International Journal of Molecular Sciences

Article Synthesis and Antiradical Activity of Isoquercitrin Esters with Aromatic Acids and Their Homologues

Eva Heˇrmánková-Vavˇríková, Alena Kˇrenková, Lucie Petrásková, Christopher Steven Chambers, Jakub Zápal, Marek Kuzma, KateˇrinaValentová * and Vladimír Kˇren Laboratory of Biotransformation, Institute of Microbiology, Czech Academy of Sciences, Vídeˇnská 1083, CZ-142 20 Prague, Czech Republic; [email protected] (E.H.-V.); [email protected] (A.K.); [email protected] (L.P.); [email protected] (C.S.C.); [email protected] (J.Z.); [email protected] (M.K.); [email protected] (V.K.) * Correspondence: [email protected]; Tel.: +420-296-442-509

Academic Editor: Yujiro Hayashi Received: 27 April 2017; Accepted: 13 May 2017; Published: 17 May 2017

Abstract: Isoquercitrin, (IQ, quercetin-3-O-β-D-glucopyranoside) is known for strong chemoprotectant activities. Acylation of flavonoid glucosides with carboxylic acids containing an aromatic ring brings entirely new properties to these compounds. Here, we describe the chemical and enzymatic synthesis of a series of IQ derivatives at the C-600. IQ benzoate, phenylacetate, phenylpropanoate and cinnamate were prepared from respective vinyl esters using Novozym 435 (Lipase B from Candida antarctica immobilized on acrylic resin). The enzymatic procedure gave no products with “hydroxyaromatic” acids, their vinyl esters nor with their benzyl-protected forms. A chemical protection/deprotection method using Steglich reaction yielded IQ 4-hydroxybenzoate, vanillate and gallate. In case of p-coumaric, caffeic, and , the deprotection lead to the saturation of the double bonds at the phenylpropanoic moiety and yielded 4-hydroxy-, 3,4-dihydroxy- and 3-methoxy-4-hydroxy-phenylpropanoates. Reducing capacity of the cinnamate, gallate and 4-hydroxyphenylpropanoate towards Folin-Ciocalteau reagent was significantly lower than that of IQ, while other derivatives displayed slightly better or comparable capacity. Compared to isoquercitrin, most derivatives were less active in 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, but they showed significantly better 2,20-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid, ABTS) scavenging activity and were substantially more active in the inhibition of tert-butylhydroperoxide induced lipid peroxidation of rat liver microsomes. The most active compounds were the hydroxyphenylpropanoates.

Keywords: isoquercitrin; aromatic acid; ; ; antioxidant activity; Novozym 435; lipase; DPPH; lipoperoxidation

1. Introduction Plant are attractive for researchers and food producers due to their free radical scavenging and other, mostly positive, biological activities. Generally low toxicity of the polyphenols is another positive feature. One of the most popular flavonoids with a large number of health benefits is quercetin [1]. Some controversies concerning its possible mutagenic effects (positive Ames test) were closed and this substance now has a Generally Recognized As Safe (GRAS) status from the United States Food & Drug Administration (FDA) [2]. Its glucosylated form is isoquercitrin (1, IQ, quercetin-3-O-β-D-glucopyranoside, Figure1), which can be found in fruits (apples, berries), vegetables (onions), medicinal plants (St. John’s worth) and in various plant-derived beverages (tea, wine) [3,4]. IQ is known as a strong chemoprotectant [4] against cardiovascular diseases [5], asthma [6], and diabetes [7]. Recently, an efficient method of biocatalytic production of pure IQ from rutin was

Int. J. Mol. Sci. 2017, 18, 1074; doi:10.3390/ijms18051074 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2017, 18, 1074 2 of 14 Int. J. Mol. Sci. 2017, 18, 1074 2 of 14 wasdeveloped developed [8,9 ][8,9] based based on on the the use use of of thermophilic thermophilicα α-L--rhamnosidaseL-rhamnosidase [ [10]10] fromfrom Aspergillus terreus terreus heterologouslyheterologously expressed expressed in in PichiaPichia pastoris pastoris,, which which affords affords 11 inin very very high high yields yields and and purity purity (typically (typically 97–99%).97–99%).

β FigureFigure 1. IsoquercitrinIsoquercitrin ( (11,, quercetin quercetin 3- 3-O--β--DD-glucopyranoside).-glucopyranoside).

AcylationAcylation of of flavonoid glucosides glucosides with with carboxylic carboxylic acids acids containing containing an aromatic an aromatic ring brings ring structuralbrings structural diversity diversityto the flavonoid to the skeleton flavonoid and often skeleton alters and the physico-chemical often alters the (co-pigmentation, physico-chemical increased(co-pigmentation, stability) increasedand physiological stability) (UV and resistivity, physiological radical (UV scavenging resistivity, capacity) radical scavenging properties capacity) of these naturalproperties and of thesesemi-synthetic natural andmolecules semi-synthetic [11,12]. moleculesFor example, [11,12 ].the For presence example, of the p-coumaroyl- presence of glucopyranosylp-coumaroyl-glucopyranosyl units in delphinidin-type units in delphinidin-type anthocyanidins anthocyanidins from Dianella from Dianellaberries wasberries found was foundto be responsibleto be responsible for their for intensive their intensive blue color, blue at color, physiological at physiological pH values, pH values,due to co-pigmentation due to co-pigmentation related bathochromicrelated bathochromic shift and shiftpigment and stabilization pigment stabilization [13]. Similarly, [13]. chrysin Similarly, glucoside chrysin acylated glucoside with acylated methyl pwith-methoxycinnamic methyl p-methoxycinnamic acid showed more acid efficient showed complexation more efficient of complexationAl3+ and stronger of Albinding3+ and to stronger bovine serumbinding albumin to bovine (BSA) serum than albuminthe parent (BSA) compound than the [14] parent. Tiliroside compound (p-coumaroyl [14]. Tiliroside kaempferol (p-coumaroyl glucoside) fromkaempferol Tilia glucoside)argentea from(Linden)Tilia argenteaexhibited(Linden) a exhibitedstrong hepatoprotective a strong hepatoprotective effect effectagainst against D- galactosamine/lipopolysaccharideD-galactosamine/lipopolysaccharide induced induced liver liver injury injury in in mice mice [15]. [15]. Also, Also, a a semi-synthetic introductionintroduction of of aromatic aromatic acyl acyl groups groups into into the the sugar sugar moiety moiety of of IQ IQ resulted resulted in in improved improved thermostability thermostability andand light-sensitivity light-sensitivity [11]. [11]. AcylAcyl derivatives derivatives of of flavonol flavonol gluc glucosidesosides can can be be prepared prepared chemically chemically or orenzymatically. enzymatically. The The use use of chemicalof chemical acylation acylation methods methods generally generally proceeds proceeds via via complex complex multistep multistep synthesis synthesis with with several protection/deprotectionprotection/deprotection strategies, strategies, while while the the enzy enzymatic-catalyzedmatic-catalyzed reactions reactions are are more more practical practical and and straightforwardstraightforward due due to to regioselectivity regioselectivity at at primar primaryy alcoholic alcoholic groups groups on on sugar sugar moieties moieties and and mild mild reactionreaction conditions. conditions. Different Different enzymes enzymes were were used used for for acylation acylation of of IQ: IQ: CandidaCandida antarctica antarctica lipaselipase B B [16], [16], subtilisinsubtilisin [17] [17] or or enzymatic reaction system from cultured cells of IpomoeaIpomoea batatasbatatas [[18].18]. Quercetin Quercetin (isoquercitrin(isoquercitrin aglycone) aglycone) is is usually usually the the starting starting material material for forchemical chemical preparation preparation of IQ of or IQ other or otherO-β- DO-glucosylated-β-D-glucosylated or O- orβ-DO-glucuronidated-β-D-glucuronidated quercetin quercetin derivatives derivatives [19]. The [19]. chemical The chemical synthesis synthesis of IQ coumaroylof IQ coumaroyl was also was initiated also initiated from quercetin from quercetin [20]. [20]. InIn this this work, work, we we aimed aimed to to study chemical or or chemoenzymatic modifications modifications of of IQ IQ that that lead lead to to alteredaltered biological biological activity. activity. We We have have prepared prepared a acomplex complex panel panel of ofIQ IQ derivatives derivatives 2–112–,11 substituted, substituted at C-6at C-6″ OH00 OH with with carboxylic carboxylic acids acids containing containing an an aromatic aromatic ring ring (benzoic (benzoic (12 (12),), phenylacetic phenylacetic ( (1313),), phenylpropanoicphenylpropanoic ( (1414),), cinnamic cinnamic ( (1515),), 4-hydroxybenzoic 4-hydroxybenzoic ( (1616),), vanillic vanillic ( (1717),), gallic gallic ( (1818),), pp-coumaric-coumaric ( (1919),), caffeiccaffeic ( (2020),), ferulic ferulic ( (2121)))) using using IQ IQ as as the the starting starting material material for for both both enzymatic enzymatic and and chemical chemical approaches. approaches. TheThe antiradical and anti-lipoperoxidant activities of all derivatives were determined.

