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2 Bioorgnic & Medicinl Chemistry 20 (2012) Contents lists vilble t SciVerse ScienceDirect Bioorgnic & Medicinl Chemistry journl homepge: Lyer-by-Lyer coted tyrosinse: An efficient nd selective synthesis of ctechols Meliss Guzzroni, Cludi Crestini b, Rffele Sldino, Deprtment of Agrobiology nd Agrochemistry, University of Tusci, Vi S. Cmillo de Lellis, Viterbo, Itly b Deprtment of Chemicl Science nd Technology, University of Rome Tor Vergt, Vi dell Ricerc Scientific 1, Rome, Itly rticle info bstrct Article history: Received 29 July 2011 Revised 7 November 2011 Accepted 11 November 2011 Avilble online 20 November 2011 Keywords: Bioctlysis Immobilized tyrosinse Phenol oxidtion Bioctive ctechols Lyer-by-Lyer Agricus bisporous tyrosinse ws immobilized on commercil vilble epoxy-resin Eupergit Ò C250L nd then coted by the Lyer-by-Lyer method (LbL). The two novel heterogeneous bioctlysts were chrcterized for their morphology, ph nd storge stbility, kinetic properties (K m, V mx, V mx /K m ) nd reusbility. These bioctlysts were used for the efficient nd selective synthesis of bioctive ctechols under mild nd environmentl friendly experimentl conditions. Ascorbic cid ws dded in the rection medium to inhibit the formtion of ortho-quinones, thus voiding the known enzyme suicide inctivtion process. Ctechols were obtined mostly in quntittive yields nd conversion of substrte. Tyrosinse immobilized on Eupergit Ò C250L nd coted by the LbL method showed better ctlytic ctivities, higher ph nd storge stbility, nd reusbility with respect to immobilized uncoted tyrosinse. Since chemicl procedures to synthesize ctechols re often expensive nd with high environmentl impct, the use of immobilized tyrosinse represents n efficient lterntive for the preprtion of this fmily of bioctive compounds. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Tyrosinse (Tyro; EC ), polyphenol oxidse widely diffused in nture, ctlyzes the oxidtion of phenols to ctechols (cresolse or monophenolse ctivity) nd tht of ctechols to corresponding ortho-quinones (ctecholse or diphenolse ctivity). 1 Both ctechols nd ortho-quinones derivtives re chrcterized by severl biologicl ctivities, including ntioxidnt nd ntitumorl properties. 2 The trnsformtion of phenols to ctechols nd quinones is usully difficult to perform by chemicl methods under environmentl friendly conditions. 3 For this reson, tyrosinse received gret ttention s useful green lterntive to chemicl tretments. 4 The ppliction of tyrosinse in bioctlysis is prtilly limited due to the formtion of rective ortho-quinones tht cn covlently bond to the enzyme or utoxidize, producing brown pigments. 5 This drwbck cn be overcome working in reducing conditions in the presence of scorbic cid or NADH. 6 Moreover, the possibility to immobilize tyrosinse on stble nd low cost supports further increse the interest for this enzyme in industril pplictions. 7 Exmples of the immobiliztion of tyrosinse on crbon nnotube, 8 copolymer mtrices, 9 chitosn, 10 gold nnoprticles, 11 lumin sol-gel, 12 membrne lginte, polycrylmide, nd geltine gels 13 hve been reported in the production Corresponding uthor. Tel.: E-mil ddress: sldino@unitus.it (R. Sldino). of L-dop, 14 in the removl of phenolic compounds from wste wter, 15 nd in other industril pplictions. 16 However, some of these immobiliztion methods re rther complicted nd do not give good enzyme stbility or retention. 17 Recently, the Lyer-by- Lyer (LbL) technique ws reported s generl nd verstile tool for the controlled productions of multilyer surfce cotings on lrge vriety of surfces. 18 This method is bsed on the consecutive deposition of lterntively chrged polyelectrolytes onto surfce. 19 The polyelectrolyte films hve the bility to protect encpsulted protein from high-moleculr-weight denturing gents or bcteri nd to llow regultion of the permebility towrds smll substrtes, which cn enter the multilyer nd rect with the ctlytic site of the enzyme. 20 In this study we describe the synthesis of two novel tyrosinse bioctlysts: one bsed on the chemicl immobiliztion of mushroom tyrosinse on the epoxy-resin Eupergit Ò C250L, nd the other bsed on the chemo-physicl procedure consisting, first, in the immobiliztion of tyrosinse on Eupergit Ò C250L nd then in the coting of the bioctlyst by the LbL technique. The novel bioctlysts were pplied for the selective synthesis of ctechol derivtives by oxidtion of lrge pnel of substituted phenols, including biologiclly ctive compounds, under friendly environmentl conditions. The rections were performed in buffer t room temperture with dioxygen s primry oxidnt in the presence of scorbic cid (AA) to void the formtion of ortho-quinones. 6 The role of AA in the process is still under discussion. It cts s n internl reducing gent to trnsform the /$ - see front mtter Ó 2011 Elsevier Ltd. All rights reserved. doi: /j.bmc

