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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: (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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Historical fiction
|Material Type:
|Fiction, Audio book, etc.
|Document Type:
|Sound Recording
|All Authors / Contributors:
Toni Morrison
|9780739342275 0739342274
|OCLC Number:
|Compact discs.
|Read by the author.
|10 audio discs (12 hr.) : digital ; 4 3/4 in.
|by Toni Morrison.
Staring unflinchingly into the abyss of slavery, this spellbinding novel transforms history into a story as powerful as Exodus and as intimate as a lullaby. Sethe, its protagonist, was born a slave and escaped to Ohio, but eighteen years later she is still not free. She has too many memories of Sweet Home, the beautiful farm where so many hideous things happened. And Sethe's new home is haunted by the ghost of her baby, who died nameless and whose tombstone is engraved with a single word: Beloved. Filled with bitter poetry and suspense as taut as a rope, Beloved is a towering achievement. After the Civil War ends, Sethe longingly recalls the two-year-old daughter whom she killed when threatened with recapture after escaping from slavery 18 years before.
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subset: commoncrawl


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Best Western Cassino properties are provided below. Search for cheap and discount Best Western hotel rooms in Cassino, Italy for your personal or business trip. We list the best Best Western Cassino hotels & motels so you can review the Cassino Best Western hotel list below to find the perfect place.
Our technology will also provide discounted Cassino, Italy properties that are comparable to the quality of Best Western.
How should we direct your call?
When you stay at Best Western Hotel La Perla in Castel Volturno, you'll be close to Liternum Ruins.
Located in Fiuggi, Best Western Hotel Fiuggi Terme Resort & Spa is in a rural location and minutes from Piazza Spada and Fiuggi L'Acqua di Bonifacio VIII.


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Please Note: We do not mail or email you tickets once you have purchased them. (Many times your ticket is actually a wristband.) Your name is placed on a "Will Call" list. Please check in at admissions with your last name to receive your tickets. Please call the office if you have any questions about tickets 732-919-3500.
Join us in celebrating George Washington's Birthday. In the early 1800's there was no holidays that the new nation could bond over more so than George Washington's Birthday. There were dances and festivities in almost every town. Join the workers and their families of the Howell Iron Works in a day of celebration! There will be a special reenactment in the Chapel at 1pm. There will be hearth cooking demonstrations, craft demonstrations, and house tours! This event is a great time to get out of the house during winter and explore the village!
The Historic Village at Allaire, the non profit living history museum receives no funding from the State of New Jersey and so relies on donations, admissions, and purchases made at the General Store and Bakery to continue teaching the public about life in New Jersey in 1836


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Much interest in the 20th UiTM 2013
20th Ukraine International Travel Market (UITM 2013) will be held from 23rd to 25th October in Kiev in International Exhibition Centre. Two months before the happening, tourist boards of Brazil, Bulgaria, Hungary, Dominican Republic, Dubai, Egypt, India, Cyprus, Palestine, Poland, Slovakia, Thailand, Turkey, Finland, Sri Lanka and other countries have already reserved exhibition place. Emirates and Flydubai planes participate in UITM for the first time. Italians , Vietnam and Maldives will prolong participation.
Key question at the fair this year is gastro-tourism- travelling which combines museums, sights and architecture with the enjoyment in charm of home made cuisine. Options on the screen saver of the UITM 2013 for lovers of travelling and food perfectly complements with the ordinary activities: winter tourism, exotic travelling, holidays and luxury tourism.
Winter resorts will be presented by Finish, Turkish, Slovakian and Bulgarian tour operators, and exotic travelling will be presented by Brazil, Dominican Republic, India, Thailand and Sri Lanka. Ukraine is one of the most perspective markets for Sri Lanka. East Europe is responsible for the biggest growth of tourist figures on this island. Those who want to escape from winter in summer and get yourself warm on the beach, can choose hotels and travelling companies in Egypt, Turkey, Cyprus and Dubai. Excursions to Hungary, Poland, Bulgaria, India and Turkey are among the options for off-season wellness and relaxing holiday in spas, clinics with herbal mud and natural sources.
Most of Ukraine exhibitors offer luxury, green and tourism of region of Ukraine, while communities and commercial companies have active role at the exhibition. One of the major problems with which the Ukraine holiday resorts are faced is prolonging of the season. How can you attract tourists between holiday seasons? For majority of settlements and boarding houses on Crimea and Carpathian region, off-season lasts almost whole year, with the exception on New year, ten days of May's holiday and period of summer time. Accommodation service in Ukraine have special infrastructure, professional staff, and the most important ready to lower the prices. But, Ukraine business community still believes that the holidays in Ukraine are chance for offering high prices and low quality. As a result of that, they like to organize business events abroad.
Work shop "Create your event" is considered to break the stereotype. Exhibitors from other events organized by Premier Expo- big car producers, building companies, medical and cosmetic companies, traders and other-will be called on the hotel managers meeting, travel agencies and event organizers. Work shop will be held in business coffee place, and pleasant working atmosphere will be ideal for presentation of possibility of meeting organisation, motivating travelling and corporative happenings which combine business and holiday.
Medical tourism is the area of growing popularity in last few years, and it can be seen on new exhibition Medical tourism Kiev, which will also be held in October along with UITM 2013 and Public health 2013, dedicated to travellers and medical experts.
UITM 2013 is held under the organisation of ITE group and Premier Expo. It must be noticed that on last year's UITM 2012 on 4800 square meters participated 44 countries, it was organized over 70 programs, and the exhibition was visited by 13 800 visitors.« Vrati se na prethodnu stranicu


