Theoretical and Experimental Investigation of Acidity of the

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Theoretical and Experimental Investigation of Acidity of the Glutamate Receptor Antagonist 6,7-Dinitro-1,4-Dihydro-Quinoxaline-2,3Dione (DNQX) and Its Possible Implication in GluA2 Binding Gutto Raffyson Silva de Freitas, Sara E. Coelho, Norberto K.V. Monteiro, Jannyely Moreira Neri, Livia Nunes Cavalcanti, Josiel B Domingos, Davi S. Vieira, Miguel A. F. de Souza, and Fabrício G. Menezes J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/acs.jpca.7b07775 • Publication Date (Web): 13 Sep 2017 Downloaded from http://pubs.acs.org on September 15, 2017

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The Journal of Physical Chemistry A is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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The Journal of Physical Chemistry

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1

Theoretical and Experimental Investigation of Acidity of

2

the Glutamate Receptor Antagonist 6,7-Dinitro-1,4-

3

Dihydro-Quinoxaline-2,3-Dione (DNQX) and Its Possible

4

Implication in GluA2 Binding

5

6 7

Gutto R. S. de Freitasa, Sara E. Coelhob, Norberto K. V. Monteiroa, Jannyely

8

Moreira Neria, Lívia Nunes Cavalcantia, Josiel B. Domingosb, Davi S. Vieiraa,

9

Miguel A. F. de Souzaa, and Fabrício G. Menezesa*

10 11 12

a

13

Grande do Norte, 59072-970, Natal-RN, Brazil

Química Biológica e Quimiometria, Instituto de Química, Universidade Federal do Rio

14 15

b

16

Federal de Santa Catarina, 88040-900, Florianópolis-SC, Brazil

Laboratório de Catálise Biomimética, Departamento de Química, Universidade

17 18 19 20

* Corresponding author:

21

FG Menezes ([email protected], phone: +55 84 3342 2323)

22 23

ACS Paragon Plus Environment


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1

Abstract

2

The acidity of organic compounds is highly relevant to understanding several

3

biological processes. Although the relevance and challenges in estimating pKa

4

values of organic acids is recognized by several reported works in the literature,

5

there is a lack in determining the acidity of amides. This paper presents an

6

experimental/theoretical combined investigation on the acid dissociation of the

7

compound

8

established antagonist of ionotropic glutamate receptor GluA2. DNQX was

9

synthesized and its two acidic constants were determined by UV-vis

10

spectroscopy. The experimental pKa of 6.99 ± 0.02 and 10.57 ± 0.01 indicate

11

that DNQX mainly exists as an anionic form (DNQXA1) in physiological media,

12

which was also confirmed by 1H NMR analysis. Five computational methods

13

were applied for estimating the theoretical pKa values of DNQX, including

14

B3LYP, M06-2X, ωB97XD and CBS-QB3, which were able to provide

15

reasonable estimates for pKa associated to DNQX. Molecular dynamics studies

16

have demonstrated that DNQXA1´ binds more effectively to the pocket of the

17

GluA2 than neutral DNQX, and this fact is coherent to the interactions between

18

amidic oxygens and Arg845 being the main interactions of this host-guest

19

system. Moreover, interaction of GluA2 with endogenous glutamate is stronger

20

than with DNQXA1, which is in agreement with literature. To the best of our

21

knowledge, we report herein an unprecedented approach involving acidity of the

22

antagonist DNQX, as well as the possible implications in binding to GluA2.

6,7-dinitro-1,4-dihydro-quinoxaline-2,3-dione

23 24 25


(DNQX),

a

well-

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1

1. Introduction

2

Organic acids play crucial roles in several biochemical reactions as well

3

as chemical processes in bench and industrial scales, and in this context the

4

ability to measure the dissociation of organic acids in water is of great interest

5

for the scientific community.1 The pKa values of organic acids can be

6

experimentally determined by several methods, such as potentiometry along

7

with UV-vis, fluorescence and NMR spectroscopies, among others.2 On the

8

other hand, advancement of computational methods has allowed estimation of

9

pKa values based on the calculated free energy of the starting acids and their

10

conjugate basis, in addition to the previously determined proton energy.3–5 In

11

fact, estimating a theoretical pKa is challenging since a small difference in its

12

value leads to high variation in the free energy of the system. Due to this

13

complexity and the particularities of each system, the choice of computational

14

methodology is crucial for obtaining accurate results.

15

In literature, there are several interesting publications about the

16

determination of experimental and theoretical pKa values for many organic.1,5–11

17

However, studies concerned with amides are very limited, as for example,

18

phthalimide (pKa 8.3212), which is a highly versatile reagent for obtaining

19

elaborated amides via Gabriel synthesis; and quinoxaline-2-one (pKa 9.0813),

20

which is a scaffold of heterocyclic compounds comprising several biological

21

activities and pharmaceutical applications. This gap can by justified by the fact

22

that most amides do not dissociate into a proton and a conjugate base in

23

neutral to slightly basic aqueous media. However, there are some exceptions.

