The acidity of organic compounds is highly relevant to understanding several biological processes. Although the relevance and challenges in estimating pK_a values of organic acids is recognized by several reported works in the literature, there is a lack in determining the acidity of amides. This paper presents an experimental/theoretical combined investigation on the acid dissociation of the compound 6,7-dinitro-1,4-dihydroquinoxaline-2,3-dione (DNQX), a well-established antagonist of ionotropic glutamate receptor GluA2. DNQX was synthesized, and its two acidic constants were determined by UV–vis spectroscopy. The experimental pK_a of 6.99 ± 0.02 and 10.57 ± 0.01 indicate that DNQX mainly exists as an anionic form (DNQXA1) in physiological media, which was also confirmed by ¹H NMR analysis. Five computational methods were applied for estimating the theoretical pK_a values of DNQX, including B3LYP, M06-2X, ωB97XD, and CBS-QB3, which were able to provide reasonable estimates for pK_a associated with DNQX. Molecular dynamics studies have demonstrated that DNQXA1′ binds more effectively to the pocket of the GluA2 than neutral DNQX, and this fact is coherent to the interactions between amidic oxygens and Arg845 being the main interactions of this host–guest system. Moreover, interaction of GluA2 with endogenous glutamate is stronger than that with DNQXA1, which is in agreement with literature. To the best of our knowledge, we report herein an unprecedented approach involving acidity of the antagonist DNQX, as well as the possible implications in binding to GluA2.

中文翻译：

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谷氨酸受体拮抗剂6,7-二硝基-1,4-二氢喹喔啉-2,3-二酮酸度的理论和实验研究及其对GluA2结合的影响

有机化合物的酸度与理解几种生物学过程高度相关。尽管文献中已报道的几篇著作认识到了估计有机酸的p K _a值的相关性和挑战，但仍缺乏确定酰胺酸度的方法。本文提供了对化合物6,7-二硝基-1,4-二氢喹喔啉-2,3-二酮（DNQX）的酸解离的实验/理论组合研究，DNQX是离子型谷氨酸受体GluA2的公认拮抗剂。合成了DNQX，并通过紫外可见光谱法确定了它的两个酸性常数。实验性p K _a6.99±0.02和10.57±0.01的数值表明DNQX在生理介质中主要以阴离子形式（DNQXA1）存在，这也通过¹ H NMR分析得到了证实。应用了五种计算方法来估计DNQX的理论p K _a值，包括B3LYP，M06-2X，ωB97XD和CBS-QB3，它们能够为p K _a提供合理的估计。与DNQX相关联。分子动力学研究表明，DNQXA1'与中性DNQX的结合更有效地结合到GluA2的口袋上，这一事实与酰胺氧和Arg845之间的相互作用相一致，这是该宿主-客体系统的主要相互作用。此外，GluA2与内源性谷氨酸的相互作用比与DNQXA1的相互作用更强，这与文献一致。据我们所知，我们在此报告了一种前所未有的方法，该方法涉及拮抗剂DNQX的酸性，以及与GluA2结合的可能含义。