Effects of conjugation metabolism on radical scavenging and transport properties of quercetin – In silico study

Accepted Manuscript Effects of conjugation metabolism on radical scavenging and transport properties of quercetin – In silico study Višnja Stepanić, Sara Matić, Ana Amić, Bono Lučić, Dejan Milenković, Zoran Marković PII:

S1093-3263(18)30535-7

DOI:

https://doi.org/10.1016/j.jmgm.2018.10.023

Reference:

JMG 7261

To appear in:

Journal of Molecular Graphics and Modelling

Received Date: 19 July 2018 Revised Date:

22 October 2018

Accepted Date: 24 October 2018

Please cite this article as: Viš. Stepanić, S. Matić, A. Amić, B. Lučić, D. Milenković, Z. Marković, Effects of conjugation metabolism on radical scavenging and transport properties of quercetin – In silico study, Journal of Molecular Graphics and Modelling (2018), doi: https://doi.org/10.1016/j.jmgm.2018.10.023. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Effects of conjugation metabolism on radical scavenging and transport

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properties of quercetin – in silico study

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Višnja Stepanić a*, Sara Matić,a Ana Amić,b Bono Lučić,a Dejan Milenković,c Zoran Markovićd

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a

Ruđer Bošković Institute, Bijenička 54, 10000 Zagreb, Croatia

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Department of Chemistry, Josip Juraj Strossmayer University of Osijek, Cara Hadrijana 8a,

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31000 Osijek, Croatia

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c

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Kragujevac, Serbia

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d

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Bioengineering Research and Development Center, Prvoslava Stojanovića 6, 34000

Department of Chemical-Technological Sciences, State University of Novi Pazar, Vuka

Karadžića bb, 36300 Novi Pazar, Serbia

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Corresponding Author

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*(V.S.) Phone: +385 1 457 1248. Fax: +385-1-4561-010.

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E-mail: [email protected]

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ORCID

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Višnja Stepanić: 0000-0001-9518-4153

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ACCEPTED MANUSCRIPT ABSTRACT

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Quercetin (Q) is a natural polyphenol with high radical scavenging capacity, but low in vivo

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bioavailability. It is extensively transformed by host phase II metabolism and microbiota.

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Herein, effects of major in vitro and in vivo conjugation transformations of Q on its radical

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scavenging capacity and human serum albumin (HSA) binding were studied by using

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appropriate computational approaches, DFT (U)B3LYP/6-31+G(d,p) and molecular docking,

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respectively. With regard to radical scavenging capacity of Q, conjugation transformations

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generally reduce its antioxidant capacity including regeneration efficiency through

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disproportionation of an intermediate radical species since these structural modifications

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occur mainly at its radical scavenging –OH groups. They were also found to alter dominant

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radical scavenging mechanism in a specific way dependent upon conjugation type and site.

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Concerning distribution by HSA, binding to this main plasma transporter protein may not be

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dominant transport mechanism for Q and its metabolites in vivo. Like Q aglycon, most of its

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metabolites are bound non-specifically at multiple binding sites of HSA, with relatively weak

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affinities. Only sulfo-conjugates including plasma abundant isomer Q-3’-O-SO3–, were

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predicted to bind specifically in warfarin-like manner, but also with relatively low binding

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affinity.

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Keywords: quercetin; conjugation metabolism; radical scavenging; disproportionation,

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serum albumin

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Abbreviations: BDE, bond dissociation enthalpy; BS I, binding site I; ETE, electron transfer enthalpy;

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FE, free energy; HAT, hydrogen atom transfer; HSA, human serum albumin; LGA, Lamarckian genetic

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algorithm; Q, quercetin; QBM, quercetin binding mode; RS, radical scavenging; SA, serum albumin;

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SPLET, sequential proton loss electron transfer; WBM, warfarin binding mode

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1. Introduction Natural compounds are notable sources of drugs and determine functional properties of

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food and beverages. Quercetin (Q) is one of the most studied natural compounds. It is an

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abundant plant polyphenol from the subgroup of flavonols with average dietary intake in EU

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exceeding 20 mg/day [1]. It has been known mainly as an antioxidant with beneficial

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cardioprotective and chemopreventive health effects [2].

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In various fruits and vegetables, Q is present in the form of diverse glycosides. Glycosides

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undergo moderately fast cleavage of sugar fragment(s) by the action of bacterial β-

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glucosidase(s) and intestinal absorption (the absorption half-time t1/2 is ~ 1 h) [3].

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Subsequently, the aglycon Q is extensively metabolised in two ways, through: (i) conjugation

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by the host phase II metabolic enzymes mainly in intestine and liver and (ii) C-ring fission by

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host enterobacteria [3]. Consequently, after oral consumption, the plasma level of Q is very

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low, generally in sub-μM range and the free aglycon is present in the amount less than 10%

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of the total plasma Q. After the extensive first-pass metabolism, Q and its metabolites are

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slowly excreted with the elimination half-time t1/2 longer than 15 h and largely determined

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by dietary source. Disposition of Q in human is complex and the great portion of Q (from

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23.0–81.1% of a dose) is reported to be totally metabolized to carbon dioxide and exhaled

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into air [4].

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The main metabolites of Q found in human plasma are Q-3-O-β-D-glucuronide (Q-3-O-Glr–),

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Q-3’-O-sulfate (Q-3’-O-SO3–) and a hybrid diconjugate 3’-O-methyl-3-O-glucuronide (3'-O-

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Me-Q-3O-Glr–) [5]. While the conjugated metabolites circulate in plasma, the bacterial

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catabolites - small molecular weight phenolic acids such as caffeic acid, hydrocaffeic acid,

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homoprotocatechuic acid and homovanillic acid, are also present in urine and feces [6]. Q is

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also metabolised in specific ways in vitro, in cellular cultures [7]. Upon 24 h incubation of Q

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ACCEPTED MANUSCRIPT (50 μM and 100 μM) with human colon carcinoma cell line HT29 or rat hepatocellular

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carcinoma cell line H4IIE, the major identified Q metabolites were Q-4’-O-Glr– (40%) and Q-

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3-O-Glr– (27%) in HT29 cells, and Q-7-O-Glr– (67%) in H4IIE cells with the residual level of

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aglycon present in the incubation media less than 1% [8]. In vitro cellular effects of Q are

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greatly determined by its concentration and by cell type implying plausible impacts of

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specific cellular metabolic transformation on its activity [9].

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Fig. 1. Quercetin and its conjugated metabolites detected in vivo and/or in vitro and considered here in silico.

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The metabolic transformations of Q have considerable impact on its antioxidative

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properties. They modify its direct radical scavenging (RS) capacity [10,11], its potency to

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inhibit low-density lipoprotein (LDL) [12] or protein oxidation [13], as well as its inhibitory

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activity to enzymes involved in endogenous free radical production such as xanthine oxidase,

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lipoxygenase or NADPH oxidase activity of generating superoxide radical anion [14,15].