24

Amides are very important to the contemporary world owing to their broad

25

spectrum of applications, which include synthetic polymers (e.g. nylon) and



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1

drugs (e.g. paracetamol). Organic compounds containing amide unities are also

2

applied in environmental and biological fields such as chemosensors.14–16

3

Further, the relevance of amides to the biochemical world is huge due to their

4

roles as key constituents of proteins and most enzymes, as well as other

5

biologically relevant chemical species. Significantly, many of these functions

6

and applications of amides are dependent on their acidity, especially due to the

7

supramolecular interactions involving both carbonyl oxygen and amidic

8

hydrogen.

9

The compound 6,7-dinitro-1,4-dihydroquinoxaline-2,3-dione (DNQX) is

10

well established as a potent antagonist at the ionotropic glutamate (Glu)

11

receptors,17 which are directly related to learning and memory processes. Thus,

12

DNQX have been extensively reported in biochemical studies, including

13

experimental and theoretical works evaluating specific interactions with its

14

natural receptor, as well as a model for developing bioactive species.17–27

15

Although structural aspects of DNQX inside the biological environment have

16

been richly reported in literature, to the best of our knowledge there are only two

17

reports focusing on their primary structural aspects. Yu et al. reported DNQX

18

proving to be a chemosensor for anionic species in DMSO because of its H-

19

donor ability.15 Kubicki and coauthors reported X-ray crystallographic data of

20

DNQX,28 and Madden and coauthors reported an infrared spectroscopy-based

21

study focused on dissociation of DNQX in aqueous medium in an attempt to

22

improve the understating of its binding mechanism to the glutamate receptor.21

23

In fact, this latter work proved to be interesting due to some additional

24

information about electronic configurations that are not possible to be extracted

25

from X-ray crystallographic data alone.


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1

In this paper, we present an experimental/computational combined study

2

based on structural aspects of DNQX. Firstly, DNQX was synthesized and

3

adequately characterized, including determination of their pKa values from UV-

4

vis spectroscopy titrations. Then, a computational approach based on the well-

5

established B3LYP/6-311++G(d,p)29,30 method was employed in attempt to

6

investigate the preferable tautomers of DNQX and its monoanion based on

7

structural and electronic properties. Further, estimation of theoretical pKa values

8

for sequential dissociation of DNQX into both mono- and di-anionic species

9

were performed by applying five different methods: B3LYP/6-311++G(d,p) and

10

B3LYP/6-311++G(3df,3pd),29,30

M06-2X/6-311++G(d,p),31

11

311++G(d,p) and CBS-QB3.32 In all cases, conductor-like polarizable continuum

12

mode (CPCM) was employed to simulate aqueous media due to the broad

13

range of systems covered by this solvation model.33,34 Lastly, the results found

14

for the first dissociation of the target-substrate in aqueous media led us to carry

15

out a molecular dynamics (MD) simulation study focused on binding both

16

neutral DNQX and its monoanion derivative to the glutamate receptor GluA2-

17

S1S2 in attempt to collect new information related to the referred binding

18

mechanism.

ωB97XD/6-

19 20

2. Materials and methods

21

2.1. General

22

All reagents and solvents were purchased from commercial sources

23

(Sigma-Aldrich, Merck, Tedia and Cambridge Isotope) in adequate analytical

24

grade and used without further purification. Melting points were measured in a

25

Microquímica MQAPF-301 and were not corrected. Elemental analyses were



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1

performed in a Carlo Erba E-1110 instrument. Infrared spectra were obtained in

2

a Perkin-Elmer model 283 equipment or in a Shimadzu FTIR-8400S. NMR

3

analysis were performed in a Varian Mercury Plus 400-MHz instrument.

4 5

2.2. Synthesis

6

2.2.1. 1,4-Dihydroquinoxaline-2,3-dione (QXDO)

7

A solution of o-phenylenediamine (10.0 g; 92.5 mmol) and dihydrated

8

oxalic acid (11.65 g; 92.5 mmol) in 3 M hydrochloric acid (150 ml) was refluxed

9

over three hours. Next, water (500 ml) was added and the precipitate was

10

collected by filtration, washed with water, cold ethanol and dried. The pure

11

product was obtained as a beige solid. Mp > 250oC. IR (KBr) υmax (cm-1): 3154,

12

3075, 2946, 1735, 1607, 1398, 1348, 898, 843, 797, 662, 1H NMR (500 MHz,

13

DMSO-D6) δ (ppm) 11.91 (br, 2H, NH), 7.15-7.10 (m,2H, CH, Ar), 7.09-7.04 (m,

14

2H, CH, Ar).

15

115,6. Elemental analysis for C8H6N2O2: C, 59.26; H, 3.73; N, 17.28; found: C,

16

56.30; H, 3.79; N, 15.32. Yield: 85%.

13

C NMR (100 MHz, DMSO-D6) δ (ppm) 155.7, 126.0, 123.4,

17 18

2.2.2. 6,7-Dinitro-1,4-dihydroquinoxaline-2,3-dione (DNQX)

19

QXDO (6.00g, 36.9 mmol) was added to a solution of concentrated

20

sulfuric acid (30 mL) in an ice bath, and this system was kept under magnetic

21

stirring until total homogenization. Then 65% nitric acid (12 mL) was added by

22

drops, and the final solution was maintained under stirring for one hour while

23

raised to ambient temperature, and then for more five hours under stirring. After

24

that, the final solution was poured onto crushed ice and the solid was then

25

filtered and washed with water and cold ethanol to afford pure product.


