Base pairs with 5-chloroorotic acid and comparison with the natural nucleobase. Structural and spectroscopic study, and three suggested antiviral modified nucleosides

Abstract A structural and spectroscopic study of 5-chloroorotic acid (5-ClOA) biomolecule was carried out by IR and FT-Raman and the results obtained were compared to those achieved in 5-fluoroorotic acid and 5-aminoorotic acid compounds. The structures of all possible tautomeric forms were determined using DFT and MP2 methods. To know the tautomer form present in the solid state, the crystal unit cell was optimized through dimer and tetramer forms in several tautomeric forms. The keto form was confirmed through an accurate assignment of all the bands. For this purpose, an additional improvement in the theoretical spectra was carried out using linear scaling equations (LSE) and polynomic equations (PSE) deduced from uracil molecule. Base pairs with uracil, thymine and cytosine nucleobases were optimized and compared to the natural Watson-Crick (WC) pairs. The counterpoise (CP) corrected interaction energies of the base pairs were also calculated. Three nucleosides were optimized based on 5-ClOA as nucleobase, and their corresponding WC pairs with adenosine. These modified nucleosides were inserted in DNA:DNA and RNA:RNA microhelices, which were optimized. The position of the -COOH group in the uracil ring of these microhelices interrupts the DNA/RNA helix formation. Because of the special characteristic of these molecules they can be used as antiviral drugs. Communicated by Ramaswamy H. Sarma


Introduction
Orotic acid (uracil-6-carboxylic acid) or vitamin B13 is an important biological molecule, playing an important role as precursor of pyrimidine nucleosides.Among its synthetic derivatives with large pharmacological activity, 5-aminoorotic acid (5-HAOA) inhibits the metabolism of the cardioprotective drug dexrazoxane (Schroeder et al., 2008) and their complexes with Pt(II), zinc(II), Nd(III) and Ce(III) metal ions have shown noticeable cytotoxic activity (Burrows et al., 1996;Kostova et al., 2006Kostova et al., , 2008;;Lalioti et al., 1998;Ortiz et al., 2012).5-fluoroorotic acid  is a potent anti-cancer drug when it is metabolized to 5-fluoro-2 0 -desoxiuridine monophosphate  and incorporated into RNA causing cessation of its synthesis (Riviere et al., 2011;Wellington & Rustchenko, 2005).In addition, 5-FOA is a potent inhibitor of the metalloproteins of human malaria parasite (Muregi et al., 2011) and it is used in yeast genetics and in crystal engineering among many other applications.
5-Chloroorotic acid  is also a synthetic derivative that inhibits the conversion of orotic acid to uridine nucleotide, and with similar molecular structures that of 5-FOA.However, it has been little studied (Singh et al., 2019).Therefore, taking into account the possible importance of this molecule for pharmaceutical chemistry, in special in synthesizing drugs for virus or for cancer treatment, and because its IR and Raman spectra have not been interpreted, neither an explanation of why it can interrupt the RNA virus or that in cancer cells, the present work is focused on these points.To undertake these tasks in 5-ClOA molecule, first of all, a tautomerism study was carried out to know the tautomer form present in the solid state, which was simulated its crystal unit cell by a dimer and tetramer forms in several of these tautomeric forms.The IR and Raman spectroscopic study will confirm the tautomer form in the solid state as well as its molecular arrangement.The most stable tautomer form found was used for the next point.
The next point to be considered was the potential interaction of 5-ClOA with DNA nucleobases in their WC base pairs, in order to know how can it interacts and how strong is this interaction.According to this interaction, three types of modified nucleosides appear possible based on 5-ClOA as nucleobase, which were optimized as well as their WC pairs with adenosine.Finally, we have further in this study and these nucleosides were inserted in several RNA and DNA microhelices, which were optimized.The objective is to know if the helix is significantly deformed or its growing is interrupted in the RNA viral when 5-ClOA modified nucleoside is used.
The present study is aimed at the following points: i) To analyze the structure and stability of the 5-ClOA conformations using DFT and MP2 methods. ii) To study the tautomerism of this molecule.iii) To optimize the dimeric and tetrameric forms in several tautomer forms.iv) To compare and assign the IR and Raman spectra in the isolated state, as well as in its dimeric and tetrameric forms, with experimental FT-IR and FT-Raman spectra.v) To identify the main tautomer form present in the solid state.vi) To know the effect of 5-ClOA when it is bonded to natural nucleobases and when modified nucleosides based on 5-ClOA are inserted into DNA and RNA microhelices.
