H/D Exchange Processes in Flavonoids: Kinetics and Mechanistic Investigations

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Article H/D Exchange Processes in Flavonoids: Kinetics and Mechanistic Investigations

Federico Bonaldo 1, Fulvio Mattivi 2 , Daniele Catorci 1,†, Panagiotis Arapitsas 3 and Graziano Guella 1,*

1 Bioorganic Chemistry Laboratory, Department of Physics, University of Trento, 38123 Trento, Italy; [email protected] (F.B.); [email protected] (D.C.) 2 Department of Cellular, Computational and Integrative Biology-CIBIO and C3A, University of Trento, 38123 Povo Trento, Italy; [email protected] 3 Department of Food Quality and Nutrition, Research and Innovation Centre, Fondazione Edmund Mach (FEM), 38098 San Michele all’Adige, Italy; [email protected] * Correspondence: [email protected] † D. Catorci passed away on 27 June 2020 while this manuscript was in the latest steps of preparation.

Abstract: Several classes of flavonoids, such as anthocyanins, flavonols, flavanols, and flavones, undergo a slow H/D exchange on aromatic ring A, leading to full deuteration at positions C(6) and C(8). Within the flavanol class, H-C(6) and H-C(8) of catechin and epicatechin are slowly exchanged

in D2O to the corresponding deuterated analogues. Even quercetin, a relevant flavonol representative, shows the same behaviour in a D2O/DMSOd6 1:1 solution. Detailed kinetic measurements of these H/D exchange processes are here reported by exploiting the time-dependent changes of their peak areas in the 1H-NMR spectra taken at different temperatures. A unifying reaction mechanism is also proposed based on our detailed kinetic observations, even taking into account pH and solvent effects. Molecular modelling and QM calculations were also carried out to shed more light on several molecular details of the proposed mechanism. Keywords: H/D isotopic exchange; flavonoids; kinetics; reaction mechanism; NMR spectroscopy Citation: Bonaldo, F.; Mattivi, F.; Catorci, D.; Arapitsas, P.; Guella, G. H/D Exchange Processes in Flavonoids: Kinetics and Mechanistic Investigations. Molecules 2021, 26, 1. Introduction 3544. https://doi.org/10.3390/ Flavonoids are polyphenolic compounds that are diverse in both chemical structure molecules26123544 and properties. Since flavonoids are naturally present in fruits, vegetable, and tea, they are an integral part of the human diet. It is generally accepted that diary flavonoids have Received: 20 April 2021 important biological effects [1,2]. Accepted: 8 June 2021 Flavonoids often demonstrate phytoalexin and antioxidant properties, important for Published: 10 June 2021 promoting abiotic and biotic stress tolerance. The antioxidant activity of flavonoids depends upon the arrangement of functional groups on the molecular skeleton [3,4]. Flavonoids are Publisher’s Note: MDPI stays neutral divided into several classes of compounds based on substituents and the functional groups with regard to jurisdictional claims in pattern on the shared backbone (Scheme1). published maps and institutional affil- The key feature that gives flavonoids most of their characteristic properties seems to iations. be an unusually high local concentration of ortho-phenolic hydroxyl groups [5]. In later years, great importance has been given to the reactivity of the aromatic rings of flavonoids, due to their peculiar hydroxyl substitution pattern towards other organic and inorganic molecules. Two o-hydroxyl groups and an ether-oxygen atom are attached to ring A, in an Copyright: © 2021 by the authors. alternated fashion, and two or more hydroxyl groups are attached to ring B generating a Licensee MDPI, Basel, Switzerland. very unique reactivity on the carbon sites of the aromatic rings. Based on polyhydroxyl This article is an open access article configuration on the benzene rings of flavonoids, the B ring is considered to be prone to distributed under the terms and redox processes (catechol to o-quinone conversion) [6,7], whilst ring A would be much conditions of the Creative Commons more reactive to electrophilic substitutions at the strongly activated nucleophilic sites C(6) Attribution (CC BY) license (https:// and C(8). Among the flavonoids, structural changes in the B ring are also considered to creativecommons.org/licenses/by/ influence the antioxidant activity. 4.0/).

Molecules 2021, 26, 3544. https://doi.org/10.3390/molecules26123544 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 3544 2 of 16 Molecules 2021, 26, x FOR PEER REVIEW 2 of 17

Scheme 1. Scheme showing the studied chemical species from ﬂavanolflavanol (1–2)) andand ﬂavonolflavonol ((33)) classes.classes.

TheWe recentlykey feature described, that gives within flavonoids the class most of flavan-3-ols,of their characteristic an interesting properties case ofseems ring to C bereactivity an unusually towards high sulfite local addition concentration leading toof theortho corresponding-phenolic hydroxyl 4-sulfonated groups analogues [5]. In later [8]. years,During great this importance investigation, has we been also given observed to the the reactivity slow disappearance of the aromatic of therings NMR of flavonoids, signals of dueH-C(6) to their and H-C(8) peculiar in ringhydroxyl A when substitution D2O was usedpattern as atowards solvent forother the organic flavan-3-ol and substrates,inorganic molecules.evidently due Two to o solvent-mediated-hydroxyl groups H/D and exchangean ether- atoxygen these positions.atom are attached In fact, during to ring kinetic A, in anmeasurements alternated fashion, of sulfonation and two andor more thermal-induced hydroxyl groups isomerization are attached of to these ring flavanolsB generating [8], wea very observed unique that reactivity H/D exchangeon the carbon occurred, sites of within the aromatic a daytime rings. scale, Based on both on polyhydroxyl the aromatic configurationprotons of ring on A. the benzene rings of flavonoids, the B ring is considered to be prone to redoxWe processes were not (catechol the first to to o observe-quinone this conversion) phenomenon [6,7] in, whilst polyphenols. ring A would Several be studies much morealready reactive reported to electrophilic on the modification substitutions of the at the H-NMR strongly spectra activated of different nucleophilic flavonoids sites C(6) in andthe presenceC(8). Among of deuterated the flavonoids, solvent structural [9]. This exchangechanges in was the firstB ring observed are also in considered catechin-like to influencesubstrates the by antioxidant Kolar et al. [activity.10], after cleavage of methylated procyanidin in a D2O-dioxane solventWe mixture. recently It described, was also reported within the that class specifically of flavan sites-3-ols, 6 and an 8interesting of ring A of case pelargonidin of ring C reactivityand malvidin towards undergo sulfite the addition same exchangeleading to process the corresponding in acidified 4 D-sulfonated2O and/or analogues acidified CD OD, apparently following a keto-enol tautomerism pathway, while sites in other rings [8]. 3During this investigation, we also observed the slow disappearance of the NMR sig- remain unlabelled [11–13]. nals of H-C(6) and H-C(8) in ring A when D2O was used as a solvent for the flavan-3-ol The reactivity of ring A can be chemically related to that of phloroglucinol (1,3,5- substrates, evidently due to solvent-mediated H/D exchange at these positions. In fact, trihydroxybenzene), which is well-known to undergo both O- and C- alkylation indicating during kinetic measurements of sulfonation and thermal-induced isomerization of these an aromatic or carbonylic structure of phloroglucinol, respectively. The position of this flavanols [8], we observed that H/D exchange occurred, within a daytime scale, on both keto-enol equilibrium implies several tautomeric forms and species distribution depends the aromatic protons of ring A. on specific medium and reaction conditions [14,15]. Similarly, deuteration at C(6) and/or We were not the first to observe this phenomenon in polyphenols. Several studies C(8) on ring A can be interpreted, in acidic media, as a first electrophilic attack of D O+ already reported on the modification of the H-NMR spectra of different flavonoids in3 the (or of D2O in neutral conditions) on activated sites of the ring A aromatic structure or as presence of deuterated solvent [9]. This exchange was first observed in catechin-like sub- the result of solvent-mediated acid-base exchange at the α-carbonyl positions of the ring A strates by Kolar et al. [10], after cleavage of methylated procyanidin in a D2O-dioxane sol- keto structure. Totlani et al. [16] suggested that catechin-like substrates could essentially ventreact mixture. via electrophilic It was also aromatic reported substitution that specifically leading sites to H/D6 and exchange 8 of ring [A17 of,18 pelargonidin]. Moreover, andthe two malvidin active undergo sites (6 and the 8) same on ring exchange A have process been proven in acidified to have D different2O and/or behaviours acidified CDtowards3OD, apparently H/D exchange. following Other a keto reports-enol havetautomerism found different pathway, rates while of sites exchange in other for rings the remaintwo sites unlabelled [12,19,20 ],[11 which–13]. can reflect differences in their electron density, triggered by the acidityThe reactivity of the hydroxyl of ring groupsA can be in chemically position 5 andrelated 7. Nam to that et al. of [ 21phloroglucinol] demonstrated (1,3,5 that- trihydroxybenzene), which is well-known to undergo both O- and C- alkylation indicating flavonoids undergo an exchange reaction in deuterated protic solvents (such as CD3OD an aromatic or carbonylic structure of phloroglucinol, respectively. The position of this or D2O) under acidic conditions. This reaction occurs much more slowly under neutral ketoconditions-enol equilibrium as observed implies by following several the tautomeric reaction pathway forms and during species storage distribution of the samples depends at on−20 specific◦C over medium a period and of 12reaction months conditions [21]. These [14 results,15]. Similarly, suggested deuteration the existence at C(6) of different and/or C(8)intermediate on ring A species can be interpreted, (mono-deuterated in acidic species) media, inas additiona first electrophili to the initialc attack substrate of D3O+ and (or ofdi-deuterated D2O in neutral product. conditions) Moreover, on activated other studies sites of convergedthe ring A onaromatic the same structure main outcomeor as the resultleading of to solvent different-mediated rates for acid the-base process exchange at the twoat the sites α-carbonyl C(6) and C(8),positions indicating of the aring quite A ketodifferent structure. electron Totlani density et al on. [ them16] suggested [22]. that catechin-like substrates could essentially react However,via electrophilic since detailedaromatic kinetic substitution data on leading this H/D to H/D exchange exchange process [17,18 for]. Moreover, flavanols thein pure two Dactive2O at sites neutral (6 and pH 8) have on ring never A beenhave reported,been proven we decidedto have different to exploit behaviour the NMRs towardstechnique H/D to obtain exchange robust. Other kinetic reports measurements have found that different would rates allow of us, exchange in turn, for not the only two to sitesreliably [12,19,20] assess the, which kinetic can parameters reflect differences of the process in their but alsoelectron to shed density, light on triggered the mechanistic by the details of the process.

