Intramolecular tautomerization of the quercetin molecule due to the proton transfer: QM computational study.

Quercetin molecule (3, 3', 4', 5, 7-pentahydroxyflavone, C15H10O7) is an important flavonoid compound of natural origin, consisting of two aromatic A and B rings linked through the C ring with endocyclic oxygen atom and five hydroxyl groups attached to the 3, 3', 4', 5 and 7 positions. This molecule is found in many foods and plants, and is known to have a wide range of therapeutic properties, like an anti-oxidant, anti-toxic, anti-inflammatory etc. In this study for the first time we have revealed and investigated the pathways of the tautomeric transformations for the most stable conformers of the isolated quercetin molecule (Brovarets' & Hovorun, 2019) via the intramolecular proton transfer. Energetic, structural, dynamical and polar characteristics of these transitions, in particular relative Gibbs free and electronic energies, characteristics of the intramolecular specific interactions-H-bonds and attractive van der Waals contacts, have been analysed in details. It was demonstrated that the most probable process among all investigated is the proton transfer from the O3H hydroxyl group of the C ring to the C2' carbon atom of the C2'H group of the B ring along the intramolecular O3H…C2' H-bond with the further formation of the C2'H2 group. It was established that the proton transfer from the hydroxyl groups to the carbon atoms of the neighboring CH groups is assisted at the transition states by the strong intramolecular HCH…O H-bond (~28.5 kcal∙mol-1). The least probable path of the proton transfer-from the C8H group to the endocyclic O1 oxygen atom-causes the decyclization of the C ring in some cases. It is shortly discussed the biological importance of the obtained results.

Introduction

Quercetin molecule is a compound of natural origin, which is found in different foods and plants [1, 2]. This compound has attracted a lot of attention, since it is suggested to have a wide range of properties, such as an anti-oxidant, anti-toxic, anti-inflammatory etc. [3-20]. The structure of the quercetin contains two (A+C) and B rings and also has five hydroxyl groups at the 3, 3′, 4′, 5, 7 positions. So, due to these structural features it can acquire different conformations [21-26] and perform structural transitions between them due to the mutual rotations of the (A+C) and B rings around the C2-C1′ bond and also of the hydroxyl groups OH around the exocyclic C-O bonds [27]. Thus, investigations of the conformational transformations through the rotations around the C2-C1′ bond have been presented in literature [28-30]. In particular, in our recent works we have investigated in details conformational variety [26] and also conformational transitions of the quercetin molecule via the rotations of its rings [31] by using the quantum-mechanical (QM) calculations at the MP2/6-311++G(d,p)//B3LYP/6-311++G(d,p) level of theory and Bader’s quantum theory of “Atoms in Molecules” (QTAIM). Altogether, as a result of the study it was revealed 48 stable conformers (24 planar and 24 non-planar) with relative Gibbs free energies within the range of 0.0–25.3 kcal∙mol-1 under normal conditions, stabilized by the H-bonds (both classical OH…O and so-called unusual CH…O and OH…C) and attractive van der Waals contacts O…O, which have been divided into four different subfamilies by their structural properties [26]. Conformers of the quercetin molecule have been established to be polar structures with a dipole moment, which varies within the range from 0.35 to 9.87 Debay. We have also found out the interconversions of the 24 pairs of the conformers of the quercetin molecule via the rotation of its practicallay non-deformable (A+C) and B rings around the C2-C1' bond through the quasi-orthogonal transition states with Gibbs free energies of activation in the range of 2.17–5.68 kcal·mol−1 at normal conditions [31]. It was also provided comprehensive analysis of the 123 prototropic tautomers of the quercetin molecule [32, 33].

Also, quercetin can potentially acquire different prototropic tautomeric forms, but these data are weakly presented in the literature yet [34-38], despite the continuous comprehensive research of the quercetin molecule during the last decades [21-38].

It is widely known from the literature data that proton transfer is important biochemical phenomenon and plays important role in the biochemical reactions [39-45]. Thus, it was established that even movement of the single proton (SP) from the one site to another can cause significant changes of the energetic, structural and dynamical properties of the molecule, thus changing its functionality. In particular, tautomerization via the single (SPT) or double (DPT) proton transfer has been established for the canonical or non-canonical DNA base pairs [46-55], by the participation as of classical DNA bases [56], so by the participation of modified bases such as hypoxanthine [57-59], 5-bromouracil [60] and 2-aminopurine [61-64] molecules.

Currently, there are only some studies in the literature, devoted to the prototropic tautomerism of the quercetin molecule, in particular keto-enol tautomerism [34-38]. Their importance is caused by the relevance of the quercetin tautomers to the hydrogen↔deuterium (H↔D) exchange processes of its CH-groups [36], irreversible structural changes of the quercetin molecule at the increasing of the temperature [34-35] and tautomerization of the quercetin molecule at the transition to an excited electronic state [38]. However, possible ways of the formation of the rare prototropic tautomers of the quercetin molecule have not been carefully considered.

So, the aim of this study is to reveal and investigate the possible pathways of the prototropic transformations of the isolated quercetin molecule [65].

As a result of this scrupulous investigation we have revealed possible ways of tautomerization of the quercetin molecule via the single proton transfer, which are entangled with the following phenomena (Fig 1):

Fig 1

Schematic representations of the possible mechanisms of the intramolecular proton mobility in the quercetin molecule.

Red arrows denote the directions of the proton transfer, yellow–rotations of the hydroxyl groups around the C-O bond by 180 degree.

Proton transfer from the C8H group to the neighboring O1 oxygen atom.

Transition of the proton from the O7H/O3′H hydroxyl groups to the carbon atoms of the neighboring C6H/C2′H groups.

Migration of the proton from the O7H/O5H/O3H/O4′H hydroxyl groups to the carbon atoms of the neighboring C8H/C6H/C2′H/C5′H groups, preceded by the rotations of the hydroxyl groups around the C7O7/C5O5/C3O3/C4′O4′ bonds by 180 degree.

Proton transfer from the O3H hydroxyl group to the neighboring O4 oxygen atom.

Transition of the proton from the O7H/O5H hydroxyl groups to the C6 carbon atom of the neighboring C6H group.

Number of the important physico-chemical parameters of these transformations, in particular relative Gibbs free and electronic energies, characteristics of the intramolecular H-bonds and attractive van der Waals interactions have been analysed in details. Especial attention has been focused on the processes of the intramolecular tautomerization by proton transfer, which are more or less likely to occur. Possible chemical and biological roles of the obtained results have been shortly outlined.

