Substituted adenine quartets: interplay between substituent effect, hydrogen bonding, and aromaticity

Adenine, one of the components of DNA/RNA helices, has the ability to form self-organizing structures with cyclic hydrogen bonds (A4), similar to guanine quartets. Here, we report a computational investigation of the effect of substituents (X = NO2, Cl, F, H, Me, and NH2) on the electronic structure of 9H-adenine and its quartets (A4-N1, A4-N3, and A4-N7). DFT calculations were used to show the relationships between the electronic nature of the substituents, strength of H-bonds in the quartets, and aromaticity of five- and six-membered rings of adenine. We demonstrated how the remote substituent X modifies the proton-donating properties of the NH2 group involved in the H-bonds within quartets and how the position of the substituent and its electronic nature affect the stability of the quartets. We also showed the possible changes in electronic properties of the substituent and aromaticity of adenine rings caused by tetramer formation. The results indicate that the observed relationships depend on the A4 type. Moreover, the same substituent can both strengthen and weaken intermolecular interactions, depending on the substitution position.


Introduction
Purine derivatives play a signicant role in medicinal chemistry and they are also important intermediates for the synthesis of biologically-active nucleoside analogues, and frequently exhibit antiviral or antitumor properties. 1 The nature and position of the substituents determine their reactivity and biological activity. Adenine (6-amino substituted purine derivative), one of the building blocks of DNA/RNA helices, can participate in intermolecular interactions which may lead to substantial changes in its electronic structure and, in consequence, to changes in its chemical/physicochemical/biochemical properties. In this perspective, the knowledge of the changes in the electronic structure of adenine caused by well-dened factors, such as introduction of substituents with specic electron donating/accepting properties, is of fundamental importance. It should be stressed that the effects of the intermolecular interactions (H-bonding, p-stacking, etc.) on the electronic structure and molecular properties of nucleic acid bases have long been a subject of wide and intensive studies. 2 However, the effects of substituents on the properties of nucleic acid bases are much less represented in the literature. It was reported that in the synthesis of deoxyribonucleosides, substituents at the C8 position of purine can greatly affect the rate of deoxyribosyl transfer to the base and the nature of the nucleoside formation. 3 An electron-donating substituent directs deoxyribosyl transfer to N9 of the base, while an electron-withdrawing substituent leads to the N3-product. Halogen substituents at the C8 position of adenine do not change the tautomeric preference in the gas phase but induce signicant changes in vibrational frequencies and Raman intensities. 4 Substituent effects on hydrogen bonding were most frequently studied in structurally-modied Watson-Crick base pairs (adenine-thymine and guaninecytosine). [5][6][7][8][9][10][11] One of the interesting features of DNA/RNA bases is their ability to form self-assembled structures (clusters). Among them, the most commonly studied are cyclic hydrogen-bonded quartets (tetramers), 12,13 which occur naturally in telomeres, 14 immunoglobulin gene switch regions, 15 and certain gene promoters. 16 The guanine quartet is the most well-known for a long time, for reviews see ref. 17 and 18. Although adenine quartets (A 4 ) have been studied experimentally [19][20][21][22] and have also been the subject of computational investigations, 23-26 the effect of remote substituents on hydrogen bonds in adenine tetramers has not yet been studied. Introduction of additional functionalities to the adenine with subsequent formation of quartet with intermolecular adenine/adenine hydrogen-bond interactions is one of the design strategies to enrich chemistry of purine derivatives with ability to bind many metal ions. 27 Since the stabilization of such complexes depends on the functional groups, the simulation of adenine quartets with certain substituents of different electronic nature is a good step toward design of synthetic functional materials. Moreover, changes in aromaticity of the adenine rings in the tetramer could be used to ne-tune p-p stacking interactions.
In this work, we have investigated computationally the effect of substituents (X ¼ NO 2 , Cl, F, H, Me, NH 2 ) on electronic structure of 9H-adenine and its quartets (A 4 -N1, A 4 -N3, and A 4 -N7) with dispersion-corrected Density Functional Theory. We have demonstrated how the position of the substituent and its electronic nature affect the stability of the quartets. Additionally, we have analyzed the inuence of the electronic nature of the substituents on the strength of H-bonds in the quartets and aromaticity of veand six-membered rings of adenine.