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acidity of the hydroxyl groups in position 5 and 7. Nam et al. [21] demonstrated that flavonoids undergo an exchange reaction in deuterated protic solvents (such as CD3OD or D2O) under acidic conditions. This reaction occurs much more slowly under neutral conditions as observed by following the reaction pathway during storage of the samples at - 20 °C over a period of 12 months [21]. These results suggested the existence of different intermediate species (mono-deuterated species) in addition to the initial substrate and di- deuterated product. Moreover, other studies converged on the same main outcome leading to different rates for the process at the two sites C(6) and C(8), indicating a quite different electron density on them [22]. However, since detailed kinetic data on this H/D exchange process for flavanols in pure D2O at neutral pH have never been reported, we decided to exploit the NMR tech- nique to obtain robust kinetic measurements that would allow us, in turn, not only to Molecules 2021, 26, 3544 3 of 16 reliably assess the kinetic parameters of the process but also to shed light on the mechanistic details of the process.

2. Results and Discussion 2.1. H/D Isotopic Exchange on R Ring-Aing A P Protonsrotons at P Positionsositions C(6) and C(8) of F Flavanolslavanols Although thethe solubilitysolubility in in water water of of simple simple ﬂavanols, flavanols, such such as as catechin catechin (1) ( or1) epicatechinor epicate- (chin2) is ( low2) is ( ≈low10 ( mM),≈10 mM), aqueous aqueous homogeneous homogeneous solutions solutions can becan prepared be prepared and investigatedand investi- gatthroughed through NMR withoutNMR without any apparent any apparent phase separation phase separation along several along days.several When days. they When are theydissolved are dissolved in deuterated in deuterated water (D 2waterO), all (D phenolic2O), all O-Hphenolic protons O-H are protons quickly are exchanged quickly ex- by changeddeuterium by ions deuterium leading toions4a leadingor 5a O-D to labelled4a or 5asubstrates O-D labelled (Scheme substrates2). At the(Scheme concentrations 2). At the ≈ concof theentrations homogenous of the solutions homogenous of 1 and solutions2 achievable of 1 inand D2 2O( achievable10 mM) thein D pH2O was(≈10 measured mM) the 1 pHto be was 6.9 measured (pD = 7.3) to using be 6.9 H-NMR(pD = 7.3) signals using 1 ofH- imidazoleNMR signals as pHof imidazole indicator as [23 pH]. We indicator found [that23]. catechin We found4a thatundergoes catechin a 4a slow undergoes process aof slow deuterium process incorporation of deuteriumleading incorporation to the leadingfully deuterated to the fully ring deuterated A species ring4d through A species the 4d partially through deuterated the partially intermediates deuterated 4b-4cinter-. Similarly, the O-D labelled epicatechin species 5a in D O is slowly converted into 5d, mediates 4b-4c. Similarly, the O-D labelled epicatechin species2 5a in D2O is slowly con- vertedthrough into the 5d intermediates, through the5b intermediates-5c. 5b-5c.

Scheme 2. CatecCatechinhin derivatives: derivatives: O O-D-D labelled labelled (4a (4a), ),mono mono-deuterated-deuterated at atC-8 C-8 (4b (),4b mono), mono-deuterated-deuterated at C at-6 C-6 (4c) (,4c and), and di- di-deuterateddeuterated at C at-6 C-6 and and C-8 C-8 (4d ().4d Similar). Similar structures structures for for epicatechin epicatechin-derivatives-derivatives are are named named 5a–5ad–. d.

0 Although the standard Gibbs free energyenergy differencedifference ((∆∆GG0) of the overall equilibrium process (1):(1): 4a + 2D2O 4d + 2HDO (1) ퟒ퐚 + 2D2O ⇄ ퟒ퐝 + 2HDO (1) is expected to be about zero due to self-compensating enthalpic and entropic effects, the is expected to be about zero due to self-compensating enthalpic and entropic effects, the equilibrium position is completely shifted towards the products since D2O is present in the equilibriumreacting system position in a much is completely larger molar shifted excess towards than 4a ,the thus products leading since to complete D2O is irreversible present in theconversion reacting of system4a into in4d a .much Since larger the free molar energy excess of activation than 4a, thus (∆G‡ leading) of the processto complete (1) is irre- not versibleso high asconversion it would beof 4a expected into 4d for. Since any the C-H/C-D free energy exchange, of activation its rate ( can∆G‡ be) of evaluated the process at (r.t.1) is (or not within so high a smallas it would T range be aroundexpected it) for by any following C-H/C- theD e timexchange, (t) and its rate temperature can be eval- (T) uateddependence at r.t. (or of within the 1H-NMR a small signals T range of around ring A it) aromatic by following protons. the time (t) and temperature (T) dependenceIn fact, at T =of 310 the K, 1H4a-NMRwas observedsignals of to ring undergo A aromatic in a few protons. hours a signiﬁcant D-labelling at C(6) and C(8) as highlighted in Figure1a (blue dotted boxes) by the signiﬁcant decrease 1 of the H-NMR signals H-C(6) (δH6 = 6.13) and H-C(8) (δH8 = 6.05). The same behaviour was shown (Figure1b, blue dotted box) by the corresponding signals of epicatechin ( 5a) (δH6 = 6.15 and δH8 = 6.13). For the overall process 4a → 4d (Figure2a) the time-varying amount of ( 4a + 4b) and

(4a + 4c) were obtained from the NMR area integration of the signals H-C(6) and H-C(8), respectively, and were normalized to the unperturbed area of the ring B protons. Molecules 2021, 26, x FOR PEER REVIEW 4 of 17

In fact, at T = 310 K, 4a was observed to undergo in a few hours a significant D-label- Molecules 2021, 26, x FOR PEER REVIEW 4 of 17 ling at C(6) and C(8) as highlighted in Figure 1a (blue dotted boxes) by the significant decrease of the 1H-NMR signals H-C(6) (δH6 = 6.13) and H-C(8) (δH8 = 6.05). The same behaviour was shown (Figure 1b, blue dotted box) by the corresponding signals of epicatechin In(5a fact,) (δH6 at = T6.15 = 310 and K, δ 4aH8 =was 6.13). observed to undergo in a few hours a significant D-labelling at C(6) and C(8) as highlighted in Figure 1a (blue dotted boxes) by the significant Molecules 2021, 26, 3544 4 of 16 decrease of the 1H-NMR signals H-C(6) (δH6 = 6.13) and H-C(8) (δH8 = 6.05). The same behaviour was shown (Figure 1b, blue dotted box) by the corresponding signals of epicatechin (5a) (δH6 = 6.15 and δH8 = 6.13).