2.2. Results Results and and Discussion Discussion

2.1.2.1. Preparation Preparation of of Isoquercitrin Isoquercitrin Esters Esters ( (2–5)) by by Enzymatic Enzymatic Approach Approach WhileWhile the the chemical chemical modifications modifications of of flavonoids wi withth numerous numerous reactive reactive hydroxyl hydroxyl groups groups require require multistepmultistep protection/deprotection protection/deprotection strategies, strategies, often often including including aggressive aggressive and and toxic toxic chemicals, chemicals, lipases lipases preferprefer stereoselective stereoselective acylation acylation of of primary primary alcoholic alcoholic groups groups [21]. [21 ].Lipase-catalyzed Lipase-catalyzed preparation preparation of C- of 00 ® 6C-6″ acylacyl derivatives derivatives of IQ of ( IQ2–5 ()2 was–5) wasaccomplished accomplished by Novozym by Novozym 435®—( 435Candida—(Candida antarctica antarctica lipase lipaseCAL- B immobilized on an acrylic resin, Scheme 1). We have successfully used vinyl esters of benzoic, phenylacetic, phenylpropanoic, cinnamic acids 22–25 [22,23] (Figure 2), as donors, and therefore have Int. J. Mol. Sci. 2017, 18, 1074 3 of 14

CAL-B immobilized on an acrylic resin, Scheme1). We have successfully used vinyl esters of benzoic, Int.Int. J.J. Mol.Mol. Sci.Sci. 20172017,, 1818,, 10741074 33 ofof 1414 phenylacetic, phenylpropanoic, cinnamic acids 22–25 [22,23] (Figure2), as donors, and therefore have achievedachieved aa faster faster lipase-catalyzed lipase-catalyzed reactionreaction withwith aa stronger stronger shift shift of of reaction reaction equilibrium equilibrium towards towards the the productsproducts 22––55... TheThe ratioratio betweenbetween between IQIQ IQ andand and vinylvinyl vinyl esterester ester ofof of respecrespec respectivetivetive aromaticaromatic aromatic acidacid acid waswas was 1:51:5 1:5 inin in drydry dry acetone.acetone. acetone.

OO

RORO

1515 RR == H H 25 R = CH=CH 25 R = CH=CH22 FigureFigureFigure 2.2.2. AromaticAromaticAromatic acidsacids acids 121212–––151515 andandand theirtheir their activatedactivated activated formsforms 2222––2525 usedused inin thethe enzymaticenzymaticenzymatic approach approachapproach [ 22[22,23].[22,23].,23].

IQ derivatives 2, 4, and 5 were prepared previously [11] using Chirazyme L-2 or Amano-PS as IQ derivatives 2, 4, and 5 were prepared previously [[11]11] usingusing ChirazymeChirazyme L-2 or Amano-PSAmano-PS as catalysts and vinyl esters of respective aromatic acids were used as acyl donors. However, formation catalysts and vinyl esters of respective aromatic acidsacids were used as acyl donors. However, formation of these products was confirmed only by 1313C NMR and MS, but 11H NMR was missing. NMR and MS of these products was confirmedconfirmed onlyonly byby 13C NMR and MS, but 1H NMR was missing. NMR and MS data are missing completely in the work of Nakajima et al. [18], who claimed the preparation of IQ data are missing completely in the work of Nakajima et al. [[18],18], who claimed the preparation of IQ cinnamate (5) with an enzymatic system from cultured cells of Ipomoea batatas. In contrast, we present cinnamate (5) with an enzymatic system from cultured cells of Ipomoea batatas.. In contrast, we present here full structural data of all these esters (see Section 3.4.1 and Tables S1–S3 and Figures S1–S8 in here fullfull structuralstructural datadata ofof all all these these esters esters (see (see Section Section 3.4.1 3.4.1 and and Tables Tables S1–S3 S1–S3 and and Figures Figures S1–S8 S1–S8 in the in the Supplementary Materials). Supplementarythe Supplementary Materials). Materials).

22,, 2222 RR == 33,, 2323 RR == 44,, 2424 RR == 55,, 2525 RR ==

SchemeScheme 1.1. PreparationPreparationPreparation ofof isoquercitrinisoquercitrin estersesters 22––55 byby enzymaticenzymatic methodology.methodology.

