This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and

Size: px
Start display at page:

Download "This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and"

Transcription

1 This rticle ppered in journl published by Elsevier. The ttched copy is furnished to the uthor for internl non-commercil reserch nd eduction use, including for instruction t the uthors institution nd shring with collegues. ther uses, including reproduction nd distribution, or selling or licensing copies, or posting to personl, institutionl or third prty websites re prohibited. In most cses uthors re permitted to post their version of the rticle (e.g. in Word or Tex form) to their personl website or institutionl repository. Authors requiring further informtion regrding Elsevier s rchiving nd mnuscript policies re encourged to visit:

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. R.; Brumghim, J. L. Cell. Biochem. Biophys. 2009, 53, 75; (c) Ginj Teixeir, J.; Brrocs Dis, C.; Mrtins Teixeir, D. Electronlysis 2009, 21, ( Xio, Z.-P.; M, T.-W.; Fu, W.-C.; Peng, X.-C.; Zhng, A. H.; Zhu, H.-L. Eur. J. Med. Chem. 2010, 45, 5064; (b) Bnsl, V. K.; Kumr, R.; Prsd, R.; Prsd, S.; Nirj J. Mol. Ctl. A: Chem. 2008, 284, ( Mrín-Zmor, M. E.; Rojs-Melgrejo, F.; Grcí-Cánovs, F.; Grcí-Ruiz, P. A. J. Biotechnol. 2009, 139, 163; (b) Myer, A. M. Phytochemistry 2006, 67, ( Rmsden, C. A.; Strtford, M. R. L.; Riley, P. A. rg. Biomol. Chem. 2009, 7, 3388; (b) Muñoz-Muñoz, J. L.; Grcí-Molin, F.; Grcí-Ruiz, P. A.; Molin- Alrcón, M.; Tudel, J.; Grcí-Cnovs, F.; Rodriguez-Lopez, J. N. Biochem. J. 2008, 416, ( Espín, J. C.; Soler-Rivs, C.; Cntos, E.; Tomás-Brberán, F. A.; Wichers, H. J. J. Agric. Food Chem. 2001, 49, 1187; (b) Brown, R. S.; Mle, K. B.; Luong, J. H. T. Anl. Biochem. 1994, 222, Sheldon, R. A. Adv. Synth. Ctl. 2007, 349, Mjid, Z. A.; Sbri, N. A. M.; Bung, N. A.; Shhir, S. J. Fundm. Sciences 2010, 6, Arsln, A.; Kirlp, S.; Toppre, L.; Ygci, Y. Int. J. Biol. Mcromol. 2005, 35, Jfr, A.; Mus, A.; Ndrjh, K.; Lee, Y. H.; Hmidh, S. Sens. Actutors, B 2006, 114, Snz, V. C.; Men, M. L.; Gonzlez-Cortes, A.; Ynez-Sedeno, P.; Pingrron, J. M. Anl. Chim. Act 2005, 528, Zejli, H.; Hidlgo-Hidlgo de Cisneros, J. L.; Nrnjo-Rodriguez, I.; Liu, B.; Temsmni, K. R.; Mrty, J. L. Anl. Chim. Act 2008, 612, ( Munjl, N.; Swhney, S. K. Enzyme Microb. Technol. 2002, 30, 613; (b) Durn, N.; Ros, M. A.; D Annible, A.; Ginfred, L. Enzyme Microb. Technol. 2002, 31, Tuncgil, S.; Kyhn, S. K.; Byrmoglu, G.; Aric, M. Y.; Toppre, L. J. Mol. Ctl. B: Enzym. 2009, 58, ( Yin, H.; Zhou, Y.; Xu, J.; Ai, S.; Cui, L.; Zhu, L. Anl. Chim. Act 2010, 659, 144; (b) Hnifh, S. A.; Heng, L. Y.; Ahmd, M. Anl. Sci. 2009, 25, ( Anghileri, A.; Lntto, R.; Kruus, K.; Arosio, C.; Freddi, G. J. Biotechnol. 2007, 127, 508; (b) Thlmnn, C. R.; Lötzbeyer, T. Eur. Food Res. Technol. 2002, 214, 276; (c) Chen, T.; Embree, H. D.; Wu, L. Q.; Pyne, G. F. Biopolymers 2002, 64, 292; (d) Hung, T. H.; Kuwn, T.; Wrsinke, A. Biosens. Bioelectron. 2002, 17, Yhsi, A.; Shin, F.; Demirel, G.; Tumturk, H. Int. J. Biol. Mcromol. 2005, 36, ( Arig, K.; Lvov, Y. M.; Kwkmi, K.; Ji, Q.; Hill, J. P. Adv. Drug. Deliv. Rev. 2011, 63, 762; (b) Arig, K.; Ji, Q.; Hill, J. P. Adv. Polym. Sci. 2010, 229, 51; (c) Decher, G. Nchr. Chem. Tech. Lb. 1993, 41, Decher, G.; Schmitt, J. Prog. Colloid Polym. Sci. 1992, 89, Held, C.; Kndelbuer, A.; Schroeder, M.; Cvco-Pulo, A.; Gubitz, G. M. Environ. Chem. Lett. 2005, 3, ( de Fri, R..; Rotuno-Moure, V.; Lopes de Almeid, M. A.; Krieger, N.; Mitchell, D. A. Food Technol. Biotechnol. 2007, 45, 287; (b) Andrwis, A.; Vrd, K. J. Food Biochem. 1990, 14, ( Ho, P. Y.; Chiou, M. S.; Cho, A. C. Appl. Biochem. Biotechnol. 2003, 111, 139; (b) Ros, J. R.; Rodriguez-Lopez, J. N.; Grcí-Cánovs, F. Biochim. Biophys. Act 1995, 1204, ( Brtto, L.; Cndido, A.; Mrzorti, M.; Sgui, F.; Riv, S.; Dnieli, B. J. Mol. Ctl. B: Enzym. 2006, 39, 3; (b) Ruch, P.; Ferri, E. N.; Girotti, S.; Ruchov, H.; Crre, G.; Bovr, R.; Fini, F.; Rod, A. Anl. Biochem. 1997, 245, Msmoto, Y.; Iid, S.; Kubo, M. Plnt Med. 1980, 40, Mrtın, M. T.; Plou, F. J.; Alclde, M.; Bllesteros, A. J. Mol. Ctl. B: Enzym. 2003, 21, Knezevic, Z.; Milosvic, N.; Bezbrdic, D.; Jkovljevic, Z.; Prodnovic, R. Biochem. Eng. J. 2006, 30, ( Aytr, B. S.; Bkir, U. Process Biochem. 2008, 43, 125; (b) Achry, C.; Kumr, V.; Sen, R.; Kundu, S. C. Biotechnol. J. 2008, 3, nd, M.; Arig, K.; Kunitke, T. J. Biosci. Bioeng. 1999, 87, Linewever, H.; Burk, D. JACS 1934, 56, ( Hung, C. L.; Cheng, W. C.; Yng, J. C.; Chi, M. C.; Chen, J. H.; Lin, H. P.; Lin, L. L. J. Ind. Microbiol. Biotechnol. 2010, 37, 717; (b) Chitrngd, A.; Veerendr, K.; Rmkrishn, S.; Subhs, C. K. Biotechnol. J. 2008, 3, Sukhorukov, G.; Fery, A.; Möhwld, H. Prog. Polym. Sci. 2005, 30, Espin, G. J. C.; Toms, B. F.; Grci, V.M.C.; Ferreres, A. F.; Soler, R. C.; Wichers, H. J. US , EP A1, Kzndjind, R.; Klibnov, A. M. J. Am. Chem. Soc. 1985, 107, Moreno-Álvrez, S. A.; Mrtínez-Cstñón, G. A.; Niño-Mrtínez, N.; Reyes- Mcís, J. F.; Ptiño-Mrín, N.; Loyol-Rodríguez, J. P.; Ruiz, F. J. Nnoprt. Res. 2010, 12, Chnwitheesuk, A.; Teerwutgulrg, A.; Kilburnb, J. D.; Rkriythm, N. Food Chem. 2007, 100, Sto, Y.; Itgki, S.; Kurokw, T.; gur, J.; Kobyshi, M.; Hirno, T.; Sugwr, M. Int. J. Phrm. 2011, 403, Costi, R.; Di Snto, R.; Artico, M.; Mss, S.; Rgno, R.; Loddo, R.; L Coll, M.; Trmontno, E.; L Coll, P.; Pni, A. Bioorg. Med. Chem. 2004, 12, Tuerker, B. H.; Ilhmi, G.; Abdullh, M.; Sueleymn, G.; Ertn, S. J. Enzyme Inhib. Med. Chem. 2010, 25, Colon, M.; Guevr, P.; Gerwick, W. H. J. Nt. Prod. 1987, 50, Yngui, T.; Dhouib, A.; Rhoum, A.; Sydi, S. Food Chem. 2009, 117, Hmden, K.; Alloucheb, N.; Dmkb, M.; Elfeki, A. Chem. Biol. Interct. 2009, 180, ( Bertelli, A.; Ds, D. J. Crdiovsc. Phrmcol. 2009, 54, 468; (b) Rietjens, S. J.; Bst, A.; de Vente, J.; Henen, G. R. M. M. Am. J. Physiol. Hert Circ. Physiol. 2007, 292, H González Corre, J. A.; López-Villodres, J. A.; Asensi, R.; Esprtero, J. L.; Rodríguez-Gutiérez, G.; De L Cruz, J. P. Br. J. Nutr. 2009, 101, Fki, I.; Shnoun, Z.; Sydi, S. J. Agric. Food Chem. 2007, 55, Cruso, F.; Furlong, D. N.; Arig, K.; Ichinose, I.; Kunitke, T. Lngmuir 1998, 14, Hn, J.; Tlorete, T. P. N.; Ymd, P.; Isod, H. Cytotechnol. 2009, 59, Mteo, C.; Abin,.; Fernndez-Lorente, G.; Predoche, J.; Fernndez-Lfuente, R.; Guisn, J. M. Biotechnol. Prog. 2002, 18, ( Perzzini, R.; Sldino, R.; Guzzroni, M.; Crestini, C. Bioorg. Med. Chem. 2011, 19, 440; (b) Tiourin,. P.; Antipov, A. A.; Sukhorukov, G. B.; Lrionov, N. I.; Lvov, Y.; Möhwld, H. Mcromol. Biosci. 2001, 1, ( Sedmk, J. J.; Grossberg, S. E. Anl. Biochem. 1977, 79, 544; (b) Brdford, M. M. Anl. Biochem. 1976, 72, 248.