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Phase II metabolism also alters various other biological activities of Q including interactions

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with membrane transporters and effects on angiogenesis [16].

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The metabolic conjugations of Q may alter its interactions with serum albumin (SA), the

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main transporter protein of endogenous and exogenous substances in blood [17]. According

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to in vitro studies [18], Q and most of its main metabolites are bound by human serum

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albumin (HSA) what may impact their in vivo bioavailability and distribution [12,19].

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Scheme 1 Assumed RS mechanisms of Q and its metabolites: (a) hydrogen atom transfer (HAT) and (b)

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sequential proton loss electron transfer (SPLET). In double HAT pathway (a), aryloxyl monoradical Q-O is an

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intermediate and quinoid specie Q(=O)2 is a final product. (c) Disproportionation of intermediate radical Q-O

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regenerates the parent reducing form Q-OH.

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The purpose of our study was to explore the effects of main metabolic transformations of Q,

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their types and positions, on its RS capacity and HSA binding by using appropriate

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computational methods. The RS computational models as well as molecular docking

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calculations have already been applied for study of polyphenols [20]. The reliable and

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insightful results obtained for polyphenol aglycons as well as similar (semi)synthetic

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compounds, encouraged us to apply in silico approaches on quercetin metabolites. The

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effects of conjugation transformations on biological properties of Q have not been studied

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enough so far. In addition to the major in vivo metabolites Q-3-O-Glr−, Q-3’-O-SO3− and 3'-O-

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Me-Q-3O-Glr– found in human plasma [5], the conjugated 3’-O- (Q-3’-O-Me, isorhamnetin)

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and 4’-O- (Q-4’-O-Me, tamarixetin) methyl ethers, 3-O- (Q-3-O-SO3−) and 7-O- (Q-7-O-SO3−)

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sulfates as well as 4’-O-β-D-glucuronide (Q-4’-O-Glr−) were also included into the studied set

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of Q metabolites (Fig. 1). Some of these metabolites were detected in tissues of pigs and/or

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rats [21] or in cellular cultures [7,22]. For some of the metabolites, in vitro data on RS activity

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[11] and SA binding have been reported [19]. In this extensive study, quercetin and its

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metabolites were considered in their neutral (Q-OH) and anionic (Q-O–) reduced states,

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neutral aryloxyl monoradical intermediate state (Q-O•) as well as in doubly oxidized quinoid

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states (Q(=O)2) (Scheme 1). In particular, effects of metabolic transformations on the double

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sequential RS processes and on disproportionation of radical intermediate were investigated

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to explore their contributions to the unusually high RS extent of Q [9]. The results obtained

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by relatively inexpensive in silico methods, provide valuable mechanistic insights at

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molecular level which can be further used for better understanding redox activity of

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extensively metabolised polyphenol Q under in vitro and in vivo conditions. It is shown that

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RS properties of most of conjugates are considerably changed as compared with the aglycon

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Q. They are determined by the type and site of conjugation. Regarding ways of

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transportation through the body, conjugates of Q are expected to be distributed by other

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ways than serum albumin transport.

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2. Materials and methods

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2.1 Quantum-chemical calculations of radical scavenging activities

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The influence of methylation, sulfurylation and glucuronidation on RS efficiency and

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mechanism of Q, was studied by applying quantum-mechanical density functional theory

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(DFT) approach. The DFT model (U)B3LYP/6-31+G(d,p) was used in combination with

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polarizable continuum model of solvation (IEFPCM) for the estimation of RS parameters

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(Scheme 1) for Q and its major metabolites (Fig. 1) (Table 1) [23,24,25,26,27,28,29].

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Recently, the similar analysis has been done for the set of Q catabolic ring fission phenolic

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acid products (Supplementary Table S1) [6]. Sulfate (-OSO3–) and glucuronide (-OGlr–)

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conjugates were considered with deprotonated, negatively charged -SO3– and -COO– groups,

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respectively. Equilibrium structures in neutral, anionic and quinoid closed-shell ground

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electronic states as well as in monoradical open-shell doublet ground electronic state, were

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fully optimized in the gas phase. The minima were confirmed by no imaginary vibrational

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frequencies. The expectation values of the operator calculated by the (U)B3LYP/6-

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31+G(d,p) model for the open-shall radical species FlO• were within the range 0.760-0.784.

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The NBO spin density analysis was performed by using the NBO 6.0 software (Fig. S1) [30].

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Calculated bond dissociation enthalpy (BDE) for hydrogen atom transfer (HAT) mechanism

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[31] and proton affinity (PA) / electron transfer enthalpy (ETE) values for sequential proton

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loss electron transfer (SPLET) pathway [32] in the gas phase as well as corresponding free

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energies (BDFEaq and ∆Gbasicity (i.e. pKa) / ETFEaq, respectively) in aqueous phase, were used

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ACCEPTED MANUSCRIPT as parameters for prediction of relative RS activities (Scheme 1) [27]. These thermodynamic

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reaction parameters were estimated at temperature of 298.15 K, pressure of 1 atm for the

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gas phase and standard state of 1 M for aqueous phase [27]. In the case of a set of flavonols,

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significant linear correlation between aqueous parameters BDFEaq and ETFEaq was found,

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demonstrating that BDFEaq can be used as a general parameter for estimating relative RS

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capacities of phenolic compounds regardless underlying RS mechanism [27]. Hence, herein

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we considered only double HAT to estimate relative capacity of a metabolite molecule to

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scavenge two free radicals. DFT calculations were done by using Gaussian 03 software [33].

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2.2 Molecular docking into human serum albumin

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Rigid, non-covalent molecular docking of Q and its conjugated metabolites including their

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differently deprotonated species and doubly oxidized quinoid species, was performed into

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HSA by using the program AutoDock 4.2 [34]. The crystal structure 2BXD of a complex of HSA

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with R-warfarin was downloaded from Protein Data Bank [17]. The initial 3D conformations

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of Q and its metabolites were taken from DFT calculations. The structure of a hybrid

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conjugate 3'-O-Me-Q-3-O-Glr– and all reference ligands (Table S2) were generated by the

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program Marvin 6.0.0 using Dreiding force field [35]. AutoDockTools 1.5.6 was applied for

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preparation of compounds and HSA for molecular docking [36]: B-chain and ligands were

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removed from the structure 2BXD; nonpolar hydrogens and lone pairs were merged and

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Gasteiger partial atomic charges were assigned to HSA as well as to each compound

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considered. The binding site was defined as the grid map centred at the point (4,-11,3), with

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60x60x80 points spaced by 0.375 Å. Using the Lamarckian genetic algorithm (LGA), 100

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docking calculations were performed per molecule with default values of other algorithm

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parameters. Two clusters of binding conformations - the most stable cluster (E1) and most

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ACCEPTED MANUSCRIPT populated cluster (Emp) with the binding affinity window (Emp – E1) not greater than 2

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kcal/mol, were analysed for finding representative binding mode(s) of a studied compound.