The M06-2X method shows good results for a broad range of interactions, including non-covalent interactions (Zhao & Truhlar, 2008b).It appears also as one of the best options among the meta-generalized gradient functionals for analyzing dispersion-bound systems (Riley et al., 2010;Riley & Hobza, 2011), especially in biological systems such as in nucleobase stacking (Zhao & Truhlar, 2008a;van Mourik, 2009).Therefore, it was used to optimize the stacking interactions in one of the tetrameric forms of 5-ClOA.In addition, it was also used in the optimization of the DNA:DNA and RNA:RNA microhelices (or minihelices), due to in previous computations on related microhelices this method led to the best results (Alcolea Palafox, 2019) with an excellent concordance of its interaction energy with the reported values using CCSD(T)/CBS methods (Hunter & van Mourik, 2012).
Several basis sets were used with these methods starting from 6-31G(d,p) to 6-311þþG(3df,pd), but the 6-31G(d,p) basis set was mainly used in the present work because it needs noticeable lower computational cost than with the 6-311þþG(3df,pd) basis set, but with only a slight improvement in the accuracy of the results.The optimum molecular geometry was determined by minimizing the energy with respect to all geometrical parameters without imposing molecular symmetry constraints.Berny's optimization algorithm was applied under the standard convergence criterion.The calculations of the vibrational wavenumbers were carried out in the harmonic approximation to obtain the Raman values.This vibrational calculation was also necessary to get the Gibbs energy (G) and entropy (S).No-imaginary wavenumbers appeared in the computed spectra by DFT.Natural NBO atomic charges (Carpenter & Weinhold, 1988) were also calculated.These charges are one of the most accurate to correlate molecular properties.
All these methods and basis sets are used in the GAUSSIAN16 program package (Frisch et al., 2019).The UNIX version with the standard parameters of this package has been running in the alpha-supercomputer 'Brigit' at the Computer Center of the Complutense University of Madrid.
DNA and RNA microhelices (Alcolea Palafox, 2019) were studied with the 5-ClOA molecule instead of uracil (RNA) or thymine (DNA) nucleobases.Only 5 0 -TTT-3 0 and 5 0 -UUU-3 0 microhelices were considered (Palafox et al., 2022).They were optimized at the M06-2X/6-31G (d,p) level in a simplified model with only three nucleoside pairs and including an overall negative charge on the helix of À 4. When this microhelix was optimized with four sodium atoms and 16 water molecules distributed around the phosphate groups, a zero charge on the helix was assigned.The notation used in the microhelices is that reported in previous publications (Alcolea Palafox, 2019;Palafox et al., 2022).The ends H5 0  -1 and H3 0 1 hydrogen atoms of each strand of the helix were especially oriented to prevent false intermolecular/ intramolecular H-bonds formation.
The relative Raman intensities (I i ) were determined from the Raman scattering activities (S i ) through the well known relationship (Srivastav et al., 2019).
where, m o is the frequency (in cm À 1 ) of exciting radiation, and � i corresponds to the vibrational frequency of the i th normal mode.f is an appropriated selected scale factor to be used for all peak intensities.The remaining physical parameters correspond to: h: Planck's constant, c: speed of light, k: Boltzmann's constant, and T: absolute temperature.

Interaction energy calculations in 5-ClOA
They are determined in the dimer structure of anti conformer of 5-ClOA, as well as in the two most stable tetramer forms.In addition, the interaction energies in the three possible Watson-Crick(WC) pairs of 5-ClOA with adenine nucleobase were calculated, and the obtained energies were contrasted to those determined in the natural thymi-ne���adenine WC base pair.The calculations in the dimer and tetramer forms were performed at the B3LYP/6-311þþG(3df,pd) level, while the calculations with the nucleobasis were done at the MP2/6-31G(d,p) level.All the energies obtained were corrected by the Boys & Bernardi procedure reported for basis set superposition error (Boys & Bernardi, 1970;Hunter & van Mourik, 2012).Therefore, the total counterpoise (CP) corrected interaction energy, DE CP AB , in a system (AB) was calculated by the following relationship: where (AB) represents to all the molecules of the system.Therefore, according to the studied system, the equations used in the present work to calculate the interaction energies E int (AB) were the followings: i.In the dimer form of 5-ClOA, the interaction energy E int (AB) is between both molecules of dimer, called molecules A and B, but because they are the same in dimer form, it can be renamed as E int (AA) and the equation used can be simplified to: where E AA AA AA ð Þ is the electronic energy of the dimer form (AA), while E AA A (AA) corresponds to the electronic energy of the monomer form used in this dimer (AA).