ChemoenzymaticChemoenzymatic preparation preparation of of isoquercitrin isoquercitrin esters esters with with both both cinnamic, cinnamic, pp-coumaric-coumaric and and ferulicferulic acidsacids has has been been described described previously previously asas aa three-stepthree-stthree-stepep synthesissynthesissynthesis [[17].[17].17]. First, First, methyl methyl malonate malonate was was introducedintroduced toto to thethe the primaryprimary primary OHOH OH ofof of isoquercitrinisoquercitrin isoquercitrin inin in thethe the presencepresence presence ofof ofthethe the proteaseprotease protease subtilisin,subtilisin, subtilisin, followedfollowed followed byby hydrolysisbyhydrolysis hydrolysis ofof the ofthe the methoxycarbonymethoxycarbony methoxycarbonylll groupgroup group catalyzedcatalyzed catalyzed byby by biophinebiophine biophine eses esterase.terase.terase. TheThe The finalfinal final stepstep step waswas was the the KnoevenagelKnoevenagel reactionreactionreaction with withwith the thethe corresponding correspondingcorresponding aldehyde, aldehyde,aldehyde, which whichwhich condensed condensedcondensed with malonic withwith IQmalonicmalonic monoester IQIQ monoestertomonoester afford IQ totop afford-coumarateafford IQIQ pp-coumarate-coumarate or ferulate. oror ferulate.ferulate. ToTo obtain obtain IQ IQ estersesters withwith acidsacids containingcontaining containing aa a hydroxyaromatichydroxyaromatic hydroxyaromatic moietymoiety moiety (4-hydroxybenzoic,(4-hydroxybenzoic, (4-hydroxybenzoic, vanillic,vanillic, vanillic, gallic,gallic, pp-coumaric,-coumaric, caffeic,caffeic,caffeic, and andand ferulic, ferulic,ferulic,16 1616––21–2121, Figure,, FigureFigure3) 3)3) CAL-B CAL-BCAL-B catalyzed catalyzedcatalyzed reactions reactionsreactions (up (up(up to 12 toto days, 1212 days,days, 45 ◦ 4545C) °C)were°C) werewere tested testedtested with withwith intact intactintact acids, acids,acids, their theirtheir vinyl vinylvinyl esters, estersesters acetyl-protected,, acetyl-protectedacetyl-protected vinyl vinylvinyl esters estersesters andbenzyl-protected andand benzyl-protectedbenzyl-protected acids. acids.However,acids. However,However, the enzymatic thethe enzymaticenzymatic reactions reactionsreactions did not diddid afford notnot afaf anyfordford products anyany productsproducts with IQ withwith and IQIQ the andand non-protected thethe non-protectednon-protected acids acids16acids–21 .1616 In––21 contrast21.. InIn contrastcontrast to the previously toto thethe previouslypreviously published publishedpublished results with resultsresultsp-coumaric withwith pp-coumaric-coumaric acid (no spectroscopic acidacid (no(no spectroscopicspectroscopic data given dataatdata all) givengiven [24], vinylestersatat all)all) [24],[24], ofvinylestersvinylesters the acids 16ofof– the21thedid acidsacids not 1616 afford––2121 diddid the notnot expected affordaffordproducts thethe expectedexpected with products IQproducts (Novozym withwith 435, IQIQ (Novozymup(Novozym to 12 days, 435,435, 45 upup◦ C). toto 1212 When days,days, the 4545 aromatic°C).°C). WhenWhen OHs thethe aromwerearomatic protectedatic OHsOHs werewere by acetylation, protectedprotected byby the acetylation,acetylation, vinyl esters thethe yielded vinylvinyl esterswithesters isoquercitrin yieldedyielded withwith (Novozymisoquercitriisoquercitrin 435,n (Novozym(Novozym 2 days, 435, 45435,◦ C)22 days,days, only 4545 undesired °C)°C) onlyonly undesiredundesired isoquercitrin isoquercitrinisoquercitrin 300,600-di-O 33-acetate″″,6,6″″-di--di- O(confirmedO-acetate-acetate (confirmed(confirmed by MS and by NMR).by MSMS Usingandand NMR).NMR). the benzyl-protected UsinUsingg thethe benzyl-protectedbenzyl-protected 4-hydroxybenzoic 4-hydroxybenzoic4-hydroxybenzoic (26)[25], vanillic ((2626 (27)) )[[25],[25],26], vanillicgallicvanillic (28 ((2727)[))27 [26],[26],], p -coumaric gallicgallic ((2828)) ([27],[27],29), caffeicpp-coumaric-coumaric (30)[ (28(2929]),), and caffeiccaffeic ferulic ((3030)) ( 31[28][28])[ 29andand] acids, ferulicferulic prepared ((3131)) [29][29] according acids,acids, preparedprepared to the accordingpreviousaccording procedures toto thethe previousprevious (Figure proceduresprocedures3, Section (Figure2.2(Figure), enzymatic 3,3, SectSectionion reactions 2.2),2.2), enzymaticenzymatic (2 days, 45reactionsreactions◦C) were (2(2 days,days, also unsuccessful, 4545 °C)°C) werewere alsoinalso this unsuccessful,unsuccessful, case probably inin due thisthis to casecase bulk probablyprobably acyl donors. duedue Chemoenzymatic toto bulkbulk acylacyl donors.donors. synthesis ChemoenzymaticChemoenzymatic is therefore not synthesissynthesis completely isis thereforetherefore notnot completelycompletely versatileversatile andand IQIQ estersesters withwith aromaticaromatic acidsacids 1616––2121 andand therefore,therefore, theirtheir estersesters couldcould notnot bebe preparedprepared byby thisthis method.method. ThThus,us, thethe chemicalchemical approachapproach forfor compoundcompound 66––1111 synthesissynthesis waswas chosen.chosen. Int. J. Mol. Sci. 2017, 18, 1074 4 of 14 versatile and IQ esters with aromatic acids 16–21 and therefore, their esters could not be prepared by thisInt.method. J. Mol. Sci. 2017 Thus,, 18, the1074 chemical approach for compound 6–11 synthesis was chosen. 4 of 14

Int. J. Mol. Sci. 2017, 18, 1074 4 of 14

FigureFigure 3. 3.Aromatic Aromatic acidsacids 16–21 and their their protected protected forms forms 2626–31–31 usedused for for the the chemical chemical approach. approach.

2.2.2.2. Preparation Preparation of of Isoquercitrin Isoquercitrin EstersEsters (6 –11) by by Chemical Chemical Approach Approach ProtectionFigure of3. Aromaticthe phenolic acids 16OH–21 andgroups their ofprotected the acids forms 16 26––2131 (seeused forSection the chemical 2.1) and approach. of IQ (1) was Protection of the phenolic OH groups of the acids 16–21 (see Section 2.1) and of IQ (1) was required for the chemical synthesis of IQ esters 6–11. Selective protection of complex polyphenols, required2.2. Preparation for the chemical of Isoquercitrin synthesis Esters of (6 IQ–11 esters) by Chemical6–11. Approach Selective protection of complex polyphenols, such as isoquercitrin, is complicated and protection groups have to be chosen very carefully due to such as isoquercitrin, is complicated and protection groups have to be chosen very carefully due to the presenceProtection of many of thehydroxy- phenolic grou OHps groups with different of the acids reactivity. 16–21 First,(see Section the primary 2.1) and alcoholic of IQ (group1) was of theglucose presencerequired was of forselectively many the chemical hydroxy- protected synthesis groups using of with tertIQ esters-butyldimethylsilyl different 6–11. reactivity.Selective chlorideprotection First, the (TBDMSCl) of primary complex alcoholicin polyphenols, the presence group of glucoseof imidazole.such was as isoquercitrin, selectively Silylated isoquercitrin protected is complicated using 32 andwastert protection then-butyldimethylsilyl benzylated groups withhave chloridebenzylto be chosen bromide (TBDMSCl) very to carefully afford in the33 due in presence 37%to ofyield. imidazole.the Fully presence protected Silylated of many isoquercitrin hydroxy- grou 3332ps waswas with desilylated then different benzylated reactivity. with tetrabutylammonium with First, benzyl the primary bromide alcoholic fluoride to afford group to 33afford ofin 37% yield.34 inglucoseFully 82% yield. protectedwas selectivelyProtection isoquercitrin protectedwith benzoyl33 usingwas group tert desilylated-butyldimethylsilyl (BzCl) proved with tetrabutylammonium to bechloride impracticable (TBDMSCl) due fluoride into thethe presencemigration to afford 34 inof 82% Bzof yield.groupsimidazole. Protection to C-6Silylated″ OH with afterisoquercitrin benzoyl desilylation group32 was (Scheme (BzCl)then benzylated proved2). to with be impracticablebenzyl bromide due to afford to the 33 migration in 37% of yield. Fully protected00 isoquercitrin 33 was desilylated with tetrabutylammonium fluoride to afford Bz groupsFree toC-6 C-6″ OHOH afterof compound desilylation 34 (Scheme was then2). esterified by the Steglich reaction (N,N′- 34 in 82% yield. Protection with benzoyl group (BzCl) proved to be impracticable due to the migration dicyclohexylcarbodiimideFree C-600 OH of (DCC), compound 4-dimethylaminopyridine34 was then esterified (DMAP)) with by theacids Steglich26–31 yielding reaction of Bz groups to C-6″ OH after desilylation (Scheme 2). (Nrespective,N0-dicyclohexylcarbodiimide intermediates 35–40. Final (DCC), deprotection 4-dimethylaminopyridine was accomplished by (DMAP)) hydrogenolysis with with acids Pd-C26– 31 Free C-6″ OH of compound 34 was then esterified by the Steglich reaction (N,N′- yielding 6–11 (Figure 4). In the case of compounds 6–8, their expected structures were confirmed by yieldingdicyclohexylcarbodiimide respective intermediates (DCC),35 4-dimethylaminopyridine–40. Final deprotection (DMAP)) was accomplished with acids by26– hydrogenolysis31 yielding withNMR.respective Pd-C NMR yielding andintermediates MS6– have11 (Figure revealed35–404. Final). that In deprotection the du casering hydrogenolytic of compoundswas accomplishe debenzylation6–d8, by their hydrogenolysis expected of 38–40 structures ,with the doublePd-C were confirmedbondsyielding at bythe 6 NMR.– phenylpropenoic11 (Figure NMR 4). and In the MS moieties case have of compounds revealedwere hydrogenated that 6–8 during, their expected forming hydrogenolytic structures the saturated debenzylation were confirmedproducts of 9by–3811 – 40, the(Scheme doubleNMR. 2). bondsNMR and at the MS phenylpropenoic have revealed that moieties during hydrogenolytic were hydrogenated debenzylation forming of the 38– saturated40, the double products 9–11 (Schemebonds at 2the). phenylpropenoic moieties were hydrogenated forming the saturated products 9–11 (Scheme 2).