Computational methods

We have used the DFT B3LYP/6-311++G(d,p) level of theory [66-69], incorporated into Gaussian’09 program package [70], to provide the calculations of the geometrical structures and vibrational spectra of the prototropic tautomers of the quercetin molecule and transitions states (TSs) between them, which have been localized by Synchronous Transit-guided Quasi-Newton method [66]. This level of theory has been successfully approved for the calculations of the heterocyclic compounds [71-78]. Scaling factor of 0.9668 has been applied for the correction of the harmonic frequencies for the investigated structures [79, 80]. Electronic and Gibbs free energies under normal conditions have been calculated by single point calculations at the MP2/6-311++G(2df,pd) level of theory [81-83].

The Hessian-based predictor-corrector integration algorithm [84] has been applied for obtaining the IRC pathways in the forward and reverse directions from each TS.

The time τ, which is necessary to reach 99.9% of the equilibrium concentration of the reactant and product, the lifetime τ (1/k) of the prototropic tautomers, the forward k and reverse k rate constants have been obtained by the well-known formulas of physico-chemical kinetics [85], respectively: where quantum tunneling effect has been accounted by Wigner’s tunneling correction Γ [86-88]: where k−Boltzmann’s constant, h–Planck’s constant, ΔΔG−Gibbs free energy of activation for the conformational transition in the forward (f) and reverse (r) directions, ν−magnitude of the imaginary frequency associated with the vibrational mode at the TSs.

The distribution of the electron density has been analyzed by application of the program package AIM’2000 [89] with all default options and wave functions obtained at the B3LYP/6-311++G(d,p) level of theory for geometry optimisation. The presence of the (3,-1) bond critical point (BCP), bond path between hydrogen donor and acceptor and positive value of the Laplacian at this BCP (Δρ>0) have been considered as criteria for the formation of the H-bond and attractive van der Waals contact [62–63, 90–92].

Energies of the unusual intramolecular CH⋯O and OH⋯C H-bonds and attractive O⋯O and C⋯O van der Waals contacts have been obtained using Bader's quantum theory of Atoms in Molecules [93] by the empirical Espinosa-Molins-Lecomte (EML) formula [94, 95], based on the electron density distribution at the (3,-1) BCPs of the H-bonds: where V(r)–value of a local potential energy at the (3,-1) BCP.

It should be noted, that for the CH…O H-bonds, which are strong with energy that exceeds 10 kcal∙mol-1, their energy have been estimated by the Brovarets’-Yurenko-Hovorun formula [96, 97], considered in the literature [98, 99]:

The energies of the classical intramolecular OH⋯O H-bonds have been calculated by the Nikolaienko-Bulavin-Hovorun formula [100]: where ρ–the electron density at the (3,-1) BCP of the H-bond.

All calculations have been performed for the tautomeric transitions of the quercetin molecule as their intrinsic property, that is adequate for modeling of the processes occurring in real systems [101-106].

In this work standard numeration of atoms has been used [2]. At this, prototropic tautomers of the quercetin molecule have been designated by the asterisk; subscript corresponds to the localization of the mobile protons. Numeration of the conformers (highlighted in bold) is the same, as in the previous work [26].

Results and discussion

In the process of this study we have suggested different ways of the formation of the prototropic tautomers of the most stable conformer 1 [26] of the quercetin molecule. Then, by using the method of “trials and errors” we have localized TSs for these tautomeric transformations, occurring via the intramolecular proton transfer. However, only some of the suggested tautomeric transformations have been confirmed, while others of them have been modified in the course of the investigation.

So, in this study we have considered the following mechanisms of the tautomerization of the quercetin molecule, in particular of the most stable conformer 1, that can proceed in the different ways through the intramolecular proton transfer (see Figs 1 and 2, Tables 1 and 2).

Fig 2

Reaction pathways for the intramolecular proton transfer in the isolated quercetin molecule; initial and terminal states with TSs between them have been obtained at the MP2/6-311++G(2df,pd) // B3LYP/6-311++G(d,p) level of QM theory (low index near formed tautomers denotes the site of the localization of the transferred proton).

Gibbs free ΔG and electronic ΔE energies (kcal∙mol-1), imaginary frequencies vi at the TS and dipole moments μ (Debay) are provided below reaction paths. Dotted lines indicate intramolecular specific interactions. Red arrows denote the intramolecular transition of the proton, while yellow arrows–rotations of the hydroxyl groups. See also Tables 1 and 2.

Table 1

Energetic and kinetic characteristics of the tautomeric transformations by the intramolecular proton transfer in the isolated quercetin molecule obtained at the MP2/6-311++G(2df,pd)//B3LYP/6-311++G(d,p) level of QM theory under normal conditions (see also Fig 1).

Tautomerictransition	νia	ΔGb	ΔEc	ΔΔGTSd	ΔΔETSe	ΔΔGf	ΔΔEg	kfh	kri	τ99.9%j	τk
(a) Proton transfer from the C8H group to the O1 oxygen atom
1↔1*O1H	1150.3	87.11	90.07	96.31	100.00	9.20	9.93	3.09∙10−58	2.43∙106	2.84∙10−6	4.12∙10−7
4↔4*O1H	1264.1	91.76	92.64	92.66	95.39	0.90	2.75	1.64∙10−55	3.31∙1012	2.09∙10−12	3.02∙10−13
7↔7*O1H	1157.2	87.23	90.20	96.26	99.75	9.03	9.55	3.58∙10−58	3.26∙106	2.12∙10−6	3.07∙10−7
10↔10*O1H	1151.7	87.13	90.18	96.27	99.95	9.14	9.77	3.31∙10−58	2.69∙106	2.57∙10−6	3.72∙10−7
(b) Proton transfer from the O7H/O3′H groups to the C6/C2′ carbon atoms of the C6H/C2′H groups
1↔1*C6H2	1970.0	20.80	22.30	65.30	68.98	44.50	46.68	3.53∙10−35	6.37∙10−20	1.08∙1020	1.57∙1019
1↔1*C2'H2	2116.5	17.77	17.98	68.15	71.67	50.38	53.69	3.21∙10−37	3.47∙10−24	1.99∙1024	2.88∙1023
(c) Proton transfer from the O7H/O5H/O3H/O4′H groups to the C8/C6/C2′/C5′ carbon atoms of the C8H/C6H/C2′H/C5′H groups
5↔5*C8H2	1983.6	13.72	14.99	20.46	21.98	6.74	6.99	2.77∙10−2	3.21∙108	2.15∙10−8	3.12∙10−9
25↔25*C6H2	2008.3	13.27	14.49	61.63	64.96	48.36	50.47	1.79∙10−32	9.69∙10−23	7.13∙1022	1.03∙1022
20↔1**C2'H2	1370.9	29.63	31.10	34.71	36.93	5.08	5.83	5.79∙10−13	3.14∙109	2.20∙10−9	3.19∙10−10
10↔1*C5'H2	2087.7	22.85	24.62	70.59	74.59	47.74	49.97	5.09∙10−39	2.93∙10−22	2.35∙1022	3.41∙1021
(d) Proton transfer from the O3H group to the O4 atom
1↔1*O5H/O4H	892.1	14.30	14.70	13.10	15.20	-1.20	0.50	2.62∙103	8.09∙1013	8.54∙10−14	1.24∙10−14
(e) Proton transfer from the O7H/O5H groups to the C6 carbon atom of the C6H group
1**O5H/O4H/O3H↔1*O5H/O4H/O3H	1766.3	30.82	30.25	49.01	51.67	18.19	21.42	2.66∙10−23	1.07	6.43	0.93
1**O4H/O3H↔1*O5H/O4H/O3H	1791.5	57.65	58.54	75.24	79.43	17.59	20.89	1.56∙10−42	3.02	2.29	0.33