In the WC base pairs of 5-ClOA, the interaction energy DE CP AB corresponds to both molecules (AB) of the base pair, but in this case molecule A (�5-ClOA) and B (�adenine), and in the canonical WC pair, molecule A (�thymine) and B (�adenine).Similarly, in the WC with nucleosides, molecule A corresponds to the modified nucleoside formed with the nucleobase 5-ClOA, and B (� adenosine).In the tetramer form of 5-ClOA, (AB) represents to the four tetramer molecules, that we have labeled as molecules A, B, C and D. Therefore, equation ( 2) was rewritten as: On the other hand, the deformation energy E def (AB) can be calculated as sum of the deformation energies of each molecule of the system.Therefore, the following equations were used: i.In the dimer form E def (AB) can be renamed as E def (AA) and the equation used was as follow: E def ðAAÞ ¼ 2 E def A ðAAÞ where E def A corresponds to the deformation of the monomer form A in the dimer formation AA, which can be calculated as follow: where the parentheses show whether the calculations carried out on dimer form have been at optimized molecular geometry of the monomer form A or the dimer form (AA). Superscripts indicate whether the computations have been performed with the basis set of monomer (A), or with those of dimer form AA, while subscripts specify the monomer form A.
In the WC base pairs, E def (AB) was as follows: where correspond to each molecule of the WC pair and can be calculated as: where X (�A or B), and where the parentheses show whether the calculations in the WC pair have been carried out at optimized molecular geometry of each monomer (A or B) or that of the WC pair (AB).Superscripts indicate whether the computations have been performed with the basis set of monomer (X), or with those of WC pair, while subscripts specify the molecular system analyzed.
In the tetramer form of 5-ClOA, equation ( 4) was rewritten according to the four molecules of this tetramer (labelled as A, B, C and D) as follows:

Scaling the vibrational wavenumbers
The scaling procedure reduce the errors in the calculated wavenumbers, and allowing a precise relationship with the experimental bands to make the assignments accurately.Two procedures of scaling the computed harmonic wavenumbers were used in the present study (Alcolea Palafox, 2018, 2000;Alcolea Palafox & Rastogi, 2002): by a linear-scaling-equation (LSE), and by a polynomic-scaling-equation (PSE).These are the two best procedures because they provide better accuracy and they are simpler over other scaling procedures.Using them, the error found (Dm) in the comparison of the scaled frequencies (m scal. ) with the experimental ones (m exp ) was in general lower than 3%.It allows a good relationship of m scal with the experimental bands and therefore, an accurate assignment of them.The scaling equations used in the present work were determined previously from uracil molecule (Alcolea Palafox, 2018Palafox, , 2000;;Alcolea Palafox & Rastogi, 2002;Rastogi et al., 2000) at different levels.From them, the equations used for the ring modes at the B3LYP/6-311þþG(3df,pd) level and with the LSE procedure were the following: and with the polynomic equation (PSE): where m cal correspond to the harmonic calculated wavenumbers, and m scal to the scaled values that can be compared to the experimental ones.
Although with these equations is obtained a remarkable improvement in the predicted wavenumbers of the monomer form, but large differences are observed for N-H, C ¼ O and O-H groups involved in intermolecular hydrogen bonds with neighboring molecules, in particular in the stretching and in-plane bending regions.To avoid that, dimer and tetramer structures were optimized.

Experimental
5-ClOA of spectral grade (solid powder) was purchased from Aldrich Chemical Co, USA and used as such without any further purification.The FTIR spectrum was recorded in the 4000-500 cm -1 range (limited by transmission of ZnSe prism) on a FTIR spectrometer iS5 (Nicolet) at room temperature with 32 scans.The spectrum was recorded with 200 mg KBr þ 1 mg sample.The MIRacle single reflection horizontal ATR accessory was from PIKE.
The Raman spectrum was recorded in the 4000-0 cm -1 range on Multispec FT-Raman spectrometer (Bruker), Laser Denicafc Klastech of 1000 mW, and 512 scans.Due to the strong fluorescence, another Raman spectrometer RXN1 Microprobe (Kaiser Optical Systems) at 785 nm radiation line power of an Invictus laser was used as the excitation source, with 100x / NA 0.9 Nikon objective lens, with 3 accumulations, 60 sec.intergration time, and 30mW laser power.The slit width at the entrance was 380 mm, the time constant was 0.5 s, the scanning speed was 5, 1 exposure of 2 s and in the spectral range of 1860-215 cm -1 .In addition and for comparison purposes, a Raman spectrometer with the 1064 nm (IPS) radiation line was also used, with fiber probe (Wasatch), 1 exposure of 2 s and in the spectral range of 1860-215 cm -1 .