Scheme 2. Preparation of isoquercitrin esters 6–11 by chemical methodology. Reagents and conditions: (i) imidazole, tert-butyldimethylsilyl chloride, dimethylformamide (DMF), 25 °C, 24 h; SchemeScheme 2. Preparation 2. Preparation of isoquercitrin of isoquercitrin esters esters6–11 by6–11 chemical by chemical methodology. methodology. Reagents Reagents and conditions: and (ii) NaH, BnBr, DMF, 25 °C, 24 h; (iii) tetrabutylammonium fluoride , tetrahydrofuran (THF) 25 °C, 24 (i) imidazole,conditions:tert (i)-butyldimethylsilyl imidazole, tert-butyldimethylsilyl chloride, dimethylformamide chloride, dimethylformamide (DMF), 25 ◦(DMF),C, 24 h; 25 (ii) °C, NaH, 24 h; BnBr, h; (iv)(ii) perBnNaH, BnBr,aromatic DMF, acids 25 °C, 26 –2431 h;, 4-dimethylaminopyridine (iii) tetrabutylammonium fluoride , N,N′-dicyclohexylcarbodiimide , tetrahydrofuran (THF) 25 °C,(DCC), 24 DMF, 25 ◦C, 24 h; (iii) tetrabutylammonium fluoride , tetrahydrofuran (THF) 25 ◦C, 24 h; (iv) perBn 25 °C,h; (iv) 24 h;perBn (v) H aromatic2-Pd/C, acidsEtOH:THF 26–31, 4-dimethylaminopyridine1:1, 25 °C, 12 h. , N,N′-dicyclohexylcarbodiimide (DCC), aromatic acids 26–31, 4-dimethylaminopyridine , N,N0-dicyclohexylcarbodiimide (DCC), 25 ◦C, 24 h; 25 °C, 24 h; (v) H2-Pd/C, EtOH:THF◦ 1:1, 25 °C, 12 h. (v) H2-Pd/C, EtOH:THF 1:1, 25 C, 12 h. Int. J. Mol. Sci. 2017, 18, 1074 5 of 14 Int. J. Mol. Sci. 2017, 18, 1074 5 of 14

OH OH 3´ OH 4´ OH 8 HO O 2 HO O OH 2´´ OH 6 4 HO 3´´ 4´´ O 3 O OH o HO O O i m O OH OH O 6´´ O O 1´´ 5´´ 1´´´ OH O O p 2 3 OH OH OH OH

HO O HO O OH OH O O O HO OH O HO O 3´´´ O OH OH O O 1´´´ OH O 2´´´ O 4 5

OH OH OH OH HO O HO O OH OH HO O O O OH O HO O O O OH OH O O OH O O 7 OH 6 OH

OH OH OH OH

HO O HO O OH O OH O HO OH HO O O OH O OH OH O O O OH O O 9 OH OH 8 OH OH OH OH OH

HO O HO O OH OH O HO O O HO OH O OH O OH O O OH O O OH O O 10 OH 11 OH Figure 4. Proposed structures of the isoquercitrin esters 2–11. Figure 4. Proposed structures of the isoquercitrin esters 2–11. Following methods for selective deprotection of compounds 38–40 were tested to preserve the Followingdouble bond: methods (1) Inspired for selectiveby partial deprotection successful preparation of compounds of isoquercitrin38–40 were p-coumarate tested to from preserve the doubleperbenzylated bond: intermediate (1) Inspired [20], by partialpreservation successful of the double preparation bond via of catalytic isoquercitrin hydrogenationp-coumarate using from perbenzylated1,4-cyclohexadiene/Pd-C intermediate was [20 tried.], preservation However, debenzylation of the double failed bond even viaafter catalytic48 h; (2) TiCl hydrogenation4 in dry usingDCM 1,4-cyclohexadiene/Pd-C at −20 °C under Ar for was1 h, tried.previously However, used for debenzylationsynthesis of 1,2,3,4,6-penta-caffeoyl- failed even afterD- 48 h; glucopyranose with intact olefinic ◦bonds [30], gave only the saturated product 10 in our hands; (3) (2) TiCl4 in dry DCM at −20 C under Ar for 1 h, previously used for synthesis of Different polyhydroxycinnamic acids were reported to be debenzylated using AlCl3/DMAP in dry 1,2,3,4,6-penta-caffeoyl-D-glucopyranose with intact olefinic bonds [30], gave only the saturated DCM (0 °C, 48 h) [31], debenzylation of our compound 39 using this approach was unsuccessful as productno reaction10 in our occurred. hands; (3)Therefore, Different we polyhydroxycinnamic were unable to obtain acidsrespective were cinnamic reported acid to bederivatives. debenzylated ◦ usingNevertheless, AlCl3/DMAP the compounds in dry DCM 9–11 (0 (FigureC, 48 4) h) obtained [31], debenzylation have not been reported of our compound to date, isoquercitrin39 using this approach4-hydroxyphenylpropanoate was unsuccessful as no( reaction9), 3,4-dihydroxyphenylpropanoate occurred. Therefore, we were (10 unable) and to obtain4-hydroxy- respective cinnamic3-methoxyphenylpropanoate acid derivatives. Nevertheless, (11) were thus the included compounds into our9 panel–11 (Figure of isoquercitrin4) obtained derivatives have notwith been reportedaromatic to date, acids isoquercitrin and their homologues 4-hydroxyphenylpropanoate for the determination of ( 9their), 3,4-dihydroxyphenylpropanoate antioxidant activity. (10) and 4-hydroxy-3-methoxyphenylpropanoate (11) were thus included into our panel of isoquercitrin 2.3. Antiradical Activity derivatives with aromatic acids and their homologues for the determination of their antioxidant activity. Complex antioxidant activity of all isoquercitrin esters 2–11 (Figure 4) was evaluated and 2.3. Antiradicalcompared with Activity that of isoquercitrin (1, Table 1). The reducing capacity of IQ cinnamate (5), gallate (8) and 4-hydroxyphenylpropanoate (9) towards Folin-Ciocalteau reagent (FCR), which is performed Complex antioxidant activity of all isoquercitrin esters 2–11 (Figure4) was evaluated and in aqueous milieu at alkaline pH, was approximately half of that of 1 (0.5 vs. 1.1 gallic acid comparedequivalents, with thatGAE). of This isoquercitrin is probably (1 related, Table to1). th Thee increased reducing lipophilicity capacity of these IQ cinnamate compounds. ( 5 ),On gallate the (8) and 4-hydroxyphenylpropanoate (9) towards Folin-Ciocalteau reagent (FCR), which is performed in aqueous milieu at alkaline pH, was approximately half of that of 1 (0.5 vs. 1.1 gallic acid equivalents, GAE). This is probably related to the increased lipophilicity of these compounds. On the other hand, IQ phenylacetate (3), phenylpropanoate (4) vanillate (7) and 4-hydroxy-3-methoxyphenylpropanoate Int. J. Mol. Sci. 2017, 18, 1074 6 of 14