Methodology
Quartets of substituted 9H adenine tautomer were chosen as models for studying interrelationships between electronic nature of substituents, aromaticity of transmitting moiety, and intermolecular H-bonding between individual adenine molecules. Three types of adenine quartets were considered: A 4 -N1, A 4 -N3 and A 4 -N7. They are formed by one hydrogen bond between two neighbouring bases as shown in Fig. 1. These quartets have a non-planar S 4 -symmetric structure as the global minimum. However, they are forced into planarity by stacking interactions which compensate for the energy spent on planarization. The planarization energy of A 4 -N3 and A 4 -N7 was found to be 0.2 and 11 kcal mol À1 , respectively, while for A 4 -N1 system it was 22.3 kcal mol À1 . 28 The low stability of planar A 4 -N1 quartet is probably a main reason why this type of quartet has not been characterized by X-ray crystallography in contrast to A 4 -N3 29 and A 4 -N7. 20 Selected substituents, varying in their electronic properties (X ¼ NO 2 , Cl, F, H, Me, NH 2 ), were inserted into each adenine molecule at the same position within a certain quartet. For A 4 -N1 and A 4 -N3 tetramers, it was done either at the C8 or N9 position, whereas in the case of A 4 -N7 tetramer they were introduced either at the C2 or N9 position, resulting with 33 systems to be analysed. Although N9 substitution is less common, some organic ligands with functional groups can be incorporated into the adenine molecule at the N9 position. 27 Geometry optimization of each system was performed using ADF program 30-32 at the BLYP/TZ2P level of theory [33][34][35] with Grimme D3 dispersion correction. 36,37 To ensure that the obtained geometries are in energy minima, the vibrational analysis was performed and no imaginary frequencies were found.
The bonding energy of the tetramers, or hydrogen bond energy, E HB , was calculated as sum of interaction and preparation energies.
Interaction energy, E int , was calculated as the difference between the energy of the tetramer and the sum of the energies of its components (adenines) in the tetramer geometry.
Preparation energy, E prep , was calculated as the difference between energies of adenine monomers in the tetramer geometry and adenine monomers in equilibrium geometry.
Analysis of intermolecular interactions within the framework of Bader's Quantum Theory of Atoms in Molecules (QTAIM) was performed using the AIMAll program package. 38 Such parameters as the electron density in bond critical points (BCPs), r BCP , its Laplacian, V 2 r BCP , density of the total electron energy in BCP, H BCP , and its two components, potential and kinetic electron energy densities, V BCP and G BCP , respectively, were taken into account.
Substituent effect was described using cSAR (charge of the Substituent Active Region) concept. 39,40 The cSAR(X) value was calculated as the sum of Voronoi charges 41 of the atoms of substituent X and ipso C-or N-atom of adenine.
The choice of VDD charges is based on our previous research into the electronic structure of the nitro group in parasubstituted nitrobenzene derivatives 42 and studies of adenine systems substituted in C-X and N-X positions. 43 Electron delocalization was characterized using geometrybased aromaticity index HOMA (Harmonic Oscillator Model of Aromaticity), 44,45 which is dened as: where n is the number of bonds taken into the summation; a i is a normalization constant (for CC and CN bonds, a CC ¼ 257.7 and a CN ¼ 93.52) xed to give HOMA ¼ 0 for a model non-aromatic system and HOMA ¼ 1 for the system, in which all bonds are equal to the optimal value R opt,i assumed to be realized for fully aromatic systems (for CC and CN bonds R opt,CC ¼ 1.388Å and R opt,CN ¼ 1.334Å) and R i denotes bond lengths taken into calculation. The local HOMA index was calculated for both veand sixmembered adenine rings. This aromaticity descriptor was previously successfully used to describe the aromaticity changes in adenine rings due to H-bonding and complexation of metal ions. 46 The obtained values of the bonding energies and all substituent effect descriptors are gathered in Tables S1-S4. † cSAR and HOMA parameters were calculated for each individual adenine molecule in the tetramer. Because their values are similar within a given tetramer for all molecules, the mean values are given in the Tables S2 and S4, † respectively.

