Short CommunicationEnzymatic routes for the production of mono-and di-glucosylated derivatives of hydroxytyrosolAntonio Trincone,Eduardo Pagnotta,Annabella Tramice ⇑Istituto di Chimica Biomolecolare,Consiglio Nazionale delle Ricerche,Via Campi Flegrei 34,80072Pozzuoli,Naples,Italya r t i c l e i n f o Article history:Received 30August 2011Received in revised form 20October 2011Accepted 21October 2011Available online 30October 2011Keywords:a -Glucosidase Aplysia fasciata Transglycosylation Hydroxytyrosol Biocatalysisa b s t r a c tIn this work,a new eco-friendly procedure for the synthesis of hydroxytyrosol and tyrosol a -glycosidic derivatives was proposed by using the marine a -glucosidase from Aplysia fasciata ,and a commercial tyrosinase from mushroom for the bioconversion of tyrosol glycosidic derivatives into the corresponding hydroxytyrosol products.New hydroxytyrosol mono-and di-saccharide derivatives were synthesized at final concentrations of 9.35and 10.8g/l of reaction,respectively,and their antioxidant activity was evaluated by DPPH test.The best antioxidant agent resulted the (3,4-dihydroxyphenyl)ethyl-a -D -gluco-pyranoside;it showed a radical scavenging activity similar to that of the hydroxytyrosol,together with an increased hydrosolubility.This molecule could be a good response to many food industry demands,always in search of cheap antioxidants with nutritional properties to improve the nutritional value and the quality of foods.Ó2011Elsevier Ltd.All rights reserved.1.IntroductionOlive biophenols have attracted the attention of food and phar-maceutical industries first of all for their well-acquainted antioxidant activity (Obied et al.,2005).2-(4-Hydroxyphenyl)etha-nol (tyrosol,1)and 3,4-dihydroxyphenyl ethanol (hydroxytyrosol,2)represent the most abundant oil phenols (Vissers et al.,2004).Hydroxytyrosol,as well as being a powerful antioxidant and scavenger of free radicals,reduces,in fact,the risk of coronary heart disease and atherosclerosis (Visioli et al.,1995,2002)and it is involved in a mechanism of protection against oxidative DNA damage (Waterman and Lockwood,2007).Differently,tyrosol shows milder antioxidant properties (Damiani et al.,2003).Never-theless,it exerts a powerful protective effect against oxidative inju-ries in cell systems and improves the intracellular antioxidant defence systems (Mateos et al.,2008).In spite of their potential applications in the nutraceutical and pharmaceutical fields,few methods have been developed for synthesizing tyrosol and hydroxytyrosol glycosidic derivatives.In this paper,we screened the possibility to perform glucosyla-tion reactions of various phenolic compounds,including tyrosol and hydroxytyrosol and their structurally analogous compounds,by using the marine a -glucosidase from Aplysia fasciata .Interesting glucosylations at phenolic sites of some selected acceptors were ob-served,especially considering that phenolic hydroxyls are ineffi-ciently glycosylated by glycosidases (van Rantwijk et al.,1999).Among all molecules tested,tyrosol and hydroxytyrosol glycosyla-tion procedures were more deeply investigated.Tyrosol a -glycosidic derivatives were efficiently produced by direct glucosylation and in a second enzymatic step,these molecules were regioselectively oxi-dized by a commercial mushroom tyrosinase to give the hydroxyty-rosol a -glycosyl derivatives,possessing interesting radical scavenging activities.These results appeared of great interest when compared to enzymatic synthesis of salidroside,monoglucuronides derivatives of hydroxytyrosol,and tyrosol,previously reported in literature (Tong et al.,2004;Khymenets et al.,2006).2.Methods 2.1.GeneralTLC solvent systems:(A)(CH 3CN:H 2O,8:2,v/v);(B)(CH 3CN:H 2O,9:2,v/v).Compounds on TLC plates were visualized under UV light or charring with a -naphthol reagent.Other technical information were reported in Supplementary Section S.1.2.2.Enzyme sourceA clear enzymatic homogenate from A.fasciata visceral mass was prepared as previously described by Andreotti et al.