11 166 M. Guzzroni et l. / Bioorg. Med. Chem. 20 (2012) ( Rodríguez-López, J. N.; Gómez-Fenoll, L.; Penlver, M. J.; Grcí-Ruíz, P. A.; Vrón, V.; Mrtínez-rtíz, F.; Grcí-Cánovs, F.; Tudel, J. Biochim. Biophys. Act 2001, 1548, 238; (b) Sweely, C. C.; Bentlry, R.; Mkit, M.; Wells, W. W. J. Am. Chem. Soc. 1963, 85, Chernyk, N.; Dudnik, A. S.; Hung, C.; Gevorgyn, V. J. Am. Chem. Soc. 2010, 132, Nkym, S.; Iked, F. US Ptent , , Pezzell, A.; List, L.; Npolitno, A.; d Ischi, M. Tetrhedron Lett. 2005, 46, Silke, P.; Spitelle, P. J. Nt. Prod. 2006, 69, Deng, L.; Sundriyl, S.; Rubio, V.; Shi, Z.-Z.; Song, Y. J. Med. Chem. 2009, 52, Pouységu, L.; Syll, T.; Grnier, T.; Rojs, L. B.; Chrris, J.; Deffieux, D.; Quideu, S. Tetrhedron 2010, 66, 5908.