Results and discussion
The optimized A 4 -N3 and A 4 -N7 structures with substituents resemble at geometry in symmetry close to C 4h , while the substituted A 4 -N1 quartets are more distorted and adopt a similar pattern to that described by the S 4 point group. The unconstrained optimized geometries of systems with different substituents do not change signicantly within one quartet type ( Fig. S1 †). The obtained H-bond lengths for unsubstituted A 4 -N3 (d N/N ¼ 3.06Å) and A 4 -N7 (d N/N ¼ 2.93Å) are close to the experimental values, 3.05 and 2.91Å for A 4 -N3 and A 4 -N7, respectively. 20,29 The three quartets have similar stabilities. Our calculations show that unsubstituted A 4 -N3 tetramer is the most stable followed by A 4 -N7 at only 0.4 kcal mol À1 and A 4 -N1 at 2.7 kcal mol À1 (Table S1 †). However, substituents affect the electronic structure of the tetramers and change this stability order. In general, among N9-substituted tetramers the A 4 -N3 is the most stable quartet. For C2/C8-substituted quartets, the A 4 -N3 tetramer is more favourable only with H and Me substituents, while for the other studied substituents the A 4 -N7 tetramer is preferred. It should be noted that the most stable substituted A 4 -N7 tetramers (except X ¼ H and Me) have the shortest hydrogen bonds (d N/H < 1.9Å). According to the substitution position, C2/C8-substituted tetramers are considerably more stable than the N9-substituted ones.
Intramolecular interactions, the so-called substituent effect, can be considered from three points of view, as classical and reverse substituent effects, as well as the inuence of the substituent on the properties of transmitting moiety. The classical substituent effect, introduced and quantied by Hammett, 47,48 characterizes how a substituent X affects properties of a xed group Y (so-called "reaction site") in a substituted system X-R-Y (Rtransmitting moiety). In our case, properties of the reaction sitethe amino groupcan be described either by the cSAR(NH 2 ) value or by the strength of H-bonds.

Substituent effect on H-bondingclassical substituent effect
An illustrative example of the classical substituent effect is the dependence of cSAR(NH 2 ) on cSAR(X) shown in Fig. 2. The dependence was considered not only for quartets but also for substituted adenine monomers. Slopes of linear equations and determination coefficients are presented in Table 1. Note that correlations are acceptable or even good with R 2 > 0.84, except for C2-substituted A 4 -N7 series, where very narrow range of cSAR(NH 2 ) values was found. In all cases the slope a is negative meaning that electron-donating properties of the amino group decrease with increasing electron-donating ability (more positive cSAR(X) value) of substituent X. According to the slope a value, the C8 position in monomer is the most sensitive to the electronic nature of substituent X. This is consistent with results for adenine based on other substituent effect descriptors. 49 As expected, cSAR(NH 2 ) values are higher in monomers (triangles) than in quartets (circles) where amino group is involved in H-bond. The reason is accumulation of negative charge on the N atom of proton-donating -NH part of the amino group during formation of hydrogen bond. As can be seen in Fig. 2, electron-donating power of the NH 2 group is less affected by the tetramer formation in A 4 -N3 quartets than in the others, because the formed H-bonds in A 4 -N3 are the longest among all quartets, d N/H > 2.0Å. This observation was also conrmed by direct comparison of cSAR(NH 2 ) of NH 2 attached to C6 in monomers and tetramers, see Fig. S2 and Table S5. † In A 4 -N3, cSAR(NH 2 ) values of monomers and tetramers are comparable, while for A 4 -N1 and A 4 -N7, cSAR(NH 2 ) in tetramers are signicantly smaller than in monomers. Moreover, A 4 -N3 quartet is the most responsive to the electronic nature of substituent X either at C8 or N9 position.
As a consequence of the substituent effect on the properties of the amino group, H-bond energy in quartets also changes. Fig. 3 shows the dependence of bonding energy, E HB , in A 4 -N1, A 4 -N3, and A 4 -N7 tetramers on the electronic properties of remote substituents, where substituent X is described by cSAR(X) value. More positive cSAR values indicate better electron-donating properties of substituents. According to Fig. 2, substituent X changes properties of the amino group in the same direction regardless to the quartet type. However, the substituent effect on H-bond energy is different. This means that in addition to the substituent effect on the amino group, the substituent X also affects the proton-accepting abilities of N atoms of both rings. Positive and negative slopes of linear equations (Table 2) show the direction of the substituent effect, whereas their magnitude displays a sensitivity of intermolecular interactions to electronic properties of the substituents X.
Two groups can be distinguished in the studied systems: (i) with positive slope, where H-bond weakens with increase in electron-donating properties of substituent X, and (ii) with negative slope, where H-bond strengthens with the same substituent change. The rst group consists of all adenine quartets with the substituents at the carbon atom (either C2 or C8) and N9-substituted A 4 -N3 quartet. The second group is represented by A 4 -N1 and A 4 -N7 quartets with the N9substituents. In the case of A 4 -N3 and A 4 -N7 tetramers (R 2 > 0.92), the substituents notably change the energy of the intermolecular interactions. The largest changes were found in the N9-substituted A 4 -N3 system. This is consistent with observation of substituent effect X on properties of the NH 2 group (Fig. 2). On the other hand, for substituted A 4 -N1 quartets the   . 3 Relationships between the bonding energies, E HB , and cSAR(X) of substituent for the studied adenine quartets. Paper RSC Advances changes in the H-bonds strength caused by the substituent X at any position are very small (slopes a are only 0.413 and À4.468, respectively) and R 2 of these regressions are less than 0.25. Such low coefficients of determination (Table 2) can be explained by very narrow energy range. This is due to the fact that both C8 and N9 substitution positions in A 4 -N1 quartets are far from the atoms participated in the H-bond formation and the substituent effect is not transmitted so far. Different slopes for the A 4 -N7 substituted tetramers can be explained as follows: when substituent X is attached to the N9 position it changes the electron density on the N7 atom and modies its protonaccepting power to a greater extent, increasing E HB for electron-donating X groups, whereas the substitution at the C2 position affects mostly the proton-donating ability of the amino group decreasing E HB for electron-donating X groups. Thus, depending on the place of substitution the same substituent X can either weaken or enhance the intermolecular H-bonds in A 4 -N7 tetramers. Summarizing, the strong electron-accepting substituents contribute to strengthening of H-bonds in A 4 -N3 (at the C8 and N9 position) and A 4 -N7 (at the C2 position) quartets. The electron-donating substituents make H-bonds stronger only in A 4 -N7 quartet if they are located at the N9 position. Introducing different substituents into A 4 -N1 quartet has relatively small effect.