(2006).Since the most abundant hydrolytic enzyme in A.fasciata visceral mass extract was an a -D -glucosidase activity,this enzy-matic solution (8.1mg total protein/ml;1.2U/mg,using p -nitro-0960-8524/$-see front matter Ó2011Elsevier Ltd.All rights reserved.doi:10.1016/j.biortech.2011.10.073Corresponding author.Tel.:+390818675070;fax:+390818041770.E-mail address:atramice@r.it (A.Tramice).phenyl a-D-glucopyranoside as substrate)was considered useful for this work purpose.One unit of a-D-glucosidase activity was defined as that amount of enzyme required to catalyze the release of1.0l mol of p-nitro-phenol per minute.Commercial mushroom tyrosinase(3900U/mg)from Sigma was used without further purification.2.3.Substrate specificity in transglycosylation reactions2-Phenylethanol,tyrosol and its similar compounds such as 2-(2-hydroxyphenyl)ethanol,2-(3-hydroxyphenyl)ethanol,hydro xytyrosol,(±)-1-phenyl-1,2-ethanediol,R(À)-and S(+)-1-phenyl-1,2-ethanediol,4-(2-methoxyethyl)phenol,N-acetyl-L-tyrosine ethyl ester monohydrate were tested as acceptors in analytical-scale transglycosylation reactions.Visceral mass homogenate from A.fasciata(16l l,0.153U of a-D-glucosidase activity)was added to1.5ml of10mM acceptorsolutions in50mM K-acetate buffer at pH 5.5,containing 150mM maltose.Enzymatic reactions were conducted under mag-netic stirring at34°C for48h up to maltose consumption and reg-ularly monitored by TLC analysis(system solvent A).2.4.Optimization of tyrosol and hydroxytyrosol glycosylation reactionsGlycosylation reactions were performed at different maltose (donor)molar excesses with respect to tyrosol(1)or hydroxytyro-sol(2)(acceptors).All reactions were carried out on analytical scale(total volume 1.5ml)in K-acetate buffer(50mM,pH5.5)at34°C,under mag-netic stirring,for48h and in presence of0.72U of a-D-glucosidase from A.fasciata per mmol of maltose,aiming to maintain a con-stant ratio between the enzymatic units and the mmol of initial maltose.Aliquots of200l l of each reaction were withdrawn at different time intervals(from t0to48h),stopped by enzyme denaturation at 100°C for2min and lyophilized.Collected aliquots were dissolved in D2O and analyzed by1H NMR experiments.In afirst set of reactions,acceptor(comp.1or2)concentration wasfixed at5mM and maltose amount was varied according to specific molar excesses with respect to acceptor.In tyrosol exper-iment,maltose:tyrosol molar ratios were 1.5,15,30,60;in hydroxytyrosol experiment,maltose:hydroxytyrosol molar ratios became5,15,30,60.In a second set of reactions,tyrosol was se-lected as sole acceptor and used at increasing concentrations(5, 10,30,60and180mM);transglycosylation reactions were performed at a maltose:tyrosolfixed molar ratio of15:1.2.5.Preparative enzymatic reactionsProduction of tyrosol and hydroxytyrosol glycosylated deriva-tives and their isolation and spectroscopic characterization were described in detail in Supplementary(Sections S.2.1and S.2.2). Tyrosol glycosides were produced starting from a tyrosol solution 180mM containing maltose with a molar excess of15(2.7M) and in presence of 1.94U of a-glucosidase from A.fasciata (0.2ml of enzymatic homogenate).2.6.Oxidation reactionsHydroxytyrosol(2)production was performed using commer-cial mushroom tyrosinase from Sigma,as reported in a previous paper(Espín et al.,2001).Process details were described in Supplementary(Section S.3.1).2-(4-Hydroxyphenyl)ethyl-a-D-glucopyranoside(1a),2-(4-hyd roxyphenyl)ethyl-a-D-maltoside(1b),and2-(4-hydroxyphenyl) ethyl-a-D-isomaltoside(1c),were oxidized to the corresponding hydroxytyrosol derivatives by a synthetic procedure reported in Supplementary Section S.3.2.2.7.Free-radical scavenging activityThe scavenging activity of hydroxytyrosol and its a-glucosyl,a-maltosyl and a-isomaltosyl derivatives was analyzed by using1,1-diphenyl-2-picrylhydrazyl(DPPH)essay.The reaction mixture contained0.75ml of substrate methanolic solutions at differentfi-nal concentrations(16.7,10,5and1.67l M)and1.5ml of50mM DPPH(final concentration33l M).After shaking,reaction systems were kept for10min in the dark,and then the absorbance at 517nm was measured.Experiments were carried out in triplicate and always on freshly made up solutions.Appropriate controls were performed by mixing0.75ml of MeOH(blank solution)with 1.5ml of DPPH solution.EC50of molecules,the amount of antiox-idant needed to decrease by50%the initial radical concentration, were evaluated.All experimental data of absorption decrease showed a linear correlation as to the concentration of the antioxi-dant compounds(straight line resulting from thefit by linear regression,r20.99–0.97);each point was the average of three determinations(SD<0.05).3.Results and discussion3.1.Substrate specificity in transglycosylation reactionsIn thefirst part of this work,the ability of a-D-glucosidase from A.fasciata to glycosylate various naturally occurring phenolic com-pounds with alcoholic and/or phenolic hydroxyls in their molecular skeleton(Section2.3),was analyzed