Study on enzyme-assisted aqueous extraction of oil from soybean

Study on enzyme-assisted aqueous extraction of oil from soybean 8 Journl Scientific & Industril Reserch J SCI IND RES VOL 69 NOVEMBER 2010 Vol. 69, November 2010, pp. 8-865 Study on enzyme-ssisted queous extrction oil from soyben Jun-Qing Qin*, De-Hui Qin, Xing-Mo

More information

Rate and Activation Energy of the Iodination of Acetone

Rate and Activation Energy of the Iodination of Acetone nd Activtion Energ of the Iodintion of Acetone rl N. eer Dte of Eperiment: //00 Florence F. Ls (prtner) Abstrct: The rte, rte lw nd ctivtion energ of the iodintion of cetone re detered b observing the

More information

** Dpt. Chemical Engineering, Kasetsart University, Bangkok 10900, Thailand

** Dpt. Chemical Engineering, Kasetsart University, Bangkok 10900, Thailand Modelling nd Simultion of hemicl Processes in Multi Pulse TP Experiment P. Phnwdee* S.O. Shekhtmn +. Jrungmnorom** J.T. Gleves ++ * Dpt. hemicl Engineering, Ksetsrt University, Bngkok 10900, Thilnd + Dpt.hemicl

More information

TITLE THE PRINCIPLES OF COIN-TAP METHOD OF NON-DESTRUCTIVE TESTING

TITLE THE PRINCIPLES OF COIN-TAP METHOD OF NON-DESTRUCTIVE TESTING TITLE THE PRINCIPLES OF COIN-TAP METHOD OF NON-DESTRUCTIVE TESTING Sung Joon Kim*, Dong-Chul Che Kore Aerospce Reserch Institute, 45 Eoeun-Dong, Youseong-Gu, Dejeon, 35-333, Kore Phone : 82-42-86-231 FAX

More information

Treatment Spring Late Summer Fall 0.10 5.56 3.85 0.61 6.97 3.01 1.91 3.01 2.13 2.99 5.33 2.50 1.06 3.53 6.10 Mean = 1.33 Mean = 4.88 Mean = 3.

Treatment Spring Late Summer Fall 0.10 5.56 3.85 0.61 6.97 3.01 1.91 3.01 2.13 2.99 5.33 2.50 1.06 3.53 6.10 Mean = 1.33 Mean = 4.88 Mean = 3. The nlysis of vrince (ANOVA) Although the t-test is one of the most commonly used sttisticl hypothesis tests, it hs limittions. The mjor limittion is tht the t-test cn be used to compre the mens of only

More information

Himaja M et al / IJRAP 2010, 1 (1) 147-152

Himaja M et al / IJRAP 2010, 1 (1) 147-152 imj M et l / IJRAP 2010, 1 (1) 147-152 Reserch Article Avilble online through www.ijrp.net SYTESIS AD ATIMICRBIAL ACTIVITY F DICTMI A AALGS imj Mlipeddi *, Mlipeddi Venktrmn b, Shoo Atish Kumr c, Annd

More information

Experiment 6: Friction

Experiment 6: Friction Experiment 6: Friction In previous lbs we studied Newton s lws in n idel setting, tht is, one where friction nd ir resistnce were ignored. However, from our everydy experience with motion, we know tht

More information

INVESTIGATION OF THE EXTINGUISHING FEATURES FOR LIQUID FUELS AND ORGANIC FLAMMABLE LIQUIDS ATOMIZED BY A WATER FLOW

INVESTIGATION OF THE EXTINGUISHING FEATURES FOR LIQUID FUELS AND ORGANIC FLAMMABLE LIQUIDS ATOMIZED BY A WATER FLOW EPJ Web of Conferences 110, 01083 (2016) DOI: 10.1051/ epjconf/ 201611001083 C Owned by the uthors, published by EDP Sciences, 2016 INVESTIGATION OF THE EXTINGUISHING FEATURES FOR LIQUID FUELS AND ORGANIC

More information

REVIEW QUESTIONS Chapter 16

REVIEW QUESTIONS Chapter 16 Chemistry 102 ANSWER EY REVIEW QUESTIONS Chpter 16 1. A buffer is prepred by dding 20.0 g of cetic cid (HC 2 H O 2 ) nd 20.0 g of sodium cette (NC 2 H O 2 ) in enough wter to prepre 2.00 L of solution.

More information

THERMAL EXPANSION OF TUNGSTEN

THERMAL EXPANSION OF TUNGSTEN . THERMAL EXPANSION OF TUNGSTEN S515 By Peter Hidnert nd W. T. Sweeney ABSTRACT This pper gives the results of n investigtion on the therml expnsion of tungsten (99.98 per cent) over vrious temperture

More information

COMPONENTS: COMBINED LOADING

COMPONENTS: COMBINED LOADING LECTURE COMPONENTS: COMBINED LOADING Third Edition A. J. Clrk School of Engineering Deprtment of Civil nd Environmentl Engineering 24 Chpter 8.4 by Dr. Ibrhim A. Asskkf SPRING 2003 ENES 220 Mechnics of

More information

The International Association for the Properties of Water and Steam. Release on the Ionization Constant of H 2 O

The International Association for the Properties of Water and Steam. Release on the Ionization Constant of H 2 O The Interntionl Assocition for the Properties of Wter nd Stem Lucerne, Sitzerlnd August 7 Relese on the Ioniztion Constnt of H O 7 The Interntionl Assocition for the Properties of Wter nd Stem Publiction

More information

An Undergraduate Curriculum Evaluation with the Analytic Hierarchy Process

An Undergraduate Curriculum Evaluation with the Analytic Hierarchy Process An Undergrdute Curriculum Evlution with the Anlytic Hierrchy Process Les Frir Jessic O. Mtson Jck E. Mtson Deprtment of Industril Engineering P.O. Box 870288 University of Albm Tuscloos, AL. 35487 Abstrct

More information

Helicopter Theme and Variations

Helicopter Theme and Variations Helicopter Theme nd Vritions Or, Some Experimentl Designs Employing Pper Helicopters Some possible explntory vribles re: Who drops the helicopter The length of the rotor bldes The height from which the

More information

The Velocity Factor of an Insulated Two-Wire Transmission Line

The Velocity Factor of an Insulated Two-Wire Transmission Line The Velocity Fctor of n Insulted Two-Wire Trnsmission Line Problem Kirk T. McDonld Joseph Henry Lbortories, Princeton University, Princeton, NJ 08544 Mrch 7, 008 Estimte the velocity fctor F = v/c nd the