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1

Mp > 230oC. IV (KBr) υmax (cm-1) 3154, 3075, 2946, 1706, 1558, 1398, 1348,

2

898, 662. RMN 1H (400 MHz, DMSO-D6) δ (ppm) 12.50 (s, 2H, NH); 7.72 (s,

3

2H, CH, Ar).

4

112.4. Elemental analysis for C8H4N4O6: C, 38.11; H, 1.60; N, 22.22; found: C,

5

38.18; H, 1.67; N, 22.27. Yield: 80 %.

13

C NMR (100 MHz, DMSO-D6) δ (ppm) 155.2; 137.3; 130.3;

6 7

2.3 Experimental pKa determination The

8

acidity

constants

(pKa)

for

DNQX

were

determined

by

9

spectrophotometric titration, carried out using an Orion Expandable Ion

10

Analyzer EA 920 pH meter fitted with a glass-combined electrode (Ag/AgCl) and

11

a Varian Cary 50 Bio UV-vis spectrophotometer. The titration temperature was

12

kept constant at 25.00 ± 0.05oC using a thermostatic bath. Deionized water was

13

used to prepare the titration media H2O/DMSO (95:5 v/v) and the ionic strength

14

of all solutions were kept constant at 0.1 mol L-1 by using KCl. The experiments

15

were performed at minimum in duplicate in a magnetically stirred double-wall

16

glass titration vessel and started by titrating 20 mL of solutions containing 2x10-

17

3

18

DNQX, in the pH range of 4.18 to 11.46. The pKa values were calculated as an

19

average value from all wavelengths through Equation 1; where the absorbance

20

value (Ai) is the absorbance corresponding to a given wavelength of the

21

spectrum recorded in the intermediate pH buffer solution (pHi), and the

22

absorbance values (Aac and Abas) for the fully protonated and fully deprotonated

23

species, respectively.35,36

24

‫ܭ݌‬௔ = ‫ܪ݌‬௜ − ݈‫ ݃݋‬ቂ஺ ೌ೎ ି஺

mmol of DNQX, with a solution of 0.1 mol L-1 NaOH containing 0.1 mmol L-1

஺

ି஺೔

೔

್ೌೞ

ቃ

(1)



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1

2

2.4 NMR study The

1

H NMR spectra were recorded on a Varian Oxford AS-400

3

spectrometer. For tautomeric and structural resonance analysis, 0.5 mL NMR

4

samples were prepared by dissolving 18 mg of the DNQX in 1.5 mL of

5

D2O/DMSO-d6 (95:5 v/v) and adding D2O solution of NaOD (0.1 mol L-1) until

6

pD 6, 8 and 12. Spectra were obtained collecting 32 scans per 1H NMR

7

spectrum. Experimental 1H NMR chemical shift data were fitted using the

8

ACD/Labs Version 12.01 program.

9

2.5. Theoretical quantum calculations

10

All calculations were carried out using a Gaussian 09W program.37

11

Optimization of the geometric and electronic properties of each structure was

12

performed using B3LYP/6-311++G(d,p), firstly in gas phase, and then using

13

CPCM as an implicit model to predict the effect in aqueous solution. The

14

stationary points were characterized properly from the eigenvalues of the

15

Hessian matrix and none of calculations presented negative vibrational

16

frequencies. Optimized structures of DNQX and their monoanion (DNQXA1)

17

and dianion (DNQXA2) were used as input for determining theoretical pKa

18

values using B3LYP/6-311++G(d,p), B3LYP/6-311++G(3df,3pd), M06-2X/6-

19

311++G(d,p), ωB97XD/6-311++G(d,p) and CBS-QB3.

20

Calculation of theoretical pKa values for dissociation of DNQX into its

21

DNQXA1 form, as well as from this latter into DNQXA2 were based on

22

Equations 2 and 3:


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1 2

‫ ܪ ⇆ ܣܪ‬ା + ‫ିܣ‬

3

(2)

4

p‫ܭ‬௔ =

5

(3)

∆ீ(ೌ೜,ಹಲ) ଶ.ଷ଴ଷோ்

6 7

where HA is the acidic form, and A- is the conjugate base, ∆G is the free energy

8

of the equilibrium, Ka is the acidity constant, R is the universal gas constant and

9

T is the temperature.

10

The theoretical Gibbs free energy variation involved in the dissociation

11

processes were obtained by considering solvation contribution to the gas phase

12

value according to Equations 4-6, which are extracted from the thermodynamic

13

cycle presented in Scheme 1. The most acceptable value for proton free energy

14

in gas phase (G(gas,H+)) and proton free energy in aqueous medium (G(solv,H+))

15

were considered -6.28 kcal mol-1 and –265.63 kcal mol-1) 3–5.