aThe imaginary frequency at the TS of the tautomeric transition, cm-1.

bThe Gibbs free energy of the initial relatively the terminal structure of the tautomerisation reaction (T = 298.15 K), kcal∙mol-1.

cThe electronic energy of the initial relatively the terminal structure of the tautomerisation reaction, kcal∙mol-1.

dThe Gibbs free energy barrier for the forward tautomerisation reaction, kcal∙mol-1.

eThe electronic energy barrier for the forward tautomerisation reaction, kcal∙mol-1.

fThe Gibbs free energy barrier for the reverse tautomerisation reaction, kcal∙mol-1.

gThe electronic energy barrier for the reverse tautomerisation reaction, kcal∙mol-1.

hThe rate constant for the forward tautomerisation reaction, s-1.

iThe rate constant for the reverse tautomerisation reaction, s-1.

jThe time necessary to reach 99.9% of the equilibrium concentration between the reactant and the product of the tautomerisation reaction, s.

kThe lifetime of the product of the tautomerisation reaction, s.

Table 2

Energetical, electron-topological and geometrical characteristics of the intramolecular specific contacts–H-bonds and attractive van der Waals (vdW) contacts, polar and geometrical parameters of the investigated structures of the isolated quercetin molecule obtained at the B3LYP/6-311++G(d,p) level of QM theory (see also Fig 1).