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The docking procedure (binding site definition, use of LGA for conformational sampling and

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choice of docking parameter values) was validated by docking warfarin and other known

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strong and weak ligands of binding site I (BS I), and comparison of the obtained in silico

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results with the wet HSA binding data available in literature (Table S2). The program PyMOL

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was used for visualization and preparation of figures [37].

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3. Results and discussion

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3.1 Influence of phase II metabolic transformations on radical scavenging capacity of

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quercetin

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Q is a very efficient direct free radical scavenger with generally high stoichiometric

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coefficient (∼5) [11,38]. The high free RS capacity of Q implies multi-step process and is

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commonly ascribed to the presence of more RS OH groups in Q and to ability of Q to

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regenerate the active reducing forms, the parent form or aryloxyl radical intermediate. The

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redox active groups of Q, the catecholic group (3’-OH and 4’-OH) and 3-OH group are

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coupled by the extended π electron conjugation involving the double C2=C3 bond, and

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additionally stabilized by intramolecular H-bonds (Fig. 1 and Fig. S1). Structural modification

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that impairs any of these structural features generally diminishes the RS capacity of Q.

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Accordingly, conjugation reactions of phase II metabolism– glucuronidation, sulfation and

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/or methylation, have been found to reduce the RS capacity of Q [10,12]. Herein, by means

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of computations, the metabolic structural transformations were found to affect not only

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capacity, but also dominant RS mechanism of Q, in great dependence upon the position of

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conjugation (Table 1).

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ACCEPTED MANUSCRIPT As a direct radical scavenger, Q may act through HAT [31] or SPLET [32] mechanism, in great

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dependence upon the medium polarity and pH. In the gas phase, which may well

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approximate effects of non-polar environment, HAT is a dominant RS mechanism of Q. The

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4’-OH group is the 1st RS centre of Q considering that the aryloxyl radical Q-4’-O• is more

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stable than the Q-3-O• one, with more delocalized spin density (Fig. S1) and accordingly

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BDEg,1(4’-OH) of 74.2 kcal/mol lower than BDEg,1(3-OH) of 80.8 kcal/mol (Table 1). However,

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in aqueous phase, the prevalent RS mechanism is SPLET since Q is deprotonated at its 4’-OH,

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3-OH or 7-OH group [27]. In anionic state, Q is most prone to react as a radical scavenger at

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3-O position. The formation of Q-3-O• radical was predicted to be favourable oxidation

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process of Q in water: ETFEaq(3-OH) < ETFEaq (4’-OH) and BDFEaq,1(3-OH) < BDFEaq,1 (4’-OH)

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(Table 1) [27]. In environment of either type, the intermediate aryloxyl radicals Q-4’-O• and

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Q-3-O• even more readily release H-atom (BD(F)E2 < BD(F)E1, Table 1) and transform into the

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corresponding quinoid form as a final product of double RS process (Scheme 1) [6].

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It’s well known that any compound with the catechol moiety may exert RS activity [39]. High

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RS activity of the catechol group is ascribed to the stabilisation of an aryloxyl radical through

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donating H-bond by the adjacent OH group (Scheme 1) [31]. However, the RS mechanism of

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the catechol fragment is highly dependent upon the rest of the structure. The acidity of the

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4’-OH group is greater for quercetin (pKa ~ 7.5) than for the simple phenolic acids with the

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catecholic moiety (pKa > 9), and thus SPLET is significant RS mechanism for Q, but not for its

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catabolic products at the physiological pH (Table S1) [6]. Nonetheless, HAT parameter values

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(BDEg, BDFEaq) of phenolic acidic catabolites (Table S1) were estimated to be comparable

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with those of Q (Table 1). Greater RS efficiency of Q [11] may, hence, be at least partially

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ascribed to the SPLET mechanism since deprotonation strongly accelerates the RS reaction in

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polar solvents [32]. For most of Q conjugated metabolites, SPLET is also predicted to be a

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ACCEPTED MANUSCRIPT dominant RS mechanism in polar environment (ETFEaaq < BDFEaaq,1, Table 1), what is in

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accordance with the in vitro results showing that their RS efficiencies in the ABTS assay at pH

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7.4 are generally greater than in the FRAP assay at the acidic pH 3.6 [10]. As for Q glycoside

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isomers [28], it has also evidenced herein that for successful interpretation of RS activities of

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Q metabolites, it is essential to take into account molecular acidity and solvent effects.

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For the in vivo abundant metabolite Q-3-O-Glr–, BDFEaq,1 is significantly higher than ETFEaq

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(Table 1) indicating that SPLET is its main RS mechanism in polar medium. Accordingly its RS

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activity may be considerably influenced by medium polarity as it has already been observed

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in vitro [10]. In difference, for its isomer Q-4’-O-Glr– the predicted RS mechanism is HAT

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since 4’-O-glucuronidation considerably reduces the molecular acidity (Table 1) and

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consequently the RS activity of this isomer should be quite independent upon medium

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polarity and pH. Since for Q-4’-O-Glr– double RS and disproportionation reactions are also

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cancelled by 4’-O substitution, this metabolite can act only as a single H-atom donor. The in

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vitro RS activity of Q-4’-O-Glr– is considerably lower than for the Q-3-O-Glr– isomer what has

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been illustrated by lag times of copper(II)-induced LDL oxidation decrease in the order Q-7-

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O-Glr– ≈ Q > Q-3-O-Glr– ≈ Q-3-glucoside >> Q-4’-O-Glr– (at concentrations of 2 μM) [12].

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Analogous trend in RS activities was observed for 3-O- and 4’-O glycoside isomers of Q [11].

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Due to reduced molecular acidity, the main RS mechanism of 7-O-sulfo conjugate Q-7-O-SO3–

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is also predicted to be HAT. The place of the 1st HAT in Q-7-O-SO3– depends upon the

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medium polarity: in non-polar/polar medium it should happen at 4’-OH / 3-OH group

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according to DFT calculations (Table 1). For Q-3-O-SO3–, the prevailing RS mechanism may

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also be HAT, in difference to the corresponding glucuronide isomer 3-O-Glr–. In opposite to

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the 3-O-Glr– group, the electron accepting 3-O-SO3– group, reduces electron donating

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capacity of the active 4’-O site. The most abundant human plasma sulfo-conjugate Q-3’-O-

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ACCEPTED MANUSCRIPT SO3– [5], is predicted to act as a single free radical scavenger at 3-OH group since its RS

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activity at 4’-OH site and disproportionation propensity are considerably weaker than for

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aglycon Q (Table 1) [10]. Its RS mechanism should also be determined by medium polarity

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and free radical type, as well. The analogous, but much less reducing effect on the RS activity

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of 4’-O position is observed in the case of neutral and relatively small methyl group at 3’-O-

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position in isorhamnetin. For isorhamnetin, the RS process is also predicted to start at 3-OH

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group and corresponding BDFEaq and ETFEaq values are comparable with those for Q (Table

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1). However, the 2nd HAT process at 4’-OH position is predicted to be somewhat less efficient

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than for Q, which may at least partially explain experimentally observed smaller RS capacity

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of isorhamnetin than for the non-modified aglycon [11]. In comparison, the 4’-O-methylated

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isomer tamarixetin has relatively weak RS activity because double reducing and

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disproportionation properties are cancelled by 4’-O-conjugation. Since Q metabolites

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modified at the 4’-OH group may not form quinone forms, this metabolic transformation

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generally considerably attenuate not only RS, but also pro-oxidative and associated cytotoxic

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effects of Q.