16 17

∆‫(ܩ‬௔௤,ு஺) = ∆‫(ܩ‬௚௔௦,ு஺) + ∆∆‫(ܩ‬௦௢௟௩,ு஺)

18

(4)

19

∆‫(ܩ‬௚௔௦,ு஺) = ‫(ܩ‬௚௔௦,஺ି) + ‫(ܩ‬௚௔௦,ுା) − ‫(ܩ‬௚௔௦,ு஺)

20

(5)

21

∆∆‫(ܩ‬௦௢௟௩,ு஺) = ∆‫(ܩ‬௦௢௟௩,஺ି) + ∆‫(ܩ‬௦௢௟௩,ுା) − ∆‫(ܩ‬௦௢௟௩,ு஺)

22

(6)

23 24



Page 10 of 37

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A-(gas)

HA(gas)

∆G(solv,A-)

-∆G(solv,HA)

1

HA(aq)

H+(gas)

+

A-(aq)

+

∆G(solv,H+)

H+(aq)

2

Scheme 1. Thermodynamic cycle used for rationalizing the calculation of

3

theoretical pKa considering solvent effect.

4 5

2.6. Molecular dynamics

6

The X-ray crystal structure of glutamate receptor (GluA2) ligand binding core

7

(S1S2J) in complex with the antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX)

8

was obtained from the Protein Data Bank (PDB ID code: 1FTL).17 Protons were

9

added to the protein structure using CHARMM27 force field.38. MD simulations

10

were performed by GROMACS 5.1.4 program39 for each DNQX species, neutral

11

and monoanion (Figure 1), using CHARMM27 force field with TIP3P water

12

molecules.38,40 DNQX topology was generated by the SwissParam web

13

server.41 Counter ions were added to neutralize charge and maintain the system

14

at a concentration of 150 mmol L-1 in a triclinic box. Covalent bonds in the

15

protein were constrained using the LINCS algorithm.42 Electrostatic forces were

16

calculated with the PME implementation of the Ewald summation method and

17

the Lennard-Jones interactions were calculated within a cut-off radius of 1.0 nm.

18

The system was equilibrated according to the following protocol: solvated

19

DNQX-GluA2 complex geometry was minimized by the steepest descent

20

algorithm for 50000 steps with tolerance of 10 KJ mol-1 nm-1 and by a gradient


Page 11 of 37


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1

conjugated algorithm for 50000 steps with tolerance of 10 KJ mol-1 nm-1. In

2

order to eliminate the repulsion energy eventually present in the system, a

3

400ps simulation was performed in the NVT ensemble at 310K, followed by

4

another 400ps simulation in the NpT ensemble with controlled pressure at

5

1.0bar by Berendsen pressure coupling algorithm.43 Finally, 20-ns production

6

MD simulation using NVT ensemble was performed for each DNQX species

7

(neutral and monoanion) in order to evaluate the stability of the system.

8

Interaction potential energy (IPE) can be defined as the total interaction

9

energy between protein A and protein B, and it was computed according to the

10

equation:

11

IPE = ∑∑ Ei , j

NA NB i

(7)

j

12

where Eij is the interaction energy between an atom (i ) from Protein A and

13

an atom ( j ) from Protein B, and NA and NB are the total number of protein A

14

and B atoms, respectively.

15 16

3. Results and discussion

17

3.1 Synthesis of DNQX

18

DNQX was prepared according to the literature44 via a two-step protocol,

19

as demonstrated in Scheme 2. Firstly, o-phenylenediamine and oxalic acid

20

undergo acid-catalyzed condensation to afford the 1,4-dihydroquinoxaline-2,3-

21

dione (QXDO), which is then doubly nitrated at positions 6 and 7 to afford the

22

target-molecule in good yields. The product was adequately characterized by

23

elemental analysis along with IR and NMR spectroscopies.



Page 12 of 37

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O OH

HO O +

NH2

1 2

HCl, H2O, reflux, 2h (85%)

NH2

H N

O

HNO3, H2SO4, 0 oC to RT, 6h

N H

O

(80%)

QXDO

O2N

H N

O

O2N

N H

O

DNQX

Scheme 2. Synthetic protocol for obtaining DNQX.

3 4

3.2 Determination of the experimental values for pKa1 and pKa2 of DNQX

5

The experimental acidity constants (pKa1 and pKa2) of DNQX were

6

determined by spectrophotometric titration in the pH range from 4.18 to 11.46 in

7

H2O/DMSO (95:5 v/v) and the ionic strength was adjusted to 0.1 mol L-1 with

8

KCl. Figure 1a shows the UV-vis spectra evolution over the pH change during

9

the titration. All threeneutral (DNQX), monoanionic (DNQXA1) and dianionic

10

(DNQXA2) species presented different absorption in a wide wavelength range,

11

and also defined isosbestic points. As can be seen in the inset of Figure 1a, the

12

isosbestic points at 387 and 318 nm express each deprotonation process,

13

revealing the selective appearance of DNQXA1 and DNQXA2, respectively.

14

Thus, from these spectra evolution it was possible to isolate the spectra change

15

for both dissociation processes (Figure S1, Supporting Information); and by

16

using Equation 1, the respective acidity constants were determined to be pKa1 =

17

6.99 ± 0.02 and pKa2 = 10.57 ± 0.01 for the formation of DNQXA1 and

18

DNQXA2, respectively. Based on these pKa values, the relative species

19

distribution was calculated and is shown in Figure 1b. As one can observe, at

20

the physiological pH (around 7.4), 28% of the species correspond to DNQX,

21

and 72% to DNQXA1.