Conformer,TS and tautomer	AH⋯B H-bond /A⋯B vdW contact	EAH⋯B / EA··B a	ρb	Δρc	100∙εd	dA⋯Be	dH⋯Bf	∠AH⋯Bg	ΔC3-C2-C1′-C6′h	μi
(a) Transition of the proton from the C8H group to the O1 oxygen atom
1↔1*O1H
1	O5H . . .O4	6.71	0.041	0.124	1.52	2.655	1.770	147.3	180.0	0.35
	O3H . . .O4	3.36	0.027	0.103	60.55	2.625	2.009	119.0
	C2'H . . .O3	4.01*	0.018	0.076	0.92	2.883	2.137	123.8
TS1↔1*O1H	O5H . . .O4	2.89	0.025	0.082	1.10	2.830	1.990	142.9	-164.8	3.38
	O3H . . .O4	3.84	0.029	0.109	38.00	2.600	1.963	120.4
	C2'H . . .O3	3.32*	0.016	0.063	10.19	2.924	2.221	120.6
1*O1H [32]	O5H . . .O4	9.10	0.051	0.148	2.33	2.565	1.672	147.8	-155.3	1.83
	O3H . . .O4	5.75	0.037	0.134	17.66	2.509	1.853	121.6
	C9 . . .O1	6.47*	0.029	0.090	2.46	2.895	-	-
	C2'H . . .O3	3.38*	0.016	0.064	26.96	2.856	2.253	113.1
4↔4*O1H
4	O5H . . .O4	6.47	0.040	0.124	1.46	2.659	1.776	147.3	180.0	5.33
	O3H . . .O4	3.36	0.027	0.103	60.63	2.624	2.009	118.9
	C6'H . . .O3	3.83*	0.018	0.073	0.80	2.895	2.159	123.3
TS4↔4*O1H	O5H . . .O4	2.65	0.024	0.081	1.20	2.835	1.995	143.0	162.2	7.77
	O3H . . .O4	3.84	0.029	0.109	38.52	2.601	1.965	120.2
	C6'H . . .O3	3.07*	0.015	0.058	12.45	2.942	2.261	119.2
4*O1H [32]	O5H . . .O4	5.04	0.034	0.112	1.32	2.705	1.842	145.2	154.4	8.88
	O3H . . .O4	4.32	0.031	0.113	30.34	2.579	1.939	120.5
	C6'H . . .O3	2.57*	0.012	0.048	22.18	2.975	2.358	114.7
7↔7*O1H
7	O5H . . .O4	6.71	0.041	0.125	1.50	2.652	1.767	147.3	180.0	5.05
	O3H . . .O4	3.12	0.026	0.102	68.01	2.630	2.020	118.5
	C2'H . . .O3	3.83*	0.018	0.073	0.02	2.886	2.160	122.4
TS7↔7*O1H	O5H . . .O4	2.89	0.025	0.082	1.09	2.828	1.988	142.9	-162.0	4.84
	O3H . . .O4	3.60	0.028	0.107	42.14	2.606	1.977	119.8
	C2'H . . .O3	3.03*	0.014	0.058	13.43	2.936	2.267	118.2
7*O1H [32]	O5H . . .O4	9.10	0.051	0.147	2.27	2.565	1.673	147.8	-151.5	4.65
	O3H . . .O4	5.28	0.035	0.131	19.50	2.516	1.869	121.0
	C9 . . .O1	6.54*	0.029	0.091	2.45	2.374	-	-
	C2'H . . .O3	2.98*	0.014	0.056	36.27	2.876	2.327	109.7
10↔10*O1H
10	O5H . . .O4	6.71	0.041	0.124	1.51	2.654	1.770	147.3	180.0	2.99
	O3H . . .O4	3.12	0.026	0.103	61.90	2.626	2.011	118.9
	C2'H . . .O3	3.98*	0.018	0.075	1.02	2.889	2.141	124.1
TS10↔10*O1H	O5H . . .O4	2.89	0.025	0.082	1.09	2.830	1.990	142.9	-164.7	2.34
	O3H . . .O4	3.84	0.029	0.109	38.76	2.601	1.966	120.3
	C2'H . . .O3	3.27*	0.015	0.062	10.11	2.932	2.227	120.8
10*O1H [32]	O5H . . .O4	9.10	0.051	0.148	2.31	2.565	1.673	147.8	-154.8	2.24
	O3H . . .O4	5.51	0.036	0.133	18.06	2.510	1.857	121.5
	C9 . . .O1	6.52*	0.029	0.091	2.51	2.375	-	-
	C2'H . . .O3	3.30*	0.015	0.062	27.26	2.865	2.263	113.1
(b) Transition of the proton from the O7H/O3′H groups to the neighboring C6/C2′ carbon atoms of the C6H/C2′H groups
1↔1*C6H2
TS1↔1*C6H2	HC6H . . .O7	28.46**	0.116	0.059	20.81	2.211	1.388	105.3	179.3	3.96
	O5H . . .O4	8.62	0.049	0.136	1.51	2.595	1.692	148.4
	O3H . . .O4	3.12	0.026	0.101	69.93	2.633	2.025	118.4
	C2'H . . .O3	3.97*	0.018	0.075	1.12	2.886	2.141	123.8
1*C6H2 [32]	O5H . . .O4	8.86	0.050	0.137	1.47	2.587	1.687	147.5	180.0	5.34
	O3H . . .O4	2.65	0.024	0.099	96.94	2.646	2.048	117.7
	C2'H . . .O3	4.14*	0.019	0.078	1.09	2.872	2.126	123.9
1↔1*C2'H2
TS1↔1*C2'H2	O5H . . .O4	6.47	0.040	0.124	1.37	2.657	1.775	147.0	-176.8	3.94
	O3H . . .O4	2.89	0.025	0.100	76.39	2.637	2.032	118.1
	C2'H . . .O3	3.05*	0.014	0.058	39.78	2.833	2.322	106.5
	HC2'H . . .O3'	26.97**	0.110	0.072	28.82	2.244	1.409	105.0
1*C2'H2 [32]	O5H . . .O4	6.47	0.040	0.124	1.43	2.657	1.775	147.1	180.0	3.80
	O3H . . .O4	3.12	0.026	0.102	68.85	2.631	2.022	118.4
	O3 . . .C2'	2.90*	0.012	0.056	374.04	2.807	-	-
	O4'H . . .O3'	2.65	0.024	0.098	139.26	2.650	2.071	116.2
(c) Transition of the proton from the O7H/O5H/O3H/O4′H groups to the carbon atoms of the C8H/C6H/C2′H/C5′H groups, preceded by the rotations of the hydroxyl groups around the C7O7/C5O5/C3O3/C4′O4′ axes by 180 degree
5↔5*C8H2
5	O5H . . .O4	6.47	0.040	0.123	1.47	2.660	1.777	147.2	180.0	3.01
	O3H . . .O4	3.36	0.027	0.104	58.22	2.623	2.004	119.1
	C2'H . . .O3	4.01*	0.018	0.076	0.91	2.883	2.138	123.8
TS5↔5*C8H2	O5H . . .O4	8.35	0.048	0.136	1.28	2.607	1.700	149.6	177.3	4.60
	O3H . . .O4	6.78	0.026	0.103	61.23	2.623	2.010	118.6
	C2'H . . .O3	3.87*	0.018	0.073	1.71	2.894	2.151	123.6
	HC8H . . .O7	30.20**	0.123	0.033	17.10	2.209	1.363	105.4
5*C8H2 [32]	O5H . . .O4	7.19	0.043	0.130	1.07	2.642	1.746	149.1	180.0	6.69
	O3H . . .O4	3.36	0.027	0.104	62.47	2.620	2.008	118.5
	C2'H . . .O3	3.84*	0.018	0.073	0.71	2.897	2.154	123.7
25↔25*C6H2
25	O3H . . .O4	4.56	0.032	0.117	31.44	2.571	1.921	121.2	180.0	3.55
	O5 . . .O4	2.91*	0.012	0.049	13.37	2.765	-	-
	C2'H . . .O3	3.93*	0.018	0.074	0.62	2.890	2.145	123.8
TS25↔25*C6H2	HC6H . . .O5	31.44**	0.128	0.012	15.09	2.208	1.347	105.3	179.7	2.51
	O3H . . .O4	4.16	0.030	0.112	2.00	2.590	1.950	120.5
	C2'H . . .O3	3.87*	0.018	0.073	0.56	2.895	2.151	123.8
25*C6H2 [32]	O5 . . .O4	2.55*	0.010	0.041	188.30	2.903	-	-	179.6	4.46
	O3H . . .O4	4.80	0.033	0.118	28.83	2.564	1.909	121.5
	C2'H . . .O3	3.75*	0.017	0.071	0.38	2.906	2.162	123.8
20↔1**C2'H2
20	O5H . . .O4	8.38	0.048	0.136	1.36	2.600	1.700	148.6	135.5	3.62
	O3H . . .C2'	2.36*	0.012	0.045	251.5	3.033	2.242	138.5
TS20↔1**C2'H2	O5H . . .O4	8.71	0.049	0.138	1.11	2.591	1.692	148.5	148.4	4.07
	HC2'H . . .O3	27.22**	0.111	0.085	3.56	2.509	1.398	141.0
1**C2'H2 [32]	O5H . . .O4	8.62	0.049	0.138	0.89	2.594	1.697	148.2	180.0	3.40
	C2' . . .O3	4.47*	0.018	0.075	458.16	2.695	-	-
10↔1*C5'H2
TS10↔1*C5'H2	O5H . . .O4	6.47	0.040	0.124	1.48	2.657	1.774	147.1	-175.7	3.21
	O3H . . .O4	3.36	0.027	0.104	57.17	2.621	2.003	119.1
	C2'H . . .O3	4.28*	0.019	0.080	2.57	2.885	2.107	126.5
	HC5'H . . .O4'	27.47**	0.112	0.070	25.78	2.232	1.407	104.6
1*C5'H2 [32]	O5H . . .O4	6.47	0.040	0.123	1.37	2.661	1.780	146.8	-173.4	4.69
	O3H . . .O4	3.36	0.027	0.106	52.39	2.614	1.994	119.2
	C5'H . . .O4'	4.11*	0.019	0.077	3.93	2.894	2.125	125.8
(d) Transition of the proton from the O3H group to the O4 oxygen atom
1↔1*O5H/O4H
TS1↔1*O5H/O4H	O5H . . .O4	2.65	0.024	0.090	0.90	2.802	1.964	143.0	180.0	3.79
	O4H . . .O3	22.48	0.107	0.082	1.04	2.354	1.408	136.1
	C2'H . . .O3	3.75*	0.018	0.066	3.88	2.958	2.175	127.2
1*O5H/O4H [32]	O5H . . .O4	2.89	0.025	0.100	4.07	2.754	1.923	142.1	180.0	4.06
	O4H . . .O3	7.43	0.044	0.128	12.54	2.503	1.783	125.2
	C2'H . . .O3	4.53*	0.021	0.077	3.16	2.903	2.114	127.3
(e) Transition of the proton from the O7H group to the C6 carbon atom of the C6H group
1**O5H/O4H/O3H↔1*O5H/O4H/O3H
1**O5H/O4H/O3H [32]	O4H . . .O5	5.04	0.034	0.128	6.17	2.634	1.806	140.7	180.0	8.58
	O3H . . .O4	2.17	0.022	0.100	66.71	2.613	2.047	115.4
	C2'H . . .O3	4.15*	0.019	0.078	1.36	2.868	2.130	123.2
TS1**O5H/O4H/O3H↔1*O5H/O4H/O3H	O4H . . .O5	5.75	0.037	0.135	6.00	2.613	1.765	142.5	180.0	7.99
	O3H . . .O4	2.17	0.022	0.099	79.70	2.617	2.055	115.1
	C2'H . . .O3	4.11*	0.019	0.077	1.28	2.871	2.133	123.2
1*O5H/O4H/O3H [32]	O4H . . .O5	6.47	0.040	0.138	5.72	2.599	1.740	143.4	180.0	6.68
	O3H . . .O4	1.93	0.021	0.097	126.99	2.633	2.076	114.7
	C2'H . . .O3	4.21*	0.019	0.079	1.29	2.863	2.124	123.3
1↔1*O5H/O4H/O3H
TS1**O4H/O3H↔1*O5H/O4H/O3H	O4H . . .O5	4.47	0.032	0.100	1.67	2.735	1.856	145.7	180.0	6.05
	C2'H . . .O3	4.32*	0.019	0.081	1.39	2.686	2.143	113.9
1*O5H/O4H/O3H [32]	O4H . . .O5	6.47	0.040	0.138	5.72	2.599	1.740	143.4	180.0	6.68
	O3H . . .O4	1.93	0.021	0.097	126.99	2.633	2.076	114.7
	C2'H . . .O3	4.21*	0.019	0.079	1.29	2.863	2.124	123.3