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Q has ability to regenerate its reducing parent form [40]. The aryloxyl radical intermediate

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Q-O• was postulated to quickly disproportionate to regenerate the parent Q and produce a

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quinone form Q(=O)2 (Scheme 1). A produced quinoid tautomer may further bind

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nucleophilic solvent molecule (e.g. methanol) resulting in regeneration of catechol at the

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ring B which may again exert RS activity [40]. According to the values of reaction parameters

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predicted for formation of aryloxyl radical and its disproportionation in gas and water (Table

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1), recycling to the parent reducing form by disproportionation mechanism, is most

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favourable for Q. In general, at least one of parameters calculated for metabolites (i.e.

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BDFEaq, pKa, ETFEaq, or disproportionation reaction energy) is less favourable than for

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ACCEPTED MANUSCRIPT aglycon Q (Table 1). Such in silico results are in accordance with considerably less in vitro RS

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efficiency of metabolites as compared to their aglycon Q [11,38].

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The disproportionation process favours formation of para-quinone methide in comparison

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with ortho-quinone (Table 1). The four possible tautomers of the doubly oxidized Q may be

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formed and herein we considered only two, ortho-quinone and para-quinone-methide which

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can be formed directly by sequentially donating two H-atoms from the most active 4’- and 3-

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hydroxyl groups (Scheme 1) [9]. The para-quinone-methide is estimated to be energetically

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more favourable tautomer and thus it may be formed also by isomerization of ortho-quinone

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tautomer. The significant role of para-quinone methide in prolongation of RS activity of Q,

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may represent the additional significant contribution of 3-OH group and conjugated C2=C3

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bond to the high RS capacity of Q as well as of Q metabolites with these structural

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characteristics non-modified [41].

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3.2 Influence of phase II metabolic transformations on human serum albumin binding of

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quercetin

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Q and its metabolites were docked into the rigid structure of the extended Sudlow’s BS I

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[17]. The docking space comprised BS I and the adjacent cleft located on another side of the

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fluorescent residue Trp214, among sub-domains IIA, IIB and IIIA [42] (Fig. 2). The ligand

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warfarin in the 2BXD structure of HSA is placed within BS I and its binding mode is denoted

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as warfarin binding mode (WBM). In vitro displacement studies by fluorescence

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spectroscopy, has shown that Q binds via non-covalent interactions to HSA in area close to

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its amino acid residue Trp214, but in different region than warfarin [18,43,44]. The positively

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charged hydrophilic cleft has been suggested to be a binding site of Q and Q-3-O-Glr– [43].

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According to our results, in the predicted binding mode with the most favourable docking

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ACCEPTED MANUSCRIPT score E1 (Table 2), Q is also placed within the cleft with its catechol ring B interacting with

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amino acid residues Ser202 and Trp214 of domain II, and its benzopyranone A/C making H-

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bonds and van der Waals contacts with the amino acid residues Ser454, Leu457, Cys461,

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Leu481, Arg484, Arg485 of the sub-domain IIIA (Fig. 2). However, in the most populated

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binding mode (predicted by 66 of 100 LGA runs) with only somewhat less favourable binding

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score Emp (Table 2), Q is located within BS I and its quercetin binding mode (QBM) partially

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overlaps with WBM (Fig. 2). Thus, Q is expected to be bound by HSA in multiple ways. In

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addition, the binding score Emp for QBM is quite higher than for the strong BS I ligand

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warfarin (Table 2). Hence, Q is expected to be easily replaced by warfarin as it has already

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been noticed in many in vitro studies [43]. The binding score of Q is also less favourable than

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the binding scores for other strong BS I binders of comparable molecular weight:

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indomethacin, piroxicam and ciprofloxacin (Table S2). The calculated binding scores for all

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strong BS I ligands including warfarin are lower than -8.7 kcal/mol. For Q, its three anions as

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well as for its two di-oxidized quinone forms considered (Fig. 1), the most populated modes

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QBM have binding scores quite higher than -8.7 kcal/mol (Table 2).

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Fig. 2. a) Electrostatic surface of HSA illustrating positively charged cleft and upper entrance into BS I. b) The

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extended BS I of HSA (PDB: 2BXD) with binding poses of Q (QBM green; in the cleft cyan) and warfarin (WBM

303

red). Interacting amino acid residues and a few atom distances (---, Å) are marked.

304

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301

305

Q and its metabolites are analogous compounds sharing the same flavone scaffold. Hence,

306

binding scores normalized by molecular weights were used for estimation of contribution of

307

metabolic substituents to binding affinities of HSA for metabolites in reference to the parent

308

aglycon Q (Table 2) [45]. HSA binds Q and its metabolites with generally similar affinity [12].

13

ACCEPTED MANUSCRIPT Deprotonation and methylation of OH groups are estimated to weakly strengthen binding of

310

Q to HSA in agreement with in vitro findings [16]. Among other metabolites, only for the

311

most abundant plasma sulfo-conjugate Q-3’-O-SO3–, somewhat stronger binding to BS I has

312

been predicted. The computed relative binding scores are consistent with the in vitro results

313

according to which the binding affinity of HSA is similar for 3-O- and 7-O-sulfo-conjugates

314

[19], and decreases in the order Q-3’-O- SO3–> Q-3- Glr–> Q-4’- Glr– [12,16].

315

Q, Q-3’-O-SO3– and Q-3-O-Glr– have been demonstrated to show protective effects against

316

peroxynitrite-induced nitration of tyrosine amino acid residues in HSA [13]. Since simple

317

phenolic acids did not inhibit the nitrotyrosine formation, it was assumed that the inhibitory

318

properties of Q, Q-3’-O-SO3– and Q-3-O-Glr– imply their binding to HSA. Herein, it is

319

predicted that sulfo-conjugates and 3-O-glycuronide may bind within BS I (Table 2) what

320

indicates their tentative protective effects against peroxynitrite-induced nitration of tyrosine

321

amino acid residue Tyr150 in the BS I of HSA. Predicted binding modes of Q-3’-O-SO3– and Q-

322

3-O-Glr– within BS I are, however, different pointing to their different protective reaction

323

mechanism. Q-3-O-Glr– is bound within BS I like Q in QBM with the redox active catechol ring

324

B placed close to Tyr150, while Q-3’-O-SO3–is bound in WBM with the B ring positioned at

325

the open side entrance and its redox active 3-OH group placed close to Tyr150 (Fig. 3).