Page 13 of 37


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1 2

Figure 1. (a) Variation in UV-vis absorption spectra of DQNX (black line) over

3

the pH increment, the yellow lines correspond to DNQXA1 formation and the

4

blue lines to DNQXA2 formation. The inset shows the pH dependence of the

5

absorption at 387 nm (yellow square) and at 318 nm (blue circle), the isosbestic

6

points for the selective appearance of DNQXA1 and DNQXA2, respectively. (b)

7

Plot of the relative species distributions, DNQX (black triangle), DNQXA1

8

(yellow square) and DNQXA2 (blue circle) as a function of pH.



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1 2

3.3. Theoretical quantum calculations

3

3.3.1 Tautomeric equilibrium

4

Several organic compounds are subjected to undergo tautomerism in

5

solution. The most popular are definitely aldehydes and ketones,45 however

6

many heterocylic derivatives also present this kind of proton transfer, including

7

those derived from benzimidazole,46,47 quinoxaline45,48,49 and pyridine50–53 to cite

8

a few. There is evidence for tautomerism in amides, however, the iminol form is

9

very unfavorable,54,55 even when there is aromatic character in some extent,

10

such as in the case of 2-pyridone/2-hydroxipyridine system. DNQX is suggested

11

to coexist with two other tautomers, 3-hydroxy-6,7-dinitro-1H-quinoxalin-2-one

12

(T1a) and 2,3-dihydroxy-6,7-dinitroquinoxaline (T2a), which are able to adopt

13

two other main conformations, T1b and T2b, respectively (Scheme S1).

14

The stability of DNQX and their referred tautomers were evaluated from

15

energetic parameters in water under implicit solvation (CPCM model), and the

16

results of relative free energy of chemical structures shown in Scheme S1 are

17

presented in Table S1.

18

The more stable tautomer of DNQX in aqueous environment is the

19

diamide form of 7.94 kcal mol-1 in relation to structure T1a. In fact, the

20

preference for amide over aminol form is coherent to some similar systems,

21

such as 1,4,-dihydroquinoxaline (QXDO)49 and 2-pyridone.45,50 Differently from

22

QXDO, there is a slight preference for the conformer T1a over T1b in the

23

studied medium (c.a. 1 kcal mol-1), and this additional stabilization can be

24

attributed to intramolecular hydrogen bonding involving sp2-hydridized oxygen

25

of the carbonyl group and hydrogen of the hydroxyl. Although contribution of


Page 14 of 37

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15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

both di-iminol tautomers can be assumed to be negligible to the equilibrium, the

2

T2b structure was found to be more stable than T2a by approximately 0.51

3

kcal.mol-1, and this can be originated from a more symmetric charge distribution

4

in the molecule.

5 6

3.3.2 Dissociation of DNQX into DNQXA1 and DNQXA2

7

Interaction of DNQX with iGluR in biological media takes place by several

8

hydrogen bonding events which involve effective participation of both amide and

9

nitro groups.17,20,24,27 Since electronic density of whole molecules plays a crucial

10

role in the binding mechanism, it becomes interesting to understand H-donor

11

properties of DNQX. In general, neutral amides are very weak acids, however,

12

some activated species may lead to the formation of anionic species, especially

13

due to the presence of the strong electron-withdrawing nitro groups and/or the

14

possibility to generate (hetero)aromatic species. In theory, when DNQX losses

15

an amidic proton to the media, the DNQXA1a monoanion can be stabilized by

16

resonance, i.e. the excessive electronic density is delocalized into the carbonyl

17

group or para nitro group to undergo canonical DNQXA1b and DNQXA1c

18

forms, respectively (Scheme 3). Similarly, after a second deprotonation, the

19

additional electronic density of the dianion can be dispersed by resonance in

20

both carbonyl and nitro directions, generating several canonical forms.



Page 16 of 37

16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

O2N O2N

H N N H DNQX

O O

-H+ O2N O N O

O

O2N

N

O

O2N

N O H DNQXA1c

O2N

N O H DNQXA1a

O2N

N

N

O

N O H DNQXA1b

-H+

O2N

N

O

(futher canonical forms) O2N

1

N

O

DNQXA2a

O2N

N

O

O2N

N

O

H

DNQXA1d

2

Scheme 3. Illustrative representation of the two-consecutive dissociation of

3

DNQX and their possible stabilization forms.

4 5

Figure 2 presents the electrostatic potential maps for DNQX and their

6

monoanion and dianion species, DNQXA1 and DNQXA2. Clearly, the more

7

positive regions in DNQX are those associated to the acidic N-H groups. On the

8

other hand, carbonyl oxygen has the most electronic rich center due to the well-

9

established resonance effect of amides. The aromatic hydrogen is also

10

considerably positive due to the presence of strong electron-withdrawing nitro

11

group in the para position of the benzene ring. There is a considerable increase

12

in the electronic density in the whole molecule after the first deprotonation, but

13

more pronounced in N2 and O2, which strongly supports amide group

14

resonance being the main responsible for stabilization of anionic DNQX.

15

Although DNQXA1c tautomer could be stabilized by an intramolecular hydrogen


Page 17 of 37


17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

bond between hydroxyl of iminol and iminoxy groups, this tautomer was found

2

to be less stable than DNQXA1a by 5.20 kcal mol-1.

