing for another 1 h. Triethylammonium bromide was removed by passing the hot solution through a sintered glass funnel with a 2 cm thick layer of diatomeous earth. After 4 h incubation at room temperature, the solution was warmed to 50³C, treated with 40 g charcoal, and ¢ltered again over a new layer of diatomeous earth; the ¢ltercake was washed with a small amount of hot toluene. The combined solutions were left overnight at 4³C to crystallise the product and toluene was removed by ¢ltration. Crude Br^PEG was dissolved in 1.0 l of absolute ethanol at 60³C and treated again with 20 g charcoal. After ¢ltration through diatomeous earth the solution was kept at 4³C overnight to re-crystallise the Br^PEG. The solid material was separated by ¢ltration, washed with cold ethanol and dried in a vacuum over P2 O5 . The pale yellow product (282 g, yield 80%) had a substitution grade of 75% as determined by 13 C-NMR. K-(2-Mesyl-ethyl)-g-monomethoxy-poly(oxyethylene) (Mesyl^PEG, 3) was synthesised from the corresponding PEG (Mw 5000) by a modi¢ed method of Harris et al. [12]. 50 mmol of PEG were dissolved in 500 ml dichloromethane containing 10 g triethanolamine and chilled on ice. A solution of methanesulfonyl chloride (28 g) in 500 ml dichloromethane was added slowly and the reaction mixture stirred for 3 h at 0³C. The solution was reduced to about 500 ml using a rotatory evaporator and the product was precipitated from a viscous solution by the addition of 1000 ml distilled diethylether followed by 500 ml petroleum ether (boiling point 40^60³C). The precipitate was separated by ¢ltration, dissolved in a small amount of dichloromethane and then washed successively with 100 ml 0.1 N HCl, 100 ml saturated sodium hydrogencarbonate, and water until neutral. The product was precipitated with diethyl ether, dissolved in a small amount of dichloromethane and precipitated again. After drying in a vacuum Mesyl^PEG was obtained as a white powder (218 g, yield
86%). The substitution grade was determined after hydrolysis of 1 g sample with 10 ml 0.1 N NaOH (16 h at 90³C) followed by titration with 0.1 N HCl to be s 95%. K-(1-Pyrene-butyl)-g-monomethoxy-poly(oxyethylene) (pyrene^PEG, 4) was obtained from the Mesyl^PEG (or Br^PEG) and 1-pyrenebutanol. 10 mmol 3 were dissolved in dry tetrahydrofuran to obtain a 10% solution. 1-Pyrenebutanol (1), in a small excess amount with respect to the functionalised part of PEG, was dissolved in dry tetrahydrofuran (ca. 20 ml per 1 g PAH) and an excess of potassium-tert-butylate (ca. 5 g per 1 g of PAH) was added. The solution was stirred for 30 min at room temperature under argon and slowly added to a solution of (3). The reaction mixture was stirred at 50³C for 70 h under a re£ux condenser with a moisture trap. Progress of the reaction was monitored by thin layer chromatography (TLC) using Silicagel-F254 plates (Merck) with chloroform/methanol (9:1) as the eluent and inspected under UV ; PEG was detected with iodine vapour. Finally, an excess of acetic acid was added and after addition of 1 g charcoal, the mixture was stirred for 15 min at 50³C. The suspension was ¢ltered through 1 cm layer of diatomeous earth and the ¢ltercake washed with dichloromethane. The ¢ltrate was concentrated using a rotatory evaporator and the product precipitated twice with an excess of diethyl ether. Pyrene-PEG (4) was dissolved in dichloromethane, passed through a small silica gel column (3U10 cm) and eluted with 10% methanol in dichloromethane. The eluate was concentrated and the pyrene^PEG precipitated again with diethyl ether, dissolved in chloroform and puri¢ed on a silica gel column (3U40 cm, Type 60, Merck). 4 was eluted with chloroform, 10% and 50% methanol in chloroform successively (450 ml each) while being inspected with a UV-detector. Fractions containing 4 (inspected by TLC) were pooled and chloroform was evaporated in vacuum. After dissolving in water the product was washed in an Amicon cell with Y3 membrane and the concentrated solution was freeze-dried resulting in a 60% yield. The structure of the pyrene^PEG was con¢rmed by UV-vis- and 13 C-NMR spectroscopy and compared to the data obtained from pyrene and PEG alone. The amount of bonded PAH was measured by absorption in 20% methanol. This indicated an 18% substitution degree. The purity of the product was checked with TLC and HPLC, indicating, in addition to 4, only free PEG and a small amount of oxidation products; 1-pyrenebutanol was not detected.