3 158 M. Guzzroni et l. / Bioorg. Med. Chem. 20 (2012) Tyrosinse 1/ 2 2 H 2 o-diphenol 1/ 2 2 H 2 o-quinone CH 2 CH H ortho-quinone formed during the oxidtion into the desired ctechols (Fig. 1). It is lso known tht tyrosinse cn be inhibited t reltively high concentrtions of AA. 21 For this reson, the AA concentrtion requires to be optimized for good compromise between n high enzyme ctivity nd high yield in ctechols. 22 The comprison between the efficiency nd selectivity of tyrosinse with nd without the LbL procedure, s well s the possibility to recycle the bioctlysts for more runs, re lso reported. 2. Results nd discussion Dehydroscorbic cid Tyrosinse 2.1. ptimiztion of tyrosinse immobiliztion The immobiliztion of tyrosinse ws performed with the commercilly vilble epoxy-crylic resin Eupergit Ò C250L using modifiction of previously reported procedures. 23 The rtio between the enzyme nd the resin (mg/g), the incubtion time nd ph were investigted to optimize the yield of immobiliztion. As generl procedure, the enzyme ( mg, IU) ws suspended in N-phosphte buffer (ph ) ( ml) in the presence of defined mount of resin (1.0 g) for h t room temperture. The immobilized tyrosinse (Tyro/E) ws wshed with wter to remove excess of protein nd treted with glycine to block residul epoxy-groups (Fig. 2, pnel A). The effectiveness of the immobiliztion procedure ws investigted in terms of immobiliztion yield (Eq. 1, where U is the totl ctivity of enzyme dded in the solution nd U r is the ctivity of the CH 2 CH H Ascorbic cid Figure 1. Role of scorbic cid in the oxidtion of phenols by tyrosinse. Tble 1 Tyrosinse immobiliztion on Eupergit Ò C250L t different conditions Entries Rtio Tyro/ support (mg/g) Incubtion time (h) residul enzyme recovered in the wshing solutions, nd ctivity yield (Eq. 2, where U x is the ctivity of the immobilized enzyme ssyed by dopchrome method. 24 Imm: Yield ð%þ ¼½ðU U r ÞU 1 Š100 ð1þ Activity Yield ð%þ ¼½U x ðu U r Þ 1 Š100 ð2þ As reported in Tble 1, the ctivity yield incresed with the mount of the enzyme in contct with the resin (entries 1 5), reching the mximum vlue of 38% (tht corresponds to n immobiliztion yield of 66%) t 5:1 enzyme/support rtio (entry 4). When the mount of enzyme ws further incresed, the recovered ctivity yield decresed to 31%; nevertheless, the bound protein ws slightly higher (entry 5 vs entry 4). Similr results were obtined for the immobiliztion of cyclodextrin glucosyltrnsferse 25 nd lipse 26 on Eupergit Ò, probbly becuse the close pcking of the enzymes on the support surfce limits the ccess of substrte. nce defined the optimum vlue of the enzyme/support rtio, the immobiliztion ws performed t different times (24 nd 48 h). Dt reported in Tble 1 show the highest immobiliztion yield t 48 h (77% nd 66%), even if longer incubtion time led to reduction of the ctivity yield (30% nd 38%, respectively) (entry 6 vs entry 4). The influence of ph on the immobiliztion procedure ws lso studied in the rnge of The optimum binding ws chieved with sodium phosphte buffer t ph 7.0 (Tble 1 entries 5 vs 7 9). With the im to further increse the stbility of Tyro/E, the LbL technique ws pplied by coting Tyro/E through sequentil deposition of chrged polyelectrolytes. Briefly, Tyro/E ws first suspended in ph %Immobiliztion yield (mg bounded Tyro) (0.74) (1.52) (2.80) (3.30) (4.08) (3.85) (2.90) (3.00) (2.80) 26 Activity yield (%) Figure 2. Schemtic representtion of Tyro/E (pnel A) nd Tyro/E-LbL (pnel B) preprtion. Figure 3. SEM imges of Tyro/E (pnel A nd B) nd Tyro/E-LbL (pnel C nd D) t different mgnifiction.

4 M. Guzzroni et l. / Bioorg. Med. Chem. 20 (2012) positively chrged polyllylmine hydrochloride (PAH) (2 mg/ml in 0.5 M NCl), nd then treted with negtively chrged polystyrene sulfonte (PSS) (2 mg/ml in 0.5 M NCl). The procedure ws repeted until the formtion of three lyers (Fig. 2, pnel B). The immobilized LbL enzyme (Tyro/E-LbL) retined bout 87% of the ctivity with reference to Tyro/E. A set of scnning electron microscopy (SEM) photogrphs showing the morphology of the surfce of Tyro/E nd Tyro/E-LbL prticles re reported in Figure 3. Tyro/E shows prticles with regulr shpe nd n verge dimeter vlue of lm(fig. 3, pnel A). A low number of irregulr frgments ws observed, which re probbly formed by mechnicl dmge of prticles during the smple preprtion. At lrger mgnifiction the prticles show n irregulr surfce chrcterized by grumes of different dimension (Fig. 3, pnel B). The SEM photogrphs of group of prticles (verge dimeter of lm) nd mgnifiction of the cross-section of single prticle of Tyro/E-LbL re shown in Fig. 3, pnel C nd D. In this ltter cse the ultrthin coting lyers cover the surfce of the prticle. Both immobilized tyrosinses were chrcterized for their ph nd storge stbility, kinetic properties nd reusbility Effect of ph on tyrosinse ctivity The ph/ctivity curves relted to free (Tyro) nd immobilized tyrosinse (Tyro/E nd Tyro/E-LbL) t 25 C re shown in Figure 4. Irrespective to immobiliztion procedure, tyrosinse showed the optimum ph 7.0 tht is the sme vlue selected for the immobiliztion of the enzyme. Noteworthy, Tyro/E nd Tyro/E-LbL were more ctive thn the free enzyme in the rnge of ph studied. Chnges in ph-ctivity profile fter immobiliztion, eventully involving the shift of the optiml ph vlue, hve been reported during immobiliztion of tyrosinse on others support. 