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 ferulic acid, 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; gallic acid; cinnamic acid; antioxidant activity; Novozym 435; lipase; DPPH; lipoperoxidation 1. Introduction Plant polyphenols 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 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 flavonoids wi withth numerous numerous reactive reactive