More information

Graphs on Logarithmic and Semilogarithmic Paper

Graphs on Logarithmic and Semilogarithmic Paper 0CH_PHClter_TMSETE_ 3//00 :3 PM Pge Grphs on Logrithmic nd Semilogrithmic Pper OBJECTIVES When ou hve completed this chpter, ou should be ble to: Mke grphs on logrithmic nd semilogrithmic pper. Grph empiricl

More information

Module 2. Analysis of Statically Indeterminate Structures by the Matrix Force Method. Version 2 CE IIT, Kharagpur

Module 2. Analysis of Statically Indeterminate Structures by the Matrix Force Method. Version 2 CE IIT, Kharagpur Module Anlysis of Stticlly Indeterminte Structures by the Mtrix Force Method Version CE IIT, Khrgpur esson 9 The Force Method of Anlysis: Bems (Continued) Version CE IIT, Khrgpur Instructionl Objectives

More information

DlNBVRGH + Sickness Absence Monitoring Report. Executive of the Council. Purpose of report

DlNBVRGH + Sickness Absence Monitoring Report. Executive of the Council. Purpose of report DlNBVRGH + + THE CITY OF EDINBURGH COUNCIL Sickness Absence Monitoring Report Executive of the Council 8fh My 4 I.I...3 Purpose of report This report quntifies the mount of working time lost s result of

More information

Plotting and Graphing

Plotting and Graphing Plotting nd Grphing Much of the dt nd informtion used by engineers is presented in the form of grphs. The vlues to be plotted cn come from theoreticl or empiricl (observed) reltionships, or from mesured

More information

All pay auctions with certain and uncertain prizes a comment

All pay auctions with certain and uncertain prizes a comment CENTER FOR RESEARC IN ECONOMICS AND MANAGEMENT CREAM Publiction No. 1-2015 All py uctions with certin nd uncertin prizes comment Christin Riis All py uctions with certin nd uncertin prizes comment Christin

More information

Eleni Kalogria Athanasia Varvaresou Spyridon Papageorgiou Evaggelia Protopapa Ioannis Tsaknis Alexios Matikas Irene Panderi

Eleni Kalogria Athanasia Varvaresou Spyridon Papageorgiou Evaggelia Protopapa Ioannis Tsaknis Alexios Matikas Irene Panderi Chromtogrphi (2014) 77:1275 1281 DOI 10.1007/s10337-014-2722-9 ORIGINAL Pre Column Derivtiztion HPLC Procedure for the Quntittion of Aluminium Chlorohydrte in Antiperspirnt Crems Using Quercetin s Chromogenic

More information

Controlled Synthesis of Block Copolymers containing N-isopropylacrylamide by Reversible Addition-Fragmentation Chain-Transfer (RAFT) Polymerization

Controlled Synthesis of Block Copolymers containing N-isopropylacrylamide by Reversible Addition-Fragmentation Chain-Transfer (RAFT) Polymerization J. Mex. Chem. Soc. 2011, 55(1), 21-32 Controlled Article Synthesis of Block Copolymers contining N-isopropylcrylmide by RAFT Polymeriztion 2011, Sociedd Químic de México 21 ISSN 1870-249X Controlled Synthesis

More information

Economics Letters 65 (1999) 9 15. macroeconomists. a b, Ruth A. Judson, Ann L. Owen. Received 11 December 1998; accepted 12 May 1999

Economics Letters 65 (1999) 9 15. macroeconomists. a b, Ruth A. Judson, Ann L. Owen. Received 11 December 1998; accepted 12 May 1999 Economics Letters 65 (1999) 9 15 Estimting dynmic pnel dt models: guide for q mcroeconomists b, * Ruth A. Judson, Ann L. Owen Federl Reserve Bord of Governors, 0th & C Sts., N.W. Wshington, D.C. 0551,

More information

Econ 4721 Money and Banking Problem Set 2 Answer Key

Econ 4721 Money and Banking Problem Set 2 Answer Key Econ 472 Money nd Bnking Problem Set 2 Answer Key Problem (35 points) Consider n overlpping genertions model in which consumers live for two periods. The number of people born in ech genertion grows in

More information

RP-HPLC method development and validation for estimation of rivaroxaban in pharmaceutical dosage forms

RP-HPLC method development and validation for estimation of rivaroxaban in pharmaceutical dosage forms Brzilin Journl of Phrmceuticl Sciences vol. 49, n. 2, pr./jun., 2013 Article RP-HPLC method development nd vlidtion for estimtion of rivroxbn in phrmceuticl dosge forms Mustf Çelebier, Tub Reçber, Engin

More information

Small Business Networking

Small Business Networking Why network is n essentil productivity tool for ny smll business Effective technology is essentil for smll businesses looking to increse the productivity of their people nd business. Introducing technology

More information

Unit 29: Inference for Two-Way Tables

Unit 29: Inference for Two-Way Tables Unit 29: Inference for Two-Wy Tbles Prerequisites Unit 13, Two-Wy Tbles is prerequisite for this unit. In ddition, students need some bckground in significnce tests, which ws introduced in Unit 25. Additionl

More information

2. Transaction Cost Economics

2. Transaction Cost Economics 3 2. Trnsction Cost Economics Trnsctions Trnsctions Cn Cn Be Be Internl Internl or or Externl Externl n n Orgniztion Orgniztion Trnsctions Trnsctions occur occur whenever whenever good good or or service

More information

Health insurance marketplace What to expect in 2014

Health insurance marketplace What to expect in 2014 Helth insurnce mrketplce Wht to expect in 2014 33096VAEENBVA 06/13 The bsics of the mrketplce As prt of the Affordble Cre Act (ACA or helth cre reform lw), strting in 2014 ALL Americns must hve minimum

More information

Why is the NSW prison population falling?