14, Storge stbility The storge stbility of free nd immobilized tyrosinse ws evluted by storing the enzyme in N-phosphte buffer t 20, 4 nd 25 C for 25 dys (Fig. 5, pnels A, B nd C, respectively). The ctivity ws then mesured t specific times t room temperture by the dopchrome method. At ech of the temperture studied, Tyro/E nd Tyro/E-LbL were more stble thn free enzyme, being the Tyro/E-LbL the most stble bioctlyst. To further confirm the integrity of Tyro/E-LbL over time, novel SEM nlysis of prticles t time zero (just prepred) nd fter two months upon storge t 4 C ws crried out t higher mgnifiction (15,000). As reported in Figure 6, the surfce morphology of the prticles Figure 4. ph optim of free (Tyro) nd immobilized tyrosinse (Tyro/E nd Tyro/E- LbL). Tyrosinse ctivity ws determined using L-Tyrosine s substrte, ph (pnels A nd B) is not significntly ltered during the storge period. In ddition, the stbility of the system ws confirmed by Trnsmission Electron Microscopy (TEM) nlysis of section of the prticles fter two months of storge, which shows substntil integrity of the lyers (Fig. 6, pnel C). This fct, coupled with the mintennce of the ctlytic ctivity for multiple subsequent rections (see below) confirms substntil stbility of the system. The enhnced stbility of enzyme ctivities within LbL ssembly ws previously discussed in detils by nd et l. 28 These uthors reported tht glucose oxidse ssembled lterntely with polyions retined bout 80% of ntive enzyme ctivity, thus suggesting tht the LbL procedures did not cuse significnt denturtion. Also in tht cse, fter the LbL coting, the enzyme mintined its ctivity for severl dys Kinetic ssy Kinetic prmeters of free nd immobilized tyrosinses were exmined by mesuring the enzyme ctivity t different concentrtions of L-tyrosine (L-Tyr; rnge lm) nd plotting dt to double reciprocl plot (Linewever Burk plot). 29 Irrespective to procedures used for the immobiliztion, V mx decresed nd K m incresed for supported tyrosinses, leding to prtil reduction of the ctlytic efficiency (V mx /K m ) with respect to free enzyme (Tble 2). Similr trends in K m vlues were reported for tyrosinse immobilized on other crriers nd re ttributed to ltertion of three-dimensionl structure nd mss trnsfer limittions Recycle nd reusbility Recycle nd reusbility ssy ws performed using L-Tyr s substrte. The oxidtions were followed spectrophotometriclly t 475 nm. After reching bsorbnce plteu, the immobilized bioctlyst ws recovered, wshed nd reused with fresh dded substrte. ne unit of enzyme ctivity ws defined s the increse in bsorbnce of t defined wvelength, temperture nd ph. For successive runs, the enzyme ctivity mesured in the first oxidtion ws used s the reference vlue. As shown in Tble 3, Tyro/ E-LbL ws more stble thn Tyro/E, retining 75% of ctivity fter 5 runs. This behvior suggest tht the LbL coting process effectively stbilizes the enzyme from inctivting gents. Different exmples of the stbiliztion effect of LbL re reported xidtion of phenols With the im to evlute the synthetic relevnce of immobilized tyrosinses, lrge pnel of phenols (Figure 7) ws oxidized, including pr-cresol 1, 4-ethyl phenol 2, 4-tert-butyl phenol 3, 4-sec-butyl phenol 4, 2,4-di-tert-butyl phenol 5, met-cresol 6, 3,4-dimethyl phenol 7, 4-chloro phenol 8, 4-chloro-2-methyl phenol 9, 2-methoxy-4-methyl phenol 10, 2-methoxy phenol 11, 3- (4-hydroxyphenyl)propionic cid 12, 4-hydroxyphenylcetic cid 13, bis(4-hydroxyphenyl)methne 14 nd tyrosol 15. The oxidtion of 1 (0.05 mmol) with free tyrosinse (Tyro; 263 IU) in buffer (5.0 ml) ws performed in the presence of AA in previously optimized concentrtion (1.5 equiv). 32 For low soluble phenols 3, 5, 14, the substrtes were dissolved in CH 3 CN (1.0 ml) nd then dded to the rection mixture (see next). Rections were performed t room temperture for 24 h. Under these experimentl conditions ctechol 1 ws obtined s the only recovered product in quntittive yield nd conversion of substrte (Scheme 1, Tble 4, entry 1) Tyro/E nd Tyro/E-LbL performed in similr wy ffording 1 in quntittive yield nd conversion of substrte (Scheme 1, Tble 4, entries 2 nd 3). Thus, the rectivity nd selectivity of tyrosinse

5 160 M. Guzzroni et l. / Bioorg. Med. Chem. 20 (2012) Figure 5. Storge stbility of free (Tyro) nd immobilized tyrosinse (Tyro/E nd Tyro/E-LbL) t (A) 20 C, (B) 4 C nd (C) 25 C in N-phosphte buffer 0.1 M, ph 7. Figure 6. Pnels A nd B: SEM imges of Tyro/E-LbL t higher mgnifiction (15,000) t time zero (pnel A) nd fter two months (pnel B) upon storge t 4 C. Pnel C: TEM imge of section of Tyr/E-LbL prticle upon storge for two months t 4 C. LBL lyers re clerly visible in the upper prt of the picture. ws completely retined fter the immobiliztion procedures. The oxidtion of 2 confirmed the high rectivity of immobilized tyrosinses, the ctechol 2 being gin obtined s the only recovered product in quntittive yield nd conversion of substrte (Scheme 1, Tble 4, entries 4 6). Noteworthy, Tyro/E nd Tyro/E-LbL were efficient nd selective bioctlysts lso in the oxidtion of pr-lkyl substituted phenols chrcterized by high steric hindrnce, s in the cse of bulky substituted phenols 3 nd 4. As reported in Tble 4, the oxidtion of 3 required the ddition smll mount of CH 3 CN (0.1 ml) to increse the solubility of substrte. In these ltter cses, irrespective to experimentl conditions, ctechols 3 nd 4 were synthesized in yield higher thn 90% (Scheme 1, Tble 4, entries