(a) (b)

Figure 1. Downfield region of 1H-NMR spectra of 10 mM solutions of (a) 4a and (b) 5a in D2O taken at different times (T = 310 K).

(a) For the overall process 4a → 4d (Figure 2a) the time-varying(b) amount of (4a + 4b) and

(4a1 + 4c) were obtained from the NMR area integration of the signals H-C(6) and H-C(8), Figure 1. DownfieldDownﬁeld region of of 1HH-NMR-NMR spectra spectra of of 10 10 mM mM solutions solutions of of (a ()a 4a) 4a andand (b ()b 5a) 5a inin D D2O Otaken taken at atdifferent different times times (T respectively, and were normalized to the unperturbed area 2of the ring B protons. (T= 310 = 310 K). K).

For the overall process 4a → 4d (Figure 2a) the time-varying amount of (4a + 4b) and (4a + 4c) were obtained from the NMR area integration of the signals H-C(6) and H-C(8), respectively, and were normalized to the unperturbed area of the ring B protons.

(a) (b)

1 FigureFigure 2.2. HH-NMR-NMR kinetics kinetics data data at at T T = =310 310 K K for for the the deuteration deuteration of of(a) ( acatechin) catechin in inD2 DO2 (O(4a →4a 4d→) and4d) and(b) epicatechin (b) epicatechin in D2 inO (5a → 5d); red-blue points refer to C(8) deuteration whilst green-black points refer to C(6) deuteration. D2O(5a → 5d); red-blue points refer to C(8) deuteration whilst green-black points refer to C(6) deuteration.

(a) By integratingintegrating thethe areaarea of of these these signals, signals, we we evaluated evaluated not( bnot) only only the the time time dependence dependence of theof the disappearance disappearance of 4aof but4a but also also of the of the appearance/disappearance appearance/disappearance of the of the monodeuterated monodeuter- Figure 2. 1H-NMR kinetics data at T = 310 K for the deuteration of (a) catechin in D2O (4a → 4d) and (b) epicatechin in D2O (5a → 5d); red-blue points referD8ated (to4b D8 C(8)) and(4b deuteration) theand monodeuterated the monodeuteratedwhilst green- D6black intermediates D6points intermediates refer to ( 4cC(6)). (deuteration.4c). In fact, at a given time of any kinetics run (Figure3, t = 500 min, T = 305 K), the peakBy deconvolution integrating the of area the signal of these of signals, H-C(6) atweδ Hevaluated= 6.07 allows not only establishing the time dependence the relative contributionsof the disappearance of the residual of 4a but H-C(6) also of of the4a appearance/disappearance(doublet, J = 2.1 Hz) and a of new the singletmonodeuter- signal (centredated D8 ( at4b almost) and the the monodeuterated same frequency D6 as theintermediates former) attributable (4c). to D-8 monodeuterated species 4b.

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In fact, at a given time of any kinetics run (Figure 3, t = 500 min, T = 305 K), the peak deconvolution of the signal of H-C(6) at δH = 6.07 allows establishing the relative contri- Molecules 2021, 26, 3544 butions of the residual H-C(6) of 4a (doublet, J = 2.1 Hz) and a new singlet signal (centred5 of 16 at almost the same frequency as the former) attributable to D-8 monodeuterated species 4b.

FigureFigure 3. 3.1H-NMR 1H-NMR spectral spectral deconvolution deconvolution (GSD)(GSD) ofof peaks corresponding to to sites sites C(6) C(6) (6.07 (6.07 ppm) ppm) and and C(8) C(8) (5.98 (5.98 ppm). ppm). 1H- 1H-NMRNMR spectrum spectrum taken taken at at t t~ ~ 500 500 min min from 4a kinetics experimentexperiment atat TT == 305305 K.K. Red, Red, blue, blue and, and green green curves curves represent represent the the experimentalexperimental1H-NMR, 1H-NMR, deconvoluted deconvoluted peaks, peaks and, and residuals, residuals, respectively. respectively.

ByBy normalizing normalizing to to the the initial initial concentration concentration of of4a 4a, the, the % % molar molar fraction fraction of of these these two two speciesspecies (x (x4a 4a+ + x 4bx4b))was was evaluatedevaluated toto bebe 60% 60% and and peak peak deconvolution deconvolution leads leads to to x x4a4a ≈≈ 22xx44bb. . Correspondingly,Correspondingly, the the peak peak deconvolution deconvolution of of the the signal signal of of H-C(8) H-C(8) at δatH δ=H 5.98= 5.98 allows allows establishingestablishing the the relative relative contribution contribution ofof thethe residual H H-C(8)-C(8) of of 4a4a (doublet,(doublet, J =J 2.1= 2.1 Hz) Hz) and anda new a new singlet singlet signal signal attributable attributable to 4c to. T4che. % The molar % molar fraction fraction of these of two these species two species (x4a + x4c (x4a= 52%)+ x4c can= 52% be) obtained can be obtained by the byrelative the relative area integration area integration,, whilst whilst the peak the peak deconvolution deconvo- lutionleads leads to x4a to ≈ x44ax4c≈. 4x4c. ToT fulfilo fulfil the the mass mass balance balance constrain, constrain, the the overall overall normalized normalized % % distribution distribution was was eval- eval- uateduated to to be be x x4a4a:x:x4b4b:x:x4c:x:x4d4d == 42:20:1:28, 42:20:1:28, indicating indicating different different specific specific rate rate constants constants (i.e. (i.e.,, regi- regioselectivity)oselectivity) at atthe the two two different different sites sites with with a afaster faster H/D H/D exchange exchange at at C(8) C(8) than than at at C(6). C(6). In Inprinciple, principle, the the rela relativetive amount amount of of 4a4a–4–d4d speciesspecies can can be beevaluated evaluated at any at any time time and and anyany fixed 1 fixedtemperature temperature during during the the kinetic kinetic run run.. A Actually,ctually, several several 1HH-NMR-NMR spectraspectra takentaken in in kinetic kinetic runsruns carried carried out out at at higher higher temperature temperature do do not not have have sufficient sufficient resolution resolution to to allow allow an an ade- ade- quatequate deconvolution deconvolution of of H(6) H(6) and and H(8) H(8) signals, signals, but but this this outcome outcome did did not not hinder hinder our our analysis analy- sosis much. so much. The peakThe peak deconvolution deconvolution of the of H-C(6) the H and-C(6) H-C(8) and H signals-C(8) signals allowed allowed us to separateus to sep- the time-dependent contribution of the intermediates 4b and 4c, reaching maximum values arate the time-dependent contribution of the intermediates 4b and 4c, reaching maximum of x4b ≈ 0.20 and x4c ≈ 0.10 at about the same time (Figure4a tmax ≈ 500 min). More values of x4b ≈ 0.20 and x4c ≈ 0.10 at about the same time (Figure 4a tmax ≈ 500 min). More importantly, a single exponential fitting (Figure4) of time, the varying x 4a (t), was obtained importantly, a single exponential fitting (Figure 4) of time, the varying x4a (t), was obtained × −5 −1 with an observed rate constant (T = 305 K) kobs (4a) = 3.90 10−5 −1s (t 1 (4a) = 17,800 s) with an observed rate constant (T = 305 K) kobs (4a) = 3.90 × 10 s (t½ (4a2 ) = 17,800 s) sug- suggestinggesting that that all all the the processes processes contributing contributing to the to the disappearance disappearance of 4a of are4a are(pseudo) (pseudo) first- first-orderorder processes, processes, only only depending depending on onthe the initial initial concentration concentration of of4a 4a(Figure(Figure 4b4).b).

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(a) (b)

FigureFigure 4.4. Experimental kinetic kinetic data data and and best best fitting ﬁtting curves curves for for the the deuteration deuteration processes processes of of4a4a (a)( aat) atT = T 300 = 300 and and (b) (atb) T at = T305 = 305K. K.