(11) displayed slightly better (1.5 GAE) reducing capacity than isoquercitrin and the rest of the derivatives were not significantly different from 1. Compared to isoquercitrin, compounds 3, 9, and 10 had comparable activity (IC50, i.e., the concentration exhibiting 50% effect, of 1.4–1.9 µM) in 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and all other derivatives were less active (IC50 2.2–11.5 µM with compound 6 being the least effective). In contrast, most of the derivatives showed significantly better ABTS scavenging activity than isoquercitrin (1.98 trolox equivalents, TE). The loss of activity was observed only in the case of IQ phenylacetate (3) and cinnamate (5). Compounds 2, 7, 9, 10 and 11 displayed the strongest activity (over 7 trolox equivalents (TE)), followed by IQ gallate (8) with 5.7 TE. Finally, isoquercitrin derivatives, except for 5, were substantially more active than isoquercitrin (IC50 = 972 µM) in the inhibition of lipid peroxidation of rat liver microsomes induced by tert-butylhydroperoxide. Compounds 8 (IC50 = 6.6 µM) and 3 (IC50 = 6.8 µM) were the most active, but strong activity was noted also for originally unwanted compounds 9–11 (IC50 ≈ 9.4 µM). These results are most likely influenced by the spatial arrangement of the aromatic moieties in the presence of the water-lipid interphase [32].

Table 1. Radical scavenging and anti-lipoperoxidant activity of isoquercitrin and its conjugates 2–11.

FCR DPPH ABTS Lpx Compounds (GAE) (IC50, µM) (TE) (IC50, µM) Isoquercitrin (1, IQ) 1.11 ± 0.30 a 1.40 ± 0.06 d 1.98 ± 0.07 f 972 ± 11 IQ benzoate (2) 1.24 ± 0.05 a 2.85 ± 0.22 7.34 ± 0.01 g 8.29 ± 0.39 h IQ phenylacetate (3) 1.45 ± 0.03 b 1.89 ± 0.04 d 1.10 ± 0.20 6.68 ± 0.39 i IQ phenylpropanoate (4) 1.47 ± 0.04 b 3.31 ± 0.22 3.65 ± 0.18 10.5 ± 0.7 IQ cinnamate (5) 0.51 ± 0.13 c 4.64 ± 0.16 0.64 ± 0.05 295 ± 14 IQ 4-OH benzoate (6) 0.90 ± 0.05 a 11.5 ± 0.4 1.81 ± 0.25 f 12.6 o ± 0.3 j IQ vanillate (7) 1.50 ± 0.04 b 2.18 ± 0.04 e 7.05 ± 0.07 g 14.1 ± 0.6 j IQ gallate (8) 0.52 ± 0.15 c 2.28 ± 0.18 e 5.27 ± 0.20 6.64 ± 0.27 i IQ 4-OHPh propanoate (9) 0.59 ± 0.06 c 1.77 ± 0.06 d 7.18 ± 0.07 g 9.39 ± 0.29 h IQ 3,4-diOHPh propanoate (10) 0.88 ± 0.04 a 1.67 ± 0.09 d 7.20 ± 0.15 g 9.41 ± 0.28 h IQ 4-OH-3-OMePh propanoate (11) 1.48 ± 0.03 b 2.22 ± 0.11 e 7.33 ± 0.05 g 9.36 ± 0.22 h Data are presented as means ± SE from at least three independent experiments performed in triplicate. a-j The values marked with the same letter are not significantly different. FCR: Folin-Ciocalteau reduction; GAE: gallic acid equivalents; DPPH: 1,1-diphenyl-2-picrylhydrazyl radical scavenging; ABTS: 2,20-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical cation scavenging; TE: trolox equivalents; Lpx: lipid peroxidation; IC50: the concentration of the tested compound that inhibited the reaction by 50%.

The results obtained for isoquercitrin are in a good agreement with our previous study [16]. From the compounds described in the present work, some antioxidant activity was previously reported only for IQ-600-gallate and isolated from Euphorbia supina [33] and Pemphis acidula [34], respectively. Peroxynitrite (ONOO−)[33] and DPPH scavenging activity, inhibition of methyl linoleate oxidation and inhibition of oxidative cell death of IQ-600-gallate was enhanced compared to IQ [34]. This is in accordance with the present study. Isomeric IQ-200-gallate, isolated from Persicatia lapathifolia inhibited peroxynitrite and superoxide production from RAW 264.7 macrophages, but its effect was compared only with quercetin (which was more efficient) and not isoquercitrin [35]. Compared to previously reported isoquercitrin esters with mono- and dicarboxylic aliphatic acids from our laboratory [16], the derivatives containing an aromatic ring presented here generally displayed similar DPPH scavenging potency and were less able to reduce FCR. On the other hand, the esters 2, 4 and 7–11 especially exhibited more pronounced ABTS scavenging and proved to be much stronger inhibitors against lipid peroxidation.

3. Materials and Methods

3.1. Chemicals and Reagents Isoquercitrin (purity 96%) was prepared by enzymatic deglycosylation of rutin using our procedure [8,9]. Lipase B from Candida antarctica immobilized on acrylic resin (Novozym 435) was Int. J. Mol. Sci. 2017, 18, 1074 7 of 14 purchased from Novo-Nordisk (Copenhagen, Denmark). Folin-Ciocalteau reagent was purchased from Merck (Prague, Czech Republic). Benzoic (12), phenylacetic (13), phenylpropanoic (14), cinnamic (15), 4-hydroxybenzoic (16), vanillic (17), gallic (18), p-coumaric (19), caffeic (20) and ferulic (21) acids, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, antioxidant assay kit (CS0790); pooled microsomes from male rat liver (M9066); trolox and other chemicals were obtained from Sigma-Aldrich (Prague, Czech Republic). Vinyl esters (12, 15)[22], (13, 14)[23], and benzylated acids 16 [25], 17 [26], 18 [27], 19, 20 [28], and 21 [29] were prepared as described previously.

3.2. NMR and MS Methods NMR spectra were recorded on a Bruker Avance III 700 MHz spectrometer (700.13 MHz for 1H, 176.05 MHz for 13C), a Bruker Avance III 600 MHz spectrometer (600.23 MHz for 1H, 150.93 MHz for 13C) and Bruker Avance III 400 MHz (400.00 MHz for 1H, 100.59 MHz for 13C, all spectrometers ◦ from Bruker BioSpin, Rheinstetten, Germany) in DMSO-d6 (99.8% atom D) at 30 C or acetone-d6, (99.8% atom D, VWR International, Stˇríbrná Skalice, Czech Republic) at 20 ◦C. The residual signal of the solvent was used as an internal standard (for DMSO-d6: δH 2.500 ppm, δC 39.60 ppm, for acetone-d6: δH 2.050 ppm, δC 30.50 ppm). The following experiments were performed and processed using the manufacturer’s software (Topspin 3.2, Bruker BioSpin, Rheinstetten, Germany): 1H NMR, 13C NMR with power-gated 1H decoupling, ge-2D COSY, ge-2D multiplicity-edited 1H–13C HSQC, ge-2D 1H–13C HMBC and 1D TOCSY. 1H NMR and 13C NMR spectra were zero filled to four-fold data points and multiplied by a window function before the Fourier transformation. The two-parameter double-exponential Lorentz–Gauss function was applied for 1H to improve resolution, and line broadening (1 Hz) was applied to get a better 13C signal-to-noise ratio. The multiplicity of the 13C signals was resolved using the ge-2D multiplicity-edited 1H–13C HSQC. Chemical shifts are given in δ-scale with digital resolution justifying the reported values to three decimal places for δH or two decimal places for δC. Mass spectra were obtained using the Shimadzu Prominence system (Shimadzu, Kyoto, Japan) equipped with an electrospray ion source. The samples were dissolved in acetonitrile and introduced into the mobile phase flow (acetonitrile, 0.4 mL/min).