Conformational study
By rotation of the carboxylic group around the C6-C7 bond in 5-ClOA, two possible conformers appear, Figure 1.They differ in the position of the carboxyl OH group with respect to the halogen atom, denoting this position with the anti (OH close to halogen) nomenclature and syn (farthest halogen side), in accordance to that reported in 5-FOA (Cuellar et al., 2014).To refer to conformers, these will be denoted with the nomenclature anti and syn, and this nomenclature will be maintained throughout the work.
The anti form was the most stable one (Figure 1) because it appears stabilized by an intramolecular interaction of H1 with the neighboring O7 that is stronger than in syn form.This higher stability of anti conformer is confirmed by reported X-ray data on 5-AOA (Guidoni et al., 2009;Portalone, 2008), as well as in the theoretical study carried out in 5-FOA (Cuellar et al., 2014) and 5-AOA (Ortiz et al., 2012).The energy difference between the two conformers is very small, 6.0 kJ/mol at B3LYP/6-31G(d,p) level (4.4 kJ /mol by MP2/6-31G(d,p)), in accordance to the calculated value of 9.0 kJ /mol in 5-FOA (Cuellar et al., 2014) and 6.0 kJ/mol in orotic acid (Hilal et al., 2004), Table 1.This low energy difference shows that at room temperature both syn and anti forms can appear in the gas-phase spectrum.However, the different location of the carboxylic proton leads to significant higher dipole moment in syn form (4.292 D by MP2) than in syn form (3.775 D), which indicates that the syn form can be more stable in water environment.
The optimized bond lengths and angles of both forms were listed in Table 1-SUP (Supplementary Information Section).The labeling of atoms was included in Figure 1.Due to the anti form is slightly more stable than syn form, the study of the following sections was mainly carried out with this anti form of 5-ClOA.
Compared to 5-FOA, the C4-C5 bond length is slightly larger in  than in 5-FOA (1.469 Å) (Cuellar et al., 2014), as well as C5 ¼ C6 bond length with a lengthening of its value, 1.367 Å vs. 1.359Å in 5-FOA.It is because the negative charge on C5 atom in 5-ClOA is À 0.183e by MP2 vs. the positive value in 5-FOA (0.376e) that shortens the adjacent bonds.The C4-C5 ¼ C6 angle is slightly lower in 5-ClOA (119.4 � ) than in 5-FOA (121.2 � ), which can be interpreted by the greater volume of the chlorine atom, which slightly closes this angle to avoid its steric interaction with the neighbour atoms.
In the anti form, the conformation resulted by the H8 rotation towards the chlorine atom was not considered because in all carboxylic acids, the H8 proton always appears in cis form related to the carbonyl O7 oxygen to form intermolecular H-bonds with neighboring molecules in the solid state.Although, a very weak interaction between the chlorine atom (0.077e at MP2/6-31G(d,p) level, Table 2-SUP) and H8 proton could be established, however it is repulsive and hence, this conformation will be less stable and it is not expected to be observed in the solid state, and in gas phase.
Several thermodynamic parameters including Gibbs energy (G) and entropy (S) were calculated using Gaussian16 at different levels in the monomer and dimer forms of 5-ClOA molecule and collected in Table 1.The energy difference between the anti and syn forms appear reduced with the Gibbs values, which were used to obtain the zero-point vibration energy (ZPVE).A remarkable reduction in the rotational constant values was observed in the dimer form, which present an almost zero dipole moment indicating its null solubility in water environments.
Since related molecules to ours with pharmacological activity, such as 5-aminoorotic acid and 5-fluoroorotic acid, can present several tautomeric forms, in the present manuscript we have carried out a similar study (Cuellar et al., 2014;Ortiz et al., 2012) on 5-ClOA.Because the most stable tautomeric form on this molecule it is not known, first a theoretical study on the possible tautomeric forms was carried out, and then a theoretical/experimental vibrational study to confirm the tautomeric form present in the solid state, as well as its molecular arrangement.

Tautomerism in 5-ClOA
Several tautomeric forms of 5ClOA are possible, differing from each other by both: whether a single proton or two protons migrate, and bind to ring oxygen or nitrogen atoms.
As each tautomer has two different possibilities depending on the spatial arrangement of the migrating hydrogen atom, the possible combinations are expanded to 11 tautomers: one diketo form and ten enol and dienol forms, Figure 2.Only these tautomers were found stable.Although the tautomerism in both conformers syn and anti was studied, but Figure 2 and Table 2 show only the optimized structures found in the most stable anti conformer.The notation used for these tautomers was based on that reported in 5-AOA (Ortiz et al., 2012) and 5-FOA (Cuellar et al., 2014) molecules.These tautomers were always found more stable than the related ones in syn form and for this reason they were the only ones studied here.The exception was tautomer T4c, which shows a rotated -COOH group but with a orientation as syn conformer, however, this tautomer is not being stable in anti form.