6 M. Guzzroni et l. / Bioorg. Med. Chem. 20 (2012) Tble 2 Kinetic prmeters of free (Tyro) nd immobilized (Tyro/E, Tyro/E-LbL) tyrosinse Entry Enzyme K m (lm) V mx (10 3 ) V mx /K m (10 6 ) 1 Tyro Tyo/Eup Tyro/E-LbL V mx ws defined s dabs/min lg enzyme. Tble 3 Reusbility of Tyro-immobilized systems Run Tyro/E Tyro/E-LbL Reusbility is expressed s percentge of ctivity in ech runs respect to tht mesured in the first reference oxidtion Cl Cl 9 10 CH ). n the other hnd, 2,4-di-tert-butyl phenol 5 ws stble under ll of the conditions tested, probbly due to known inhibition effect exerted by the steric encumbering of the ortho-substituent. 33 A substituent in the met-position on the romtic ring, s in the cse of 3-methyl phenol 6 nd 3,4-dimethylphenol 7, showed slightly inhibitory effect. In these ltter cses, twice mount of enzyme ws required (526 IU) to produce ctechols 1 nd 7 in 42 48% nd 80 84% yield, respectively, Tyro/E-LbL being the best bioctlyst (Scheme 2, Tble 5, entries 2, 3, 5 nd 6).The ctechols were obtined s the only recovered products. The highest yield observed in the cse of 7 suggests tht the inhibitory effect of the met-substituent cn be prtilly blnced by the presence of pr-substituent with n inductive electrondonor effect. Next we nlyzed the oxidtion of chloro phenol 11 5 CH Figure 7. Phenols selected for the oxidtion with free nd immobilized tyrosinses Tble 4 xidtion of pr-lkyl substituted phenols 1 4 Entry Substrte Bioctlysts Products Conversion (%) Yield (%) 1 1 Tyro 1 >99 > Tyro/E Tyro/E-LbL Tyro 2 >99 > Tyro/E Tyro/E-LbL Tyro 3 b >99 > Tyro/E 3 b Tyro/E-LbL 3 b Tyro Tyro/E Tyro/E-LbL Rection conditions: substrte (0.05 mmol), AA (1.5 equiv) nd tyrosinse (263 IU) were tken in 5.0 ml of phosphte buffer solution for 24 h. b xidtion performed in N-phosphte buffer/ch 3 CN. derivtives, 4-chloro phenol 8 nd 4-chloro-2-methyl phenol 9. Tretment of 8 with Tyro/E nd Tyro/E-LbL (263 IU) fforded ctechol 8 in quntittive yield nd conversion of substrte (Scheme 2, Tble 5, entries 8, 9). A similr result ws obtined with the free enzyme (Tble 5, entry 7). As expected, twice mount of tyrosinse (526 IU) ws required for the oxidtion of 9, due to the presence of the ortho-substituent. Despite this request, the ctechol 9 ws isolted in high yield (Scheme 2, Tble 5, entries 11 nd 12), confirming beneficil role of the electron-donor substituent in the pr-position of the romtic ring. Note tht, phenol derivtives chrcterized by n electron-donor ortho-substituent, s in the cse of 2-methoxy-4-methyl phenol 10 nd 2-methoxy phenol 11, while requesting twice mount of enzyme (526 IU), fforded the corresponding ctechols 10 nd 11 in significnt yield (Scheme 3, Tble 6, entries 2, 3, 5 nd 6). Agin, the Tyro/E-LbL ws the best bioctlyst. ur ttention ws next focused on the synthesis of ctechol derivtives chrcterized by potentil biologicl ctivity. Since cidic ctechols shows ntibcteril, 34 ntimicrobil 35 nd ntioxidnt 36 ctivities, we first evluted the oxidtion of two phenolic cid derivtives, 3-(4-hydroxyphenyl)propionic cid 12 nd 4-hydroxyphenylcetic cid 13. Irrespective to experimentl conditions used, the ctechols 12 nd 13 were obtined in high yield s the only recovered products, confirming the generlity of the procedure (Scheme 3, Tble 6, entries 8, 9, 11 nd 12). In similr wy, the oxidtion of bis(4-hydroxyphenyl)methne 14 proceeded with high conversion of substrte to fford the mono-ctechol nd R 2 6, 7 b) 1: =Me, R 2 =H; 6: =Me, R 2 =H 7: =R 2 =Me R 2 1, 7 1,2,3,4 b) or c) 1: =Me 2: =Et 3: =tert-c 4 H 9 4: =sec-c 4 H 9 1,2,3,4 Cl 8, 9 8: =H 9: =Me b) Cl 8, 9 Scheme 1. xidtion of phenols 1 4. Regents nd conditions: ( Tyro-bsed systems, 2, AA; (b) N-phosphte buffer; (c) N-phosphte buffer/ch 3 CN. Scheme 2. xidtion of phenols 6 9. Regents nd conditions: ( Tyro-bsed systems, 2, AA; (b) N-phosphte buffer.

7 162 M. Guzzroni et l. / Bioorg. Med. Chem. 20 (2012) Tble 5 xidtion of phenols 6 9 Entry Substrte Bioctlysts Products Conversion (%) Yield (%) 1 6 Tyro Tyro/E Tyro/E-LbL Tyro Tyro/E Tyro/E-LbL Tyro 8 b >99 > Tyro/E 8 b 94 > Tyro/E-LbL 8 b 97 > Tyro 9 >99 > Tyro/E Tyro/E-LbL Rection conditions: substrte (0.05 mmol), AA (1.5 equiv) nd tyrosinse (526 IU) were tken in 5.0 ml of phosphte buffer solution for 24 h. b xidtion performed with 263 IU of tyrosinse. Tble 6 xidtion of phenols Entry Substrte Bioctlysts Products Conversion (%) Yield (%) 1 10 Tyro 10 b Tyro/E 10 b Tyro/E-LbL 10 b Tyro 11 b Tyro/E 11 b Tyro/E-LbL 11 b Tyro Tyro/E Tyro/E-LbL Tyro Tyro/E Tyro/E-LbL ,11 10: =Me 11: =H 12, 13 12: n=1 13: n=2 n b) b) Rection conditions: substrte (0.05 mmol), AA (1.5 equiv) nd tyrosinse (263 IU) were tken in 5.0 ml of phosphte buffer solution for 24 h. b xidtion performed with 526 IU of tyrosinse. bis-ctechol derivtives 14 nd 14b in pprecible yield (Scheme 4, Tble 7, entries 1 3). In ccordnce with the selectivity of the free enzyme, immobilized tyrosinses fforded 14 s the min rection product, Tyro/ E-LbL being the best bioctlys. Moreover, the rection performed with twice mount of enzyme nd for longer rection time (48 h) produced 14 s the only recovered product in quntittive yield (Tble 7, entries 4 nd 5). This trnsformtion is of synthetic interest becuse polyhydroxylted diphenylmethne derivtives re chrcterized by ntivirl, 37 ntioxidnt, 38 nd ntimicrobil ctivities , 11 12, 13 Scheme 3. xidtion of phenols Regents nd conditions: ( Tyro-bsed systems, 2, AA; (b) N-phosphte buffer. n Finlly, we studied the synthesis of 3,4-dihydroxyphenylethnol (hydroxytyrosol), low moleculr weight component in virgin olive oil nd in mill wstes. 