3.3. HPLC Analysis All analytical HPLC analyses were performed with the Shimadzu Prominence System (Shimadzu Corporation, Kyoto, Japan) consisting of a DGU-20A3 mobile phase degasser, two LC-20AD solvent delivery units, a SIL-20AC cooling auto sampler, a CTO-10AS column oven and SPD-M20A diode array detector. The Chromolith Performance RP-18e monolithic column (100 mm × 3 mm i.d., Merck, DE) coupled with a guard column (5 mm × 4.6 mm i.d., Merck, DE) was employed. Mobile phase acetonitrile/water/formic acid (5/95/0.1, v/v/v, phase A) and acetonitrile/water/formic acid (80/20/0.1, v/v/v, phase B) were used for the analyses; gradient: 0–10 min 7–80% B; 10–12 min 80% B, 12–14 min 80–7% B at 25 ◦C; the flow rate was 1.2 mL/min. The PDA data were acquired in the 200–450 nm range and signals at 360 nm were extracted. Chromatographic data were collected and processed using Shimadzu Solution software (version 5.75 SP2, Shimadzu Corporation, Tokyo, Japan) at a rate of 40 Hz and detector time constant of 0.025 s.

3.4. Chemistry

3.4.1. Synthesis of Compounds 2–5 Dried isoquercitrin (1, 300 mg, 0.65 mmol, 1 equivalents (eq.)) and vinyl ester of the respective acid (22–25, 3.23 mmol, 5 eq.) were dissolved in anhydrous acetone (15 mL). Novozym 435® (400 mg) and molecular sieves Å4 (300 mg) were added to the solution. The reaction mixture was incubated at 45 ◦C, 220 rpm. Part of IQ dissolved gradually during the reaction, which was terminated after 12 days Int. J. Mol. Sci. 2017, 18, 1074 8 of 14 by filtering off the enzyme and the solvent was evaporated in vacuo. The crude product was purified by silica gel flash chromatography (chloroform/methanol 95:5). ((2R,3S,4S,5R,6S)-6-((2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl)oxy)-3,4,5-trihydro xytetrahydro-2H-pyran-2-yl)methyl benzoate (2, IQ benzoate): Yellow solid (yield 29%, 106 mg, 0.19 mmol). For 1H and 13C NMR data see Tables S1, S2 and Figures S1, S2 in the Supplementary − Materials. MS-ESI m/z: [M − H] calcd for C28H23O13 567.1; found 567.1 (Figure5). ((2R,3S,4S,5R,6S)-6-((2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl)oxy)-3,4,5-trihydro xytetrahydro-2H-pyran-2-yl)methyl 2-phenylacetate (3, IQ phenylacetate): Yellow solid (35%, 133 mg, 0.23 mmol). For 1H and 13C NMR data see Tables S1, S2 and Figures S3, S4 in the Supplementary − Materials. MS-ESI m/z: [M − H] calcd for C29H25O13 581.1; found 581.1 (Figure5). ((2R,3S,4S,5R,6S)-6-((2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl)oxy)-3,4,5-trihydro xytetrahydro-2H-pyran-2-yl)methyl 3-phenylpropanoate (4, IQ phenylpropanoate): Yellow solid (yield 33%, 128 mg, 0.22 mmol). For 1H and 13C NMR data see Tables S1, S3 and Figures S5, S6 in the − Supplementary Materials. MS-ESI m/z: [M − H] calcd for C30H27O13 595.1; found 595.1 (Figure5). ((2R,3S,4S,5R,6S)-6-((2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl)oxy)-3,4,5-trihydro xytetrahydro-2H-pyran-2-yl)methyl cinnamate (5, IQ cinnamate): Yellow solid (yield 39%, 152 mg, 0.26 mmol). For 1H and 13C NMR data see Tables S1, S2 and Figures S7, S8 in the Supplementary − Materials. MS-ESI m/z: [M − H] calcd for C30H25O13 593.1; found: 593.1 (Figure5).

3.4.2. Synthesis of Compounds 32–34 (Structures in Figure S21 in the Supplementary Materials)

3-(((2S,3R,4S,5S,6R)-6-(((tert-Butyldimethylsilyl)oxy)methyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2- yl)oxy)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-chromen-4-one (32): To a stirred solution of 1 (2.0 g, 4.31 mmol) and imidazole (586.6 mg, 8.61 mmol, 2.0 eq.) in dry DMF (20 mL) at 0 ◦C and under Ar, a solution of TBDMSCl (1.3 g, 8.63 mmol, 2.0 eq.) in DMF (20 mL) was added dropwise. After stirring at RT for 17 h the reaction mixture was diluted with H2O (100 mL) and extracted with EtOAc (3 × 300 mL). Combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography using gradient (EtOAc/MeOH from 50:1 to 25:1) to produce 32 as yellow oil (yield 82%, 2.0 g, 3.46 mmol). For 1H and 13C NMR data see Tables S7, S8 and Figures S22, S23 in the Supplementary Materials. MS-ESI m/z: [M − H]− calcd for C27H33O12Si 577.2; found 577.2. 5,7-bis(Benzyloxy)-2-(3,4-bis(benzyloxy)phenyl)-3-(((2S,3R,4S,5R,6R)-3,4,5-tris(benzyloxy)-6-(((tert- butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-4H-chromen-4-one (33): To an argon-purged solution of 32 (2.0 g, 3.46 mmol) and NaH (60% suspension in mineral oil, 2.3 g, 57.5 mmol, 16.6 eq.) in dry DMF (80 mL) at 0 ◦C, a solution of BnBr (29.0 mL, 24.38 mmol, 7.0 eq.) was added dropwise. The solution was stirred at RT for 24 h and then extracted with EtOAc (3 × 300 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (toluene/EtOAc = 20:1) to produce title compound as a brownish oil (yield 37%, 1.5 g, 1.24 mmol). For 1H and 13C NMR data see Tables S7, S8 and Figures S24, S25 in + the Supplementary Materials. MS-ESI m/z: [M + H] calcd for C76H77O12Si 1209.5; found 1209.5. 5,7-bis(Benzyloxy)-2-(3,4-bis(benzyloxy)phenyl)-3-(((2S,3R,4S,5R,6R)-3,4,5-tris(benzyloxy)-6-(hydroxyme- thyl)tetrahydro-2H-pyran-2-yl)oxy)-4H-chromen-4-one (34): To an argon-purged flask containing a solution of 33 (1.5 g, 1.24 mmol) in anhydrous THF (30 mL) at 0 ◦C a solution of tetrabutylammonium fluoride in THF (1 M, 7.8 mL) was added dropwise. Reaction mixture was stirred under an Ar atmosphere at 0 ◦C for 30 min and then at RT for next 17 h. The reaction mixture was diluted with H2O (100 mL) and then extracted with EtOAc (3 × 300 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (toluene/EtOAc = 10:1) to yield yellow oil (82%, 1.1 g, 1.01 mmol). For 1H and Int. J. Mol. Sci. 2017, 18, 1074 9 of 14

13C NMR data see Tables S7, S8 and Figures S26, S27 in the Supplementary Materials. MS-ESI m/z: + [M + H] calcd for C70H63O12 1095.4; found 1095.4.