Why is the NSW prison population falling? NSW Bureu of Crime Sttistics nd Reserch Bureu Brief Issue pper no. 80 September 2012 Why is the NSW prison popultion flling? Jcqueline Fitzgerld & Simon Corben 1 Aim: After stedily incresing for more thn

More information

Reversing Medications That Cause Bleeding

Reversing Medications That Cause Bleeding Reversing Medictions Tht Cuse Bleeding Dine M. Birnbumer, M.D., FACEP Professor of Medicine University of Cliforni, Los Angeles Senior Fculty Deprtment of Emergency Medicine Hrbor-UCLA Medicl Center The

More information

Project 6 Aircraft static stability and control

Project 6 Aircraft static stability and control Project 6 Aircrft sttic stbility nd control The min objective of the project No. 6 is to compute the chrcteristics of the ircrft sttic stbility nd control chrcteristics in the pitch nd roll chnnel. The

More information

Understanding Life Cycle Costs How a Northern Pump Saves You Money

Understanding Life Cycle Costs How a Northern Pump Saves You Money Understnding Life Cycle Costs How Nrn Pump Sves You Money Reference: Hydrulic Institute (www.s.g) Introduction Wht Life Cycle Cost (LCC) Clculting Totl LCC LCC Components Wht Life Cycle Cost Life Cycle

More information

ClearPeaks Customer Care Guide. Business as Usual (BaU) Services Peace of mind for your BI Investment

ClearPeaks Customer Care Guide. Business as Usual (BaU) Services Peace of mind for your BI Investment ClerPeks Customer Cre Guide Business s Usul (BU) Services Pece of mind for your BI Investment ClerPeks Customer Cre Business s Usul Services Tble of Contents 1. Overview...3 Benefits of Choosing ClerPeks

More information

Data replication in mobile computing

Data replication in mobile computing Technicl Report, My 2010 Dt repliction in mobile computing Bchelor s Thesis in Electricl Engineering Rodrigo Christovm Pmplon HALMSTAD UNIVERSITY, IDE SCHOOL OF INFORMATION SCIENCE, COMPUTER AND ELECTRICAL

More information

Small Business Networking

Small Business Networking Why network is n essentil productivity tool for ny smll business Effective technology is essentil for smll businesses looking to increse the productivity of their people nd processes. Introducing technology

More information

Small Business Networking

Small Business Networking Why Network is n Essentil Productivity Tool for Any Smll Business TechAdvisory.org SME Reports sponsored by Effective technology is essentil for smll businesses looking to increse their productivity. Computer

More information

Small Business Networking

Small Business Networking Why network is n essentil productivity tool for ny smll business Effective technology is essentil for smll businesses looking to increse the productivity of their people nd business. Introducing technology

More information

Small Business Networking

Small Business Networking Why network is n essentil productivity tool for ny smll business Effective technology is essentil for smll businesses looking to increse the productivity of their people nd processes. Introducing technology

More information

JaERM Software-as-a-Solution Package

JaERM Software-as-a-Solution Package JERM Softwre-s--Solution Pckge Enterprise Risk Mngement ( ERM ) Public listed compnies nd orgnistions providing finncil services re required by Monetry Authority of Singpore ( MAS ) nd/or Singpore Stock

More information

Introducing Kashef for Application Monitoring

Introducing Kashef for Application Monitoring WextWise 2010 Introducing Kshef for Appliction The Cse for Rel-time monitoring of dtcenter helth is criticl IT process serving vriety of needs. Avilbility requirements of 6 nd 7 nines of tody SOA oriented

More information

ACID-BASE BASICS Stephen Wright, Ph.D. Dept of Physiology

ACID-BASE BASICS Stephen Wright, Ph.D. Dept of Physiology Foundtions Lerning Objectives for the self-lerning module on ACID-BASE Bsics 1. Define ph. 2. Clculte the hydrogen ion concentrtion from ph nd vice vers. 3. List the mjor body buffer systems. ACID-BASE

More information

Physics 43 Homework Set 9 Chapter 40 Key

Physics 43 Homework Set 9 Chapter 40 Key Physics 43 Homework Set 9 Chpter 4 Key. The wve function for n electron tht is confined to x nm is. Find the normliztion constnt. b. Wht is the probbility of finding the electron in. nm-wide region t x

More information

Effect of viscosity on C sugar in Beet sugar factories

Effect of viscosity on C sugar in Beet sugar factories ANNUAL TRANSACTIONS OF THE NORDIC RHEOLOGY SOCIETY, VOL. 16, 2008 Effect of on C sugr in Beet sugr fctories Mohmmd Hojjtoleslmy 1, Rez Shokrni 2 nd Ahmd Krsi 3 1 Deprtment of food technology, College of

More information

Health insurance exchanges What to expect in 2014

Health insurance exchanges What to expect in 2014 Helth insurnce exchnges Wht to expect in 2014 33096CAEENABC 02/13 The bsics of exchnges As prt of the Affordble Cre Act (ACA or helth cre reform lw), strting in 2014 ALL Americns must hve minimum mount

More information

2006 IPCC Software for National Greenhouse Gas Inventories: Application and use for India

2006 IPCC Software for National Greenhouse Gas Inventories: Application and use for India 2006 IPCC Softwre for Ntionl Greenhouse Gs Inventories: Appliction nd use for Indi Presenttion for NGGIP Side Event Bonn, June 8, 2013 Prof. Amit Grg (mitgrg@iimhd.ernet.in), INDIA GHG Inventory Softwre

More information

Learner-oriented distance education supporting service system model and applied research

Learner-oriented distance education supporting service system model and applied research SHS Web of Conferences 24, 02001 (2016) DOI: 10.1051/ shsconf/20162402001 C Owned by the uthors, published by EDP Sciences, 2016 Lerner-oriented distnce eduction supporting service system model nd pplied

More information

Small Business Networking

Small Business Networking Why network is n essentil productivity tool for ny smll business Effective technology is essentil for smll businesses looking to increse the productivity of their people nd processes. Introducing technology

More information

THE PARAMETERS OF TRAPS IN K-FELDSPARS AND THE TL BLEACHING EFFICIENCY

THE PARAMETERS OF TRAPS IN K-FELDSPARS AND THE TL BLEACHING EFFICIENCY GEOCHRONOMETRIA Vol. 2, pp 15-2, 21 Journl on Methods nd Applictions of Asolute Chronology THE PARAMETERS OF TRAPS IN K-FELDSPARS AND THE TL BLEACHING EFFICIENCY ALICJA CHRUŒCIÑSKA 1, HUBERT L. OCZKOWSKI

More information

Advanced Treatment of Wastewater from Food Waste Disposer in Modified Ludzack-Ettinger Type Membrane Bioreactor