6 This compound shows severl biologicl ctivity, including ntimicrobil, 40 hypoglycemic, 41 ntioxidnt, 41 crdiovsculr properties, 42 inhibition of pltelet ggregtion 43 nd inhibition of lipoxygenses, 44 or induction of poptosis. 45 When 2-(4-hydroxy phenyl)ethnol (tyrosol) 15 ws treted with Tyro/E nd Tyro/E-LbL under previously reported conditions, ctechol 15 ws obtined in 70% nd 77% yield, respectively (Scheme 4, Tble 7, entries 7 nd 8). In this ltter cse, free enzyme showed rectivity slightly higher thn immobilized bioctlysts (Tble 7, entry 6 vs entries 7 nd 8). 3. Conclusion c) b) Scheme 4. xidtion of phenols 14 nd 15. Regents nd conditions: ( Tyrobsed systems, 2, AA; (b) N-phosphte buffer/ch3cn; (c) N-phosphte buffer. Tble 7 xidtion of phenols Entry Substrte Bioctlysts Products Conversion (%) Yield (%) 1 14 Tyro 14(14b) b >99 65(34) 2 14 Tyro/E 14(14b) b 95 53(41) 3 14 Tyro/E-LbL 14(14b) b 98 66(32) 4 14 Tyro/E 14 b,c >99 > Tyro/E-LbL 14 b,c >99 > Tyro Tyro/E Tyro/E-LbL Rection conditions: substrte (0.05 mmol), AA (1.5 equiv) nd tyrosinse (263 IU) were tken in 5.0 ml of phosphte buffer solution for 24 h. b xidtion performed in N-phosphte buffer/ch 3 CN. c xidtion performed with 526 IU of tyrosinse for 48 h. New heterogeneous tyrosinse bioctlysts were synthesized by immobiliztion on Eupergit Ò C250L nd LbL coting with polyelectrolyte. With respect to the free enzyme used s reference, tyrosinse retined the ctlytic ctivity nd selectivity fter immobiliztion on Eupergit Ò C250L nd successive coting by LbL technique. It is interesting to note, tht in ll of the cses studied, Tyro/E-LbL ws more efficient thn Tyro/E, suggesting stbiliztion effect exerted by the polyelectrolyte coting. Tyro/E nd Tyro/E-LbL were stble enough to perform t lest five recycling experiments with similr conversion nd selectivity. The stbility of tyrosinse under storge conditions t different tempertures ws lso found to be incresed in the presence of the support. Agin, Tyro/E-LbL ws the most stble nd reusble ctlyst. The structure of the interction between the Eupergit-supported enzyme nd the polyelectrolyte coting hs not been studied in detil, becuse of the greter complexity of this system compred to the previously described film in which the enzyme is immobilized directly within the LbL coting. However, the mintennce of the ctlytic ctivity for multiple runs by Tyro/E-LbL, b

8 M. Guzzroni et l. / Bioorg. Med. Chem. 20 (2012) suggests tht the system does not undergo structurl chnges. 46 About the selectivity of the oxidtions, pr-substituted phenols were efficiently oxidized, even in the cse of highly encumbering lkyl pr-substituents. Met-substituted nd ortho-substituted phenols required twice mount of enzyme to yield the corresponding ctechols in high yield, probbly due both to the effect exerted by the substituent on the electronic distribution of the romtic ring 33 nd the steric encumbering for the formtion of the first intermedite with the Cu tom in the ctive site of the enzyme. Since ctechols re biologiclly ctive compounds difficult to synthesize by trditionl chemicl procedure under environmentl friendly conditions, the use of immobilized tyrosinses open novel synthetic lterntive for this interesting fmily of substnces. 4. Experimentl section Mushroom tyrosinse from Agricus bisporus (Tyro), Eupergit Ò C250L, poly(sodium 4-styrenesulfonte) (PSS, MW 70000), poly(llylmine hydrochloride) (PAH, MW 56000), L-tyrosine (L- Tyr), scorbic cid, 2,2 0 -zino-bis(3-ethylbenzothizoline-6-sulfonic cid) (ABTS), bovine serum lbumin (BSA), ethyl cette (EtAc), cetonitrile (CH 3 CN), sodium sulfte nhydrous (N 2 S 4 ), dodecne, pyridine, hesmethyldisilzne (HMDS), trimethylchlorosilne (TMCS) nd phenols were purchsed from Sigm Aldrich. All spectrophotometric mesurements were mde with Vrin Cry50 UV vis spectrophotometer equipped with single cell peltier thermosttted cell holder. Spectrophotometric dt were nlyzed with Cry WinUV softwre. All experiments were crried out in triplicte using free nd immobilized tyrosinse Tyrosinse immobiliztion on Eupergit Ò C250L The immobiliztion of tyrosinse ws performed by modifiction of literture procedures. 