3.4.3. Synthesis of Compounds 35–40 (Structures in Figure S21 in the Supplementary Materials)

Anhydrous CH2Cl2 (10 mL) was added to a flask containing 34 (200 mg, 0.18 mmol, 1 eq.), benzyl-protected acids 26–31 (0.46 mmol, 2.5 eq.), DCC (76 mg, 0.36 mmol, 2 eq.) and DMAP (11 mg, 0.09 mmol, 0.5 eq.) and the reaction mixture was stirred at RT for 48 h. Then solvent was evaporated in vacuo and the residue was purified by column chromatography using toluene/EtOAc (99:1) and products 35–40 were isolated. ((2R,3R,4S,5R,6S)-3,4,5-tris(Benzyloxy)-6-((5,7-bis(benzyloxy)-2-(3,4-bis(benzyloxy)phenyl)-4-oxo-4H -chromen-3-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl 4-(benzyloxy)benzoate (35): Yellow solid (yield 75%, 180 mg, 0.14 mmol). For 1H and 13C NMR data see Tables S9, S10 and Figures S28, S29 in the + Supplementary Materials. MS-ESI m/z: [M + H] calcd for C84H73O14 1305.5; found 1305.7. ((2R,3R,4S,5R,6S)-3,4,5-tris(Benzyloxy)-6-((5,7-bis(benzyloxy)-2-(3,4-bis(benzyloxy)phenyl)-4-oxo-4H -chromen-3-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl 4-(benzyloxy)-3-methoxybenzoate (36): Yellow solid (yield 72%, 175 mg, 0.13 mmol). For 1H and 13C NMR data see Tables S9, S10 and Figures S30, S31 in + the Supplementary Materials. MS-ESI m/z: [M + H] calcd for C85H75O15 1335.5; found 1335.8. ((2R,3R,4S,5R,6S)-3,4,5-tris(Benzyloxy)-6-((5,7-bis(benzyloxy)-2-(3,4-bis(benzyloxy)phenyl)-4-oxo-4H -chromen-3-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl 3,4,5-tris(benzyloxy)benzoate (37): Yellow solid (yield 61%, 170 mg, 0.11 mmol). For 1H and 13C NMR data see Tables S9, S11 and Figures S32, S33 in the + Supplementary Materials. MS-ESI m/z: [M + H] calcd for C98H85O16 1517.6; found 1517.9. (E)-((2R,3R,4S,5R,6S)-3,4,5-tris(Benzyloxy)-6-((5,7-bis(benzyloxy)-2-(3,4-bis(benzyloxy)phenyl)-4-oxo-4H -chromen-3-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl 3-(4-(benzyloxy)phenyl)acrylate (38): Yellow solid (yield 70%, 172 mg, 0.13 mmol). For 1H and 13C NMR data see Tables S9, S11 and Figures S34, S35 in the + Supplementary Materials. MS-ESI m/z: [M + Na] calcd for C86H74O14Na 1353.5; found 1353.7. (E)-((2R,3R,4S,5R,6S)-3,4,5-tris(Benzyloxy)-6-((5,7-bis(benzyloxy)-2-(3,4-bis(benzyloxy)phenyl)-4-oxo-4H -chromen-3-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl 3-(3,4-bis(benzyloxy)phenyl)acrylate (39): Yellow solid (yield 86%, 227 mg, 0.16 mmol). For 1H and 13C NMR data see Tables S12, S13 and Figures S36, S37 in + the Supplementary Materials. MS-ESI m/z: [M + H] calcd for C93H81O15 1437.6; found 1437.7. (E)-((2R,3R,4S,5R,6S)-3,4,5-tris(Benzyloxy)-6-((5,7-bis(benzyloxy)-2-(3,4-bis(benzyloxy)phenyl)-4-oxo-4H -chromen-3-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl 3-(4-(benzyloxy)-3-methoxyphenyl)acrylate (40): Yellow solid (yield 64%, 160 mg, 0.12 mmol). For 1H and 13C NMR data see Tables S12, S13 and Figures S38, + S39 in the Supplementary Materials. MS-ESI m/z: [M + H] calcd for C87H77O15 1362.5; found 1361.7.

3.4.4. Synthesis of Compounds 6–11

Hydrogenolysis of 35–40 (200 mg) was performed with Pd/C-H2 (200 mg, 10% w/w) in EtOH/THF 1:1 (40 mL) and was stirred for 12 h. Then reaction mixtures were filtrated and evaporated in vacuo to yield 6–11. ((2R,3S,4S,5R,6S)-6-((2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl)oxy)-3,4,5-trihydro xytetrahydro-2H-pyran-2-yl)methyl 4-hydroxybenzoate (6, IQ 4-OH benzoate): Yellow solid (yield 91%, 74 mg, 0.13 mmol). For 1H and 13C NMR data see Tables S4, S5 and Figures S9, S10 in the − Supplementary Materials. MS-ESI m/z: [M − H] calcd for C28H23O14 583.1; found 583.1 (Figure5). ((2R,3S,4S,5R,6S)-6-((2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl)oxy)-3,4,5-trihydro xytetrahydro-2H-pyran-2-yl)methyl 4-hydroxy-3-methoxybenzoate (7, IQ vanillate): Yellow solid (yield 60%, 50 mg, 0.08 mmol). For 1H and 13C NMR data see Tables S4, S5 and Figures S11, S12 in the − Supplementary Materials. MS-ESI m/z: [M − H] calcd for C29H25O15 613.1; found 613.1 (Figure5). Int. J. Mol. Sci. 2017, 18, 1074 10 of 14

((2R,3S,4S,5R,6S)-6-((2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl)oxy)-3,4,5- trihydroxytetrahydro-2H-pyran-2-yl)methyl 4-hydroxy-3-methoxybenzoate (7, IQ vanillate): Yellow solid Int.(yield J. Mol. 60%, Sci. 502017 mg,, 18 ,0.08 1074 mmol). For 1H and 13C NMR data see Tables S4, S5 and Figures S11, S12 10in ofthe 14 Supplementary Materials. MS-ESI m/z: [M − H]− calcd for C29H25O15 613.1; found 613.1 (Figure 5).