aThe energy of the AH⋯B / A⋯B specific contact, calculated by Espinose-Molins-Lecomte [94, 95] (marked with an asterisk), Brovarets-Yurenko-Hovorun [96] (marked with a double asterisk) or Nikolaienko-Bulavin-Hovorun [100] formulas, kcal∙mol-1

bThe electron density at the (3,-1) BCP of the specific contact, a.u.

cThe Laplacian of the electron density at the (3,-1) BCP of the specific contact, a.u.

dThe ellipticity at the (3,-1) BCP of the specific contact

eThe distance between the A and B atoms of the AH⋯B / A⋯B specific contact, Å

fThe distance between the H and B atoms of the AH⋯B H-bond, Å

gThe H-bond angle, degree

hThe dihedral angle ∠C3-C2-C1′-C6′, degree

iThe dipole moment of the molecule, Debay. See also Fig 1 and Table 1.

It was established that these transformations of the quercetin molecule are accompanied by the changes of their geometry, dipole moment rearrangement and breakage and formation of the intramolecular specific contacts (H-bonds and attractive van der Waals contacts).

Analysis of the investigated mechanisms and their discussion are provided further one-by-one.

a) Proton transfer from the C8H group to the O1 atom. First of the considered mechanisms consists in the intramolecular transition of the proton, localized at the C8 carbon atom, to the neighboring endocyclic oxygen atom O1, leading to the formation of the new tautomer with formed O1H hydroxyl group (Figs 1 and 2). We have analysed this tranformation for the case of the main stable conformer 1 of the quercetin molecule and also checked it for the others–conformers 4, 7 and 10: 1↔1* (ΔΔGTS = 96.31); 4↔4* (ΔΔGTS = 92.66); 7↔7* (ΔΔGTS = 96.26) and 10↔10* (ΔΔGTS = 96.27 kcal∙mol-1) (Table 1).

Finally, four new prototropic tautomers have been formed– 1* (ΔG = 9.20), 4* (ΔG = 0.90), 7* (ΔG = 9.03) and 10* (ΔG = 9.14 kcal∙mol-1) (Table 1). Notably, all of them, except the case of the conformer 4, which contains opened C-ring and new exotic strong attractive van der Waals contact C9 o…O1 (~6.5 kcal∙mol-1 (Table 2)) instead of the C9-O1 covalent bond in the C ring. In the case of the 4↔4* tautomeric transition, the covalent bond C9-O1 survives during this transformation. Notably, three lower H-bonds, stabilizing conformers–O5H …O4, O3H …O4 and C2'H …O3,–remain the same, changing only their energies during tautomerisation (Table 2).

The 1↔1* tautomerisation reaction occurs via quite high activation barrier and TS1↔1*O1H with high imaginary frequency (vi = 1150.3 cm-1). Notably, that we have checked and revealed that this transition is typical for all investigated conformers. Thus, the Gibbs free energies of activation consist ~93–96 kcal∙mol-1 for the 4↔4*, 7↔7* and 10↔10* tautomeric transformations of the non-planar conformers 4, 7 and 10 (see Fig 1 and Table 1).

At this, the tautomer 4* has been established to be dynamically-unstable (ΔΔG = 0.9 kcal∙mol-1)–its lifetime τ = 3∙10−13 s (Table 1) is less than the period of the most low-frequency torsional vibration of the rings around the C2-C1′ bond, which could not develop during this lifetime.

We have also tried to localize the tautomer with the proton, transferred to the O1 oxygen atom from the other neighboring C6H group for others conformers of the quercetin molecule [26] in the case, when these groups are closely located. However, since the stable structure could not be localized, that means that in fact this reaction would not occur.