and the regio-and chemo-selectivity in their transglycosylation processes were qualitatively examined.Reaction monitoring was carried out using a TLC system that we succeeded in discriminating between glycosylated prod-ucts(mono-or di-saccharides)in phenolic or alcoholic positions.A different qualitative distribution of products at4,24and48h was recorded.Accumulation of mono-and di-saccharidic deriva-tives was noticed for almost all acceptors up to24h.In many cases, glycosylations were not chemo-selective with the phenolic and alcoholic hydroxyls involvement,but after24h,a prevalent recov-ery of products glycosylated in alcoholic position was observed,as in the case of hydroxytyrosol,tyrosol and its regioisomers with phenolic hydroxyls in2and3aromatic positions.Furthermore,4-(2-methoxyethyl)phenol disaccharidic products were still present at24h,while monoglucosides of2-phenylethanol and N-acetyl-L-tyrosine ethyl ester monohydrate were partially or totally hydro-lyzed,respectively.Racemic1-phenyl-1,2-ethanediol and its pure enantiomers were poly-glycosylated and partially(racemic,R(À)) or totally(S+)hydrolyzed after24h.Results of this qualitative transglycosylation survey encouraged us to more deeply investigate the glycosylation reactions using tyrosol and hydroxytyrosol as acceptors.3.2.Production of tyrosol and hydroxytyrosol glycosydic derivatives: reaction conditions optimizationAiming to identify the best conditions for glycosylating tyrosol (1)and hydroxytyrosol(2),a series of reactions were conducted at different maltose concentrations(from7.5to300mM),constant acceptor concentration(5mM)and afixed enzyme amount.Ali-quots of each reaction were collected over time and analyzed by TLC(system solvent A)and1H NMR experiments.80 A.Trincone et al./Bioresource Technology115(2012)79–83Concerning tyrosol glycosylations,results were reported in Fig.1(a).Reactions B,C and D followed a similar trend up to 7h (al-most 50%of bioconversion).All reactions reached a plateau mostly at 24–31h.Process B was the most synthetically interesting reac-tion:56%of tyrosol glycosylation was achieved after 24h.Subse-quent experiments were conducted in more concentrated media to set up tyrosol glycosylation processes in which an improvement in the production efficiency of products could be obtained (Fig.1(b)).A fixed 15:1maltose:tyrosol molar ratio (B conditions,Fig.1(a)),was used.In particular,at 48h the reaction I (Fig.1(b))furnished a conversion of 70%,corresponding to 126mmol of reacted tyrosol/l of reaction.In these experimental conditions (pro-cess I),a semi-preparative reaction was performed and products were isolated and spectroscopically characterized (Supplementary Sections S.2.1and S.2.3).It was produced:(i)tyrosol monoglucosy-lated at alcoholic and phenolic positions (1a :2-(4-hydroxyphenyl)ethyl-a -D -glucopyranoside and 1b :2-(4-a -D -glucopyranosyloxy-phenyl)ethanol,in molar ratio 6.2:1respectively),with 41%of yield,corresponding to 20g of tyrosol monoglucosydes/l of reaction;(ii)53%of tyrosol diglucosylated derivatives (31g of products/l of reac-tion),of which the 78%were disaccharidic alcoholic derivatives in almost a 1:1mixture,(1c :2-(4-hydroxyphenyl)ethyl-a -D -malto-side,1d :2-(4-hydroxyphenyl)ethyl-a -D -isomaltoside)and the remaining 22%was a complex mixture of disaccharidic phenolic derivatives;(iii)6%of oligosaccharide derivatives.This final compo-sition indicated a prevalent presence of products glycosylated at primary alcoholic position,which represented the thermodynami-cally controlled products.A TLC monitoring of reaction over time showed that phenolic glucosylated product 1b was produced at the beginning of the reaction but was hydrolyzed more quickly than compound 1a .The preferential hydrolysis of phenolic monogluco-side 1b was proved by an hydrolysis experiment on a mixture 1:1of the two monosaccharidic derivatives 1a and 1b ,which was mon-itored by 1H NMR experiments,as described in Supplementary Sec-tion S.2.4.Concerning hydroxytyrosol glycosylation process,as described in Section 2.4,different results were obtained.The best conversion,with the lowest maltose molar excess (EM:30),was of 45%after 24h (data not shown).In these experimental conditions,aiming to isolate and characterize hydroxytyrosol saccharidic derivatives,we performed a semi-preparative reaction (Supplementary Section S.2.2).2.27mmol of reacted hydroxytyrosol (mono-and di-saccha-ridic derivatives)/l of reaction were produced,with a yield lower than 3.35mmol of tyrosol saccharidic