23 Dry Eupergit Ò C250L (1.0 g) ws dded to different mount of buffer 0.1 M (ph ) contining tyrosinse (Tyro, mg, U/mg). The mixture ws incubted for h t room temperture with orbitl shking. At the end of the coupling period, the resin beds were filtered, wshed (5 8 ml) with buffer until no ctivity ws detected in the wshing. The obtined beds were incubted with glycine (3.0 M) for 2 h to block residul epoxy groups, 47 then wshed with buffer nd finlly ir-dried nd stored t 4 C. The mount in milligrms nd the units of coupled tyrosinse (Tyro/ E) were clculted by the difference between the mount/units loded nd tht recovered in the wshings by conventionl Brdford nd ctivity ssy Tyrosinse immobiliztion on Eupergit Ò C250L coted with Lyer-by-Lyer method Tyro/E, synthesized using the optiml experimentl conditions described bove, ws coted with the Lyer-by-Lyer method (LbL) in ccordnce to literture procedures. 48 Briefly, PAH nd PSS solutions (2.0 mg/ml in 0.5 M NCl) were lterntely dded to Tyro/E system: ech polyelectrolyte lyer ws dsorbed for 20 min t room temperture with orbitl shking nd then wshed with 0.5 M NCl to remove excess of polyelectrolytes. The deposition of polyelectrolytes strted with PAH nd ws repeted to obtined three lyers (PAH-PSS-PAH). Immobilized tyrosinse (Tyro/E-LbL) ws ir-dried nd stored t 4 C Determintion of protein concentrtion Protein concentrtion ws determined spectrophotometriclly t 595 nm ccording to Brdford using BSA s stndrd Activity ssy Tyrosinse ssy ws performed by the dopchrome method s previously described. 24 Briefly, L-Tyr solution (1.0 ml, 2.5 mm), N-phosphte buffer 0.1 M, ph 7.0 (1.9 ml) ws incubted under vigorous stirring t 25 C for 10 min. Then, n pproprite mount of free or immobilized enzyme in N-phosphte buffer (100 ll) ws dded to the mixture nd the initil rte ws immeditely mesured s liner increse in opticl density t 475 nm, due to dopchrome formtion. ne unit of enzyme ctivity ws defined s the increse in bsorbnce of per minute t ph 7, 25 C in 3.0 ml rection mixture contining 0.83 mm of L-tyrosine nd 67 mm of N-phosphte buffer ph 7.0. The specific ctivity of bioctlysts ws lso nlyzed in the ph rnge of Kinetic ssy Kinetic prmeters, K m nd V mx nd V mx /K m, were determined by mesuring enzyme ctivity t different concentrtions of L-Tyr ( lm) nd plotting dt to double reciprocl plot (Linewever Burk plot). 29 Rections were crried out by mens of the sme procedure s for ctivity ssy, using Tyro (53 lg) nd Tyro/E (70 lg) nd Tyro/E-LbL (70 lg), nd mesuring bsorbnce t 475 nm s described bove Stbility ssy Tyrosinse (53 lg for Tyro nd 70 lg for Tyro/E nd Tyro/E-LbL) in N-phosphte buffer 0.1 M (ph 7.0) ws stored t three tempertures ( 20, +4, 25 C). At different times (0 25 dys), liquots were tken nd the ctivity ws determined t room temperture by the dopchrome method. For ech smple, tyrosinse ctivity ws expressed s reltive percentge ctivity respect to tht t time zero Trnsmission Electron Microscopy (TEM) mesurements Smples were prepred t the Interdeprtmentl Centre of Electron Microscopy, Tusci University, Viterbo, Itly, using conventionl procedures. For Trnsmission Electron Microscopy (TEM), smples were fixed with 2.5% glutrldehyde in 0.1 M ccodylte buffer ph 7.2 overnight t 4 C. After rinsing in the sme buffer, they were post-fixed in ccodylte-buffered 1% osmium tetroxide for 1 h nd then wshed in distilled wter. Specimens were dehydrted in grded ethnol series nd embedded in LRWhite resin. Thin sections were cut with Reichert Ultrcut ultrmicrotome using dimond knife, collected on copper grids, stined with urnyl cette nd led citrte, nd observed with JEL 1200 EX II electron microscope. Microgrphs were cquired by the lympus SIS VELETA CCD cmer equipped the item softwre Scnning Electron Microscopy (SEM) mesurements Smples were prepred t the Interdeprtmentl Centre of Electron Microscopy, Tusci University, Viterbo, Itly, using conventionl procedures. For Scnning Electron Microscopy (SEM), smples were sputter-coted with gold in Blzers MED 010 unit nd observed with JEL JSM 5200 electron microscope. Microgrphs were tken by Mmiy cmer pplied to the microscope using TMAX 100 ASA films Enzyme recycling Immobilized enzyme (Tyro/E nd Tyro/E-LbL) ws recycled s follow: L-tyrosine (0.83 mm), immobilized Tyro (70 lg) nd N-phosphte buffer 0.1 M, ph 7.0 (3.0 ml) were plced in vils

9 164 M. Guzzroni et l. / Bioorg. Med. Chem. 20 (2012) t 25 C. At specific time, solutions were removed to mesure bsorbnce t 475 nm nd then returned to the vils s rpidly s possible. After reching plteu, enzyme ws wshed with buffer, recycled nd reused gin. ne unit of enzyme ctivity ws defined s the increse in bsorbnce of t defined wvelength, temperture nd ph. For ech run, tyrosinse ctivity ws expressed s reltive percentge ctivity respect to tht t first run tert-Butylctechol (4-tert-butylbenzene-1,2-diol) (3 il. 1 H NMR (200 MHz, CDCl 3 ) d H (ppm) 1.33 (9H, s, CH 3 ), (3H, m, Ph-H). 