So, intramolecular proton transfer from the C8H group to O1 oxygen atom causes decyclization (opening) of the C ring of the quercetin molecule. We consider this result quite important, taking into account how much attention attracts prototropic, in particular ring-chain tautomerism [107, 108], in the modern computer-aided drug design [42, 43].

b) Transition of the proton from the O7H/O3′H hydroxyl groups to the carbon atoms of the neighboring C6H/C2′H groups. Firstly, we have considered all possible sites for the proton transfer from the hydroxyl groups to the carbon atoms of the neighboring CH groups with the formation of the CH2 group. It was revealed only two tautomerization reactions, which occur in this case–O7H→C6H and O3′H→C2′H. Investigated tautomeric transformations– 1↔1* (ΔΔGTS = 65.30) and 1↔1* (ΔΔGTS = 68.15 kcal∙mol-1)–occur via the intramolecular proton transfer, which are preceded by the rotations of the hydroxyl groups to the CH groups, with Gibbs free energy barriers of activation– 65.30 and 68.15 kcal∙mol-1, respectively. As a result of these tautomerisations, the planar tautomers 1* and 1* with relative Gibbs free energies 44.50 and 50.38 kcal∙mol-1, containing the C6H2 and C2′H2 groups have been formed, respectively (Fig 1, Tables 1 and 2).

These processes of tautomerisation are assisted by the strong intramolecular HC6H …O7 (28.46) and HC2'H …O3' (26.97 kcal∙mol-1) H-bonds at the TS1↔1*C6H2 and TS1↔1*C2'H2 transition states. All others H-bonds (O5H …O4, O3H …O4 and C2'H …O3) remain the same at the starting 1 and terminal 1* structures for the transformation 1↔1*, while the initial set of the H-bonds (O5H …O4, O3H …O4, C2'H …O3) rearranges into the terminal network of the H-bonds (O5H …O4, O3H …O4, O3 …C2', O4'H …O3') for the transformation 1↔1* (see Fig 1 and Table 2).

c) Transitions of the proton from the O7H/O5H/O3H/O4′H hydroxyl groups to the C8/C6/C2′/C5′ carbon atoms of the C8H/C6H/C2′H/C5′H groups, which are preceded by the rotations of the hydroxyl groups around the C7O7/C5O5/C3O3/C4′O4′ bonds by 180 degree. We have also surveyed other sites of the proton attachment for the possibility of the proton transfer to them. However, analysed sites require rotation of the OH hydroxyl groups around the C-O bond by 180 degree, leading to the prototropic transformations–O7H→C8H, O5H→C6H, O3H→C2′H and O4′H→C5′H. Only in this way of the initial rotation of the OH hydroxyl group of the basic tautomer 1 of the quercetin molecule [26], it is possible to form new prototropic tautomers through the intramolecular transfer of single proton. However, precise investigation of the transformations via the rotations of the OH hydroxyl groups would be the subject of the next study [27], since in this paper we are focusing exactly on the mechanisms of the intramolecular proton transfer.

Thus, it was revealed the following chains of the SPT reactions (Fig 1, Table 1): 5↔5* (ΔΔGTS = 20.46); 25↔25* (ΔΔGTS = 61.63); 20↔1** (ΔΔGTS = 34.71) and 10↔1* (ΔΔGTS = 70.59 kcal∙mol-1).

Notably, all of these reactions are assisted by the formation at the TSs of the extremely strong intramolecular HCH…O H-bond (26.97–31.44 kcal∙mol-1 (Table 2)) between the CH2 group and neighboring oxygen atom. At this, all other H-bonds remain practically unchanged at the initial and terminal states (Fig 1, Table 2). Prototropic tautomers, which are formed in this case, are planar structures (Table 2).

Notably, activation barriers for the considered 5↔5*, 25↔25* and 10↔1* tautomerisations are quite high (~62–71 kcal∙mol-1), except the cases 5↔5* (ΔG = 20.46) and 20↔1** (ΔG = 34.71 kcal∙mol-1). This relatively small value of the barrier can be explained by the formation of the six-membered ring at the TS20↔1**C2'H2 and by moving of the proton along the O3H…C2′ H-bond [26]. In those cases, when TS25↔25*C6H2, TS5↔5*C8H2 and TS10↔1*C5'H2 contains four-membered rings and proton does not move along the intramolecular H-bond–the values of the activation barriers are much higher. At this, 1** tautomer is the only one structure, which has the C2 = C1′ double bond.

d) Transition of the proton from the O3H hydroxyl group to the O4 atom.

Further we investigated structural mechanisms of the single proton transfer, occurring between the O5H and O3H hydroxyl groups. Thus, it was found that proton can transfer from the O3H hydroxyl group to the O4 oxygen atom through the 1↔1* tautomerization reaction with the barrier ΔΔGTS = 13.10 kcal∙mol-1. However, terminal localized complex is dynamically unstable–reverse Gibbs free energy barrier has negative value (ΔΔG = -1.20 kcal∙mol-1) (exactly in this case it is observed at TSs the lowest value of the imaginary frequency νi = 892.1 cm-1) (Table 1).

It is logically to think by analogy that the same intramolecular proton transfer should occur from the O5H hydroxyl group to the O4 oxygen atom. But in this case the TSs and tautomers could not be localized at all.

e) Proton migration from the O7H/O5H hydroxyl groups to the C6 atom of the C6H group.

We also considered tautomeric transformation of the 1* tautomer by the transition of the protons from the O7H/O5H hydroxyl groups to the neighboring C6 atom of the C6H group.

Thus, in the first case the 1**↔1* tautomerization reaction proceeds via the transfer of proton from the O7H hydroxyl group to the neighboring C6 atom and occurs via the quite high barrier (ΔΔGTS = 49.01 kcal∙mol-1) and leads to the dynamically stable tautomer 1* (Fig 1, Table 1).

In the second case, the intramolecular proton transfer in the 1** tautomer from the O5H hydroxyl group to the neighboring C6 carbon atom of the C6H group– 1**↔1* –occurs through the TS (ΔΔGTS = 75.24 kcal∙mol-1) and leads to the formation of the dynamically unstable 1** tautomer with relative electronic energy 14.87 kcal∙mol-1, which further causes chain transfer of the proton from the O4H hydroxyl group to the O5 oxygen atom, leading to the stable conformer 1 (Fig 1, Table 1).

It can be expected the reduction of the values of the activation barriers at the consideration of these transitions in the polar solutions or assisted by various ligands.

Conclusions and perspectives

Presented QM/QTAIM computational modeling of the tautomers formation through the intramolecular proton transfer shows that the quercetin molecule is able to tautomerise via the different routes within the framework of the classical valency rules:

Proton transfer from the C8H group to the O1 atom, leading in three cases to the breakage of the C ring: 1↔1*, 7↔7* and 10↔10*, except the case of 4↔4* reaction (ΔΔGTS ~ 93–96 kcal∙mol-1).

Transition of the proton from the O7H/O3′H hydroxyl groups to the carbon atoms of the neighboring C6H/C2′H groups: 1↔1* and 1↔1* (ΔΔGTS ~ 65–68 kcal∙mol-1).