326

Difference in binding mode may also indicate different effects of their HSA binding on their

327

RS activities [12]. In general, binding modes and affinities of doubly oxidized quinone forms

328

of Q and its metabolites are predicted to be like those of the parent species. This implies

329

that RS properties of Q and its metabolites may be retained in the HSA bound state.

AC C

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309

330

14

ACCEPTED MANUSCRIPT 331

Fig. 3. Left: Difference in non-covalent binding of the most abundant human plasma mono-conjugated Q

332

metabolites Q-3-O-Glr (pink) and Q-3’-O-SO3 (yellow) within BS I of HSA. Atom distances (---) are in Å. Right:

333

DFT conformations of the three flexible metabolites.

–

–

334

RI PT

335

In difference to Q and other metabolites, binding of the most of considered sulfo-conjugated

337

species to HSA is predicted to be only within BS I. Their top-ranked clusters with the most

338

favourable binding score are also the most populated ones. They bind in WBM in the way

339

that enables negatively charged sulfo-group to interact with positively charged lysine

340

(Lys195, Lys199) and arginine (Arg218, Arg222) residues at the side entrance to BS I (Fig. 3).

341

Since they are estimated to be bound with weaker or similar affinity than warfarin (Table 2),

342

warfarin and other strong binders are expected to displace them and, consequently, they

343

may be released in blood. Similarly, the negatively charged glucuronyl group of Q-3-O-Glr– is

344

placed at the same position as the –O-SO3– group in both QBM and WBM (Fig. 3). However,

345

the isomer 4’-O-Glr– is predicted to protrude outside BS I into adjacent cleft due to its

346

stretched shape (Fig. 3). Relatively weaker HSA binding of glucuronyl-conjugates is in

347

accordance with their distribution by blood cells. The metabolite 3-O-Glr–accumulates within

348

the activated macrophages. Macrophages transport it to the acidic inflammatory sites where

349

β-glucosidase releases the aglycon which exerts antioxidant and other biological effects [9].

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AC C

350

SC

336

351

4. Conclusions

352

Direct radical scavenging capacities and HSA binding of major mono- and di-conjugated

353

metabolites of the abundant plant polyphenol Q (Fig. 1) were studied by using DFT

354

((U)B3LYP/6-31+G(d,p)) and molecular docking computational approaches, respectively

355

[20,27,28]. The valuable mechanistic insights in contributions of the conjugated metabolites

15

ACCEPTED MANUSCRIPT to in vitro and in vivo activities of Q have been gained and supported by wet experimental

357

results available in literature [10-13,19].

358

Since phase II conjugation transformations of Q occur mainly at its radical scavenging –OH

359

groups, their attenuation effect on RS capacity of Q is primarily due to a reduced number of

360

active –OH groups, that is H-atoms that may participate in RS. However, at least one of the

361

calculated RS reaction parameters: BDFEaq (HAT), pKa, ETFEaq (SPLET), or aryloxyl radical

362

disproportionation energy (Scheme 1), was also found to be less favourable for metabolites

363

as compared to their aglycon Q (Table 1) [27]. The aryloxyl radical disproportionation

364

reaction resulting in the formation of the para-quinone methide product, was revealed to

365

contribute considerably to the extent of RS for unmodified Q. Such a finding may contribute

366

to the understanding of the importance of the free 3-OH group and conjugated C2=C3 bond

367

for unusually high RS capacity of Q [41].

368

The impact of the metabolic transformations on RS capacity and HSA binding of Q was found

369

to depend upon both, conjugation type and position. The dominant RS mechanism was

370

found to change upon conjugation. For example, in aqueous medium at physiological pH, the

371

dominant RS mechanism of Q is SPLET, while for its metabolites Q-7-O-SO3– and Q-4’-O-Glr–,

372

due to decreased OH acidity, it is HAT, which implies that these species target different free

373

radicals.

374

Most of Q metabolites were predicted to be bound by HSA in the area close to its

375

fluorescent amino acid residue Trp214, non-specifically, at multiple sites with similar and

376

relatively low affinity. Only sulfo-conjugates with anchoring interactions of their –O-SO3–

377

group with positively charged lysine and arginine residues at the side entrance to BS I, were

378

found to bind only within the BS I, similarly to warfarin, but with lower binding affinity.

379

Accordingly, albumin binding is not expected to contribute considerably to body distribution

AC C

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SC

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356

16

ACCEPTED MANUSCRIPT of Q and its metabolites. The other ways of distribution such as uptake by macrophages is

381

expected to be more important [9]. Since the double oxidized quinoid forms share the same

382

binding mode and have similar or higher binding affinities as corresponding parent reducing

383

species (Table 2), RS properties of Q and its metabolites is expected to be similar in their HSA

384

bound state.

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380

385

Supplementary material

387

The supplemental file includes radical scavenging data for phenolic acid catabolites, binding

388

parameters for known ligands of HSA binding site I and spin density distributions for neutral

389

monoradicals of Q and its metabolites. The Cartesian coordinates for DFT optimized

390

structures used for calculations of parameters given in Table 1, are provided in the sdf

391

format.

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386

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392

Conflicts of interest

394

There are no conflicts to declare.

395

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393

Acknowledgment The research was supported by the basic funding of the Croatian Ministry of

397

Science and Education, the program of The Scientific Centre of Excellence for Marine Bioprospecting

398

–BioProCro (Competitiveness and Cohesion Operational Program, European Regional Development

399

Fund KK.01.1.1.01), as well as by the projects of the Ministry of Science of the Republic of Serbia

400

(172015 and 174028). All authors are grateful to Jelena Đorović for participating in computations.

401

Croatian authors are thankful to Croatian Ministry of Science and Education for supporting the

402

computational cluster Isabella (http://www.srce.unizg.hr/en/usluge/isabella-cluster).

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403 404

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ACCEPTED MANUSCRIPT References

[1] A. Vogiatzoglou, A.A. Mulligan, M.A. Lentjes, R.N. Luben, J.P. Spencer, H. Schroeter, K.T. Khaw, G.G. Kuhnle, Flavonoid intake in European adults (18 to 64 years), PLoS One, 10 (2015) e0128132 [2] D. Del Rio, A. Rodriguez-Mateos, J.P.E. Spencer, M. Tognolini, G. Borges, A. Crozier, Dietary

chronic diseases, Antioxid. Redox Signal. 18 (2013) 1818-1892.