The calculated gas-phase relative energies with B3LYP and MP2 methods are collected in Table 2.For comparison purposes, the values reported in 5-FOA (Cuellar et al., 2014) and uracil (Alcolea Palafox et al., 2002a;Alcolea Palafox & Rastogi, 2002) molecules were also included in this table.According to DEþZPE energies, the stability trend in 5-FOA (Cuellar et al., 2014) and 5-ClOA is as follows: In general, both trends are in concordance, although with some differences.Tautomers T2a, T5a and T5b with the keto C4 ¼ O4 form are less stable in 5-ClOA than in 5-FOA, while it is reverse in the remaining tautomers with the O4-H enol form.It may be due to an intramolecular interaction of ¼ O4 higher with C' atom than with F atom, because of a slight higher NBO negative charge on ¼ O4 in 5-ClOA (Table 2-SUP) than in 5-FOA, À 0.663e and 0.654e, respectively.
Tautomers T3a, T4a and T4d with the O4-H hydroxyl hydrogen oriented to the N3 atom are slightly more stable than tautomers T3b, T4b and T4c with the O4-H hydroxyl hydrogen oriented to C' atom.We could not found an explanation of it, but in these O4-H enol tautomers the chlorine atom appears slightly tilted towards the H(O4) hydrogen atom due to a weak intramolecular interaction between both atoms.Thus, the C4-C5-C' angle is 118.8 � in T3a and 118.4 � in T3b versus 114.8 � in the keto T1 form.This interaction is lower than that observed with the fluorine atom in 5-FOA.
In general, stability of the enol tautomers is slight higher in 5-ClOA than in 5-FOA, with the exception of tautomer T2a.However, tautomers of uracil are always more stable than the related ones in 5-FOA and 5-ClOA, which can be explained by the position of -COOH group in these molecules that increments difficult to their tautomerization.
T1 keto is the most stable tautomer in the isolated state, in accordance to that found in 5-FOA and uracil molecules, being their molecular structures full planar.The next most stable T3a enol form has an energy very close to that in 5-FOA.The stability order is slight reversed in the next most stable tautomer, T4a in 5-ClOA, while it is T2a in 5-FOA, although they are very close in energy.
The intramolecular interaction between the H1 amino hydrogen and the O7 carbonyl oxygen leads to a coplanarity of the -COOH group with the uracil ring.However (with the exception of T2a tautomer), in T2b, T4a, T4b, T4c and T4d tautomers, this H1 atom has migrated and, therefore, in their optimized form the -COOH group appears noticeable rotated towards the uracil ring, as can be observed in Figure 2. At the B3LYP/6-311þþG(3df,pd) level this rotation is maximum and the -COOH group appears perpendicular to the uracil ring.Calculations at the MP2/6-31G(d,p) level lead in these tautomers to a rotated -COOH group but smaller than in the perpendicular form.The coplanar form is a saddle point with one negative value.These tautomers with the rotated -COOH group do not appear to be stable in the solid state due to the crystal packing forces.
The dipole moment l is also included in Figure 2 to know the expected form in water environments.Only T3b and T5a tautomers appear with a noticeable high value and therefore, they would be the expected forms in aqueous solution, but they have noticeable low stability.Other tautomers have also a high dipole moment, and therefore an equilibrium between the amide and imidic acid form in solution is expected.

Calculated natural NBO atomic charges
The calculated values of natural atomic charges for the most stable anti conformer are listed in Table 2-SUP.For comparison purposes, the values in the uracil molecule were also included in this table.At MP2/6-31G(d,p) level, the main effects observed in the NBO charges of the anti form of 5-ClOA related to uracil molecule were the followings: i.The C' atom appears to have a similar effect on C4 ¼ O charges as the -COOH substituent on N1-H charges.Therefore, by effect of the -C' atom, the positive charge on C4 (0.810e in 5-ClOA vs 0.820e in uracil) and the negative charge on O4 (-0.663e vs -0.694e in uracil) atoms are reduced, and this effect is similar to that due to -COOH group on the N1 charge, which is reduced (-0.728evs -0.739e in uracil) but increases the positive value on H1 (0.491e vs 0.462e).This effect of the C' atom was also confirmed by comparing 5-chlorouracil (Ortiz et al., 2014) with uracil molecule, with similar reduction in the positive charge on C4 and in the negative charge on O4.The effect of the -COOH group was also tested by comparing orotic acid (uracil-6-carboxylic acid) with uracil molecule, with a similar reduction in the negative charge on N1 and an increase in the positive charge on H1. ii.The main effect of C' atom is on the negative charge value of C5 atom, which is noticeably reduced, -0.183e vs -0.451e in uracil molecule.Similar reduction appears in 5-chlorouracil molecule, confirming this effect.This change mainly affects to the values of the C4-C5 and C5 ¼ C6 bond lengths and to the C4-C5 ¼ C6 angle.However, the effect of the -COOH substituent on the charge of C6 atom is smaller, 0.132e vs. 0.119e in uracil molecule.iii.The negative charge on N1 and N3 atoms is reduced as compared to uracil molecule, and therefore the positive charge on H1 and H3 is increased, especially on H1 due to the weak interaction with O7 atom.This feature is also observed in 5-chlorouracil and orotic acid, with lower negative charge on N1 and N3 than in uracil molecule and with higher positive charge on H1 and H3.This means, the acidity of these atoms is slightly increased in 5-ClOA.