Additional intermolecular interactions
Some interesting observation was done when considering the relationships between strength of intermolecular interactions and length of the hydrogen bonds, d N/H , shown in Fig. 4. Despite the fact that H-bond lengths in A 4 -N3 quartets are longer (d N/H > 2.0Å) than in the A 4 -N7 ones (d N/H < 1.9Å), the bonding energies in A 4 -N3 tetramers are comparable in most cases with the energies in A 4 -N7 systems or even stronger. It is worth mentioning that the two systems with the strongest and the weakest H-bonds have a common element, namely the nitro group as a substituent. These terminal cases were found in the A 4 -N3 and A 4 -N7 tetramers with the nitro substituent at the N9 position (Fig. 5). Their energies are À44.3 and À27.5 kcal mol À1 for A 4 -N3 and A 4 -N7, although H-bond lengths are 2.01 and 1.91 A, respectively. The reduced E HB in A 4 -N7 N9-NO 2 substituted tetramer is expected because of the electron-accepting character of the nitro group as discussed above, whereas the large E HB in A 4 -N3 N9-NO 2 substituted tetramer is understood not only by the electron-accepting character of the substituent (vide supra), but also by the presence of additional intermolecular interactions.
Upon a detailed examination of their structures we found two additional close contacts in A 4 -N3 quartets (Fig. 5). One of them is formed between C-H unit of one adenine molecule and N1 atom of the other adenine (CH/N), whereas the second contact is made between NO 2 group and NH 2 group (O/HN). To characterize these extra contacts quantitatively we performed QTAIM analysis for 3 systems: unsubstituted A 4 -N3, and N9-NO 2 -substituted A 4 -N3 and A 4 -N7. Particular attention was paid to CH/N interactions in A 4 -N3 quartets. The distance d H/N is 2.33Å in A 4 -N3, and 2.27Å in its N9-NO 2 -substituted analogue, whereas such d H/N in adenine-thymine pair is 2.75Å. The parameters of the corresponding BCPs are collected in Table 3. Their values conrm that CH/N and O/HN contacts are weak closed-shell interactions. Although the interactions between NO 2 and NH 2 groups are very week, the CH/N contacts in A 4 -N3 quartets can be considered as weak H-bonds, 50 and therefore additionally contribute to their stability.

Change in substituent electronic propertiesreverse substituent effect
The cSAR concept allows us to study the reverse substituent effect, 42 which describes how the electronic properties of substituents X depend on the properties of the moiety R-Y to which they are attached. Various substituent constant scales 51-53 "represent" a xed electronic structure of substituents or, in other words, its xed electron-donating/attracting power. In turn, cSAR(X) value characterizes not the xed electronic properties of substituent X but shows its properties depending on the substituted moiety. The reverse substituent effect can be illustrated by changes in cSAR(X) values for a particular substituent X when it is considered in different adenine quartets (Fig. S3 †). The range of cSAR(X) values (D in Table S4 †) documents that the properties of X depend on the moiety to which it is attached. For C-substituted systems, the largest change is observed for the NH 2 substituent, while for the Nsubstituted ones the cSAR(NO 2 ) changes the most; these changes correspond to approximately 13 and 20% of the total variation of the cSAR(X) values in the series, respectively.
The effect of tetramer formation on the electronic properties of X can be demonstrated by comparing cSAR(X) in the quartet with the values in the adenine monomer ( Fig. S4 and Table S6 †). Undoubtedly, very high determination coefficients, R 2 > 0.99 and slopes close to 1.0 indicate a large similarity of interactions between X and substituted moieties in both cases. For N9substitution in A 4 -N3 systems, the slope a is slightly larger than in other series. This means that in this case the substituent X is more responsive to tetramer formation most probably due to additional spatial interactions with the amino group of neighboring adenine molecule (Fig. 5). In adenine monomer, cSAR(X) values of all substituents are slightly greater than their values in tetramers (Table S4 †), where these values depend on the position of substituent X relative to the proton-acceptor place (N1, N3 or N7) and proton-donor group (NH 2 ). Only in N9-substituted A 4 -N7 tetramer, cSAR(X) values become more positive than in monomer due to stronger withdrawal of electronic density to the H-bonding area. Importantly, the H-bond formation may decrease the electron-attracting ability of the nitro group by $60%.