((2((2R,3,3S,4S,5R,6S)-6-((2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4HH-chromen-3-yl)oxy)-3,4,5--chromen-3-yl)oxy)-3,4,5-trihydro xytetrahydro-2trihydroxytetrahydro-2H-pyran-2-yl)methylH-pyran-2-yl)methyl 3,4,5-trihydroxybenzoate 3,4,5-trihydroxybenzoate(8, IQ gallate): (8, IQ Yellow gallate): solid Yellow (yield solid 75%, 60(yield mg, 0.1075%, mmol).60 mg, For0.101 Hmmol). and 13 ForC NMR 1H and data 13 seeC NMR Tables data S4, see S5 andTables Figures S4, S5 S13, and S14 Figures in the SupplementaryS13, S14 in the − − Materials.Supplementary MS-ESI Materials.m/z: [M MS-ESI− H] mcalcd/z: [M for − CH]28H calcd23O16 for615.1; C28H found23O16 615.1; 615.1 (Figurefound 615.15). (Figure 5). ((2((2R,3,3S,4S,5R,6S)-6-((2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4HH-chromen-3-yl)oxy)-3,4,5--chromen-3-yl)oxy)-3,4,5-trihydro xytetrahydro-2trihydroxytetrahydro-2H-pyran-2-yl)methylH-pyran-2-yl)methyl 3-(4-hydroxyphenyl)propanoate 3-(4-hydroxyphenyl)propanoate(9, IQ 4-OHPh(9, IQ 4-OHPh propanoate): propanoate): Yellow solidYellow (yield solid 71%, (yield 54 71%, mg, 54 0.09 mg, mmol). 0.09 mmol). For 1H For and 1H13 andC NMR 13C NMR data data see Tables see Tables S4, S6 S4, and S6 Figuresand Figures S15, − − S16S15, inS16 the in Supplementary the Supplementary Materials. Materials. MS-ESI MS-ESIm/z m: [M/z: [M− H] − H]calcd calcd for for C C3030HH2727OO1414 611.1; found 611.1 (Figure5 5).). ((2((2R,3,3S,4S,5R,6S)-6-((2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4HH-chromen-3-yl)oxy)-3,4,5--chromen-3-yl)oxy)-3,4,5-trihydro xytetrahydro-2trihydroxytetrahydro-2H-pyran-2-yl)methylH-pyran-2-yl)methyl 3-(3,4-dihydroxyphenyl)propanoate 3-(3,4-dihydroxyphenyl)propanoate(10, IQ 3,4-diOHPh (10, IQ propanoate):3,4-diOHPh Yellowpropanoate): solid Yellow (yield 64%,solid 60(yield mg, 64%, 0.10 60 mmol). mg, 0.10 For mmol).1H and For 131HC and NMR 13C dataNMR see data Tables see Tables S4, S6 S4, and S6 − − Figuresand Figures S17, S17, S18 inS18 the in Supplementarythe Supplementary Materials. Materials. MS-ESI MS-ESIm/z m: [M/z: [M− H]− H]calcd calcd for for C C3030HH2727OO1515 627.1; found 627.2 (Figure5 5).). ((2((2R,3,3S,4S,5R,6S)-6-((2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4HH-chromen-3-yl)oxy)-3,4,5--chromen-3-yl)oxy)-3,4,5-trihydro xytetrahydro-2trihydroxytetrahydro-2H-pyran-2-yl)methylH-pyran-2-yl)methyl 3-(4-hydroxy-3-methoxyphenyl)propanoate 3-(4-hydroxy-3-methoxyphenyl)propanoate(11, IQ (11 4-OH-3-OMePh, IQ 4-OH-3- propanoate):OMePh propanoate): Yellow solidYellow (yield solid 80%, (yield 60 80%, mg, 0.0960 mg, mmol). 0.09 mmol). For 1H andFor 113HC and NMR 13C dataNMR see data Table see S4,Table S6 − − andS4, S6 Figures and Figures S19, S20 S19, in S20 the in Supplementary the Supplementary Materials. Materials. MS-ESI MS-ESIm/z :m [M/z: −[MH] − H]calcd calcd for for C C3131HH2929OO1515 641.2; found 641.0 (Figure5 5).).

Figure 5. MS-ESI spectra of the isoquercitrin esters 2–11.. Int. J. Mol. Sci. 2017, 18, 1074 11 of 14

3.5. Antioxidant Activity Measurement Reducing capacity using Folin–Ciocalteau reagent [36], antiradical activity by DPPH [37] and ABTS• [38] radical scavenging assays, and anti-lipoperoxidant activity as inhibition of lipid peroxidation of rat liver microsomes induced by tert-butylhydroperoxide [39] were determined as described previously with modifications specified in detail in our previous work [32].

3.6. Statistical Analysis All data were analyzed with one-way ANOVA, Scheffé and Least Square Difference tests for post hoc comparisons among pairs of means using the statistical package Statext ver. 2.1 (Statext LLC, Wayne, NJ, USA). Differences were considered statistically significant when p < 0.05.

4. Conclusions The chemoenzymatic and chemical approach have been used for the esterification at C-600 of IQ with acyls containing an aromatic ring. First, the lipase catalyzed preparation of the derivatives was accomplished with Novozym 435 and the vinyl esters of benzoic, phenyl acetic, phenylpropanoic and cinnamic acids, but “hydroxyaromatic” acids or their vinyl esters were not substrates for the lipase. The reaction of protected “hydroxyaromatic” acids with selectively protected IQ lead to the fully protected C-600 esterified products. The deprotection of the benzyl groups lead to the parallel saturation of the double bond yielding respective hydroxyphenylpropanoic derivatives. Several deprotection methods were attempted to overcome this problem but they proved unsuccessful. We have therefore obtained a panel of following IQ derivatives: IQ benzoate (2), phenylacetate (3), phenylpropanoate (4), cinnamate (5), 4-OH benzoate (6), vanillate (7), gallate (8), 4-OHPh propanoate (9), 3,4-diOHPh propanoate (10), and 4-OH-3-OMePh propanoate (11). The FCR reducing and DPPH scavenging capacity of most derivatives was lower or similar to IQ. In contrast, they mostly showed significantly better ABTS scavenging activity—the compounds 2, 7, 9, 10 and 11 displaying the strongest activity. Almost all derivatives were much more efficient inhibitors of microsomal lipid peroxidation. Compounds 8 and 3 (IC50 ≈ 7 µM) were the most active, but strong activity was noted also for phenylpropanoates 9–11 (IC50 ≈ 9.4 µM), which proved to be the most active compounds from the whole series.

Supplementary Materials: Supplementary materials containing 1H, 13C NMR data of the new compounds can be found at www.mdpi.com/1422-0067/18/5/1074/s1. Acknowledgments: This work was supported by Czech Science Foundation grant GP14-14373P and project No. LD15082 from Ministry of Education of the Czech Republic (COST Action FA1403 POSITIVe). Author Contributions: Eva Heˇrmánková-Vavˇríková synthetized all the new compounds; Alena Kˇrenková and Lucie Petrásková performed the HPLC measurements; Kateˇrina Valentová was responsible for the determination of DPPH and ABTS scavenging, FCR, and inhibition of lipid peroxidation; Christopher Steven Chambers performed part of the syntheses and language editing; Marek Kuzma and Jakub Zápal measured the NMRs; Vladimír Kˇren designed and evaluated the experiments. All the authors contributed to the manuscript writing. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

ABTS 2,20-Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) BnBr Benzyl bromide CAL-B Lipase B from Candida antarctica COSY Correlation spectroscopy DCC N,N0-Dicyclohexylcarbodiimide DCM Dichloromethane DMAP 4-Dimethylaminopyridine DMF Dimethylformamide Int. J. Mol. Sci. 2017, 18, 1074 12 of 14

DMSO Dimethyl sulfoxide DPPH 1,1-Diphenyl-2-picrylhydrazyl eq. Equivalents FCR Folin-ciocalteau reduction FDA Food & drug administration GAE Gallic acid equivalents GRAS Generally recognized as safe HMBC Heteronuclear multiple-bond correlation spectroscopy HSQC Heteronuclear single-quantum correlation spectroscopy IC50 The concentration of the tested compound that inhibited the reaction by 50% IQ Isoquercitrin (quercetin-3-O-β-D-glucopyranoside) Lpx Lipid peroxidation MS Mass spectrometry MS-ESI Electron spray ionization mass spectrometry NMR Nuclear magnetic resonance RT Room temperature TBARS Thiobarbituric acid reactive substances TBDMSCl tert-Butyldimethylsilyl chloride TE Trolox-equivalents THF Tetrahydrofuran TOCSY Two-dimensional nuclear magnetic resonance spectroscopy UV Ultra violet

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