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July 01, 2010
From July 18 to 24, 2010, Hampden-Sydney College will host a Gilder Lehrman Institute Summer Seminar for K-12 teachers and National Park Service interpreters. The topic will be "Civil Rights in America."
Held at institutions throughout the United States and Great Britain, these seminars provide intellectual stimulation and practical resources and strategies to take back to the classroom. Over the past fourteen years, over 6000 educators have participated in Gilder Lehrman Seminars.
Founded in 1994, the Gilder Lehrman Institute of American History is a nonprofit organization supporting the study and love of American history through a wide range of programs and resources for students, teachers, scholars, and history enthusiasts throughout the nation.
The Institute creates and works closely with history-focused schools; organizes summer seminars and development programs for teachers; produces print and digital publications and traveling exhibitions; hosts lectures by eminent historians; administers a History Teacher of the Year Award in every state and U.S. territory; and offers national book prizes and fellowships for scholars to work in the Gilder Lehrman Collection as well as other renowned archives.


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Style: 65202
2" Eisenhower Flower
Classic- Eisenhower flower. This elegant brooch has Olivine with AB stones accents in the leaf veins. Can be worn as large rhinestone button or makes a wonderful Hat Pin. Has a vintage rhinestone look like from Grandma's closet. Features Antique Gold plating. Breathtaking!!
99 items in stock


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Whomp's Fortress is the second level in Super Mario 64 and it's remake Super Mario 64 DS. It takes one star to unlock.
A floating fortress with unstable pillars, rotating platforms, and enemies galore. The Whomp King awaits Mario at the top.
- The Whomp King
- Piranha Plants
- Whomps
- Thwomps
- Bullet Bill
- Goombas (DS)
- Bob-omb
- In Super Mario Galaxy, the Throwback Galaxy is essentially a replica of Whomp's Fortress.
|This Mario-related article is a stub. You can help by adding to it.


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Get notified when new sales are live
Subscribe to our notifications so you don't miss out on our new sales!
Staedtler LUNA Whiteboard Marker 4-Pack
$5.99 for this stationery essential!
Non-toxic | Point blocked | 4.2mm nib
- Staedtler Luna stationery
- Whiteboard marker pack
- Pack contents: 4 x markers
- Colour: Black/Blue/Green/Red
- Non-toxic
- Point blocked
- 4.2mm nib
- Erasable from glass, plastics. ceramic and steel
- Wipe off easily with dry cloth or eraser
- Quick drying
- High intensity ink - bright and vivid colours
- Replace cap after use
- Writing length (approx.): 400m
- Country of origin: China