Comparing at MP2/6-31G(d,p) level the effects of the C' atom related to those obtained with the F atom (molecule of 5-fluoroorotic acid , 5-FOA, Cuellar et al., 2014), the following was obtained: i.The C4 charge is less reduced by the C' atom (0.810e) than by F atom (0.767e) related to uracil molecule (0.820e).The negative charge on O4 is also less reduced by C' atom (-0.728e) than by F atom (-0.654e) related to uracil molecule (-0.739e).ii.The effect is smaller in the negative charge of C5 atom, which pass to a positive value (0.376e) with the F atom.This change mainly affects to the C4-C5 and C5 ¼ C6 bond lengths, with a lengthening of C4-C5 higher with C' (1.480 Å) than with F (1.469 Å) related to uracil molecule 1.455 Å.The C5 ¼ C6 bond length is also longer with C' (1.367 Å) than with F (1.359 Å) related to uracil molecule (1.351 Å).This higher lengthening with C' is consequence of the closing of the C4-C5 ¼ C6 angle 119.4 � with C' vs the opening to 121.2 � with F related to 119.9 � in uracil molecule.iii.The negative charge on N1 and N3 atoms is similar but slightly smaller with C' (-0.728e on N1 and -0.776e on N3) than with F atom (-0.729e on N1 and -0.779e on N3) related to uracil molecule (-0.739e and -0.784e on N1 and N3, respectively).This higher reduction in the nitrogen atom charges with C' leads to an increase in the positive charge of its hydrogen atoms H1 and H3, respectively.

Geometry optimization in the dimer and tetramer forms
X-ray crystal data of orotic acid, and as well of carboxylic acid derivatives, show a crystal structure where their molecules are associated in ribbons forms and stabilized by strong H-bonds.Therefore, the crystal unit cell of the solid state in 5-ClOA was considered under the simplified model of a dimer and tetramer forms through these H-bonds.Similar to the carboxylic acids, the dimer of 5-ClOA is expected to be established through the -COOH groups, Figure 3.However, the crystal of 5-ClOA forms chains stabilized in alternative way by strong O-H���O ¼ C H-bonds between the OH and C ¼ O groups of the carboxylic group, and by N-H���O ¼ C H-bonds involving NH groups and carbonyl oxygens of the uracil ring.These H-bonds can be observed in the tetramer structures of Figure 4. Three types of optimized structures corresponding to the simulation of the crystal unit cell of 5-ClOA in the solid state by a tetramer form are shown in this figure: i.A linear form: it is planar with alternative O8-H8���O7 ¼ C and N3-H3���O4 ¼ C H-bonds, as shown in Figure 4a.It is the most stable form according to its total energy þ ZPE.ii.A zig-zag form: it is formed with O8-H8���O7 ¼ C and N1-H1���O2 ¼ C H-bonds, as plotted in Figure 4b.Because of these intramolecular H-bonds, this tetramer form of 5ClOA appears also planar, and it is the most stable one according to its Gibbs energy, although the difference of DG is very small with the tetramer linear form (1.04 kJ/mol).This higher Gibbs energy of the zig-zag form can be explained due to one of the O8-H���O7¼ Hbonds of the pair appears stronger than in the linear form, although the intermolecular H-bonds N1-H���O2¼ of this tetramer form are less stronger than the N3-H���O4 of the linear form, Figure 4a.