Similar data were alsoalso obtainedobtained forfor thethe 5a5a →→ 5d5d process.process. However,However, deuterationdeuteration ofof aromatic protons of 5a inin DD22O (Figure2 2b)b) doesdoes notnot showshow anyany sitesiteselectivity. selectivity.In In fact, fact, the the overall rate of disappearance of the H-6H-6 signals (5a5a ++ 5b5b) waswas almostalmost thethe samesame asas thatthat ofof the H-8H-8 signals (5a5a ++ 5c5c). In this case, we were notnot ableable toto evaluateevaluate atat aa givengiven timetime pointpoint the relative contributioncontribution ofof monodeuteratedmonodeuterated 5b andand 5c5c byby peakpeak deconvolutiondeconvolution sincesince fromfrom one side the differencedifference ofof chemicalchemical shiftshift betweenbetween H-C(6)H-C(6) andand H-C(8)H-C(8) inin epicatechinepicatechin isis soso small to become become comparable comparable to the the JJ(6,8)(6,8) coupling coupling constant constant (leading(leading to to non-first non-first order order NMR signals) and,and, from the other side,side, the signal lineshape was not good enough to allow reliable fittings.fittings. Initially, wewe thoughtthought thatthat thethe kinetickinetic behaviourbehaviour showedshowed byby deuterationdeuteration ofof 4a4a couldcould bebe rationalized by assumingassuming twotwo competitivecompetitive pseudo-first-orderpseudo-first-order conversionsconversions startingstarting fromfrom thethe samesame substratesubstrate ((4a4a →→ 4b inin competitioncompetition withwith 4a4a →→ 4c)) followedfollowed byby twotwo convergentconvergent pseudo-first-orderpseudo-first-order processes leading to the samesame endend productproduct ((4b4b →→ 4d inin additionaddition toto 4c4c → 4d4d).). To To evaluate evaluate the the four four unknown unknown rate rate constants constants (k (k1, 1k,2 k, k2,3, k k34,), k we4), weresorted resorted to the to theKi- Kinetiscopenetiscope software software using, using, as as the the initial initial guess guess for for the the fitting, fitting, the the values values roughly estimated from ourour raw kinetic datadata shownshown inin Figure2 2.. However,However, we we soon soon realized realized that that it it was was almost almost impossible toto findfind aa setset ofof fourfour kkii able toto adequatelyadequately fulfilfulfil ourour experimentalexperimental xxii(t)(t) data.data. NoNo set of four kii was able to fitfit the general trend of these curves and to be in agreement withwith several significant significant constraints constraints imposed imposed by by experimental experimental measures, measures, such such as asthe the time time of the of thecrossing crossing points points of the of curve the curve x4d(t) x 4dwith(t) with that thatof x4a of(t) x (264a(t),800 (26,800 s) and/or s) and/or the time the of time the ofcross- the crossing point of the curve of x (t) with those of x (t) (73,000 s), x (t) (42,200 s), and ing point of the curve of x4b(t) with4b those of x4a(t) (734a,000 s), x4b(t) (424b,200 s), and x4c(t) (≈ x80,0004c(t) ( ≈s).80,000 To find s). an To optimal find an fitting optimal of fittingour data, of ouranother data, process another needed process to needed come into tocome play, intoi.e., a play, process i.e., aable process to convert able to 4a convert into 4d4a withoutinto 4d anywithout detectable any detectable intermediate. intermediate. Thus, we Thus,were somehow we were somehow forced by forcedexperimental by experimental findings to findings suppose to the suppose kinetic the pathway kinetic drawn pathway in drawnScheme in 3, Scheme based 3on, based competitive on competitive-consecutive-consecutive pseudo-first pseudo-first-order-order processes processes.. The time The de- timependence dependence of all the of stable all the species stable is species regulated is regulated by a system by aof systemlinear differential of linear differential equations equations (Scheme3, top) that, with the appropriate boundary conditions and the mass (Scheme 3, top) that, with the appropriate boundary conditions and the mass balance con- balance constrain, lead to the integrated solution xi(t) here reported (Scheme3, bottom). strain, lead to the integrated solution xi(t) here reported (Scheme 3, bottom).

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Scheme 3. Reaction scheme of the five steps involved in the deuteration of catechin by D2O (top, left); differential (top, Scheme 3. Reaction scheme of the ﬁve steps involved in the deuteration of catechin by D2O (top, left); differential (top, right)right) andand integratedintegrated (bottom)(bottom) kinetickinetic lawslaws assumingassuming pseudo-ﬁrst-orderpseudo-first-order processes.processes.

TheThe fitting fitting of of these these integrated integrated equations equations with with the thestochastic stochastic simulator simulator Kinetiscope Kinetiscope leads −5 −1 −5 −1 −5 −1 leadsto the tofollowing the following best fitting best values fitting at values T = 305 at K: T =k1 305= 1.7 K: × k101 = s 1.7, k×2 =10 0.85 ×s 10, ks2 ,= k 0.853 = 1.35× −5−5 −11 −5 −1 −5 −1 −5 −1 −5 −1 −5 −1 10× 10 ss , ,k k4 =3 2.5= 1.35 × 10×s10,ands k5 =, k2.44 =× 10 2.5 ×s ,10 fulfillings , the and condition k5 = 2.4 k×obs10 = (k1 s+ k2,+ fulfillingk3) = 3.90 −5 −1 −5 −1 the× 10 condition s . By introducing kobs = (k1 these+ k2 +values k3) = into 3.90 integrated× 10 s equations,. By introducing we found that these the values calculated into integratedcurves were equations, almost superimposable we found that to the our calculated experimental curves kinetic were d almostata (Figure superimposable 4b). to our experimentalSince, in principle, kinetic the data molar (Figure fraction4b). of every species can be evaluated at every time (and Since,temperature), in principle, we carried the molar out a fractiondetailedof kinetic every analysis species following can be evaluated the time atdepend- every timeence (andof the temperature), NMR-sensitive we species carried 4a out/4b/ a4c detailed performing kinetic 1H- analysisNMR measurements following the at time five dependencedifferent temperatures of the NMR-sensitive (300–320 K) species. Unfortunately,4a/4b/4c theperforming peak deconvolution1H-NMR measurements of the H-C(6) atand five H- differentC(8) signals temperatures was possible (300–320 only at K). 300 Unfortunately, (Figure 4a) and the peak305 K deconvolution (Figure 4b) since of the at H-C(6)higher temperatures and H-C(8) signals the peak was resolution possible only of these at 300 signals (Figure was4a) not and sufficient 305 K (Figure to allow4b) sincerepro- atducible higher deconvolutions. temperatures the peak resolution of these signals was not sufficient to allow reproducibleThus, the deconvolutions. kinetic parameters reported in Table 1 have just an empirical significance, representingThus, the the kinetic observed parameters time dependence reported (k’ inobs Table) of 1the have peak just area an of empirical the H-C(6) significance, (Figure 5a) representingand H-C(8) (Figure the observed 5b) at timefixed dependence temperatures (k’obs and) of not the the peak true area rate of theconstants H-C(6) controlling (Figure5a ) andthe time H-C(8) changes (Figure of5 b)x4a at(t). fixed In other temperatures words, k’obs and (and not the the corresponding true rate constants activation controlling energies) the timeare not changes simply of related x4a(t). to In the other true words, values k’ ofobs the(and three the processes corresponding, whereby activation 4a is converted energies) areto th note intermediates simply related 4b to and the 4c true and/or values to ofthe the final three product processes, 4d. Anyhow, whereby 4atheseis converted empirical todata the are intermediates quite useful 4bsinceand they4c and/or allow us to to the estimate final product how and4d .how Anyhow, much thesethe average empirical site dataselectivity are quite of useful deuteration since they depends allow uson to temperature. estimate how If and we how define much the the ratio average (k’(C6) site − selectivity of deuteration depends on temperature. If we define the ratio (k’(C6) − k’(C8))/