3

When a second deprotonation takes place, the resulting dianion

4

DNQXA2 is even more electronically rich, and consequently less stable.

5

Notably, charge distribution is more prominent in the N-C-O site of the

6

heterocyclic ring as consequence of the conjugation between both anionic

7

nitrogen and the carbonyl groups. In observing Figure 2, it is possible to

8

understand the contribution of the nitro group in the acidity of DNQX since the

9

region bearing both activators also becomes more negative; however, the

10

magnitude of this electronic density increasing as a result of a conjugation

11

involving nitro-aromatic systems being less expressive than conjugation to

12

carbonyl groups. As expected, DNQXA2 dianion has a symmetric charge

13

distribution in relation to DNQXA1 monoanion.

14



18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Figure 2. Electrostatic potential maps of DNQX, its monoanion (DNQXA1) and

2

dianion (DNQXA2) forms obtained using B3LYP/6-31++G(d,p) method and

3

CPCM solvation model.

4 5

Structural parameters of DNQX as well as DNQXA1 and DNQXA2 were

6

obtained from the optimized structure using B3LYP/6-31++G(d,p) method, and

7

the selected bond lengths and angles are given in Table S2. Only slight

8

differences in the structural parameters of DNQX determined by X-ray

9

crystallography28 were found by the herein reported theoretical results, which

10

indicates that the employed theory level was able to provide reliable results. In

11

this comparison, the more pronounced deviation was found as 0.165 Å and 2.3o

12

for N─H and N2─N2─C4 bonds.

13

The more relevant results found for the anionic species arrive from

14

stretching the C─O (carbonyl groups) bonds and shortening the C─N bonds of

15

the heterocyclic ring, as consequence of more pronounced single and double

16

character of these C─O and C─N bonds, resulting from the resonance effect.

17 18

3.3.3 Theoretical determination of pKa values for DNQX

19

The literature shows several interesting examples in which theoretical

20

and experimental data present good correlation for pKa values; however,

21

different methods can lead to better or worse results depending on the system.

22

Five different methods were employed for estimating the pKa values for first and

23

second dissociation of DNQX, as presented in Table 1.

24


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19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Table 1. Theoretical (B3LYP/6-311++G(d,p), B3LYP/6-311++G(3df,3pd), M06-

2

2X/6-311++G(d,p), ωB97XD /6-311++G(d,p) and CBS-QB3) and experimental

3

(UV-Vis) pKa values obtained for first and second deprotonation of DNQX.

4

5 6

Method

pKa1

pKa2

B3LYP/6-311++G(d,p)

5.39

10.43

B3LYP/6-311++G(3df,3pd)

6.40

12.04

M06-2X/6-311++G(d,p)

4.54

10.61

ωB97XD /6-311++G(d,p)

8.72

14.57

CBS-QB3

8.04

12.80

UV-Visa

6.99 ± 0.02

10.57 ± 0.01

a

Experimental data obtained from titration of DNQX from pH 4.18 to 11.46 in

H2O/DMSO (95:5 v/v) and ionic strength of 0.1 mol L-1 (KCl).

7 8

Experimental pKa for DNQX determined by UV-vis spectroscopy in 5%

9

aqueous DMSO were found to be 6.99 ± 0.02 and 10.57 ± 0.01, respectively for

10

first and second dissociation. As can be noted in Table 1, theoretical values

11

were found between 4.54-8.72 and 10.43-12.80 for pKa1 and pKa2, respectively.

12

In general, all computational methods presented reasonable results for

13

determining acidity constants for DNQX. The results were verified below or

14

above the experimental data to some extent, depending on the method. In fact,

15

these variations are expected, especially due to the complexity involved in the

16

theoretical determination of acidity constants for organic acids. B3LYP was the

17

first choice for estimating theoretical pKa values for DNQX, since this hybrid

18

functional method is the most used in computational chemistry and was found



20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

being able to successfully predict several properties for a broad spectrum of

2

both neutral and ionic organic molecules. B3LYP calculations were performed

3

using two different base sets, 6-31++G(d,p) and 6-31++G(3df,3pd), which led to

4

results close to the experimental values for both constants related to

5

dissociation of DNQX. Interestingly, the method based on 6-31++G(3df,3pd)

6

was found presenting the better value for pKa1 (6.40) of DNQX, while 6-

7

31++G(d,p) presented the closest value for pKa2 (10.43). The GGA metahybrid

8

M06-2X was applied since it presented good results for thermochemistry of the

9

main group, as well as kinetics and non-covalent interactions. Indeed, this

10

method was found presenting interesting results for the acidity constant of

11

DNQX, especially for pKa2 value (10.61). The ωB97XD is a GGA meta hybrid

12

method with empirical dispersion terms and long range corrections. Lastly, the

13

CBS-QB3 is a composite method able to provide thermodynamic properties with

14

high precision, such as deprotonation with errors smaller than 1 kcal.mol-1. Both

15

ωB97XD and CBS-QB3 were found presenting higher values of pKa for

16

dissociation of DNQX into both monoanion and dianion species.