In addition, the CP-corrected interaction energy DE CP is slightly higher in this tetramer form of 5ClOA than in its linear form, Table 3.It can be explained as due to the noticeable higher deformation energy E def , especially in molecules II and III of this zig-zag tetramer, than in the linear form.Therefore, it is the expected form in the solid state.i.A staking form: with the fourth molecule of tetramer in a staking form, Figure 4c.This structure was optimized by the M06-2X method instead of by B3LYP, since it takes long term interactions into account (Riley & Hobza, 2011;Riley et al., 2010).This staking form was stable (no negative frequencies), but it is less stable than the other two tetrameric forms described above.It is due to only one weak intermolecular H-bond C-O8-H(molecule IV)���O2(molecule II) was established.Interactions of molecule IV with molecule I also appear to be established because the linearity of the four molecules of the system in Figure 4a is not observed here, with molecule I out of the linearity with molecules II-III.Because this feature, this structure was not further considered in the present study.If the system is improved with additional molecules placed in staking form, a lower deformation is expected and therefore, this structure could be considered.
An additional tetramer structure was also optimized, Figure 1-SUP, but because of its lower stability and its nonplanarity, it was also not considered.
Additional H-bonds through the N-H, and -COOH groups with neighbouring molecules could increase the stabilization of these tetramer forms.The intermolecular H-bonds through the -COOH group are noticeably stronger than those through C2 ¼ O and N1-H, and through C4 ¼ O and N3 There are no experimental X-ray data available on this molecule, and as well on 5-FOA (Cuellar et al., 2014) and on 5-HAOA (Ortiz et al., 2012).Because one of our goals is to confirm the tautomer form present in the solid state, and as well to know the molecular arrangement, the tetramer forms with tautomers T4a and T4d were optimized in their more stable linear form, Figure 5.
In the isolated state these tautomers appear at the B3LYP/6-311þþG(3df,pd) level with the -COOH group perpendicular to the uracil ring, and this perpendicularity remains in its tetramer form.Thus, it is expected that the crystal packing forces impede this large non-planarity of the -COOH group, which destabilizes the tetramer forms of these tautomers.I.e.they are not present in the solid state.Moreover, the lower stability obtained in the tetramer forms with T4a and T4d confirm that they will not be present in the solid state.Tautomers T4b and T4c can only exit in dimer form because H-bonds through N3 cannot be established.Therefore, the keto T1 tautomer in its zig-zag or in its tetramer linear form are the most probable to appear in the solid state.
In addition to these calculations, the tautomer form and the molecular arrangement present in the crystal may be confirmed by the comparison of the theoretical IR and Raman spectra of these tetramer forms with the corresponding experimental spectra in the solid state.Therefore, the vibrational study is of great interest for the design of nucleoside analogues based on the most stable conformer and tautomer forms of 5-ClOA.

Vibrational wavenumbers
The experimental IR and Raman spectra together with the scaled ones using the scaling equation procedure in the monomer, dimer and tetramer forms appear collected in Figures 6-9.For convenience, other spectra are included in Figure 2-SUP to Figure 8-SUP (Supplementary Material Section).The theoretical vibrational bands were calculated at the B3LYP/6-311þþG(3df,pd) level under the harmonic approximation.For a better discussion of the spectra plotted they were divided in two main ranges: in IR from 3700-2700 and 1800-550 cm -1 , while in Raman they were from 3700-2700 and 1800-0 cm -1 .For comparison purposes, two conformers were considered in the monomer form.

Assignment of the ring normal modes
Figures 6-9 show a comparison of the theoretical scaled spectra with the corresponding experimental ones.In this comparison, it is noted that in the stretching region of the IR spectra, the predicted scaled spectrum of the monomer forms, in both T1 and T4a conformers, differs remarkably in the position of the bands to that found experimentally.In addition, no bands are predicted in the 3400-2800 cm -1 range in totally disagreement with the four broads bands found experimentally.This feature indicates that in the solid state 5-ClOA does not exist in the monomer form, as expected.For this reason, and for other noticeable disagreements found in Figures 6-9, the vibrational wavenumbers in the monomer forms were not considered for discussion.Therefore, the vibrational wavenumbers of dimeric form were then analyzed and compared to the experimental ones.Because the keto tautomer T1 was the most stable one, therefore, firstly the wavenumbers in this keto tautomer were calculated.The values obtained were listed in Table 4, while the scaled IR spectra were recorded in Figures 3-SUP and 4-SUP, and the Raman spectra in Figures 8-9 and Figures 6-SUP and 7-SUP.Although the spectra were remarkably improved with this dimer form, noticeable differences remained yet, and therefore they were neither considered for the discussion and assignments of the experimental bands, but included in Supplementary Material section, Figures 3-SUP, 4-SUP and 7-SUP.
For this reason, several tetramer forms were optimized (Figures 4 and 5) to be closer to the crystal unit cell, and their vibrational spectra were compared to the experimental ones.In this process, the spectra of tautomer T4a was analyzed first, and it was noted that in the predicted IR spectra in the 1800-1600 cm -1 range, Figure 7, only one band was found, but experimentally two bands and a shoulder was observed.In addition, many other noticeable differences were also found in the spectra, which means that T4a tautomer form does not exist in the solid state of 5-ClOA.