13 C NMR (50 MHz, CDCl 3 ) d C (ppm) 31.2 (3 CH 3 ), 34.5 (C), (CH), (CH), 122 (CH), (C), (C), (C). MS, m/z: 310 (M + ), 295 (M CH 3 ), 280 [M (CH 3 ) 2 ], 265 [M (CH 3 ) 3 ], 237 [M Si(CH 3 ) 3 ], 222 [M Si (CH 3 ) 3 ], 207 [M Si(CH 3 ) 4 ], 192 [M Si(CH 3 ) 5 ], 176 [M Si(CH 3 ) 6 ], 148 [M Si 2 (CH 3 ) 6 ] Phenols oxidtion A pnel of phenols (Fig. 7) were oxidized, including pr-cresol 1, 4-ethyl phenol 2, 4-tert-butyl phenol 3, 4-sec-butyl phenol 4, 2,4- di-tert-butyl phenol 5, met-cresol 6, 3,4-dimethyl phenol 7, 4- chloro phenol 8, 4-chloro-2-methyl phenol 9, 2-methoxy-4-methyl phenol 10, 2-methoxy phenol 11, 3-(4-hydroxyphenyl)propionic cid 12, 4-hydroxyphenylcetic cid 13, bis(4-hydroxyphenyl)methne 14 nd tyrosol 15. As generl procedure phenol (0.05 mmol), tyrosinses ( IU) nd AA (1.5 equiv) were plced in 0.1 M N-phosphte buffer ph 7.0 (5.0 ml) in vigorous stirring t room temperture. For insoluble queous phenols 3, 5, 14 substrtes were dissolved in CH 3 CN (1.0 ml) nd then dded to the buffer solutions. xidtions were performed using homogeneous nd heterogeneous conditions. Rections were monitored by thin lyer chromtogrphy (TLC). After the disppernce of the substrte, the rection mixture ws cidified with solution of HCl 1.0 N nd extrcted twice with EtAc. The orgnic extrcts were treted with sturted solution of NCl nd dried over nhydrous N 2 S 4, then filtered nd concentrted under vcuum to yield colored crude. In the cse of immobilized enzyme, bioctlyst ws first recovered by filtrtion nd the solution ws subjected to the sme work up described bove. The obtined colored residue ws treted with pyridine, HMDS nd TMCS (HMDS TMCS, 2:1 v/v) under vigorous stirring t room temperture for 30 min, then llowed to stnd for 5 min. 50 All products were identified by 1 H NMR, 13 C NMR nd GC MS. 1 H NMR nd 13 C NMR were recorded on Bruker 200 MHz spectrometer using CDCl 3 s solvent. All chemicl shift re expressed in prts per million (d scle). GC MS nlysis were performed on GCMS-QP5050 Shimdzu pprtus using SPB column (25 m 0.25 mm nd 0.25 mm film thickness) nd n isotherml temperture profile of 100 C for 2 min, followed by 10 C/min temperture grdient to 280 C for 25 min. The injector temperture ws 280 C. Chromtogrphy-grde helium ws used s the crrier gs with flow of 2.7 ml/min. Mss spectr were recorded with n electron bem of 70 ev sec-Butylctechol (4-(1-methylpropyl)-1,2-benzenediol) (4 il. 1 H NMR (200 MHz, CDCl 3 ) d H (ppm) 1.10 (3H, m, CH 3 ), 1.22 (3H, m, CH 3 ), 1.53 (2H, m, CH 2 ), 3.23 (1H, m, CH), (3H, m, Ph-H). 13 C NMR (50 MHz, CDCl 3 ) d C (ppm) 11.2 (CH 3 ), 22.3 (CH 3 ), 31.2 (CH 2 ), 43.1 (CH), (CH), (CH), (CH), (C), (C), (C). MS, m/z: 310 (M + ), 295 (M CH 3 ), 280 [M (CH 3 ) 2 ], 237 [M Si(CH 3 ) 3 ], 222 [M Si(CH 3 ) 3 ], 207 [M Si (CH 3 ) 4 ], 192 [M Si(CH 3 ) 5 ], 149 [M Si 2 (CH 3 ) 6 ], 133 [M 2 Si 2 (CH 3 ) 6 ] ,5-Dimethylctechol (4,5-dimethyl-1,2,-benzenediol) (7 il. 1 H NMR 53 (200 MHz, CDCl 3 ) d H (ppm) 2.20 (s, 6H,CH 3 ), 6.51 (s, 2H, Ph-H). 13 C NMR (50 MHz, CDCl 3 ) d C (ppm) 19.7 (2 CH 3 ), (2 CH), (2 C), (2xC). MS, m/z: 282 (M + ), 267 (M CH 3 ), 252 [M (CH 3 ) 2 ], 237 [M (CH 3 ) 3 ], 210 [M Si(CH 3 ) 3 ], 194 [M Si(CH 3 ) 3 ], 179 [M Si(CH 3 ) 4 ], 164 [M Si (CH 3 ) 5 ], 149 [M Si(CH 3 ) 6 ], 105 [M 2 Si 2 (CH 3 ) 6 ] Chloroctechol (4-chloro-1,2-benzenediol) (8 il. 1 H NMR (200 MHz, CDCl 3 ) d H (ppm) (3H, m, Ph- H), 8.10 (2H, br s, ). 13 C NMR (50 MHz, CDCl 3 ) d C (ppm) (CH), (CH), (CH), (C), (C), (C). MS, m/z: 288 (M + ), 273 (M CH 3 ), 258 [M (CH 3 ) 2 ], 243 [M (CH 3 ) 3 ], 215 [M Si(CH 3 ) 3 ], 199 [M Si(CH 3 ) 3 ], 184 [M Si(CH 3 ) 4 ], 169 [M Si(CH 3 ) 5 ], 126 [M Si 2 (CH 3 ) 6 ] Chloro-3-methylctechol (5-chloro-3-methyl-1,2- benzenediol) (9 il. 1 H NMR (200 MHz, CDCl 3 ) d H (ppm) 2.10 (3H, s, CH 3 ), (2H, s, Ph-H), 7.52 (2H, br s, ). 13 C NMR (50 MHz CDCl 3 ) d C (ppm) 17.2 (CH 3 ), (CH), (C), (C), (CH), (C), (C). MS, m/z: 302 (M + ), 287 (M CH 3 ), 272 [M (CH 3 ) 2 ], 229 [M Si(CH 3 ) 3 ], 213 [M Si(CH 3 ) 3 ], 198 [M Si (CH 3 ) 4 ], 168 [M Si(CH 3 ) 6 ] Methylctechol (4-methyl-1,2-benzenediol) (1 il. 1 H NMR 51 (200 MHz, CDCl 3 ) d H (ppm) 2.24 (3H, s, CH 3 ), 5.04 (1H, br s, ), 5.18 (1H, br s, ), (3H, m, Ph-H). 13 C NMR 51 (50 MHz, CDCl 3 ) d C (ppm) 20.8 (CH 3 ), (CH), (CH), (CH), (C), (C), (C). MS, (m/z): 268 (M + ), 253 (M CH 3 ), 238 [M (CH 3 ) 2 ], 223 [M (CH 3 ) 3 ], 195 [M Si(CH 3 ) 3 ], 179 [M Si(CH 3 ) 3 ], 164 [M Si(CH 3 ) 4 ], 149 [M Si(CH 3 ) 5 ], 134 [M Si(CH 3 ) 6 ], 106 [M Si 2 (CH 3 ) 6 ], 90 [M 2 Si 2 (CH 3 ) 6 ] Ethylctechol (4-ethyl-1,2-benzenediol) (2 il. 1 H NMR 52 (200 MHz, CDCl 3 ) d H (ppm) 1.04 (3H, m, CH 3 ), 2.36 (2H, m, CH 2 ), (3H, m, Ph-H). 13 C NMR (50 MHz, CDCl 3 ) d C (ppm) 15.2 (CH 3 ), 28.1 (CH 2 ), (CH), (CH), (CH), (C), (C), (C). MS, m/z: 282 (M + ), 267 [M CH 3 ], 252 [M (CH 3 ) 2 ], 237 [M (CH 3 ) 3 ], 209 [M Si(CH 3 ) 3 ], 193 [M Si(CH 3 ) 3 ], 179 [M Si(CH 3 ) 4 ], 164 [M Si(CH 3 ) 5 ], 148 [M Si(CH 3 ) 6 ], 120 [M Si 2 (CH 3 ) 6 ] Methoxy-5-methyl-1,2-benzenediol (10 il. 1 H NMR 54 (200 MHz, CDCl 3 ) d H (ppm) 2.19 (3H, s, CH 3 ), 3.80 (3H, s, CH 3 ), (2H, m, Ph-H). 13 C NMR (50 MHz, CDCl 3 ) d C (ppm) 22.0 (CH 3 ), 56.6 (CH 3 ), (CH), (CH), (C), (C), (C), (C). MS, m/z: 298 (M + ), 283 (M CH 3 ), 268 [M (CH 3 ) 2 ], 253 [M Si(CH 3 ) 3 ], 225 [M Si(CH 3 ) 3 ], 209 [M Si(CH 3 ) 3 ], 194 [M Si(CH 3 ) 4 ], 179 [M Si(CH 3 ) 5 ], 164 [M Si(CH 3 ) 6 ] Methoxy-1,2-benzenediol (11 il. 1 H NMR (200 MHz, CDCl 3 ) d H (ppm) 3.79 (3H, s, CH 3 ), (3H, m, Ph-H), 8.08 (2H, br s, ). 13 C NMR (50 MHz, CDCl 3 ) d C (ppm) 56.2 (CH 3 ), (CH), (CH), (CH), (C), (C), (C). MS, m/z: 284 (M + ), 269 (M CH 3 ), 254 [M (CH 3 ) 2 ], 239 [M (CH 3 ) 3 ], 211 [M Si(CH 3 ) 3 ], 195 [M Si (CH 3 ) 3 ], 180 [M Si(CH 3 ) 4 ], 165 [M Si(CH 3 ) 5 ], 106 [M 2 Si 2 (CH 3 ) 6 ].