derivatives/l of reaction which were obtained by the corresponding process C (67%of con-version,Fig 1(a)).Furthermore,it was discovered that hydroxytyro-sol glycosylation process was not chemo-and regio-selective at all;both alcoholic hydroxyl and two phenolic hydroxyls were involved in glycosylation processes and a mixture of all possible monogly-cosylated derivatives was identified by preparative reaction (55%of total products,2a :(3,4-dihydroxyphenyl)ethyl-a -D -glucopyran-oside,2b (3-(a -D -glucopyranosyloxy)-4-hydroxyphenyl)ethanol,2c (4-(a -D -glucopyranosyloxy)-3-hydroxyphenyl)ethanol,spectro-scopically characterized as described in Supplementary Sectiona102030405060708090100017243148Time ( h )A 1.5B 15C 30D 601020304050607080901000136242948Tyrosol conversion (%)Time ( h )bE 5F 10G 30H 60I 180tyrosol by a -D -glucosidase of Aplysia fasciata in different conditions.In (a)[tyrosol]was fixed at 5mM,working 1.5–60).In (b)maltose molar excess was fixed at 15in all reactions (E–I)conducted at different [tyrosol],calculated by the percentage ratio between the total integration value of all benzylic protonic signals of 2.76–2.68ppm for hydroxytyrosol derivatives)and the sum of integral values of the glycosylated derivatives hydroxytyrosol (2.65ppm).A.Trincone et al./Bioresource Technology 115(2012)79–8381S.2.3),together with a complex assortment of disaccharidic com-pounds.For these reasons,this synthesis was not estimated syn-thetically useful for the production of glycosydic derivative ofhydroxytyrosol.The efficient production of glucosylated hydroxytyrosol deriva-tives was planned by starting from tyrosol and by adopting a com-mercial mushroom tyrosynase as oxidizing agent of glycosylatedderivatives of tyrosol.The employed tyrosynase was used for thesynthesis of hydroxytyrosol from tyrosol,as previously described(Espín et al.,2001);however specificity of the enzyme on deriva-tives of tyrosol was unknown.3.3.Oxidation of tyrosol saccharidic derivativesStarting from glucosylated tyrosol derivatives and by using annew environmental friendly enzymatic approach,the correspond-ing catechol derivatives were pounds1a,1c,1d,re-sulted to be substrates for the adopted commercial tyrosinase andwere efficiently oxidized to2a,2d(3,4-dihydroxyphenyl)ethyl-a-D-maltoside and2e(3,4-dihydroxyphenyl)ethyl-a-D-isomalto-side)derivatives with yields of about50–60%(SupplementarySection S.3.2).Products structures were assigned by comparisonwith reagents NMR spectra.A modification of the multiplicities,chemical shifts and number of aromatic signals in the interval7.3–6.5d in1H NMR spectra proved the o-diphenol structure ofproducts(Supplementary Section S.2.3).3.4.Radical scavenging activityThe radical scavenging activity of hydroxytyrosol derivatives(compounds2a,2d,2e)was DPPH-tested and their activities com-pared with that of hydroxytyrosol(2).(3,4-Dihydroxyphenyl)ethyl-a-D-glucopyranoside(2a)showed similar radical scavenging potency to hydroxytyrosol activity(2):their EC50were10.00and 8.94l M,respectively(Fig.2).Radical scavenging activity of our products decreased upon increasing the carbohydrate chain; hydroxytyrosol a-maltosyl and a-isomaltosyl derivatives(2d,2e) possessed similar EC50value of28.69and25.15l M respectively. Nevertheless,although the antioxidant activity in disaccharidic derivatives was inferior to compound2,it was not trivial and any-way the disaccharidic units made the molecules more water-solu-ble and chemically stable.The best results were furnished by compound2a,in which,together with an increased solubility, the scavenging activity was as good as the aglycone(2).4.ConclusionsAn efficient enzymatic two-step procedure for the synthesis of hydroxytyrosol and tyrosol a-glycosidic derivatives based on a-glucosidase from A.fasciata and a commercial tyrosinase from mushroom,was described.Considering the whole enzymatic process for the formation of glycosidic derivatives of hydroxytyrosol from tyrosol,it was possi-ble to glycosylate regioselectively only the alcoholic primary posi-tion for producing hydroxytyrosol mono-and disaccharidic derivatives,with a reaction total yield which was of20%for each product.AcknowledgementsWe thank D.Melck,E.Castelluccio and A.Esposito(NMR service of Istituto di Chimica Biomolecolare,CNR,Pozzuoli,Italy)for their skilful assistance.Appendix A.Supplementary dataSupplementary data associated with this article can be found,in the online version,at doi:10.1016/j.biortech.2011.10.073. 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