Advanced Treatment of Wastewater from Food Waste Disposer in Modified Ludzack-Ettinger Type Membrane Bioreactor Environ. Eng. Res. June,7() : 59-6 Reserch Pper http://dx.doi.org/.449/eer..7..59 pissn 6-5 eissn 5-968X Advnced Tretment of Wstewter from Food Wste Disposer in Modified Ludzck-Ettinger Type Membrne Biorector

More information

Electrochemical behavior of anticancer drug 5-fluorouracil at carbon paste electrode and its analytical application

Electrochemical behavior of anticancer drug 5-fluorouracil at carbon paste electrode and its analytical application Bukkitgr nd Shetti Journl of Anlyticl Science nd Technology (2016) 7:1 DI 10.1186/s40543-015-0080-3 RESEARC ARTICLE pen Access Electrochemicl behvior of nticncer drug 5-fluorourcil t crbon pste electrode

More information

Determination of organophosphorus insecticides in natural waters using SPE-disks and SPME followed by GC/FTD and GC/MS

Determination of organophosphorus insecticides in natural waters using SPE-disks and SPME followed by GC/FTD and GC/MS Fresenius J Anl Chem (2000) 368 : 616 623 Springer-Verlg 2000 ORIGINAL PAPER D. Lmbropoulou T. Skellrides T. Albnis Determintion of orgnophosphorus insecticides in nturl wters using SPE-disks nd SPME followed

More information

Synthesis and Applications of Small Molecule Libraries

Synthesis and Applications of Small Molecule Libraries Chem. Rev. 1996, 96, 555 600 555 Synthesis nd Applictions of Smll Molecule Librries Lorin A. Thompson nd Jonthn A. Ellmn* Deprtment of Chemistry, University of Cliforni, Berkeley, Cliforni 94720 Received

More information

9 CONTINUOUS DISTRIBUTIONS

9 CONTINUOUS DISTRIBUTIONS 9 CONTINUOUS DISTIBUTIONS A rndom vrible whose vlue my fll nywhere in rnge of vlues is continuous rndom vrible nd will be ssocited with some continuous distribution. Continuous distributions re to discrete

More information

GFI MilArchiver 6 vs C2C Archive One Policy Mnger GFI Softwre www.gfi.com GFI MilArchiver 6 vs C2C Archive One Policy Mnger GFI MilArchiver 6 C2C Archive One Policy Mnger Who we re Generl fetures Supports

More information

Polynomial Functions. Polynomial functions in one variable can be written in expanded form as ( )

Polynomial Functions. Polynomial functions in one variable can be written in expanded form as ( ) Polynomil Functions Polynomil functions in one vrible cn be written in expnded form s n n 1 n 2 2 f x = x + x + x + + x + x+ n n 1 n 2 2 1 0 Exmples of polynomils in expnded form re nd 3 8 7 4 = 5 4 +

More information

According to Webster s, the

According to Webster s, the dt modeling Universl Dt Models nd P tterns By Len Silversn According Webster s, term universl cn be defined s generlly pplicble s well s pplying whole. There re some very common ptterns tht cn be generlly

More information

The Effect of Crumb Rubber Modifier (CRM) on the Performance Properties of Rubberized Binders in HMA pavements

The Effect of Crumb Rubber Modifier (CRM) on the Performance Properties of Rubberized Binders in HMA pavements The Effect of Crum Ruer Modifier (CRM) on the Performnce Properties of Ruerized Binders in HMA pvements Soon-Je Lee* Ph.D. Grdute Student Deprtment of Civil Engineering Clemson University Clemson, SC 29634-0911

More information

Small Business Cloud Services

Small Business Cloud Services Smll Business Cloud Services Summry. We re thick in the midst of historic se-chnge in computing. Like the emergence of personl computers, grphicl user interfces, nd mobile devices, the cloud is lredy profoundly

More information

WEB DELAY ANALYSIS AND REDUCTION BY USING LOAD BALANCING OF A DNS-BASED WEB SERVER CLUSTER

WEB DELAY ANALYSIS AND REDUCTION BY USING LOAD BALANCING OF A DNS-BASED WEB SERVER CLUSTER Interntionl Journl of Computers nd Applictions, Vol. 9, No., 007 WEB DELAY ANALYSIS AND REDUCTION BY USING LOAD BALANCING OF A DNS-BASED WEB SERVER CLUSTER Y.W. Bi nd Y.C. Wu Abstrct Bsed on our survey

More information

Operations with Polynomials

Operations with Polynomials 38 Chpter P Prerequisites P.4 Opertions with Polynomils Wht you should lern: Write polynomils in stndrd form nd identify the leding coefficients nd degrees of polynomils Add nd subtrct polynomils Multiply

More information

C-crcs Cognitive - Counselling Research & Conference Services (eissn: 2301-2358)

C-crcs Cognitive - Counselling Research & Conference Services (eissn: 2301-2358) C-crcs Cognitive - Counselling Reserch & Conference Services (eissn: 2301-2358) Volume I Effects of Music Composition Intervention on Elementry School Children b M. Hogenes, B. Vn Oers, R. F. W. Diekstr,

More information

Basic Analysis of Autarky and Free Trade Models

Basic Analysis of Autarky and Free Trade Models Bsic Anlysis of Autrky nd Free Trde Models AUTARKY Autrky condition in prticulr commodity mrket refers to sitution in which country does not engge in ny trde in tht commodity with other countries. Consequently

More information

FDIC Study of Bank Overdraft Programs

FDIC Study of Bank Overdraft Programs FDIC Study of Bnk Overdrft Progrms Federl Deposit Insurnce Corportion November 2008 Executive Summry In 2006, the Federl Deposit Insurnce Corportion (FDIC) initited two-prt study to gther empiricl dt on

More information

Recognition Scheme Forensic Science Content Within Educational Programmes

Recognition Scheme Forensic Science Content Within Educational Programmes Recognition Scheme Forensic Science Content Within Eductionl Progrmmes one Introduction The Chrtered Society of Forensic Sciences (CSoFS) hs been ccrediting the forensic content of full degree courses

More information

Utilization of Magnesium Hydroxide Produced by Magnesia Hydration as Fire Retardant for Nylon 6-6,6

