*Article* **Intramolecular Interactions in Derivatives of Uracil Tautomers**

**Paweł A. Wieczorkiewicz 1,\*, Tadeusz M. Krygowski <sup>2</sup> and Halina Szatylowicz 1,\***

**\*** Correspondence: pawel.wieczorkiewicz.dokt@pw.edu.pl (P.A.W.); halina.szatylowicz@pw.edu.pl (H.S.)

**Abstract:** The influence of solvents on intramolecular interactions in 5- or 6-substituted nitro and amino derivatives of six tautomeric forms of uracil was investigated. For this purpose, the density functional theory (B97-D3/aug-cc-pVDZ) calculations were performed in ten environments (1 > ε > 109) using the polarizable continuum model (PCM) of solvation. The substituents were characterized by electronic (charge of the substituent active region, cSAR) and geometric parameters. Intramolecular interactions between non-covalently bonded atoms were investigated using the theory of atoms in molecules (AIM) and the non-covalent interaction index (NCI) method, which allowed discussion of possible interactions between the substituents and N/NH endocyclic as well as =O/−OH exocyclic groups. The nitro group was more electron-withdrawing in the 5 than in the 6 position, while the opposite effect was observed in the case of electron donation of the amino group. These properties of both groups were enhanced in polar solvents; the enhancement depended on the *ortho* interactions. Substitution or solvation did not change tautomeric preferences of uracil significantly. However, the formation of a strong NO···HO intramolecular hydrogen bond in the 5-NO2 derivative stabilized the dienol tautomer from +17.9 (unsubstituted) to +5.4 kcal/mol (substituted, energy relative to the most stable diketo tautomer).

**Keywords:** substituent effect; solvent effect; hydrogen bond; tautomers; nitro group; amino group

**1. Introduction**

Uracil is a common and naturally occurring pyrimidine derivative. The best known occurrences of uracil are probably nucleic acids, as it is one of the five bases of the nucleic acid. In RNA, uracil forms a complementary base pair with adenine, while its 5 methylated derivative, called thymine, is an equivalent base in DNA [1]. Uracil and its derivatives have also found applications in other branches of biochemistry. For example, 5-fluorouracil is used in treatment of several cancer types by chemotherapy [2,3], while 5-bromo and iodo uracil derivatives are studied as radiosensitizers for radiotherapy [4–7]. In 2013, a computational study of various 5-substituted uracil derivatives (X = CN, SCN, NCS, NCO, OCN, SH, N3, NO2) was performed in order to identify the most suitable radiosensitizers for experimental studies [8]. The most promising derivatives with high electron affinities, 5-(N-Trifluoromethylcarboxy)aminouracil [9], 5-thiocyanatouracil [10] and 5-selenocyanatouracil [11], were synthesized. Among them, 5-thiocyanatouracil has already been tested against prostate cancer cells with promising results [12]. Some uracil derivatives show antifungal and antimicrobial properties, whereas others act as inhibitors of specific enzymes [13]. On the other hand, some of them are mutagenic, for example, 5-hydroxyuracil [14]. An interesting novel class of compounds that are derived from nucleic acid base molecules, including uracil, are ferrocene-like complexes in which the nitrogen base molecule is attached to one of the cyclopentadienyl ligands [15]. It is a relatively new class of compounds that may find applications in pharmacy, biology and electrochemistry.

An important issue regarding nucleic acid bases is tautomerism. Each of the bases can exist in several forms that differ in the position of the labile hydrogen atom. In general, one of these forms is more stable than the others, and most of the molecules exist in

**Citation:** Wieczorkiewicz, P.A.; Krygowski, T.M.; Szatylowicz, H. Intramolecular Interactions in Derivatives of Uracil Tautomers. *Molecules* **2022**, *27*, 7240. https:// doi.org/10.3390/molecules27217240

Academic Editor: Miroslaw Jablonski

Received: 26 September 2022 Accepted: 21 October 2022 Published: 25 October 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

<sup>1</sup> Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland

<sup>2</sup> Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland

that form [16–18]. For this reason, RNA and DNA base pairs are built only from N9H tautomer of purine bases and N1H of pyrimidine bases [1]. However, relative stability of the tautomers can significantly change upon oxidation, reduction [17], substitution of the nucleobase [19,20], polarity of the environment [17] and even interaction with a metal cation [21,22]. Tautomerism of nucleobases is of interest in knowledge of biochemical processes. Importantly, it has been proposed that the existence of rare tautomeric forms can cause mutations of genetic code recorded in the DNA or alter functions performed by different variants of RNA [23–27]. Therefore, much effort has been put into studying the properties of uracil and its tautomers, including both theoretical and experimental studies ([16,28] and references therein). As mentioned above, various uracil derivatives are used or currently being studied for medical applications, where they are introduced into the human body. For this reason, investigating which factors can affect the tautomeric equilibria of uracil (and how) is a relevant research topic.

Uracil consists of a pyrimidine ring and two attached −OH groups at the 2 and 4 positions. However, the most stable tautomeric form has both hydrogen atoms of the −OH groups attached to the nitrogen atoms in the pyrimidine ring. The four most stable uracil tautomers (**u1**–**u4**) and their two rotamers (**u5**, **u6**), along with their relative stabilities, are shown in Figure 1. Based on calorimetric experiments [29], it was found that the dienol form is 20 ± 10 kcal/mol less stable than **u1**, while **u3** by 19 ± 6 kcal/mol. In addition, both diketo (**u1**) and keto-enol tautomers (**u2**, **u3**) were identified using the dispersed fluorescence spectra, although the precise structure of the latter was not determined [30]. The most stable keto-enol tautomer was estimated to have about 9.6 kcal/mol higher energy than the diketo form (**u1**).