10 M. Guzzroni et l. / Bioorg. Med. Chem. 20 (2012) ,4-Dihydroxy-benzenepropnoic cid (12 il. 1 H NMR 55 (200 MHz, CDCl 3 ) d H (ppm) 2.47 (2H, m, CH 2 ), 2.70 (2H, m, CH 2 ), (3H, m, Ph-H). 13 C NMR (50 MHz, CDCl 3 ) d C (ppm) 31.5 (CH 2 ), 37.2 (CH 2 ), (CH), (CH), (CH), (C), (C), (C), (C). MS, m/z: 384 (M + ), 369 (M CH 3 ), 354 [M (CH 3 ) 2 ], 339 [M (CH 3 ) 3 ], 311 [M Si(CH 3 ) 3 ], 295 [M Si(CH 3 ) 3 ], 280 [M Si(CH 3 ) 4 ], 265 [M Si(CH 3 ) 5 ], 222 [M Si 2 (CH 3 ) 6 ] ,4-dihydroxy-benzenecetic cid (13 il. 1 H NMR (200 MHz, CDCl 3 ) d H (ppm) 3.56 (2H, s, CH 2 ), (3H, m Ph-H), 9.4 (3H, br s, ). 13 C NMR (50 MHz, CDCl 3 ) d C (ppm) 41.2 (CH 2 ), (CH), (CH), (CH), (C), (C), (C), (C). MS, m/z: 384 (M + ), 369 (M CH 3 ), 354 (M (CH 3 ) 2 ), 339 [M (CH 3 ) 3 ], 311 [M Si(CH 3 ) 3 ], 295 [M Si (CH 3 ) 3 ], 280 [M Si(CH 3 ) 4 ], 265 [M Si(CH 3 ) 5 ], 222 [M Si 2 (CH 3 ) 6 ] (pr-Hydroxybenzyl)-pyroctechol (4-[(4- hydroxyphenyl)methyl]-1,2-benzenediol) (14 il. 1 H NMR (200 MHz, CDCl 3 ) d H (ppm) 4.10 (2H, m, CH 2 ), (7H, m. Ph-H). 13 C NMR (50 MHz, CDCl 3 ) d C (ppm) 42.2 (CH), (CH), (2 CH), (CH), (CH), (2 C), (C), (C), (C), (C), (C). MS, m/z: 432 (M + ), 417 (M-CH 3 ), 402 [M-(CH 3 ) 2 ], 359 [M-Si(CH 3 ) 3 ], 343 [M- Si (CH 3 ) 3 ], 329 [M-Si(CH 3 ) 4 ], 314 [M-Si(CH 3 ) 5 ], 298 [M- Si(CH 3 ) 6 ] ,4 0 -Methylenedi-pyroctechol (4,4 0 -methylenebis-1,2- benzenediol) (14b) il. 1 H NMR (200 MHz, CDCl 3 ) d H (ppm) 4.12 (2H, m, CH 2 ), (6H, m. Ph-H), 8.13 (4H, s, ). 13 C NMR (50 MHz, CDCl 3 ) d C (ppm) 40.2 (CH 2 ), (2 CH), (2 CH), (2 CH), (2 C), (2 C), (2 C). MS, m/z: 520 (M + ), 505 (M CH 3 ), 447 [M Si(CH 3 ) 3 ], 431 [M Si(CH 3 ) 3 ], 417 [M Si (CH 3 ) 4 ], 343 [M 2 Si 2 (CH 3 ) 6 ], 329 [M 2 Si 2 (CH 3 ) 7 ] ,4-Dihydroxyphenylethnol (hydroxytyrosol) (15 il. 1 H NMR 56 (200 MHz, cetone-d 6 ) d H (ppm) 2.65 (2H, m, CH 2 ), 3.67 (2H, mt, CH 2 ), (3H, m, Ph-H), 7.67 (2H, br s, 2H); 13 C NMR (50 MHz, cetone-d 6 ) d C (ppm) 39.7 (CH 2 ), 64.2(CH 2 ), (CH), (CH), (CH), (C), (C), (C); MS, m/z: 386 (M + ), 371 (M CH 3 ), 356 [M Si(CH 3 ) 2 ], 341 [M Si(CH 3 ) 3 ], 269 [M Si(CH 3 ) 3 ], 253 [M Si(CH 3 ) 3 ], 238 [M Si(CH 3 ) 4 ], 223 [M Si(CH 3 ) 5 ], 208 [M Si(CH 3 ) 6 ]. Acknowledgment Itlin MIUR PRIN 2008 is cknowledged. References nd notes 1. ( Hlouli, S.; Asther, M.; Sigoillot, J.-C.; Hmdi, M.; Lomscolo, A. J. Appl. Microbiol. 2006, 100, 219; (b) Seo, S.-Y.; Shrm, V. K.; Shrm, N. J. Agric. Food Chem. 2003, 51, ( Perron, N. R.; Grcí, C. R.; Pinzón, J. R.; Chur, M. N.; Brumghim, J. L. J. Inorg. Biochem. 2011, 105, 745; (b) Perron, N. 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