Migration of the proton from the O7H/O5H/O3H/O4′H hydroxyl groups to the carbon atoms of the C8H/C6H/C2′H/C5′H groups, preceded by the rotations of the hydroxyl groups around the C7O7/C5O5/C3O3/C4′O4′ bond by 180 degree: 5↔5* (ΔΔGTS = 20.46); 25↔25* (ΔΔGTS = 61.63); 20↔1** (ΔΔGTS = 34.71) and 10↔1* (ΔΔGTS = 70.59 kcal∙mol-1).

Proton transfer from the O3H hydroxyl group to the O4 oxygen atom with the formation of the dynamically-unstable tautomer: 1↔1* (ΔΔGTS ~ 13 kcal∙mol-1).

Transition of the proton from the O7H/O5H hydroxyl group to the C6 carbon atom of the C6H group: 1**↔1* and 1**↔1* (ΔΔGTS ~ 49–75 kcal∙mol-1).

These prototropic transformations of the quercetin molecule are accompanied by the geometrical changes, dipole moment rearrangement and breakage or formation of the intramolecular specific contacts (H-bonds and attractive van der Waals contacts).

It was demonstrated that the most probable process among all investigated is the proton transfer from the O3H hydroxyl group to the C2′ carbon atom of the C2′H of the B ring along the intramolecular O3H…C2′ H-bond with the further formation of the C2′H2 group, while the least probable proton transfer occurs from the C8H group to the O1 oxygen atom–causes the decyclization of the C ring.

Obtained results can be useful for the planning of targeted chemical experiments, aimed at the acceleration of the reaction of intramolecular tautomerization of a quercetin molecule by the ligands of different structure and origin, as well as for the better understanding of the mechanisms of the course of reactions, related to the metabolism of the quercetin molecule.

1 Oct 2019

PONE-D-19-25268

Intramolecular tautomerization of the quercetin molecule via the proton transfer: QM computational study

PLOS ONE

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1. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed:

- https://www.tandfonline.com/doi/abs/10.1080/07391102.2019.1645734?journalCode=tbsd20

-https://www.tandfonline.com/doi/abs/10.1080/07391102.2019.1656671?journalCode=tbsd20

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

2. Thank you for including the following funding information within the acknowledgements section of your manuscript; "DrSci Ol’ha O. Brovarets’ expresses sincere gratitude to the U.S.-Ukraine Foundation (USUF) Biotech Initiative for a travel grant (“2018 Emerging Biotech Leader of Ukraine”; https://www.usukraine.org/biotechnology-initiative/), enabling to participate in the “63rd Annual Meeting of the Biophysical Society BPS'2019” (Baltimore, Maryland, March 2-6, 2019; https://www.biophysics.org/2019meeting#/; https://bioukraine.org/news/emerging-biotech-leader-olhabrovarets-attends-63rd-biophysical-society-meeting-in-baltimore/; https://bioukraine.org/news/emergingleader-olha-brovarets-shares-her-us-experience-with-bionity-student-biotech-club/). DrSci Ol’ha O. Brovarets’ sincere thanks for the Scholarship of Verkhovna Rada (Parliament) of Ukraine for the talented young scientists given in 2019 year."

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Reviewer #1: I do not accept the first sentence of the abstract "Quercetin molecule (3, 3′, 4′, 5, 7-pentahydroxyflvanone, C15H10O7) is

an important flavonoid compound, containing two aromatic A and B rings linked through the C ring containing oxygen and five OH hydroxyl groups attached to the 3, 3′, 4′, 5 and 7 positions. This molecule is found in many foods and plants, and is known to act as a natural drug molecule with a wide range of treatment properties, like an anti-oxidant, anti-toxic etc."

1) Quercetin is not a natural drug! So far it is not proved! The anti-oxidant, anti-toxic effects are known, but their therapeutic treatments are not proper studied.

2) The standard quercetin is the 3, 3′, 4′, 5, 7-pentahydroxyflvanone, if we use a standard numeration. The author's numeration corresponds to rotation of the B ring by 180o around C2-C1' bond in respect to the standard! Their molecule is

3, 5′, 4′, 5, 7-pentahydroxyflvanone (in respect to the standard numeration) and the choice of the calculated molecule corresponds to rotation of the B ring by 180o around C2-C1' bond axis. May be the conformer used by authors is the most stable one according to their previous studies, but this is unknown for the plain reader when he starts to read the paper. This should be mentioned from the begining.

3) The first part of the sentence is a simple tautology and needs to be removed.

4) Why proton transfer? (not H atom transfer). The corresponding polarization is not determined.

The MS is interesting and continues the previous studies of quercetin isomers. This molecule is of great biological and medical importance; it received great attention during recent years. All DFT optimizations of transition states and global minima are well documented including Bader's QTAIM analysis of bond critical points. The topology of the electron density was analyzed, using program package AIM’2000 with all default options and wave functions obtained at the level of theory

used for geometry optimisation. The presence of the (3,-1) bond critical point (BCP), bond path between hydrogen donor and acceptor and positive value of the Laplacian at this BCP (Δρ>0) were considered altogether as criteria for the formation of the H-bond and attractive van der Waals contacts. Thus, the MS is acceptable after minor revision (including abstract improvement).

The main objection concerns the influence of the solvent on predictions of this study. There are no comments on this important practical question. In page 6 we have: "All calculations were performed for the tautomeric transitions of the quercetin molecule as their intrinsic property, that is adequate for modeling of the processes occurring in real systems".

Calculations of quercetin in vacuum? with proton transfer? This is not a real system. At least, some comments are necessary.

There are some typos. Even in the first sentence of the abstract one reads"pentahydroxyflvanone"? Thus, the careful reading would be useful.

Reviewer #2: The manuscript by Brovarets’ and Hovorun addresses several intramolecular proton transfer pathways in the quercetin molecule. The authors also examined the properties of the transition states related to these transfers. The employed methods are sound and the results can be reproduced. The authors treated the literature correctly. Yet, they should avoid overcitation of their previous work not related to quercetin. In my opinion, the results of this study are not significant to the broader readership of the Journal since possible biological and chemical roles of the examined pathways are not discussed. The manuscript is not well-written and I suggest a revision of the manuscript.

Additional comments:

Page 2, the sentence “It has attracted a lot of attention last time, due to the wide range of its treatment properties, …” should be modified.