Utilization of Magnesium Hydroxide Produced by Magnesia Hydration as Fire Retardant for Nylon 6-6,6 C O M U N I C A Ç Ã O Utiliztion of Mgnesium Hydroxide Produced y Mgnesi Hydrtion s Fire Retrdnt for Nylon 6-6,6 Sôni D.F. Roch Deprtmento de Engenhri Químic, UFMG Virgíni S.T. Ciminelli Deprtmento de

More information

improved (2, 3, 4, 5, 7, 8, 9, 10, 11). Some of the

improved (2, 3, 4, 5, 7, 8, 9, 10, 11). Some of the 8 FLORIDA STATE HORTICULTURAL SOCIETY, 963 EVALUATING SPRAY EQUIPMENT FOR FLORIDA CITRUS R. F. Brooks, W. L. Thompson nd P. T. Jutrs Florid Citrus Experiment Sttion Lke Alfred The first successful, lrge,

More information

2 DIODE CLIPPING and CLAMPING CIRCUITS

2 DIODE CLIPPING and CLAMPING CIRCUITS 2 DIODE CLIPPING nd CLAMPING CIRCUITS 2.1 Ojectives Understnding the operting principle of diode clipping circuit Understnding the operting principle of clmping circuit Understnding the wveform chnge of

More information

Curve Sketching. 96 Chapter 5 Curve Sketching

Curve Sketching. 96 Chapter 5 Curve Sketching 96 Chpter 5 Curve Sketching 5 Curve Sketching A B A B A Figure 51 Some locl mximum points (A) nd minimum points (B) If (x, f(x)) is point where f(x) reches locl mximum or minimum, nd if the derivtive of

More information

Straight pipe model. Orifice model. * Please refer to Page 3~7 Reference Material for the theoretical formulas used here. Qa/Qw=(ηw/ηa) (P1+P2)/(2 P2)

Straight pipe model. Orifice model. * Please refer to Page 3~7 Reference Material for the theoretical formulas used here. Qa/Qw=(ηw/ηa) (P1+P2)/(2 P2) Air lek test equivlent to IX7nd IX8 In order to perform quntittive tests Fig. shows the reltionship between ir lek mount nd wter lek mount. Wter lek mount cn be converted into ir lek mount. By performing

More information

The Chain Rule. rf dx. t t lim " (x) dt " (0) dx. df dt = df. dt dt. f (r) = rf v (1) df dx

The Chain Rule. rf dx. t t lim  (x) dt  (0) dx. df dt = df. dt dt. f (r) = rf v (1) df dx The Chin Rule The Chin Rule In this section, we generlize the chin rule to functions of more thn one vrible. In prticulr, we will show tht the product in the single-vrible chin rule extends to n inner

More information

Assessing authentically in the Graduate Diploma of Education

Assessing authentically in the Graduate Diploma of Education Assessing uthenticlly in the Grdute Diplom of Eduction Dr Mree DinnThompson Dr Ruth Hickey Dr Michelle Lsen WIL Seminr JCU Nov 12 2009 Key ides plnning process tht embeds uthentic ssessment, workintegrted

More information

LINEAR TRANSFORMATIONS AND THEIR REPRESENTING MATRICES

LINEAR TRANSFORMATIONS AND THEIR REPRESENTING MATRICES LINEAR TRANSFORMATIONS AND THEIR REPRESENTING MATRICES DAVID WEBB CONTENTS Liner trnsformtions 2 The representing mtrix of liner trnsformtion 3 3 An ppliction: reflections in the plne 6 4 The lgebr of

More information

Math 135 Circles and Completing the Square Examples

Math 135 Circles and Completing the Square Examples Mth 135 Circles nd Completing the Squre Exmples A perfect squre is number such tht = b 2 for some rel number b. Some exmples of perfect squres re 4 = 2 2, 16 = 4 2, 169 = 13 2. We wish to hve method for

More information

Math Review 1. , where α (alpha) is a constant between 0 and 1, is one specific functional form for the general production function.

Math Review 1. , where α (alpha) is a constant between 0 and 1, is one specific functional form for the general production function. Mth Review Vribles, Constnts nd Functions A vrible is mthemticl bbrevition for concept For emple in economics, the vrible Y usully represents the level of output of firm or the GDP of n economy, while

More information

Homework #6 Chapter 7 Homework Acids and Bases

Homework #6 Chapter 7 Homework Acids and Bases Homework #6 Chpter 7 Homework Acids nd Bses 18. ) H O(l) H 3O (q) OH (q) H 3 O OH Or H O(l) H (q) OH (q) H OH ) HCN(q) H O(l) H 3O (q) CN (q) H 3 O HCN CN Or HCN(q) H (q) CN (q) H CN HCN c) NH 3(q) H O(l)

More information

Dispersion in Coaxial Cables

Dispersion in Coaxial Cables Dispersion in Coxil Cbles Steve Ellingson June 1, 2008 Contents 1 Summry 2 2 Theory 2 3 Comprison to Welch s Result 4 4 Findings for RG58 t LWA Frequencies 5 Brdley Dept. of Electricl & Computer Engineering,

More information

Enterprise Risk Management Software Buyer s Guide

Enterprise Risk Management Software Buyer s Guide Enterprise Risk Mngement Softwre Buyer s Guide 1. Wht is Enterprise Risk Mngement? 2. Gols of n ERM Progrm 3. Why Implement ERM 4. Steps to Implementing Successful ERM Progrm 5. Key Performnce Indictors

More information

ORBITAL MANEUVERS USING LOW-THRUST

ORBITAL MANEUVERS USING LOW-THRUST Proceedings of the 8th WSEAS Interntionl Conference on SIGNAL PROCESSING, ROBOICS nd AUOMAION ORBIAL MANEUVERS USING LOW-HRUS VIVIAN MARINS GOMES, ANONIO F. B. A. PRADO, HÉLIO KOII KUGA Ntionl Institute

More information

Steps in gene expression: comparison of prokaryotic and eukaryotic cells

Steps in gene expression: comparison of prokaryotic and eukaryotic cells Steps in gene expression: comprison of prokryotic nd eukryotic cells DNA 1 Trnscript RNA 2 3 mrna RNA RNA Processing Trnsport Trnscription control Primry RNA Nucleus Cytoplsm mrna Degrdtion Trnsltion Control

More information

VoIP for the Small Business

VoIP for the Small Business Reducing your telecommunictions costs VoIP (Voice over Internet Protocol) offers low cost lterntive to expensive trditionl phone services nd is rpidly becoming the communictions system of choice for smll

More information

Lecture 3 Gaussian Probability Distribution

Lecture 3 Gaussian Probability Distribution Lecture 3 Gussin Probbility Distribution Introduction l Gussin probbility distribution is perhps the most used distribution in ll of science. u lso clled bell shped curve or norml distribution l Unlike

More information

Contextualizing NSSE Effect Sizes: Empirical Analysis and Interpretation of Benchmark Comparisons

Contextualizing NSSE Effect Sizes: Empirical Analysis and Interpretation of Benchmark Comparisons Contextulizing NSSE Effect Sizes: Empiricl Anlysis nd Interprettion of Benchmrk Comprisons NSSE stff re frequently sked to help interpret effect sizes. Is.3 smll effect size? Is.5 relly lrge effect size?