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Posts Tagged 'dental emergency '
Your dentist is much more than just the person the one who cleans your teeth. Your dentist is a crucial lover with your family's healthcare workforce. Gary E. Alhadef, DDS A fantastic dentist will let you avert dental challenges, place oral cancers, and aid help your total wellbeing by preserving your tooth and gums healthful. Which is why it can be essential to discover a great loved ones dentist.
Though it is real the term "good" might be subjective, you can find objective measures by which one can assess a potential spouse and children dentist. Use these three guidelines, in addition to your personal personal criteria for your good family dentist, to locate your family's future health care lover.
3 Guidelines for Assessing a Family Dentist
one. What are the dentist's credentials and knowledge?
Dentists while in the America must graduate from an accredited dental school and pass both equally created and realistic tests. Question what qualifications the dentist has or appear on his web page to look at his credentials. Also note whether the many dentists during the observe, if it is an business with many dentists, provide the exact degree of working experience and credentials. It's also critical for dentists to continue their training right after graduation. Like any health care subject, new discoveries are being designed all the time. Your family dentist ought to be holding up with them.
Check with issues once you phone the office. Do you need a dentist who treats small children and teenagers along with grown ups? Ensure that you ask, simply because there are actually some family dentists preferring never to work with really young young children; they might only accept faculty age little ones and older as patients, and refer more youthful kids to your pediatric dentist.
Do your sons or daughters require braces and program care? Some family dental places of work provide braces as well as cleanings, fillings, and also other plan companies. This may be considered a wonderful time saver for any active relatives.
What about people who suffer from dental phobias and fears? You'll find some dentists who welcome people with phobias and fears and provide special companies like sedation dentistry, film or tunes to distract patients, nitrous oxide together with other solutions to aid them truly feel comfortable. If you want these providers, don't be afraid to request before you make your appointment! A good dentist wants you to come to feel comfy throughout your check out, and his workplace team need to welcome your concerns.
2. What is the business like?
Once you call the office, may be the receptionist impolite and rushed or pleasant and beneficial? Is he affected individual when answering thoughts or curt? Can he or she response your concerns relating to appointment moments, products and services, coverage and payments?
You'll be able to inform a good deal about an workplace from the human being staffing the front desk. That individual plays a vital purpose in any professional location, but most of all in the professional medical or dental business office. If the entrance workplace man or woman is impolite or condescending, it's possible you'll be much too intimidated to check with for whatever you want — not a good scenario if you have a dental emergency.
Some relatives dentists offer a free original appointment, and these an appointment may help you evaluate the business office. Does the dentist make use of the hottest tools? A condition with the art office environment implies a dentist who cares about studying and making use of the latest strategies, equipment and products for his patients' ease and comfort and convenience.
Last of all, could be the office environment conveniently located? You're going to be more possible to help keep appointments in case the office environment is practical for your home, school or place of work.
three. How can the dentist's office environment suit your family's requirements?
Each individual family differs. It's possible you'll have very younger young children at your house, or perhaps young children plus an getting older mother or father residing along with you. It could be useful to create a checklist within your needs for instance:
Pediatric dentistry – for my toddler and preschooler
Orthodontics for my young teen
Normal dental treatment for myself, my spouse
Dentures and also a handicapped accessible business office for my mother who life with us


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Barney Video & DVD ideas (US) is a fan-fiction thing to make.
Barney's Once Upon A Time (1997 version)
Front Cover
- Barney, Baby Bop, Stella and the kids
Back Cover
- Stella trying to open the case
- Barney, Shawn, Kristen and Tosha
Barney's Be A Friend
Front Cover
- Barney and the gang rushing to Derek
Back Cover
- Derek falling down
- Barney and Tosha
Barney's Evil Twin
An evil dinosaur named Larney kidnaps Baby Bop. Now, it's up to Barney and BJ to save Baby Bop and defeat Larney.
- Release Date: September 1, 1995.
Front Cover
- Barney facing Larney
Back Cover
- Larney falling into the water
- Barney and Baby Bop kissing.
Barney's Musical Scrapbook (1997 The Lyons Group version)
Front Cover
- Barney, Baby Bop and BJ
Back Cover
- Barney and BJ from "Hats Of To BJ"
- Barney, Stella and the kids from "Ships Ahoy"