Page 4, DNA bases are not related to their work and references 42-66 might be excluded from the manuscript

Page 4, this text is not particularly important for the presented work: “This points on the fact that proton transfer defines the quantum nature of the biological objects, so-called field of quantum biology, which was started as a separate discipline from late 1920s, when Niels Bohr, delivered an influential lecture on whether the “atomic theory” could help to solve the mystery of life.”

Page 9, “inramolecular proton”

Page 10, “in factthis”

Figure 1, middle panel “hydoxyl”

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Reviewer #1: Yes: Boris F. Minaev

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17 Oct 2019

Dear Editor, Professor Dennis Salahub!

On behalf of all the co-authors, I would like to express to you sincere appreciation for the providing of the comprehensive reviewing of our manuscript.

We made our best efforts to improve it in the strict accordance with the reviewers’ comments and remarks. Also, we have modified methods section in over to avoid the repetition.

At this all changes are highlighted in yellow.

I hope very much to receive your positive decision!

Best regards,

Professor Dmytro Hovorun

Corresponding Author

Reviewer #1:

Comment:

I do not accept the first sentence of the abstract "Quercetin molecule (3, 3′, 4′, 5, 7-pentahydroxyflvanone, C15H10O7) is an important flavonoid compound, containing two aromatic A and B rings linked through the C ring containing oxygen and five OH hydroxyl groups attached to the 3, 3′, 4′, 5 and 7 positions. This molecule is found in many foods and plants, and is known to act as a natural drug molecule with a wide range of treatment properties, like an anti-oxidant, anti-toxic etc."

1) Quercetin is not a natural drug! So far it is not proved! The anti-oxidant, anti-toxic effects are known, but their therapeutic treatments are not proper studied.

2) The standard quercetin is the 3, 3′, 4′, 5, 7-pentahydroxyflvanone, if we use a standard numeration. The author's numeration corresponds to rotation of the B ring by 180o around C2-C1' bond in respect to the standard! Their molecule is 3, 5′, 4′, 5, 7-pentahydroxyflvanone (in respect to the standard numeration) and the choice of the calculated molecule corresponds to rotation of the B ring by 180o around C2-C1' bond axis. May be the conformer used by authors is the most stable one according to their previous studies, but this is unknown for the plain reader when he starts to read the paper. This should be mentioned from the begining.

3) The first part of the sentence is a simple tautology and needs to be removed.

Reply:

We would like to sincerely thank to Reviewer for the comprehensive reviewing of our manuscript and performed comments. Below we provided the answers.

1) Using this statement we have been based on the recent literature data according quercetin. We have also added more reviews and overviews of patents on the studies according the action of quercetin. We agree with Reviewer that more studies are needed to better characterize the mechanisms of theraupeutic action of quercetin.

2), 3) We have corrected this sentence, at this a commonly used designation of quercetin has been used.

Comment:

4) Why proton transfer? (not H atom transfer). The corresponding polarization is not determined.

Reply:

In this paper we use the generally accepted in the literature term “proton transfer”. By default, it is considered that proton transfer would cause reorganization of the electronic density of the molecule.

Comment:

The MS is interesting and continues the previous studies of quercetin isomers. This molecule is of great biological and medical importance; it received great attention during recent years. All DFT optimizations of transition states and global minima are well documented including Bader's QTAIM analysis of bond critical points. The topology of the electron density was analyzed, using program package AIM’2000 with all default options and wave functions obtained at the level of theory used for geometry optimisation. The presence of the (3,-1) bond critical point (BCP), bond path between hydrogen donor and acceptor and positive value of the Laplacian at this BCP (Δρ>0) were considered altogether as criteria for the formation of the H-bond and attractive van der Waals contacts. Thus, the MS is acceptable after minor revision (including abstract improvement).

Reply:

Thank you for the comprehensive analysis and provided comments.

Comment:

The main objection concerns the influence of the solvent on predictions of this study. There are no comments on this important practical question. In page 6 we have: "All calculations were performed for the tautomeric transitions of the quercetin molecule as their intrinsic property, that is adequate for modeling of the processes occurring in real systems".

Calculations of quercetin in vacuum? with proton transfer? This is not a real system. At least, some comments are necessary.

Reply:

The aim of the present study was to investigate the intrinsically inherent properties of the quercetin molecule. Further, based on these studies on the basic properties of the quercetin molecule it would be possible to investigate more complex processes and systems, taking into account different ligands or specific environments, which could possibly accelerate these tautomerization processes.

Comment:

There are some typos. Even in the first sentence of the abstract one reads"pentahydroxyflvanone"? Thus, the careful reading would be useful.

Reply: We have removed this typos and proofread manuscript once more.

Reviewer #2:

Comment:

The manuscript by Brovarets’ and Hovorun addresses several intramolecular proton transfer pathways in the quercetin molecule. The authors also examined the properties of the transition states related to these transfers. The employed methods are sound and the results can be reproduced. The authors treated the literature correctly. Yet, they should avoid overcitation of their previous work not related to quercetin. In my opinion, the results of this study are not significant to the broader readership of the Journal since possible biological and chemical roles of the examined pathways are not discussed. The manuscript is not well-written and I suggest a revision of the manuscript.

Reply:

We are thankful to reviewer for the comprehensive analysis of our manuscript and also for the critical remarks, which we have implemented at the revision of the manuscript.

Comment:

Additional comments:

Page 2, the sentence “It has attracted a lot of attention last time, due to the wide range of its treatment properties, …” should be modified.

Page 4, DNA bases are not related to their work and references 42-66 might be excluded from the manuscript

Page 4, this text is not particularly important for the presented work: “This points on the fact that proton transfer defines the quantum nature of the biological objects, so-called field of quantum biology, which was started as a separate discipline from late 1920s, when Niels Bohr, delivered an influential lecture on whether the “atomic theory” could help to solve the mystery of life.”

Page 9, “inramolecular proton”

Page 10, “in factthis”

Figure 1, middle panel “hydoxyl”

Reply:

We have modified provided sentences and phrases.

22 Oct 2019

Intramolecular tautomerization of the quercetin molecule due to the proton transfer: QM computational study

PONE-D-19-25268R1

Dear Dr. Hovorun,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Dennis Salahub

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: As far as solvent account concens you refer to future elaboration. I should prefer to write short comment in your present version about limitation of the used vacuum approach.

This is optional deal.

Reviewer #2: I am satisfied with the corrections and thus recommend the manuscript for the publication in the present form.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: Yes: Boris F. Minaev

Reviewer #2: No

12 Nov 2019

PONE-D-19-25268R1

Intramolecular tautomerization of the quercetin molecule due to the proton transfer: QM computational study

Dear Dr. Hovorun:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

Dr. Dennis Salahub

Academic Editor

PLOS ONE