17 18

3.4 NMR study

19

A 1H NMR study was carried out in attempt to confirm the behavior of

20

DNQX in aqueous media. Figure 3 presents the 1H NMR spectra of DNQX in

21

pH 6 and pH 8. It is very clear that DNQX exists as a neutral form in pH 6,

22

characterized by a singlet at 7.69 ppm (Figure 3a). DNQX transfers one proton

23

to the media, as can be evidenced by a single singlet at 7.44 ppm (Figure 3b).

24

Both signals are only related to aromatic hydrogens as signals associated to the

25

amidic proton could not be visualized due to a fast isotopic change with the


Page 20 of 37

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21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

media. As expected, the aromatic hydrogen of the anionic specie is found at a

2

lower chemical shift value due to the higher electronic density originated from

3

the deprotonation. On the other hand, it calls attention to the fact that only one

4

signal is found in the spectrum of anionic DNQX, and this can be associated to

5

a fast equilibrium between the two main DNQXA1a and DNQXA1a’ tautomers in

6

aqueous media at pH 8 (Scheme 4).

7 8 9 10

Figure 3. 1H NMR spectra of DNQX in deuterated aqueous DMSO: pH 6 (a)

11

and pH 8 (b).

12

13 14

O2N

N

O

O2 N

H N

O

O2N

N H

O

O2 N

N

O

DNQXA1

DNQXA1´

Scheme 4. Equilibrium between two main anionic forms of DNQX in pH 8.

15



22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

3.5 Molecular dynamics: binding of DNQX and DNQXA1 to GluA2 S1-S2

2

To the best of our knowledge, we report herein the first determination of

3

acidity constant related to DNQX. From our results, the pKa 6.99 ± 0.02

4

associated to the first deprotonation of DNQX indicates an equilibrium between

5

DNQX and DNQXA1 in physiological buffer (pH around 7.4), and this fact

6

brought to light the possibility that both these neutral and anionic species could

7

be responsible for the antagonism of DNQX in the GluA2 S1-S2 system.

8

Based on the structural relationship between DNQX and Glu in binding

9

GluA2 receptor,22 monoanionic DNQX could interact with the referred receptor

10

via both DNQXA1 and DNQXA1´, i.e. N─H group next or opposite to the

11

Pro478, found as representative residue in the antagonism mechanism.17,20 In

12

order to evaluate all possibilities, interactions of GluA2 with DNQX, DNQXA1,

13

DNQXA1´ and also Glu (endogenous ligand) were investigated. The systems

14

constituted by the GluA2 and DNQX/DNQXA1/DNQXA1´/Glu complexes were

15

submitted to MD simulations in order to estimate the binding energy of DNQX

16

species and Glu to the protein target GluA2. Figure S2 presents the final

17

structures obtained from MD simulations. A total time of 20.000 ps simulations

18

were enough to equilibrate protein and ligands. RMSD (root mean square

19

deviation) were calculated over time as a parameter to estimate the structural

20

stability of the systems (see supplementary material). Structural stabilization

21

occurred around 10.000ps for all ligands, thus the potential interaction energies

22

were computed over the last 10.000ps of each simulation. The GluA2 complex

23

with DNQXA1, DNQXA1´, DNQX and Glu presented average RMSD values of

24

0.106± 0.0135 nm, 0.120 ± 0.0158 nm, 0.118 ± 0.0195 nm and 0.115 ±

25

0.0185nm, respectively, indicating that the protein is well stabilized by the


Page 22 of 37

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23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

ligands, especially by DNQXA1 and Glu. These data presumably suggest that

2

all the ligands produced similar dynamic effects on the enzyme, which

3

characterize such a molecular recognition pattern among them.

4

Using a geometric criteria for hydrogen bond detection,56 it was possible

5

to identify the intermolecular hydrogen bonds formed between the ligand

6

molecules and the neighboring amino acid residues of the protein (Figure 4). On

7

average, DNQXA1 and DNQXA1´ formed ten hydrogen bonds with GluA2, while

8

Glu formed fourteen and the neutral DNQX only formed seven. Figure 4

9

presents the main amino acid residues from the protein binding pocket which

10

interacts with ligands forming the base of the GluA2 molecular recognition. In

11

general, the anionic ligands show shorter hydrogen bonds into the protein

12

binding pocket.

13

A broader and more complete analysis of these interactions can be made by

14

IPE, which provides us with a simple way to estimate the protein-ligand binding

15

energies

16

DNQX/DNQXA1/DNQXA1´/Glu). Figure 5 clearly shows a considerable

17

difference in the IPE values between GluA2-DNQX and GluA2-DNQXA1 over

18

time. The energy profile for the anionic ligand shows more attractive values with

19

lower fluctuation than the neutral one. IPE average values are -325.483 ± 16.63

20

kJ mol-1(DNQXA1), -280.931 ± 24.28 kJ mol-1 (DNQXA1´), -339.535 ± 52.09 kJ

21

mol-1 (Glu) and -249.839 ±22.21 kJ mol-1(DNQX), indicating that GluA2 has a

22

significantly higher affinity for the anionic species. Furthermore, even in

23

considering DNQXA1 specie, these results are in agreement with the following

24

order of Glu > DNQX for electronic energy binding as previously reported.20

present

in

the

IPE

for

the

simulated

25


systems

(GluA2-


24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2

Figure 4. Binding pocket configuration for DNQX (a), DNQXA1(b), DNQXA1´(c)

3

and glutamate (d). The interatomic distances are given in Å.