More information

COMPARISON OF SOME METHODS TO FIT A MULTIPLICATIVE TARIFF STRUCTURE TO OBSERVED RISK DATA BY B. AJNE. Skandza, Stockholm ABSTRACT

COMPARISON OF SOME METHODS TO FIT A MULTIPLICATIVE TARIFF STRUCTURE TO OBSERVED RISK DATA BY B. AJNE. Skandza, Stockholm ABSTRACT COMPARISON OF SOME METHODS TO FIT A MULTIPLICATIVE TARIFF STRUCTURE TO OBSERVED RISK DATA BY B. AJNE Skndz, Stockholm ABSTRACT Three methods for fitting multiplictive models to observed, cross-clssified

More information

Reasoning to Solve Equations and Inequalities

Reasoning to Solve Equations and Inequalities Lesson4 Resoning to Solve Equtions nd Inequlities In erlier work in this unit, you modeled situtions with severl vriles nd equtions. For exmple, suppose you were given usiness plns for concert showing

More information

GFI MilArchiver 6 vs Quest Softwre Archive Mnger GFI Softwre www.gfi.com GFI MilArchiver 6 vs Quest Softwre Archive Mnger GFI MilArchiver 6 Quest Softwre Archive Mnger Who we re Generl fetures Supports

More information

Section 7-4 Translation of Axes

Section 7-4 Translation of Axes 62 7 ADDITIONAL TOPICS IN ANALYTIC GEOMETRY Section 7-4 Trnsltion of Aes Trnsltion of Aes Stndrd Equtions of Trnslted Conics Grphing Equtions of the Form A 2 C 2 D E F 0 Finding Equtions of Conics In the

More information

Performance analysis model for big data applications in cloud computing

Performance analysis model for big data applications in cloud computing Butist Villlpndo et l. Journl of Cloud Computing: Advnces, Systems nd Applictions 2014, 3:19 RESEARCH Performnce nlysis model for big dt pplictions in cloud computing Luis Edurdo Butist Villlpndo 1,2,

More information

2001 Attachment Sequence No. 118

2001 Attachment Sequence No. 118 Form Deprtment of the Tresury Internl Revenue Service Importnt: Return of U.S. Persons With Respect to Certin Foreign Prtnerships Attch to your tx return. See seprte instructions. Informtion furnished

More information

Academic Plan of Study

Academic Plan of Study Deprtment of Chemistry Chemistry: BA Degree (120 Hours); BA Degree with Secondry Teching Licensure (120 Hours); BS Degree (120 Hours), BS with Biochemistry Option (120 Hours) College of Liberl Arts nd

More information

Health insurance exchanges What to expect in 2014

Health insurance exchanges What to expect in 2014 Helth insurnce exchnges Wht to expect in 2014 33096CAEENABC 11/12 The bsics of exchnges As prt of the Affordble Cre Act (ACA or helth cre reform lw), strting in 2014 ALL Americns must hve minimum mount

More information

Chromebook Parent/Student Information

Chromebook Parent/Student Information Chromebook Prent/Student Informtion 1 Receiving Your Chromebook Student Distribution Students will receive their Chromebooks nd cses during school. Students nd prents must sign the School City of Hmmond

More information

LECTURE #05. Learning Objectives. How does atomic packing factor change with different atom types? How do you calculate the density of a material?

LECTURE #05. Learning Objectives. How does atomic packing factor change with different atom types? How do you calculate the density of a material? LECTURE #05 Chpter : Pcking Densities nd Coordintion Lerning Objectives es How does tomic pcking fctor chnge with different tom types? How do you clculte the density of mteril? 2 Relevnt Reding for this

More information

Quality Evaluation of Entrepreneur Education on Graduate Students Based on AHP-fuzzy Comprehensive Evaluation Approach ZhongXiaojun 1, WangYunfeng 2

Quality Evaluation of Entrepreneur Education on Graduate Students Based on AHP-fuzzy Comprehensive Evaluation Approach ZhongXiaojun 1, WangYunfeng 2 Interntionl Journl of Engineering Reserch & Science (IJOER) ISSN [2395-6992] [Vol-2, Issue-1, Jnury- 2016] Qulity Evlution of Entrepreneur Eduction on Grdute Students Bsed on AHP-fuzzy Comprehensive Evlution

More information

242 Z. Huang, S. Zhang / J. Chromatogr. B 792 (2003) 241 247

242 Z. Huang, S. Zhang / J. Chromatogr. B 792 (2003) 241 247 Journl of Chromtogrphy B, 792 (2003) 241 247 www.elsevier.com/ locte/ chromb Confirmtion of mphetmine, methmphetmine, MDA nd MDMA in urine smples using disk solid-phse extrction nd gs q chromtogrphy mss

More information

Humana Critical Illness/Cancer

Humana Critical Illness/Cancer Humn Criticl Illness/Cncer Criticl illness/cncer voluntry coverges py benefits however you wnt With our criticl illness nd cncer plns, you'll receive benefit fter serious illness or condition such s hert

More information

Project Recovery. . It Can Be Done

Project Recovery. . It Can Be Done Project Recovery. It Cn Be Done IPM Conference Wshington, D.C. Nov 4-7, 200 Wlt Lipke Oklhom City Air Logistics Center Tinker AFB, OK Overview Mngement Reserve Project Sttus Indictors Performnce Correction

More information