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Ask a Lawyer and Get Answers to Your Legal Questions
In Texas, there are several requirements to be eligible for the defensive driving class.
The most important to your situation is that you must not have completed a defensive driving course (for the purpose of dismissing a previous citation) within one (1) year preceding the date of the offense on your current citation.
Because you completed the course in February of 2010 and received the second citation in June of 2010, you would not be eligible for the course - only 4 months have passed since you completed the course - the minimum is 12 months.
The only way to keep this ticket off of your record is if you are found not guilty by the judge. This is unlikely if the officer clocked you with a radar.
However, one thing you can do is call the court clerk's office and ask for a continuance from the first trial setting. Often (you'd be surprised how often this happens), the officer will forget to return for court on the second setting, and if you're there and he's not, the court will dismiss the ticket.
With no defensive driving classes available to you, that's really your only choice - other than just paying the ticket.
Thanks. On what grounds can I ask for a continuance on my first trial setting. Technically I do have valid reasons since I am travelling heavily on business right now 4 days a week. Is there something that you can suggest ?


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"I'm shakin' the dust of this crummy little town off my feet and I'm gonna see the world. Italy, Greece, the Parthenon, the Colosseum. Then, I'm comin' back here to go to college and see what they know. And then I'm gonna build things. I'm gonna build airfields, I'm gonna build skyscrapers a hundred stories high, I'm gonna build bridges a mile long... "
George Bailey - It's A Wonderful Life
Travel the world...sounds like a dream! Sadly, like good ol' George Bailey, not all of us can travel the world, but we can go where we can go, right?
Anywhere sounds good to me. It is so much fun traveling to new places and seeing things you have never seen before.
You can travel by plane...
Or you can travel by train...
Or you can travel by automobile...
(see what I just did there - hee hee)
Just pack your bags, baby...
With a little...
Or with a lot!
Shake the dust off of your feet and hit the road, Jack!
There is so much to see in this big ol' world!
They say it's the journey, not the destination.
But if you ask me, sometimes the destination is mighty fine indeed :~)
Happy travels!


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ASSET announced the ScanWorks High-Speed IO (HSIO) Toolkit for PC Client, which is a web-based tool for validating and debugging circuit board designs targeting future 22nm based Intel Core processors for the desktop and mobile applications. ScanWorks HSIO Toolkit includes tools that provide detailed information on DDR3, PCI-Express Graphics (PEG), and Direct Media Interface (DMI) buses. The ScanWorks HSIO Toolkit for PC Client is available now. A 90-day license costs $1,995.
ScanWorks HSIO for PC Client Features
- Debug validation issues with detailed bit- and lane-level data
- Visualize results and filter on individual lanes to find layout problems
- Utilizes Intel's free tools (BIOS RMT, PEG and DMI executables) to unlock bit- and lane-level results
- Results are stored in a database for easy reference, searching, sorting and exporting
- 90-day license is structured to allow time for a full validation cycle at a low cost
- Options such as loop count, crosstalk, burst size ranks to be tested
- Supports Intel 22nm second generation Core microarchitecture processor (code name Ivy Bridge-HE/HM/H/M)
- Supports DDR3, PCI-Express Graphics (PEG), and Direct Media Interface (DMI) buses
- Quickly determines the poorest performing lanes or data lines on a bus and display the results of diagnostic analysis graphically
More info: ASSET InterTech


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Smith, 6-foot, 180-pounds, played wide receiver last season for Grand Rapids Community College, before heading off to Mississippi. He will spend one semester at EMCC before he will be eligible for a Division-I scholarship. He is still adding offers.
"My three latest offers are Cincinnati, Syracuse and Nebraska," the former Tennessee commit stated. "I also have offers from Tennessee, Michigan State, Illinois, Washington and West Virginia. I will be taking my five visits in the fall, but I'm not sure where just yet."
"Syracuse is a school I've been talking to a lot, and I like the coaching staff," he added. "I'm very interested in Nebraska, and they were my latest offer. I like them a lot and they are coming to see me in the spring. Cincinnati would be close to home, and I like them too. I'm still good with Tennessee, and they've been good to me all the way."