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k’(C8))/(k’(C6) + k’(C8)) at a given temperature as an empirical descriptor of this selectivity, we can notice that it significantly decreases by increasing the temperature, ranging from ≈0.2 (k’(T (C6)= 300+ K) k’(C8) until) at ≈0.1 a given (T = 320 temperature K). This is largely as an empirical expected si descriptornce increasing of this T, selectivity,the differential we canexponential notice that factors it signiﬁcantly regulating decreases the rate constants by increasing ΔΔG the‡/RT temperature, (ΔΔG‡ = ΔG ranging‡(C6) − ΔG from‡(C8)) ≈must0.2 (decrease.T = 300 K ) until ≈0.1 (T = 320 K). This is largely expected since increasing T, the differential ‡ ‡ ‡ ‡ exponential factors regulating the rate constants ∆∆G /RT (∆∆G = ∆G (C6) − ∆G (C8)) Table 1. Kinetic data for deuteriummust decrease. ion incorporation process by heavy water (D2O) on catechin and epicatechin substrates. The values of Gibbs free energy of activation (∆G‡) in the case of epicatechin are theoretically calculated based on a given Tabletemperature 1. Kinetic by datathe Eyring for deuterium equation, ion as incorporation the qualitative process contribution by heavy division water (Dinto2O) enthalpy on catechin and andentropy epicatechin of activation substrates.s. The values of Gibbs free energy of activation (∆G‡) in the case of epicatechin are theoretically calculated based on a given −1 ‡ ‡ (1) ‡ Sitetemperature T [K] by the Eyringk’obs equation, [s ] as the qualitativeEA [kJ/mol] contribution ΔH division[kJ/mol] into enthalpyΔS [kJ/mol and entropy K] of activations.ΔG [kJ/mol] 300 (1.48 ± 0.01) × 10−5 −1 ‡ ‡ (1) ‡ Site 305 T [K] (2.42 ± 0.01) k’obs × [s10−5 ]E A [kJ/mol] ∆ H [kJ/mol] ∆S [kJ/mol K] ∆G [kJ/mol] (2) C(6) 310300 (3.60(1.48 ± 0.02)± 0.01) × 10−5× 10−5 79.7 ± 5.5 77.2 ± 5.5 −0.081 ± 0.017 102.1 ± 7.6 − 315305 (6.72(2.42 ± 0.12)± 0.01) × 10−5× 10 5 (2) −5 C(6) 320310 (10.8(3.60 ± 0.5)± 0.02)× 10−5× 10 79.7 ± 5.5 77.2 ± 5.5 −0.081 ± 0.017 102.1 ± 7.6 315 (6.72 ± 0.12) × 10−5 300 (2.17 ± 0.01) × 10−5 320 (10.8 ± 0.5) × 10−5 305 (3.05 ± 0.01) × 10−5 300 (2.17 ± 0.01) × 10−5 (2) C(8) 310 (4.49 ± 0.02) × 10−5 74.4 ± 9.2 71.9 ± 9.2 −0.095 ± 0.030 101.9 ± 13.1 305 (3.05 ± 0.01) × 10−5 −5 (2) C(8) 315310 (8.91(4.49 ± 0.11)± 0.02) × 10× 10−5 74.4 ± 9.2 71.9 ± 9.2 −0.095 ± 0.030 101.9 ± 13.1 320315 (13.1(8.91 ± 0.6)± 0.11)× 10−5× 10−5 (3) C(6) 310320 (2.93 (13.1± 0.01)± 0.6)× 10×−5 10−5 / / / 102.9 (3)(3) C(8)C(6) 310310 (2.71(2.93 ± 0.01)± 0.01) × 10−5× 10−5 / // // / 102.9103.1 (3) C(8) 310 (2.71 ±(1)0.01) Values× 10 calculated−5 at T =/ 310 K. (2) Catechin /. (3) Epicatechin. / 103.1 (1) Values calculated at T = 310 K. (2) Catechin. (3) Epicatechin.

(a) (b)

FigureFigure 5.5. 1HH-NMR-NMR kinetic kinetic data, data, taken taken at at five five different different temperatures, temperatures, for for H/D H/D exchange exchange at (a at) C(6) (a) C(6) and and(b) C(8) (b) C(8)in the in cate- the chin/D2O reacting system. Here, the site-specific decreasing H-C(6) and H-C(8) signals are plotted in order to obtain k’obs; catechin/D2O reacting system. Here, the site-specific decreasing H-C(6) and H-C(8) signals are plotted in order to obtain although there is no simple relation between these observed rate constants and ki (i = 1,2,3,4,5), these data provide useful k’ ; although there is no simple relation between these observed rate constants and k (i = 1, 2, 3, 4, 5), these data provide informationobs on the site-specific deuteration kinetics. i useful information on the site-specific deuteration kinetics.

It is worth noting that the average ∆G‡ ≈ 100 kJmol−1 of the two competitive processes is quite similar and dominated by the contribution of the enthalpic term (75–80%). As already pointed out, however, these values simply represent the time dependence of the deuterium ion attack both to 4a and 4b for the H/D exchange at C(8) and both to 4a and 4c

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It is worth noting that the average ΔG‡ ≈ 100 kJmol−1 of the two competitive processes

Molecules 2021, 26, 3544 is quite similar and dominated by the contribution of the enthalpic term (75–80%). As9 of al- 16 ready pointed out, however, these values simply represent the time dependence of the deuterium ion attack both to 4a and 4b for the H/D exchange at C(8) and both to 4a and 4c for the H/D exchange at C(6). While in the early part the contributions of 4b and 4c can forbe neglected, the H/D exchange this is not at true C(6). in While the middle in the earlyor late part steps the of contributions these kinetic of runs.4b and 4c can be neglected,A deeper this isinsight not true into in the the mechanistic middle or late details steps of of the these pathway kinetic proposed runs. in Scheme 3 can beA deeperachieved insight by considering into the mechanistic the kinetic details data for of all the the pathway elementary proposed steps in obtained Scheme 3at cantwo be different achieved temperatures, by considering 300 the and kinetic 305 K. data For for the all former, the elementary the fitting steps of obtainedthe curve at x two4a (t) differentleads to temperatures,kobs (4a, T = 300 300 K) and = 2.71 305 × K. 10 For−5 s the−1 (t former,½ (4a) = the25,000 fitting s), ofwith the a curve 30% xdecrease4a (t) leads with to −5 −1 kobs (4a, T = 300 K) = 2.71 × 10 s (t 1 (4a) = 25,000 s), with a 30% decrease with respect respect to the value evaluated at T = 3052 K. The simulation of all the experimental evalu- toated the x valuei(t) affords evaluated the following at T = 305 best K. fitting The simulation values at of300 all K the: k1 experimental= 1.5 × 10−5 s−1, evaluated k2 = 0.75 × x10i(t)−5 −5 −1 −5 −1 affordss−1, k3 = the0.45 following × 10−5 s−1, bestk4 = 2.2 fitting × 10 values−5 s−1, and at 300k5 = K: 2.2 k 1×= 10 1.5−5 s×−1 (Figure10 s 4,a k).2 = 0.75 × 10 s , −5 −1 −5 −1 −5 −1 k3 = 0.45Thus,× k103 decreasess , k4 with= 2.2 the× 10 decreases , of and temperature k5 = 2.2 × 10significantlys (Figure more4a). than the other four Thus,rate constants k3 decreases pointing with theout decrease that 4a undergoes of temperature a different significantly molecular more rearrangement than the other fourbefore rate leading constants the end pointing-product out 4d. that By4a insertingundergoes these a different ki values molecular into the Eyring rearrangement equation beforewe obtained leading the the corresponding end-product 4d.ΔGBy‡, although inserting we these cannot ki values estimate into the the relative Eyring equationcontribu- ‡ wetion obtained of enthalpic the corresponding and entropic activation∆G , although terms. we cannot estimate the relative contribution of enthalpicAnother and interesting entropic feature activation is the terms. pH-dependence of these rate constants. By lowering theAnother pH (addition interesting of featureCD3COOD) is the pH-dependencein the 4a/D2O reacting of these system, rate constants. we observed By lowering as ex- thepected pH (additionthat the rate of CDof the3COOD) deuteration in the 4aprocess/D2O increase reactings system,. Thus, whilst we observed the half as-lifetime expected of that4a at the 300 rate K in of neutral the deuteration solution was process found increases. at about Thus,25 min, whilst it decrease the half-lifetimed at about 6 of min4a atat 300 K in neutral solution was found at about 25 min, it decreased at about 6 min at pH pH 3.0. However, we noticed that k3 became significantly lower than observed in neutral 3.0.pH However,conditions. we At noticed pH 3.0 that(300 kK)3 became, this effect significantly is largely lower offset than(Figure observed 6) by the in neutralincrease pH of conditions. At pH 3.0 (300 K), this effect is largely offset (Figure6) by the increase of k k1 and k2 leading to an observed rate constant significantly larger than in neutral condi-1 andtions. k2 Anotherleading tostri anking observed feature, rate related constant to the significantly pH-induced larger changes than of in these neutral rate conditions. constants, Another striking feature, related to the pH-induced changes of these rate constants, is is represented by the significant increase of the maximum of the curves x4b(t) and x4c(t), representedspeaking for bya longer the significant lifetime of increase the key- ofintermediates the maximum 4b ofand the 4c. curves x4b(t) and x4c(t), speaking for a longer lifetime of the key-intermediates 4b and 4c.