4


Page 24 of 37

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25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2

Figure 5. Potential binding energy plots of DNQX (black), DNQXA1 (red),

3

DNQXA1´ (green) and Glu (blue) with GluA2 binding pocket versus simulation

4

time. The potential binding energy values were extracted from the last 10.000ps

5

of the MD simulation.

6 7

Activation of the GluA2 system is associated to the ion channel opening,

8

which in turn is related to a high degree of ligand binding domain closure by

9

action of an agonist

20

. On the other hand, an antagonist such as DNQX does

10

not exhibit any considerable conformation changes in the binding domain in

11

relation to the apo state.17,21 From previous experimental and theoretical

12

reports, one of the main interaction sites in the binding of DNQX to GluA2

13

involves the interaction of carbonyl oxygens and the Arg485 residue.17,20 From

14

our results, DNQXA1 species bind more strongly to the GluA2 binding pocket

15

than DNQX, which is consistent with the fact that the Arg485 side chain must be



Page 26 of 37

26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

protonated in physiological conditions. Another important finding from the MD

2

study consists in the hydrogen bonding interaction between oxygen of the

3

anionic DNQX nitro group and Tyr220, which could contribute as a restriction to

4

the conformational changes required by the ion channel activation. Although

5

studies based in X-ray crystallography data pointed Pro478 relevant in the

6

binding mechanism, our results suggest that this residue is not involved in the

7

lowest energy binding pocket. In fact, at equilibrium, proteins can assume

8

several conformations and thus X-ray, as rigid model, may lead to simplified

9

interpretations of molecular recognition processes. Certainly, the elucidation of 17

10

structural data obtained from GluA2 receptor co-crystallized with DNQX

, as

11

well as other computational studies,20,23 are remarkable for understanding the

12

antagonism mechanism. However, we believe that a MD study aiming at the

13

possibility of DNQX binding to the GluA2 pocket via its anionic form DNQXA1

14

should be proposed.

15 16

4. Conclusions

17

The glutamate receptor antagonist DNQX was synthesized and had their

18

pKa experimentally determined by UV-vis analysis and estimated by quantum

19

calculations from five different computational methods. The experimental pKa

20

values for DNQX were found as 6.99 ± 0.02 and 10.57 ± 0.01 for the first and

21

second deprotonations, respectively. In general, all explored computational

22

methods

23

311++G(d,p), and ωB97XD /6-311++G(d,p) and CBS-QB3) were able to

24

provide reasonable estimations for both pKa associated to DNQX. Quantum

25

calculations indicate that DNQX mainly exists in solution in its diamide

(B3LYP/6-311++G(d,p),

B3LYP/6-311++G(3df,3pd),


M06-2X/6-

Page 27 of 37


27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

tautomer, which loses a proton to originate DNQXA1a as the main anionic

2

specie. Structural and electronic parameters indicate that this anionic specie

3

has additional electronic density which is strongly delocalized into the oxygen of

4

the carbonyl group adjacent to the original N─H moiety, and also but to a lesser

5

extent to the nitro group at para position. A second deprotonation leads to the

6

symmetrical DNQXA2a form. Additional evidence for the mono-ionic form of

7

DNQX was obtained from NMR studies, which indicates neutral DNQX existing

8

at pH 6 and the equilibrium of two main DNQXA1 and DNQXA1´ tautomers at

9

pH 8. Since DNQX has its first pKa of 6.99 ± 0.02, it is very plausible to suggest

10

that both neutral and mono-anionic species could be responsible for the

11

antagonism of DNQX. In fact, the mono-anion is 72% existing at physiological

12

pH.MD studies have indicated that interactions of DNQXA1 and DNQXA1´ with

13

GluA2 are far more favorable than DNQX, ∆ IPE around 76.0 and 35 kJ mol-1,

14

respectively; however, still less effective than natural endogenous Glu. To the

15

best of our knowledge, this is the first work reporting the existence of both

16

neutral and anionic DNQX at physiological pH, as well as the possible

17

implication of the latter species in the GluA2 antagonism mechanism.

18 19

Supporting Information

20

The supporting information of this manuscript includes UV-vis spectra of pH

21

study, final structures obtained from MD simulation, representation of

22

conformers and tautomers of DNQX as well as their computed energies,

23

andselected bond and angles of DNQX, DNQXA1 and DNQX2.

24 25



28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2

Acknowledgements

3

The authors would like to thank the Brazilian entities Coordenação de

4

Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional

5

de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de

6

Amparo à Pesquisa do Estado do Rio Grande do Norte (FAPERN) for financial

7

support.

8 9 10

References

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Shields, G. C. Absolute pKa Determinations for Substituted Phenols. J.

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Methods for the Determination of pKa Values. Anal. Chem. Insights 2013,

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Topol, I. A.; Tawa, G. J.; Burt, S. K.; Rashin, A. A. On the Structure and

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Changes in the Relative Gibbs Free Energy. Fluid Phase Equilib. 2012,

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TOC Graphic

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