Substituent effect on aromaticity
The substituent effect on aromaticity of the transmitting moiety can be demonstrated by changes in HOMA index for veand six-membered rings in the substituted quartets, as presented in Fig. S5 and Table S7. † Correlations between HOMA indices and cSAR(X) values indicate that electron-donating substituents at the C2 and C8 positions decrease the aromaticity of the vemembered rings, while at the N9 position cause the opposite effect; the latter is in line with previous studies 54 showing that N-substitution in imidazole rings increases the aromaticity for electron-donating groups. The six-membered rings are highly aromatic (HOMA > 0.9) and is slightly responsive only to the substituents at the C8 position, whereas C2-and N9substitution does not signicantly affect them. Increase in the aromaticity of the ve-membered ring by substitution, in general, goes with a reduction of the aromaticity of the sixmembered ring. Aromaticity changes by N9-substitution in the ve-membered ring translate into small variation in the sixmembered ring. Moreover, minor aromaticity changes by C2substitution are in line with previous studies indicating a high resistance of the p-electron structure to the substituent effect. 55 According to the slopes a, the highest HOMA sensitivity to the substituent effect was observed in A 4 -N3 quartets, most likely due to multiple noncovalent interactions between adenine molecules. Different slopes observed for veand sixmembered rings may depend on the number of bonds between the substituent X and the "reaction site" (NH 2 ). A similar observation is well known for meta and para substituted benzene derivatives, where the substituent effect depends on the number of bonds between X and Y. 56 Comparison of HOMA values for tetramers with values for monomers of substituted adenine (Table S3 †) clearly shows that aromaticity of both rings slightly decreases upon tetramer formation. The ve-membered rings of A 4 -N7 quartet become  Paper RSC Advances more aromatic than in monomer because their N7 atom participates in the H-bond as a proton-acceptor. Great correlations between HOMA values of ve-membered rings for substituted monomers and tetramers (R 2 > 0.94; Fig. S6 and Table S8 †) with slopes a close to 1.0 point out that aromaticity of ve-membered rings does not change much upon tetramer formation. Aromaticity of six-membered rings in tetramer becomes either more sensitive (a > 1.0) or less sensitive (a < 1.0) to the nature of substituent X in comparison with monomer.

Conclusions
The substituent X, depending on the position, modies the proton-donating properties of the NH 2 group and thus the strength of hydrogen bonds in adenine quartets. According to the ndings, to increase the stability of the A 4 -N3 quartet, it is recommended to introduce a strong electron-accepting substituent into each adenine molecule at the C8 or N9 positions. On the other hand, to stabilize A 4 -N7 quartet it is necessary to substitute C2 position with electron-accepting substituent or N9 position with electron-donating substituent. Substituent effect in the A 4 -N1 quartet is not pronounced and cannot be considered as a tool for tuning stability of the quartet.
The obtained results show that the electronic properties of the substituent X and aromaticity of veand six-membered rings are affected by the tetramer formation and are slightly different depending on the A 4 type. For C-substituted systems, the largest change in cSAR(X) values is observed for the NH 2 group, while for the N-substituted ones the cSAR(NO 2 ) changes the most. Aromaticity of both adenine rings in A 4 -N3 tetramer is more sensitive to electronic nature of the substituents than in other tetramers. Its "special properties" are most likely due to the fact that additional weak hydrogen bonds occur here as documented by the results of QTAIM analysis. In general, to increase aromaticity of the ve-membered rings the electronaccepting substituents should be introduced at the C2 or C8 positions, or the electron-donating substituent at the N9 position. In turn, six-membered rings are highly aromatic (HOMA > 0.9) and slightly responsive to the substituents at the C8 position, whereas C2-and N9-substitution does not affect them.

Conflicts of interest
There are no conicts to declare.