RI PT

(poly)phenolics in human health: Structures, bioavailability,and evidence of protective effects against

[3] J. Lee, A.E. Mitchell, Pharmacokinetics of quercetin absorption from apples and onions in healthy

humans, J. Agric. Food Chem. 60 (2012) 3874-3881.

[4] T. Walle, U.K. Walle, P.V. Halushka, Carbon dioxide is the major metabolite of quercetin in

SC

humans, J. Nutr. 131 (2001) 2648–2652.

[5] A.J.Day, F. Mellon, D. Barron, G. Sarrazin, M.R. Morgan, G. Williamson, Human metabolism of

M AN U

dietary flavonoids: Identification of plasma metabolites of quercetin, Free Radic. Res. 35, (2001) 941952.

[6] A. Amić, B. Lučić, V. Stepanić, Z. Marković, S. Marković, J.M. Dimitrić Marković, D. Amić, Free

radical scavenging potency of quercetin catecholic colonic metabolites: Thermodynamics of 2H+/2eprocesses, Food Chem. 218 (2017) 144-151.

[7] H. van der Woude, M.G. Boersma, J. Vervoort, I.M. Rietjens, Identification of 14 quercetin phase II

TE D

mono- and mixed conjugates and their formation by rat and human phase II in vitro model systems, Chem. Res. Toxicol. 17 (2004) 1520-1530.

[8] Y.Y. Lee-Hilz, M. Stolaki, W.J. van Berkel, J.M. Aarts, I.M. Rietjens, Activation of EpRE-mediated gene transcription by quercetin glucuronides depends on their deconjugation, Food Chem. Toxicol.

EP

46 (2008) 2128-2134.

[9] V. Stepanić, A. Čipak Gašparović, K. Gall Trošelj, D. Amić, N. Žarković, Selected attributes of polyphenols in targeting oxidative stress in cancer, Curr. Top. Med. Chem. 15 (2015) 496-509.

AC C

405

[10] M. Dueñas, F. Surco-Laos, S. Gonzalez-Manzano, A.M. Gonzalez-Paramas, C. Santos-Buelga, Antioxidant properties of major metabolites of quercetin, Eur. Food Res. Technol. 232 (2011) 103111.

[11] P. Goupy, C. Dufour, M. Loonis, O. Dangles, Quantitative kinetic analysis of hydrogen transfer reactions from dietary polyphenols to the DPPH radical, J. Agric. Food Chem. 51 (2003) 615-622. [12] K.M. Janisch, G. Williamson, P. Needs, G.W. Plumb, Properties of quercetin conjugates: modulation of LDL oxidation and binding to human serum albumin, Free Radic. Res. 38 (2004) 877884.

18

ACCEPTED MANUSCRIPT [13] A. Yokoyama, H. Sakakibara, A. Crozier, Y. Kawai, A. Matsui, J. Terao, S. Kumazawa, K. Shimoi, Quercetin metabolites and protection against peroxynitrite-induced oxidative hepatic injury in rats, Free Radic. Res. 43 (2009) 913-921. [14] A.J. Day, Y. Bao, M.R. Morgan, G. Williamson, Conjugation position of quercetin glucuronides and effect on biological activity, Free Radic. Biol. Med. 29 (2000) 1234-1243.

RI PT

[15] Y. Steffen, C. Gruber, T. Schewe, H. Sies, Mono-O-methylated flavanols and other flavonoids as inhibitors of endothelial NADPH oxidase, Arch. Biochem. Biophys. 469 (2008) 209-219.

[16] K. Beekmann, L. Actis-Goretta, P.J. van Bladeren, F. Dionisi, F. Destaillats, I.M. Rietjens, A stateof-the-art overview of the effect of metabolic conjugation on the biological activity of flavonoids,

SC

Food Funct. 3 (2012) 1008-1018.

[17] J. Ghuman, P.A. Zunszain, I. Petitpas, A.A. Bhattacharya, M. Otagiri, S. Curry, Structural basis of the drug-binding specificity of human serum albumin, J. Mol. Biol. 353 (2005) 38-52.

M AN U

[18] D.W. Boulton, K.U. Walle, T. Walle, Extensive binding of the bioflavonoid quercetin to human plasma proteins, J. Pharm. Pharmacol. 50 (1998) 243-249.

[19] C. Dufour, O. Dangles, Flavonoid-serum albumin complexation: determination of binding constants and binding sites by fluorescence spectroscopy, Biochim. Biophys. Acta. 1721 (2005) 164173.

TE D

[20] M. Skrt, E. Benedik, C. Podlipnik, N.P. Ulrih, Interactions of different polyphenols with bovine serum albumin using fluorescence quenching and molecular docking. Food Chem. 135 (2012) 24182424.

[21] V.C. de Boer, A.A. Dihal, H. van der Woude, I.C. Arts, S. Wolffram, G.M. Alink, I.M. Rietjens, J.

EP

Keijer, P.C. Hollman, Tissue distribution of quercetin in rats and pigs, J. Nutr. 135 (2005) 1718-1725. [22] D.J. Jones, J.H. Lamb, R.D. Verschoyle, L.M. Howells, M. Butterworth, C.K. Lim, D. Ferry, P.B. Farmer, A.J. Gescher, Characterisation of metabolites of the putative cancer chemopreventive agent

AC C

quercetin and their effect on cyclo-oxygenase activity, Br. J. Cancer. 91 (2004) 1213-1219. [23] A.D. Becke, Density-functional thermochemistry. III. The role of exact exchange, J. Chem. Phys. 98 (1993) 5648-5652.

[24] S.H. Vosko, L. Wilk, M. Nusair, Accurate spin-dependent electron liquid correlation energies for local spin density calculations: A critical analysis, Can. J. Phys. 58 (1980) 1200-1211. [25] P.C. Hariharan, J.A. Pople, The influence of polarization functions on molecular orbital hydrogenation energies, Theo. Chim. Acta. 28 (1973) 213-222. [26] M.J. Frisch, J.A. Pople, J.S. Binkley, Self-consistent molecular orbital methods. 25. Supplementary functions for Gaussian basis sets, J. Chem. Phys. 80 (1984) 3265-3269.

19

ACCEPTED MANUSCRIPT [27] V. Stepanić, K. Gall Trošelj, B. Lučić, Z. Marković, D. Amić, Bond dissociation free energy as a general parameter for flavonoid radical scavenging activity, Food Chem. 141 (2013) 1562-1570. [28] L. Lespade, S. Bercion, Theoretical investigation of the effect of sugar substitution on the antioxidant properties of flavonoids, Free Radic. Res. 46 (2012) 346-358. [29] M.T. Cancès, B. Mennucci, J. Tomasi, New integral equation formalism for the polarizable

RI PT

continuum model: Theoretical background and applications to isotropic and anisotropic dielectrics, J. Chem. Phys. 107 (1997) 3032-3304.

[30] E.D. Glendening, C.R. Landis, F. Weinhold, NBO 6.0: Natural bond orbital analysis program, J. Comput. Chem. 34 (2013) 1429-1437.