FigureFigure 6.6. ExperimentalExperimental kinetic kinetic data data and and best-ﬁtting best-fitting curves curves obtained obtained by by the the rate rate constants constants optimization optimization for the deuteration process occurring on catechin at T = 300 K in D2O with 55 mM of acetic acid for the deuteration process occurring on catechin at T = 300 K in D2O with 55 mM of acetic acid (CD3COOD), resulting in the calculated pH of 3.0. (CD3COOD), resulting in the calculated pH of 3.0.

To summarize, our explanation relies on the evidence that D2O is strong enough as To summarize, our explanation relies on the evidence that D2O is strong enough as + anan acidacid toto promotepromote deuterationdeuteration byby thethe DD+ transfertransfer toto C(8)C(8) andand strongstrong enoughenough asas aa basebase toto + acceptaccept thethe DD+ ion from DO-C(7).DO-C(7). TheThe effect of acidic catalyst seemseem toto relyrely onon (i)(i) aa relevantrelevant increase of the rate of the competitive processes 4a → 4b and 4a → 4c leading to higher relative amounts of the monodeuterated intermediates 4b and 4c, (ii) a large decrease of the rate of the process 4a → 4d, and iii) an almost unperturbed site selectivity of deuterium ion attack at C(6) or C(8).

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Finally, we carried out kinetic runs at 300 K on 4a using CD3OD in place of D2O as a deuterium ion source for the H/D exchange. The observed rate constant controlling the disappearance of 4a/CD3OD was found much lower than that of 4a/D2O.

2.2. Mechanistic Considerations In a Frontier Molecular Orbital approach (FMO), the mechanism of the rate-determining + steps can be considered as the addition of an electrophilic species (D3O /D2O) to the high- est nucleophilic centres of flavanols, i.e., C(6) or C(8) which are electrons enriched by the multiple ortho and para mesomeric effect of the oxygen atoms on ring A. This attack will occur at the nucleophilic site having the largest electron density. According to 1H and 13C-NMR experimental spectra of 4a, no matter the solvent, both H-C(8) and C(8) are slightly shielded with respect to H-C(6) and C(6) confirming their higher nucleophilicity (Figure1a). Similar data were also obtained for the 5a → 5d process (Figure1b). The main outcomes of our kinetic data on deuteration of simple flavanols such as 4a and 5a in D2O are here summarized: (i) In a first preliminary step, all O-H bonds are deuterium ion exchanged through a very fast process; (ii) at r.t., flavanols undergo slow H/D exchange at C(6) and C(8) or at both sites; (iii) the first step of deuteration occurs at a slightly higher specific rate at C(8) than at C(6); (iv) the second step of deuteration occurs at both sites with quite similar specific rates (k3 ≈ k4), but slightly faster than the first step; (v) the average ∆G‡ (≈102 kJmol−1) of all these deuteration processes is mainly determined by enthalpic effects; (vi) in other protic, but less polar, solvents (such as CD3OD), these processes occur on a significantly longer time-scale. With this kinetic information, we can propose a reasonable reaction mechanism (Scheme4). It relies on sequential elementary steps, involving a slow, solvent-mediated, C-D bond formation/O-D bond-breaking followed by a fast, solvent-mediated, C-H bond breaking/O-D bond formation where ring-A can regain its aromaticity. Although our ‡ observed rate constants (ki, i = 1, 2, 3, 4, 5) and the corresponding ∆G values are not related to true elementary reaction steps (i.e., involving single transition states), they can be considered a simple sequence of elementary steps with one of them as the rate- determining step. Assuming the well-known and largely accepted mechanism involving keto-enol tautomerism of 1,3,5 activated aromatic systems [24], we propose that deuterated keto-analogues (4ab and 4ac) could be the putative transient intermediates leading to the detectable intermediates 4b (k1) and 4c (k2) in the two competitive divergent processes 4a → 4b and 4a → 4c, respectively. Similarly, 4bd and 4cd could be the transient putative intermediates of the two convergent pathways, 4b → 4d regulated by k4 and 4c → 4d regulated by k5 leading to the end-product 4d. However, as already discussed, our data suggest a relevant role played by the direct process 4a → 4d, implying the putative intermediate 5,7-diketo derivative 4ad. Although we can only speculate about the structures of the transition states leading to the above-cited putative intermediates, since the elementary steps dealing with C-D formation/O-D breaking and loss of aromaticity are highly endothermic, they are also expected to be the rate-limiting steps and the structure/energy of the relevant TS similar to the structure/energy of the putative intermediates. Molecules 2021, 26, 3544 11 of 16 Molecules 2021, 26, x FOR PEER REVIEW 11 of 17

Scheme 4. Proposed reaction mechanism for H/D exchange at C(6) and C(8) in the catechin/D2O reacting system. Scheme 4. Proposed reaction mechanism for H/D exchange at C(6) and C(8) in the catechin/D2O reacting system. The plausibility of the termolecular elementary process 4a → 4d relies on the need to introduceAlthough another we can step only converting speculate 4aaboutinto the4d instructures addition of to the the transition bimolecular states elementary leading tosteps the ofabove mono-deuteration.-cited putative Practicallyintermediates, speaking, since the this elementary termolecular steps process dealing is feasible with C due-D formation/Oto the interaction-D breaking between and three loss moleculesof aromaticity (4a +are 2D highly2O) and endothermic, could be energetically they are also com- ex- pectedparable to (if be not the favourable) rate-limiting over steps the and sequential the structure/energy competing bimolecular of the relevant mono-deuteration TS similar to ‡ theprocesses structu [re/energy22], leading of tothe lower putative enthalpic intermediates. contribution (∆H ) of the step 4a → 4ad (assuming withinThe plausibility the Hammond of the postulatetermolecular that elementary the corresponding process transition 4a → 4d relies state on has the a structure need to introducesimilar to another4ad), only step partially converting counterbalanced 4a into 4d in byaddition a greater to the entropic bimolecular term contribution elementary ‡ steps(−T∆ ofS mono), in agreement-deuteration. with Practically the lower speaking, probability this to termolecular arrange two process D2O molecules is feasible in due the tocorrect the interaction positions between for the H/D three exchange molecules event. (4a + 2D2O) and could be energetically comparable The (if not relevant favourable) outcome over that the deuteration sequential processes competing of bimolecular4a occur much mono more-deuteration slowly in processesCD3OD than [22], in leading D2O[25 to] lower can be enthalpic explained contribution in our view by(ΔH the‡) of proton/deuterium the step 4a → 4ad acid-base (assum- ingproperties within the of flavanols Hammond in methanolpostulate that (pKa the≈ 13–14)corresponding with respect transition to water state (pK hasa ≈ a 9–10)structure [26]. similarThe lower to 4ad acidity), only of thepartia O-H(D)lly counterbalanced groups in methanol by a reflectsgreater theentropic reduced term thermodynamic contribution ability of methanol to solvate the ions produced by the proton-transfer reaction. However, (−TΔS‡), in agreement with the lower probability to arrange two D2O molecules in the correctsince the positions loss of proton/deuteriumfor the H/D exchange ions event. is also involved in all the transition states leading to the putative intermediates, we believe that it can play a relevant role also in the kinetics. The relevant outcome that deuteration processes of 4a occur much more slowly in CD2.3.3OD H/D than Isotopic in D Exchange2O [25] can on be Ring-A explained Proton in at our Position view C(8)by the of Quercetinproton/deuterium acid-base properties of flavanols in methanol (pKa ≈ 13–14) with respect to water (pKa ≈ 9–10) [26]. The solubility of quercetin (3) in water is so low (≈3 mM at 300 K) [27] to hinder the The lower acidity of the O-H(D) groups in methanol reflects the reduced thermodynamic NMR measurement of deuterium ion incorporation by D O, which is expected to be slower ability of methanol to solvate the ions produced by the proton2 -transfer reaction. However, than in flavanols. However, in D O/d -DMSO 1:1 solution, the solubility can be enhanced since the loss of proton/deuterium2 ions6 is also involved in all the transition states leading and 10 mM solutions of quercetin in this solvent mixture were appropriate to carry out to the putative intermediates, we believe that it can play a relevant role also in the kinetics. a full kinetic analysis in the temperature range T = 313–333 K. The 4-keto group on ring B strongly affects the electron density on ring-A positions, in particular at C(6) position, 2.3. H/D Isotopic Exchange on Ring-A Proton at Position C(8) of Quercetin in as much that only deuteration at C(8) can be observed in our conditions. Following the similarThe solubility approach of quercetin described (3 above) in water for flavanols, is so low we(≈3 weremM at able 300 to K) evaluate [27] to hinder the Gibbs the NMRfree energies measurement of activation of deuterium (Figure7 )ion for incorporatio H/D exchangen by at D C(8)2O, inwhich3 by is running expected the to same be slowerreaction than at different in flavanols temperatures. However, (Table in D22).O/d6-DMSO 1:1 solution, the solubility can be enhanced and 10 mM solutions of quercetin in this solvent mixture were appropriate to