**Figure 1.** Four most stable tautomers of uracil (**u1**–**u4**) and two rotamers of **u4** (**u5**, **u6**). The numbers given below are their relative energies in kcal/mol.

The aim of the research is to investigate both the intramolecular interactions in uracil derivatives and their sensitivity to solvent change, as well as their ability to change tautomeric preferences. Similar studies on adenine and purine derivatives were recently carried out [31,32]; our computational results were in agreement with the experimental NMR data of 8-halopurines obtained by other groups [19,20].

For this study, we selected the 5- and 6-substituted nitro and amino derivatives of the six tautomeric forms of uracil (Figure 1). The nitro and amino groups represent model electron-withdrawing and electron-donating substituents, respectively. In addition, the nitro group rotated by 90 degrees from the plane of the ring was taken into account. This group interacts with the substituted system only inductively, as opposed to the planar NO2 group, which acts through induction and resonance.

Two substitution positions, 5 and 6, differ in through-space *ortho* interactions and through-bond interactions with endocyclic N atoms/NH groups as well as −OH/=O groups. In position 5, depending on the tautomeric form, the substituent can interact through-space with the C4=O or C4−OH group. In turn, the substituent in position 6 can interact through-space with the N or NH group in the 1 position. Regarding the throughspace interactions, in some cases, formation of an intramolecular hydrogen bond is possible. Thus, the question arises whether it can alter tautomeric preferences.

Regarding the through-bond interactions, the 5 position is *meta*-related towards two endocyclic N/NH groups and *ortho*- and *para*-related towards two exocyclic −OH/=O groups.

Conversely, the 6 position is *meta*-related to the −OH/=O and *ortho*- and *para*-related towards N/NH. Here, it is important to mention that in pyrimidines, the position of the substituent in relation to the endocyclic N atoms has a profound influence on the substituent– substituted system interaction, which affects the electron-withdrawing/donating strength of substituents. This topic is discussed in our recent paper [33].

It should be emphasized that the −OH and =O groups have opposite electronic properties: the −OH group is an electron-donating substituent, whereas =O is an electronwithdrawing substituent. Therefore, the tautomeric form should be important for the intramolecular interactions in uracil derivatives.

## **2. Methodology**

Quantum chemical DFT calculations [34,35] were performed in the Gaussian 16 program [36]. We used the B97-D3/aug-cc-pVDZ method, in accordance with our recent research regarding purine and adenine derivatives [31,32,37]. The optimized geometries correspond to the minima on the potential energy surface since no imaginary vibrational frequencies were found. In the constrained optimization cases, i.e., systems with the NO2 group rotated by 90 degrees, one imaginary frequency corresponding to the rotation along the C-N bond was found.

Electronic properties of substituents were evaluated using the charge of the substituent active region (cSAR) parameter [38,39]. Its definition is presented in Figure 2. Positive cSAR values indicate the deficit of electrons in the substituent active region, i.e., the substituent is electron-donating. Negative values represent an excess of electrons in the active region of the substituent, indicating its electron-withdrawing properties. To allow comparison with our other results, the atomic charges used to calculate cSAR were obtained by the Hirshfeld method [40].

**Figure 2.** Definition of cSAR and interpretation of its value. *q*<sup>X</sup> is the sum of atomic charges of all atoms forming a substituent X, while *q*ipso is the atomic charge at the *ipso* atom.

In order to study solvent effects, the IEF-PCM implicit model of solvation was used [41–43]. Calculations were performed in ten media, listed in Table 1 along with their dielectric constants. It should be mentioned that the PCM has been used many times in computational studies of nucleic acid bases [6,17,19]. In the AT and GC base pairs, the molecular geometries obtained with the PCM were in good agreement with the experimental data and the calculations using the H2O microsolvation model [44].

**Table 1.** Media in which calculations were performed, and their dielectric constants, ε.


Analysis of electron density using the atoms in molecules (AIM) theory [45] was performed in the AIMAII program [46]. The main goal of this analysis was the search for possible bond critical points (BCPs) of non-covalent intramolecular interactions. When

such a BCP was present, we estimated the interaction energy according to the formula of Afonin et al. (Equation (1)) [47], derived from the Espinosa equation [48].

$$E\_{\rm HB} = 0.277 \cdot V\_{\rm BCP} + 0.450 \tag{1}$$

where *<sup>V</sup>*BCP is the potential energy density (in kcal · mol−<sup>1</sup> · bohr−3) at the BCP. This equation can be applied to OH··· O, OH··· N, OH··· halogen, NH··· O, NH··· N, CH··· O, CH··· N and CH··· halogen interactions.

Intramolecular interactions between non-covalently bonded atoms were also investigated using the non-covalent interaction index (NCI) method [49]. The nature of a given interaction was assigned and color-coded according to the value of sgn(λ2) · *ρ*(r), where λ<sup>2</sup> is the second eigenvalue of the Hessian matrix of electron density (*ρ*(r)). Points on reduced density gradient isosurfaces with a value of sgn(λ2)·*ρ*(r) > 0 indicate non-bonding (steric) contacts (in red), with sgn(λ2)·*ρ*(r)~0 indicating weakly attractive interactions (e.g., van der Waals, in green) and sgn(λ2)·*ρ*(r) < 0 indicating strongly attractive interactions (e.g., hydrogen and halogen bonding, in blue). For more information on the NCI analysis, see Johnson et al. [49]. In our case, NCI calculations were performed in Multiwfn 3.8 software [50] and the visualization in the VMD program [51].

### **3. Results and Discussion**