SC

[31] J.S. Wright, E.R. Johnson, G.A. Di Labio, Predicting the activity of phenolic antioxidants:

theoretical method, analysis of substituent effects, and application to major families of antioxidants, J. Am. Chem. Soc. 123 (2001) 1173-1183.

M AN U

[32] G. Litwinienko, K.U. Ingold, Solvent effects on the rates and mechanisms of reaction of phenols

with free radicals, Acc. Chem. Res. 40 (2007) 222-230.

[33] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, et al. (2004) Gaussian 03, Revision E.01. Wallingford CT: Gaussian, Inc.

[34] G.M. Morris, D.S. Goodsell, R.S. Halliday, R. Huey, W.E. Hart, R.K. Belew, A.J. Olson, Automated

Chem. 19 (1998) 1639-1662.

TE D

docking using a Lamarckian genetic algorithm and empirical binding free energy function, J. Comput.

[35] Marvin 6.0.0. http://www.chemaxon.com Accessed 1 December 2017. [36] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell, A.J. Olson,

EP

AutoDock4 and AutoDockTools4: automated docking with selective receptor flexiblity, J. Comput. Chem. 16 (2009) 2785-2791.

[37] The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC

AC C

[38] D. Villaño, M.S. Fernández-Pachón, M.L. Moyá, A.M. Troncoso, M.C. García-Parrilla, Radical scavenging ability of polyphenolic compounds towards DPPH free radical, Talanta 71 (2007) 230-235. [39] J. Baell, M.A. Walters, Chemistry: Chemical con artists foil drug discovery, Nature 513 (2014) 481-483.

[40] O. Dangles, G. Fargeix, C. Dufour, One-electron oxidation of quercetin and quercetin derivatives in protic and non protic media, J. Chem. Soc., Perkin Trans. 2, 12 (1999) 1387-1395. [41] P. Trouillas, P. Marsal, D. Siri, R. Lazzaroni, J.L. Duroux, A DFT study of the reactivity of OH groups in quercetin and taxifolin antioxidants: the specificity of the 3-OH site, Food Chem. 97 (2006) 679688.

20

ACCEPTED MANUSCRIPT [42] A. Bujacz, K. Zielinski, B. Sekula, Structural studies of bovine, equine, and leporine serum albumin complexes with naproxen, Proteins 82 (2014) 2199-2208. [43] H. Rimac, C. Dufour, Ž. Debeljak, B. Zorc, M. Bojić, Warfarin and flavonoids do not share the same binding region in binding to the IIA subdomain of human serum albumin, Molecules 22 (2017) 1153.

RI PT

[44] B. Sengupta, P.K. Sengupta, The interaction of quercetin with human serum albumin: a fluorescence spectroscopic study, Biochem. Biophys. Res. Commun. 299 (2002) 400-403.

[45] C. Abad-Zapatero, Ligand efficiency indices for effective drug discovery, Expert Opin. Drug

EP

TE D

M AN U

SC

Discov. 2 (2007) 469-488.

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Fig. 1. Quercetin and its conjugated metabolites detected in vivo and/or in vitro and considered here in silico.

21

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ACCEPTED MANUSCRIPT

Scheme 1 Assumed RS mechanisms of Q and its metabolites: (a) hydrogen atom transfer (HAT) and (b) •

sequential proton loss electron transfer (SPLET). In double HAT pathway (a), aryloxyl monoradical Q-O is an intermediate and quinoid specie Q(=O)2 is a final product. (c) Disproportionation of intermediate radical Q-O

AC C

EP

TE D

regenerates the parent reducing form Q-OH.

•

22

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ACCEPTED MANUSCRIPT

Fig. 2. a) Electrostatic surface of HSA illustrating positively charged cleft and upper entrance into BS I. b) The

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extended BS I of HSA (PDB: 2BXD) with binding poses of Q (QBM green; in the cleft cyan) and warfarin (WBM

AC C

EP

TE D

red). Interacting amino acid residues and a few atom distances (---, Å) are marked.

Fig. 3. Left: Difference in non-covalent binding of the most abundant human plasma mono-conjugated Q –

–

metabolites Q-3-O-Glr (pink) and Q-3’-O-SO3 (yellow) within BS I of HSA. Atom distances (---) are in Å. Right: DFT conformations of the three flexible metabolites.

23

ACCEPTED MANUSCRIPT

Table 1 In silico parameters (kcal/mol) for estimating relative RS capacities for the most reactive 4’-OH and 3-OH groups of Q and its major

a

b

BDEg,2

Q

4’

74.2

76.2/72.9

Q-3’-OCH3

4’

81.8

Q-3’-O-SO3

4’

Q-3-O-SO3

−

− Q-7-O-SO3

b

c

∆GDISPRO,aq

In vitro RS activities expressed as TEAC values

2.1/-1.2

-4.8/-6.7

408.3 ; 7.52// 3.07

-8.4

-13.2

2.13// 1.82

-24.4

-36.7

93.0/ 452.7 ; 1.83 //1.42

6.5

-0.06

Basicityaq (pKa)

71.6

66.8/64.9

8.1 (6.0)

70.8

73.4

76.7

63.6

11.8 (8.6)

72.3

93.9

69.5

89.8

53.0

7.3 (5.3)

89.8

4’

73.7

69.6

68.7

68.0

2.7 (2.0)

73.3

4’

69.2

74.3/69.8

73.3

66.8/65.6

12.8 (9.3)

67.8

5.1

-6.4

4’

81.6

71.7

72.3

68.3

8.0 (5.9)

68.2

6.2

-0.1

331.7/ 131.1 ; 3.76 //0.72

Q

3

80.8

66.3

70.3

66.2

9.5 (7.0)

68.2

-14.5

-4.1

7.52// 3.07

Q-3’-O- Me

3

80.5

74.7

70.2

70.1

8.9 (6.6)

68.6

-5.8

-0.1

2.13// 1.82

Q-4’-O- Me

3

82.1

/

69.1

/

9.1 (6.7)

67.3

/

/

3.39// 1.65

Q-3’-O- SO3

3

78.4

85.0

71.1

71.7

9.6 (7.1)

68.8

0.8

-1.6

1.83 //1.42

−

3

77.9

61.1

71.3

67.6

12.5 (9.2)

66.1

-16.8

-3.7

Q-3-O-Glr

−

−

Q-7-O- SO3

−

d

ETFEaq

∆HDISPRO,g

BDFEaq,2

−

BDFEaq,1

SC

BDEg,1

M AN U

Active OH

TE D

MOLECULE

RI PT

metabolites in gas (g, non-polar) and aqueous (aq, polar) phases as well as in vitro measured RS parameters.