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carry out a full kinetic analysis in the temperature range T = 313–333 K. The 4-keto group on ring B strongly affects the electron density on ring-A positions, in particular at C(6) position, in as much that only deuteration at C(8) can be observed in our conditions. Fol- lowing the similar approach described above for flavanols, we were able to evaluate the Molecules 2021, 26, 3544 Gibbs free energies of activation (Figure 7) for H/D exchange at C(8) in 3 by running12 the of 16 same reaction at different temperatures (Table 2).

1 1 FigureFigure 7. 7. HH-NMR-NMR kinetics kinetics data data for for H/D H/D exchange exchange at atC(8) C(8) by by D D2O2 Oat atfive ﬁve different different temperatures temperatures in in 2 d6 quercetinquercetin 3 3inin D DO/DMSO2O/DMSO 1:1d6 1:1 solution. solution.

Table 2. Kinetic data for H/D exchange at C(8) by D2O in quercetin 3 (D2O/DMSOd6 1:1 mixture). Table 2. Kinetic data for H/D exchange at C(8) by D2O in quercetin 3 (D2O/DMSOd6 1:1 mixture). −1 ‡ ‡ (1) ‡ Site T [K] k’obs [s ] −1 EA [kJ/mol] ΔH [kJ/mol]‡ ΔS [kJ/mol‡ K] (1)ΔG [kJ/mol]‡ Site T [K] k’obs [s ]EA [kJ/mol] ∆H [kJ/mol] ∆S [kJ/mol K] ∆G [kJ/mol] 313 (1.48 ± 0.01) × 10−5 313 (1.48 ± 0.01) × 10−5 318 (2.42 ± 0.01) × 10−5 318 (2.42 ± 0.01) × 10−5 −5 C(C(8)8) 323 323 (3.60 ±(3.60 0.02)± ×100.02) ×10−580.3 ± 8.780.3 ± 8.777.6 ± 8.7 77.6 ± 8.7 -0.10−0.1011 ± 0.0±20.0277 110.3 110.3 ±± 12.312.3 328328 (6.72 ±(6.72 0.12)± ×0.12) 10−5× 10−5 333333 (10.8 ±(10.8 0.5)± × 0.5)10−5× 10−5 (1) Values(1) Values calculated calculated at at T T = 323 K.K.

TheThe deuteration deuteration rate rate constants constants for for3 are3 areabout about a magnitude a magnitude order order lower lower (and the (and cor- the respondingcorresponding ΔG‡ values∆G‡ values about about 10 kJ/mol 10 kJ/mol higher) higher) than those than thosediscussed discussed above abovefor 4 and for 45.and In our5. hands, In our hands,deuteration deuteration at C(6) occurs at C(6) at occurs a so low at rate a so to low be considered rate to be considered negligible. negligible.Findings byFindings Faizi et al. by [ Faizi9] of C(6) et al. deuteration [9] of C(6) for deuteration 3 quercetin for in3 CFquercetin3COOD are in CF not3COOD in contrast are notwith in ourcontrast results with, since our in results, these acidic since conditions in these acidic deuteration conditions at C(8) deuteration is so fast at that C(8) at is the so start fast that of theat analysis the start it of was the analysisalready completely it was already exchanged. completely exchanged.

2.4.2.4. Re Resultssults from from ab ab Initio Initio DFT DFT C Calculationsalculations ToTo get get further further molecular molecular details details on onour our kinetic kinetic investigations, investigations, we resorted we resorted to density to den- functionalsity functional theory theory (DFT) calculations (DFT) calculations through through codes implemented codes implemented in the Gaussian in the Gaussian 16 soft- ware16 software [27]. To arrive [27]. To at arrivethe energy at the minima energy of minima all the structures of all the structuresof flavanols of here flavanols reported, here a reported,full conformational a full conformational search with the search aid of with the GMMX the aid add of the-on GMMX (GaussView add-on program) (GaussView was undertaken.program) was Conformational undertaken. Conformationalsearching was carried searching out, wasallowing carried both out, ring allowing C flipping both and ring C flipping and single-bond rotations through the use of the default values of the program. The MM-minimized rotamers were used as initial input structures, and the geometry was energy-minimized using the DFT/B3LYP 6–311G level of theory [28]. Regarding the catechin system, calculations carried out at the B3LYP/6-311G level of theory confirmed the higher Mulliken negative charge at C(8) (−0.20) with respect to that at C(6) (−0.17). Then, ab initio calculations (geometry optimization followed by frequency calculations) were used to estimate the relative energies of all the species participating in this pathway. A set of simulations were also performed to optimize structures and energies of the corresponding transition states. The calculated energy profile is summarized in Figure8. Molecules 2021, 26, x FOR PEER REVIEW 13 of 17

single-bond rotations through the use of the default values of the program. The MM-minimized rotamers were used as initial input structures, and the geometry was energy-minimized using the DFT/B3LYP 6–311G level of theory [28]. Regarding the catechin system, calculations carried out at the B3LYP/6-311G level of theory confirmed the higher Mulliken negative charge at C(8) (−0.20) with respect to that at C(6) (−0.17). Then, ab initio calculations (geometry optimization followed by frequency calculations) were used to estimate the relative energies of all the species participating in this pathway. A set of simulations were also performed to optimize structures and energies of Molecules 2021,the26, 3544corresponding transition states. The calculated energy profile is summarized in 13 of 16 Figure 8.