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2.3. HPLC analysis Pyrene^PEG was analysed by HPLC (HP 1090 with a DAD detector, Hewlett-Packard) using a LiChrospher 100 RP-18 5 Wm column (4U125 mm). The separation was obtained in a gradient mode from 10% acetonitrile/10% methanol (solvent A) to 50% acetonitrile/50% methanol (solvent B) with a £ow rate of 1 ml/min. After an isocratic run for 1 min in solvent A, the mobile phase was changed by a linear gradient over 15 min to solvent B and the elution was continued for an additional 2 min. Absorption spectra within a range of 200^600 nm were recorded. 2.4. Laccase and enzyme assay Laccase from Trametes versicolor was puri¢ed as previously described [7,13]. The enzyme activity was determined by the oxidation of 2,2P-azino-bis-(3-ethylbenzothiazoline6-sulphonic acid) (ABTS) [6]. 2.5. Oxidation of pyrene^PEG by laccase-mediator-systems All experiments were performed under sterile conditions using sterile-¢ltered solutions and autoclaved equipment. 2 ml of the 0.2 mM pyrene^PEG solution placed in a dialysis tube (cut-o¡ 3000; Serva) were immersed in 100 ml £asks with 20 ml of 0.1 M phosphate bu¡er (pH 4.5) containing 0.2 mM PEG5000 . Laccase (4 U/ml) and mediator compounds (2 mM ABTS or 1 mM HBT ¢nal concentrations) were added to the outer bu¡er and the system was incubated for 48 h on a rotary shaker (40 rpm). Control samples were performed as above without the addition of mediators or the enzyme. Pyrene^PEG and 1-pyrenebutanol were also incubated in the same system without a separating membrane. The samples from the outer bu¡er and from the dialysis tube were analysed by HPLC. 2.6. Oxidations by ABTS2 + and HBT ABTS dication was prepared by the oxidation of ABTS with potassium peroxodisulphate [14] as previously described [8]. HBT radical was obtained by the oxidation of HBT (saturated solution, approximately 1.5 mM) by PbO2 in water- and ethanol-free chloroform [15]. 3. Results The HPLC method made a separation of the pyrene^ PEG and 1-pyrenebutanol possible and revealed the model compound to be free of the latter. The absorption spectrum of the model compound was identical to that of pyrene or 1-pyrenebutanol (210^600 nm). The water soluble PAH-model compound, pyrene^PEG, was completely oxidised by LMS utilising ABTS or HBT. No signi¢cant degradation by laccase without a mediator
Fig. 1. Chromatogram of pyrene^PEG model compound after oxidation by LMS using ABTS (upper panel) and HBT (lower panel) as the mediator compounds in a membrane-separated system. Inserts: absorption spectra (HPLC-on-line) of pyrene^PEG, compounds I and II.