e

f

f e

f

e

f

f f f f

e

AC C

EP

111.7/258.6 Q-4’-O-Glr 3 78.2 / 69.3 / 24.2 (17.8) 75.5 / / a b Active group for the first HAT or deprotonation. The second consecutive HAT process is assumed to occur at the other of these two OH-groups (Scheme 1). BDEg and BDFEg values were calculated by using corresponding B3LYP/6-31+G** values for H-atom of -0.497912 a.u. and 0.510927 a. u., respectively. Index 1 or 2 indicates the first c θ • θ + θ − and second step of double HAT mechanism. Values pKa and ETFEaq correspond to the first SPLET process. For ΔG hyd(H ), ΔG hyd(H ) and ΔG hyd (e ), the experimental values d st nd of 27.8 kJ/mol, -1104.62 kJ/mol and -148.5 kJ/mol were used, respectively [23]. The 1 / 2 value correspond to the formation of ortho-quinone and para-quinone e − methide, respectively (Scheme 1). Lag time for LDL oxidation /min at 2.0 μM of free / HSA bound compound [12]. Values for free/ HSA bound Q-7-O-Glr and 3'-O-Me-Q-3– f O-Glr in min are 421.7 / 279.9 and 98.3 /330.9, respectively. Values determined by ABTS//FRAP assays [10]. For comparison ABTS/FRAP TEAC values for HCA are (2.83 / 0.08).

1

ACCEPTED MANUSCRIPT

metabolites. The most stable cluster C

E1

C

7

-8.09

–

C

13

Q-3- O

–

C

Q-7- O

–

-7.21

1.00

82

-7.37

1.03

83

-7.69

1.07

% conf.

1.00

QBM

66

-8.68

1.08

QBM

3

-8.12

1.01

QBM

C

5

-8.08

1.00

Q-o- quinone

C

1

-8.96

1.11

Q-p- quinone methide

QBM

68

-7.98

0.99

0.95

c

Q Q-4'-O

C

1

-8.06

–

C

1

-9.32

–

C

3

-9.36

–

Q-3'-O-Me-7- O

C

14

-8.76

Q-3'-O-Me-p-qm

QBM

65

-7.71

Q-4'-O-Me

Q-3'-O-Me-3- O

Q-3'-O-Me-4'- O

-7.63

1.06

QBM

74

-7.54

1.05

QBM

68

-7.98

1.11

QBM

57

-7.27

0.96

QBM

81

-7.83

1.04

1.11

QBM

72

-7.94

1.06

1.04

QBM

19

-7.58

1.01

0.92

QBM

65

-7.71

1.03

3

-8.55

1.01

QBM

27”

-7.43

0.98

C

2

-8.26

0.98

QBM

43

-7.94

1.06

–

QBM

31

-7.72

0.91

QBM

31

-7.72

1.03

WBM

81

-8.47

0.83

WBM

81

-8.47

0.93

–

C

13

-8.91

0.88

WBM

60

-8.5

0.94

–

WBM

88

-8.96

0.88

WBM

88

-8.96

0.99

C

5

-8.66

0.85

WBM

61

-8.24

0.91

Q-4'-O-Me-7- O

Sulfated metabolites −

Q-3-O-SO3 −

Q-3- O-SO3 4'- O O

− Q-3-O-SO3 -o-quinone

AC C

C –

Q-4'-O-Me-3- O

− Q-3-O-SO3 -7-

79

1.10

EP

Q-3'-O-Me

Emp

QBM

TE D

Methylated metabolites

2

Emp,rel

Site

Metabolite

E1,rel

b

SC

% conf.

The most populated cluster c

M AN U

Site

b

RI PT

Table 2 Predicted binding modesa and scores (kcal/mol)b as well as in vitro determined binding parameters for Q and its major conjugated

In vitro HSA binding parameters c d

5.73 ; 50

58

e

e

ACCEPTED MANUSCRIPT

inv

31

-8.24

0.81

WBM

WBM

inv

53

-8.65

0.85

WBM

WBM

inv

WBM

−

Q-7-O-SO3 -o-q − Q-7-O-SO3 -p-qm − Q-3'-O-SO3

39

-7.73

0.85

inv

53

-8.65

0.96

inv

67

-8.29

0.92

13

-8.75

0.86

WBM

WBM

47

-8.54

0.84

WBM

47

Q-3'-O-SO3 -3- O

–

WBM

40

-9.12

0.90

WBM

40

− Q-3'-O-SO3 -4'-

–

WBM

52

-9.25

0.91

WBM

52

WBM

45

-8.93

0.88

WBM

45

WBM

64

-8.76

0.85

WBM

WBM

15

-9.15

0.72

− Q-3'-O-SO3 -7

O

O

–

— Q-3'-O-SO3 -p-qm

Q-3-O-Glr

– –

QBM

50

-9.11

0.71

–

–

WBM

21

-9.47

0.74

WBM

27

-10.06

0.79

WBM

6

-6.84

0.54

QBM

36

-9.15

0.70

WBM

68

-8.83

Q-3-O-Glr -7- O –

Q-3-O-Glr -o-q Q-4'-O-Glr

–

3'-O-Me-Q-3O-Glr

–

R-Warfarin a

1.07

inv

WBM- warfarin binding mode; QBM – quercetin binding mode, C- cleft, WBM b

0.94

-9.12

1.01

-9.25

1.02

-8.93

0.98

-8.76

0.96

e

87

d

4.28

d

QBM

37

-8.11

0.71

QBM

50

-9.11

0.80

QBM

34

-8.51

0.75

QBM

29

-9.05

0.80

C

15

-6.33

0.56

20.05

QBM

36

-9.15

0.78

12.92

WBM

68

-8.83

1.20

TE D

–

Q-3-O-Glr -4' O

64

M AN U

Glucuronidated

-8.54

SC

−

inv

RI PT

−

Q-7-O-SO3

8.54

d d

– flipped WBM, with the ring B at the position of the coumarin core, and benzopyrone at

c

EP

the entrance side; E1 and Emp are the lowest binding energies in the first and most populated clusters of binding conformations, respectively. % conf. denotes the number of conformations in the corresponding cluster. The binding energies E1 and Emp normalized with molecular weights and expressed relative to corresponding normalized d

e

3

-1

AC C

values for Q, in order to estimate contributions to binding affinity per mass unit. Concentration required for 50% fluorescence quenching of Trp214 [12]. The ligand–HSA o

binding constants (10 M ) for compounds forming fluorescent intermolecular complexes (pH 7.4 phosphate–NaCl buffer, 25 C) [19].

3

ACCEPTED MANUSCRIPT Highlights Radical scavenging (RS) properties of quercetin (Q) are altered by metabolism.

-

Dominant RS mechanism of metabolites depends on conjugation type and site.

-

Disproportionation regeneration of scavenging form is most efficient for aglycon.

-

Only sulfoconjugates bind specifically to human serum albumin (HSA).

-

RS properties of Q and its metabolites may be retained in the HSA bound state.

AC C

EP

TE D

M AN U

SC

RI PT

-