Figure 8. Energy profile visualization of the putative intermediates and transition states involved in catechin/D 2O deuterationFigure process 8. Energy1 → 4aprofile→ 4d visualization. of the putative intermediates and transition states involved in catechin/D2O deuteration process 1 → 4a → 4d. Energies of the mono- and di-deuterated keto forms 4ab, 4ac, 4ad, 4bd, and 4cd were Energies of theevaluated mono- and by QMdi-deuterated calculations keto to beforms significantly 4ab, 4ac, higher4ad, 4bd than, and the 4cd corresponding were mono- and evaluated by QM di-deuteratedcalculations to enol be formssignificantl4a, 4by, 4chigher, and than4d. Importantly, the corresponding the intermediate mono- 4ad was found − and di-deuteratedmore enol stableforms (by4a, 164b kJmol, 4c, and1, even4d. Importantly, after corrections the intermediate of their different 4ad thermalwas contributions) found more stablethan (by the16 kJmol competitive−1, even intermediates after corrections4ab ofand their4ac ,different thus giving thermal further contri- support to its existence butions) than the alongcompetitive the pathway intermediat of thees overall4ab and exchange 4ac, thus process,giving further as already support guessed to by the overall ‡ ‡ ‡ its existence alongfitting the pathway of our kineticof the overall data. The exchange transition process, states as ( 4abalready, 4ac guessed, 4ad ) by of thethe three competitive −1 overall fitting of ourdeuteration kinetic data. processes The transition were found states about (4ab 130‡, 4ac kJmol‡, 4ad‡) aboveof the thethree energy com- of reactant 4a. All ‡ ‡ ‡ petitive deuterationthe processes structures were associated found toabout4ab 130, 4ac kJmol, 4ad−1 abovespecies the showed energy only of reactant one imaginary frequency 4a. All the structuresthus associated ensuring weto were4ab‡, dealing4ac‡, 4ad with‡ species true transitionshowed only states. one Interestingly, imaginary the calculations frequency thus ensuringwere also we inwere agreement dealing with true the experimentaltransition states. evidence Interestingly that specific, the cal- rates of deuteration ‡ culations were alsoare in lower agreement in deuterated with the methanol experimental than in evidence deuterated that water. specific In fact, rates the of energy of the 4ab −1 deuteration are loweranalogue in deuterated obtained methanol by deuterium than in ion deuterated transfer fromwater CD. In3 ODfact, wasthe energy found about 35 kJmol higher than that of 4ab‡ itself. The intrinsic reaction coordinate (IRC) calculation performed of the 4ab‡ analogue obtained by deuterium ion transfer from CD3OD was found about 35 † kJmol−1 higher thanon that4ab of(Supplementary 4ab‡ itself. The intrinsic Figure S1)reaction afforded coordinate reactant (IRC)4a and calculation product 4b showing that during the process there is a synchronous breaking of the bond D-OC(7) and formation of the bond D-C(8). Even the bond length O-C(7) is a fine reaction coordinate since, as expected, it undergoes a significant shortening ongoing from the enol form 4a to the keto form 4b. The calculated rate constants are of course largely lower than the experimental ones (10−10 s−1 versus 10−5 s−1) since the calculated free energies of activation are about 30 kJ/mol higher than those experimentally measured. However, we are confident that, by putting two and not only one D2O molecules in the reactive site of these TS, their energy would become significantly lower due to the well-documented assistance [29] of the 2nd D2O molecule enhancing the deuterium ion donor and deuterium ion acceptor capability of the first reacting D2O molecule. Basically, in our approach, the energy profile reported in Figure8 is aimed to just provide an approximate agreement (in terms of minimum energy of all species playing a role in the process, such as reactants, products, putative, Molecules 2021, 26, 3544 14 of 16

and isolated intermediates) with our experimental ﬁndings. Thus, activation energies of processes 4a → 4b and 4a → 4c were calculated to be almost the same (in fact k1 ≈ k2), as well as the processes 4a → 4b and/or 4c → 4d were also calculated to have quite similar activation energies as process 4a → 4d, (in fact k3 ≈ k1, k2, k4, and k5).

3. Materials and Methods Each sample was prepared directly in the NMR glass tube. In addition, 10.0 ± 0.3 mM solutions of starting material 1 and 2 were prepared by dissolving 2 mg of pure compounds in 0.750 mL of D2O with the help of a sonic bath. For compound 3, a 1:1 (0.325:0.325 mL) mixture of D2O and DMSOd6 was necessary to solubilize the pure quercetin, without altering the reaction conditions, since D2O is maintained in extremely large molar excess with respect to 3. The pH of the solution was controlled directly via NMR utilizing pure imidazole ca. 0.2 mM, following the procedure described by Orgovàn et al. [23]. Kinetics of deuteration of catechin and epicatechin by D2O at T = 300–320 K (variable temperature unit ensure δT accuracy within ± 0.1 K) were performed using Bruker Avance 400 MHz NMR spectrometer. 1H-NMR measurements were carried out using a 5 mm BBI probe with a 90◦ proton pulse length of 8.7 µs at a transmission power of 0 dB and equipped with pulsed gradient field utility. 1H-NMR spectra were recorded every ~10 min and automatic peak integration of the reference signal, which maintained the same area (100%) throughout the whole experiment and of the signals of the two hydrogen atoms that are being substituted by deuterium was performed. The chemical shift scale (δ) was calibrated on the residual proton signal of trimethylsilyl-propionic-2,2,3,3-d4 acid sodium salt (TSP) that was used as reference standard (δH = 0.00 ppm). 1 0 H-NMR of catechin (D2O, 300K): 6.86 (d, J = 1.9, 1H, H-2 ); 6.84 (dd, J = 1.9, 8.1 Hz, 1H, H-60); 6.76 (d, J = 8.1 Hz, 1H, H50); 6.01 (d, J = 2.3, 1H, H-6); 5.91(d, J = 2.3, 1H, H-8); 4.60 (d, J = 7.9, 1H, H-2); 4.06 (ddd, J = 8.3, 7.9, 5.4, 1H, H-3); 2.78 (dd, J = 16.0, 5.4, 1H, H4a); 2.42 (dd, J = 16.0, 8.3, 1H, H4b). 1 0 H-NMR of epicatechin (D2O, 300 K); 6.86 (d, J = 1.9, 1H, H-2 ); 6.84 (dd, J = 1.9, 8.1 Hz, 1H, H-60); 6.76 (d, J = 8.1 Hz, 1H, H50); 6.02 (d, J = 2.3, 1H, H-6); 6.00 (d, J = 2.3, 1H, H-8); 4.82 (brs, 1H, H-2); 4.22 (m, 1H, H-3); 2.82 (dd, J = 17.0, 4.3,1H, H4a); 2.67 (dd, J = 17.0, 2.1, 1H, H4b). The molar percentage ratio of the decrease of the former and increase of the latter was plotted for each compound analysed (catechin and epicatechin) and for each proton. Using Eyring and Arrhenius plots and kinetic equations, it was possible to obtain all the meaningful kinetic and energetic quantities (rate constants, half-life times, activation energy, and Gibbs free energy of activation). A qualitative separation of Gibbs free energy of activation into its two contributions was also carried out using the best fitting curve. The reaction can be considered a pseudo-first-order process since D2O is in large excess with respect to flavan-3-ols monomers. The same reasoning was employed also for flavonol quercetin 3 and its related kinetic analysis. The only difference for the latter is its poor solubility in pure D2O leading to the need for the use of D2O/DMSOd6 1:1 solution, as solvent. Peak deconvolution was performed using the MestReNova v12 global spectral deconvolution tool (GSD) with default parameters. Automatic peak identification and integration were also performed to obtain molar ratios of the involved structures. Rate constants guess for best curve fitting of the reaction kinetics was performed with the help of the Kinetiscope software. Molecules’ geometry, energetics and properties simulations were performed via the Gaussian 16 suite of programs [27]. All simulations were performed with DFT at the B3LYP level of theory, at first with a 6-31G basis set, which was then expanded to 6-311G to improve results accuracy. The chemistry of these molecules and processes was modelled in the solution, using the IEFPCM continuous solvent model. Standard optimization and frequency calculations were performed on all molecules, together with transition state optimization and IRC calculation to complete the computational landscape. Molecules 2021, 26, 3544 15 of 16

4. Conclusions A careful kinetic analysis of the H/D exchange processes occurring at C(6) and/or C(8) of flavanols (1 and 2) or flavonol (3) has been carried out by joining information deriving both from experimental 1H-NMR spectra taken at different temperatures, pH and protic solvents (D2O, CD3OD, D2O/DMSOd6), and from theoretical QM calculations (DFT, B3LYP 6-311G). For the first time, we report here on: (a) The existence, along the reaction coordinate leading from 1 (4a) to the 6,8 di-deuterated analogues 4d, of the monodeuterated species 4b and 4c; (b) different rates of formation of 4b and 4c as established by the peak deconvolution of the H-C(6) and H-C(8) NMR signals during the kinetics; (c) the relevant role played by termolecular processes whereby 4a is converted in 4d without passing through intermediates 4b and 4c; (d) the complex dependence of the overall rate constant on the pH imposed to the reacting system due to the opposite pH-response of the (two steps) route (4a → 4b → 4d) with respect to the (one step) route (4a → 4d).

Supplementary Materials: The following are available online, Figure S1: Qualitative reaction energy profile with reactant and putative transition state and intermediate state (a). Total energy and bond length change during the steps before and after the transition state (b). Author Contributions: F.B., F.M., P.A., D.C., and G.G. designated and conducted the experiments; F.B. and G.G. performed data analysis and wrote the paper. All authors together conceived the experiment, discussed the results, interpreted the data, and revised. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Some data obtained by DFT calculations presented in this study are available in the Supplementary Materials. Acknowledgments: Authors acknowledge the technical support given by Adriano Sterni. Conflicts of Interest: The authors declare no conflict of interest. Sample Availability: Not available.

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