was observed. To test the assumption of the mediated oxidation of pyrene by LMS without contact to the enzyme, the pyrene^PEG model (Mw 5200 Da) was separated from the enzyme using a dialysis tube with a cuto¡ of 3000 Da. Laccase and the mediator compounds, ABTS or HBT, were added to the outer bu¡er also containing the same concentration of PEG (Mw 5000 Da) as the pyrene^PEG inside the tube. Under these conditions, only the mediator compounds or their oxidised species were able to pass through the membrane. This was also visible in the case of ABTS: the blue-green radical cation was observed both outside and inside the tube. After incubation with laccase and ABTS for 48 h only 6% þ 3% of the initial pyrene^PEG were detected in the inner solution. The model compound was completely metabolised by LMS using HBT (Table 1). Two main oxidation products were detected in the inner solution: compounds I and II (Fig. 1). The same compounds were detected in reaction systems without a membrane. No pyrene^PEG or reaction products were detected in the solution outside the dialysis tube. The relative amounts of the products were approximately two times higher in the case of ABTS than in the system utilising HBT as mediator. In the latter case, small amounts of other products (III and IV) were detected, indicating further metabolisation of pyrene^PEG. The absorption spectrum of compound I in the range 210^600 nm was identical to the spectrum of pyrene^PEG (or 1-pyrenebutanol and pyrene). Compound II revealed a
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Table 1 Oxidation of pyrene^PEG (0.2 mM, 18% pyrene content) model compound by laccase from T. versicolor (4 U/ml) in presence of ABTS (2 mM) or HBT (1 mM) Membrane separation
Pyrene^PEG (% of initial)
Laccase/ABTS Laccase ABTS
3 + +
97 þ 4 6þ7 0þ5
Laccase/ABTS Laccase/HBT Laccase HBT Laccase/HBT
+ 3 + + +
94 þ 3 100 þ 0 0þ0 0þ0 100 þ 0
4. Discussion
spectrum with broad absorption bands at 240, 278, 350 nm and a shoulder at 382 nm (see inserts in Fig. 1). A detailed characterisation of the pyrene^PEG oxidation products was not possible and therefore LMS was applied in the oxidation of the synthesis substrate, 1-pyrenebutanol. The oxidation of 1-pyrenebutanol using HBT resulted in 95% removal of this compound and the appearance of two reaction products in the HPLC (data not presented). A complete oxidation of this compound was also achieved using a chemically generated HBT in chloroform (Table 2). Comparable metabolisation of 1pyrenebutanol was obtained by precipitated ABTS2 , respectively by LMS using ABTS; in the latter case only the second product was detected. One product obtained by LMS oxidation of 1-pyrenebutanol revealed an absorption spectrum identical to that of the parent compound (or pyrene); the spectrum of the second displayed broad absorption bands at 240, 278, 350 nm and a shoulder at 382 nm and corresponded to the spectrum of compound II from the pyrene^PEG oxidation. The products were analysed by direct inlet MS (EI 70 eV, Finnigan MAT 95) and GC^MS (as TMS derivatives) and are assumed to correspond to the 1-pyrene-K-furan (m/z [relative intensity] : 42[9], 101[8], 202[36], 215[22], 229[49], 239[20], 272[100]) and 1-pyrene-K-keto-butanol (EI-MS : 100[8], 201[78], 244[18], 229[100], 270[39], 288[24]; GC^MS of its TMSethers: 73[6], 75[10], 201[47], 229[50], 244[100], 345[10], 360[19] ; di-TMS ethers - two cis-trans-isomers - of the enol compound: 73[100], 147[7], 201[12], 239[71], 255[26], 314[32], 329[84], 432[19]). To con¢rm the indirect oxidation of aromatics 13 PAH Table 2 Oxidation of pyrene (25 WM) and 1-pyrenebutanol (40 WM) by laccase from T. versicolor (4 U/ml) in presence of ABTS or HBT (1 mM) and by radical/cation ; 24 h incubation Laccase Laccase/ABTS ABTS2 Laccase/HBT HBT
were oxidised using chemically generated HBT . Most of the compounds were e¡ectively oxidised in this reaction (Table 3) and considering the partially di¡erent reaction conditions (e.g. a chloroform solution of HBT ) a very similar oxidation `pattern' and reaction yield of numerous PAH in comparison to the LMS reactions were obtained and thus strengthened the supposition of an indirect oxidation of PAH by LMS system.