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¡Ask a Comida Critic! Where to Spend St. Patrick's Day Today?
The obvious question for hoy. From Forker Ann in Laguna Hills:
I want to celebrate St. Paddy's today, but I don't want to fight for a seat among a bunch of drunks. I want something classier--not Five Crowns classy. Besides, they're Brits.
Ooh, wish that I could direct you to a genteel Irish-themed pub, but "genteel" and "Irish" go together like "Mexican" and "genteel"--ain't going to happen. And nothing against our many fine Irish pubs--the Auld Dubliner and Muldoon's, Durty Nelly's and O'Malley's, among others--but they're not going to be places of civility tonight.
Besides, what do you have against the Brits? St. Patrick's Day is the perfect day to stick it to those limeys--so why not go to . . .
. . . the Crow Bar?
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It has always been a great place to eat great, inventive pub food, with a nice selection of beers. And they just got a new chef, whose name escapes me right now because I deleted the press release some hack sent to me. They used to have an Irish Car Bomb-based dessert that was--pardon the pun--bomb, but I think PC pendejos got to them. Eh, go here, order a Jameson and toast to Michael Collins in the (metaphorical) belly of the beast.
Crow Bar and Kitchen, 2325 E. Coast Hwy., Corona del Mar, (949) 675-0070; www.crowbarcdm.com.
Got a spicy question for the Comida Critic? Ask him in the comments below!
Related Location
2325 E. Pacific Coast Highway
Corona Del Mar, CA 92625
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Pop-Up Installation to Bring Animated Symphony to the Wall of the InterContinental Miami
The NEWT installation in action.
Courtesy of NEWT
Most Miamians are familiar with the Intercontinental Hotel's LED wall, thanks to the infamous "dancing lady." But next month, the display will showcase something even more impressive than those sexy moves: a music and light installation inspired by Isaac Newton's famous idea that every primary color works with a musical note.
Starting Tuesday to Sunday, November 15, the hotel's 19-story LED wall will become a digital canvas featuring bright colors and shapes, all with accompanying sounds that can be listened to via the hotel's website or on your smartphone. Dubbed NEWT Miami: Experiments in Light, Color & Sound, organizers are calling it "a temporary musical planetarium."
Through this project (which raised $10,000 on Kickstarter), NEWT co-creators Dejha Carrington and Kelly Nunes wanted to initiate a dialogue about how the arts can help Miami grow — all while utilizing existing architecture and other assets to create new ideas and endeavors.
"A few years ago, the InterContinental Miami invested in a digital facade — 364 square feet of small TV screens under 19 stories of windows — and has since been screening their own content with periodic 'interventions' by different cultural groups," Carrington says. "We saw their technology as a challenge. In exploring how this building can have an impact on its surrounding community, we began to think of the installation as a catalyst or jump-off point for mobilizing locals and visitors."
"Nunes and I were inspired by how artists such James Turrell and Carlos Cruz Diez use light and space, and the sound pieces of Ryoji Ikeda, John Whitney, and Norman McLarren," Carrington goes on to say. "One of the more poetic videos we've seen — and the video we presented in our pitch meeting with the InterContinental Miami host venue — was Sesame Street's '30 Dots Collection' from their 1960s Numbers Series. Watching old clips on YouTube, you get the sense that each dot has a distinct personality. We wanted to do something similar, but on a much larger canvas like the side of a building."
A test of the NEWT installation.
Courtesy of NEWT
The installation brings with it a whole calendar of events, including Caffeinated Conversations, a wake-up call about experiential storytelling on Tuesday, November 10; yoga in the park, NEWT edition on Wednesday, November 11; and lots more. All are free, though some require RSVP.
"All of these events are free and open to the public, and we've also published a program with editorial by O, Miami, in a special preview of their Eight Miami Poets book launch," adds Carrington. "At every juncture, we've made a concentrated effort to involve the local arts and cultural community, and are hoping others will be inspired to rethink physical spaces and actively seek collaborative opportunities."
For the complete schedule of events, visit projectnewt.com, or Instagram @projectnewt.
Related Location
100 Chopin Plaza
Miami, FL 33131
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