Pyrene (% oxidised)
1-Pyrenebutanol (% oxidised)
8þ4 6þ5 7þ4 48 þ 4 43 þ 3
63þ3 95 þ 3 88 þ 3 95 þ 3 100 þ 2
The coupling of PAH to high molecular PEG was performed to obtain a compound that can be applied in LMS without direct contact to the enzyme. Di¡erent means of synthesis and various derivatives were tested. Mesylateactivated PEG remained stable during storage ; it reacts very e¤ciently and quickly in displacement reactions [12]. Still, the results obtained were comparable to the reactions using Br-activated PEG - both alternatives are therefore presented. Alternative synthesis of PAH^PEG compounds via e.g. Grignard compounds from the corresponding PAH-bromide and PEG-bromide, or a coupling of PAH to PEG as an ester via imidazolide using the corresponding PAH-alkyl-acid (e.g. 2-naphthaleneacetic acid) and native PEG were not e¡ective enough or resulted in unstable products. The analogous napthalene^PEG was also obtained through the above synthesis from the corresponding naphthalene^alkyl derivative. Up to now, the only application of LMS demonstrating an indirect, membrane-separated oxidation using 1-hydroxyanthranilic acid as the mediator compound has been reported for the depolymerisation of a synthetic lignin model [9]. However, 1-hydroxyanthranilic acid was not e¡ective as a mediator for the oxidation of PAH by laccase from T. versicolor [7]. The formation of radical species resulting from the mediator compounds by the action of laccases and also the ability of these compounds to Table 3 Oxidation of PAH by HBT and laccase in bu¡er and HBT in chloroform PAH Naphthalene Acenaphthene Acenaphthylene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Benzo[a]anthracene Chrysene Benzo[b]£uoranthene Benzo[k]£uoranthene Benzo[a]pyrene Perylene
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Oxidation of PAH (% of control) Laccase/HBT
HBT
0 þ 3.0 99.0 þ 0.6 100.0 þ 0.0 91.9 þ 0.7 5.5 þ 2.4 98.7 þ 0.1 3.0 þ 4.1 48.1 þ 1.7 52.9þ 1.9 0.0 þ 3.1 9.0 þ 2.5 12.3 þ 4.3 91.3 þ 0.4 96.2 þ 0.1
4.7 þ 1.7 77.2 þ 1.2 94.1 þ 1.3 47.2 þ 1.3 12.1 þ 4.8 97.7 þ 1.2 16.3 þ 2.8 42.4 þ 2.0 90.8 þ 3.3 25.3 þ 4.1 43.5 þ 6.0 45.2 þ 11.1 99.0 þ 2.1 97.3 þ 3.3
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oxidise non-phenolic compounds were assumed (e.g. [3,8,16^18]) but the mediated indirect oxidation was not experimentally demonstrated. The application of a membrane separating the laccase and pyrene^PEG allowed us to study the oxidation reactions under conditions in which only the mediator compounds - respectively the radicals produced by laccase were able to react with the model compound. As expected, the mediator compound and laccase alone did not oxidise the pyrene^PEG in this system. In the system consisting of laccase and ABTS or HBT the model compound was nearly completely oxidised. The oxidation yield and the products obtained corresponded to results found by a direct reaction without a separating membrane. The results clearly demonstrated that the reactions were performed by oxidised species of the mediator compound (HBT or ABTS2 ) and that the laccase was not directly involved in the reactions. The oxidation of 1-pyrenebutanol and pyrene^PEG was more e¡ective than the oxidation of pyrene by LMS (approximately 50%). These results can not simply be explained by the lower solubility of pyrene but are probably the result of the presence of the alkyl side chain. The main reaction products detected were still coupled to PEG and were not able to permeate the membrane; minor oxidation products have not been described. The main products were characterised by their UV absorption spectra indicating that compound I possessed an unchanged aromatic system and was probably modi¢ed on the side chain only. Analogous to the results obtained with 1-pyrene-butanol, compound I was expected to be K-(1-pyrene-K-hydroxy-butoxy)-g-monomethoxy-poly(oxyethylene). The spectra of compound II indicated changes in an aromatic system or the presence of an additional conjugated double bond (e.g. carbonyl group) on the alkyl site. The UV^vis data of this compound correspond to the data of the product obtained from the oxidation of 1-pyrenebutanol and was therefore assumed to be K-(1-pyrene-K-keto-butoxy)-g-monomethoxy-poly(oxyethylene). The oxidation of the alkyl part of the substituent-activated aromatic compounds was previously reported (e.g. [3,19]). Under the reaction conditions used here only a very low oxidation of ethyl^benzene was obtained indicating that the reactions performed by laccase and mediators (ABTS and HBT) probably not proceeded via abstraction of the benzylic hydrogen atom and the aromatic structure was involved in the process. The mechanism involving an abstraction of benzylic H-atom would be theoretically possible for the reaction involving the N-oxide radical of HBT but seems to be unlikely for the ABTS2 . PAH radical cations (PAH ) formed by an abstraction of one electron are generally less reactive than the corresponding carbon-centred radicals and carbo-cations; their reactivity is dominated by the degree of coupling between the charge and the radical centres [20]. The cation centre of PAH may undergo a nucleophilic attack by water (poor nucleophile) and the resulting radical easily under-
161
Scheme 2. Proposed pathway for the oxidation of pyrene^PEG by LMS.
goes further oxidation to the hydroxy^PAH [21]. The latter compounds are also readily oxidised to the ¢nal quinones. Alternatively, disproportionation of cation radicals to the parent compound and dication lead to the same quinones but is only probable under water-free conditions. The reactivity of the radical centres of PAH and its intermediates can result in a polymerisation of the aromatic compounds [22] or a coupling with the mediator radical
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- formation of both in LMS was reported in our previous studies [6,7]. In addition to the above reaction mechanisms, the aromatic cation radicals resulting from alkyl^arenes (e.g. alkyl^PAH) can react on another pathway via proton abstraction - a very quick and irreversible process - by bases or basic nucleophiles (e.g. [23^25]). The substitution on an aromatic ring and the deprotonation of the side chain can be strongly in£uenced by the oxidant [26] and also di¡ers in oxidative biochemical systems depending on the enzymes involved [27]. Water accelerates the loss of proton and the concurrence between this pathway and the substitution on the aromatic ring is in£uenced by the pH-value of the solution (e.g. [28^30]). The deprotonation of the alkyl chain results in a radical - probably resonance stabilised - which may undergo further oxidation to the corresponding cation and ¢nally, after addition of water (or hydroxyl ion) to benzylic alcohols that can be further oxidised to the aldehydes. The above process and enhanced reactivity of substituent activated aromatics is well explained by the hypothesis of Parker [20] with regard to the decoupling of cation and radical centres. Accordingly, the deprotonation of the 4-methoxytoluene cation radical proceeds extremely fast in contrast to the relative stable methylbenzenes cation radicals. This explains the high yield of the corresponding aldehydes or K-ketones from the substituent activated alkyl^benzenes obtained by the LMS- or chemical oxidation using the same mediator compounds (e.g. [3,4,31]). According to the above mechanism, the oxidation of the alkyl^pyrene (1-pyrenebutanol or pyrene^PEG) results (via cation radical) in an alkyl cation. The subsequent addition of water (or hydroxyl ion) and further oxidations lead to the K-keto products (Scheme 2). Alternatively, an intramolecular addition of the hydroxyl-group of the 1pyrenebutanol can result in a cyclic ether: 1-pyrene-K-furan. The mechanism of the reactions may be very complex and also involve sandwich- or charge^transfer complexes of the aromatics and oxidants [32,33]. Since the LMS oxidation of pyrene^PEG proceeds through the abstraction of an electron from the aromatic site, this compound can be treated as a suitable high molecular model of PAH. It permitted an experimental demonstration of the mediated oxidation of aromatic compounds by LMS and could also be useful in intracellular/extracellular degradation studies in vivo. Based on the above results, the role of natural mediator compounds - plants and fungal metabolites - in the laccase-mediated oxidation of PAH by wood degrading fungi could be demonstrated (submitted for publication). Acknowledgements The authors thank Dr. G. Remberg for EI^MS measurements and Dr. R. Machinek for NMR measurements
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