The two keto optimized tetramer forms with full planarity were then analyzed: the linear and zig-zag forms.Therefore, their scaled IR and Raman spectra were included in Figures 6-9 and in Tables 4-7 together with the experimental ones.In a first comparison was found that the spectra with the tetramer in the zig-zag form were the most in accordance to the experimental ones.Therefore, the wavenumbers of these spectra were mainly considered for discussion.However, in the linear form several bands involved in H-bonds appear more in accordance to the experimental ones than in the zig-zag form, which indicates that strong intermolecular contacts appear between the 5-ClOA ribbons forms in the solid state.A detailed study of the vibrational bands explains these features in next sections.
This study was described in two main sections, corresponding to the carboxylic group modes and to the uracil ring modes.Thus, Table 4 lists the calculated uracil ring wavenumbers in the anti monomer form, together with those in the dimer and tetramer forms.The calculated wavenumbers in monomer are listed in the first column, Table 3.The interaction energies e int (AB), deformation energies E def , and total CP corrected interaction energy, DE CP AB in kJ mol -1 , were calculated at the B3LYP/6-311þþG(3df,pd) DFT level in the dimer, tetramers and WC pairs, where 'A' can be the nucleobase/nucleoside with 5-ClOA and 'B' can correspond to adenine/adenosine, in the tetramer forms, AB refers to their four molecules.

System
Molecule while their related IR intensities (A, in %) appear in the 2 nd column and the Raman intensities (S, in %) in 3 rd column.These values in % have been determined by normalizing the computed intensities to the strongest one found in the spectra.Raman depolarization ratios for plane (P) and unpolarized (UP) incident light were collected in columns 4 th and 5 th , respectively.For comparison purposes, the computed values for the dimer form of Figure 3 are also collected in the next two columns.Two wavenumbers appear for each normal mode corresponding to the two molecules of dimer, but the one with the highest IR intensity is shown in bold type and its intensity is listed in the 7 th column, while the one with the highest Raman intensity appears in italic type, although for simplicity, its value was not included in table.The computed values in the tetramer linear form have been collected in the next three columns.In this case, four frequencies are listed for each normal mode, which correspond to the four molecules of tetramer.Similarly to the notation used in the dimer form, that with the highest infrared intensity is indicated in bold and that with the highest Raman intensity in italic type.
Finally, the values corresponding to the tetramer zig-zag form are shown in the last four columns.The discussion is mainly focused on these wavenumbers, including the Because the theoretical values need to be compared to the experimental ones, the scaled wavenumbers were calculated by the LSE and PSE procedures in the ring modes of the tetramer in linear and zig-zag forms, Table 5.In addition, the IR and Raman spectra were recorded in different experimental conditions.Of the four wavenumbers calculated for each vibrational mode, only the one with the highest IR intensity and the one with the highest Raman intensity were scaled and included in this Table.In the tetramer forms of Figure 4, we have marked the rings with the symbols I to IV.It was done because only the vibrations corresponding to the central rings II and III were considered for the assignment of the experimental bands, and they were the only ones included in Table 4.This consideration will mainly affects to the N-H, C ¼ O and OH groups involved in Hbonds, due to their wavenumbers and their IR and Raman intensities noticeably change.This feature is explained in the following sections, but only the most important modes are discussed here.

N-H modes.
The N-H stretching modes are predicted as almost pure modes (100% PED).The m(N1-H) stretch (ring normal mode 30 of the uracil ring (Alcolea Palafox et al., 2002b) seems little affected by the chlorine atom, and therefore with the LSE procedure its scaled value at 3435 cm -1 in the monomer form appear close to that scaled in 5-FOA at 3463 cm -1 .Similarly, m(N3-H) (mode 29) is also little affected by the chlorine atom, and therefore it is predicted (scaled) in the monomer at 3452 cm -1 , close to that at 3471 cm -1 in 5-FOA.The m(N3-H) mode is calculated at slightly higher wavenumbers than the m(N1-H) mode, in accordance to that observed in 5-FOA and orotic acid (Cuellar et al., 2014).Due to the intermolecular H-bonds present in the solid state, these wavenumbers shift remarkably to lower values, ca.300 cm -1 .Therefore, in the experimental FTIR spectrum a broad band appearing at 3517.4 cm -1 can be assigned to free N-H bond, and a band appearing at 3166.4 cm -1 can be related to N-H groups involved in H-bonds.Analyzing in detail the tetramer form spectra, only the N1-H (in zig-zag tetramer) or the N3-H (in linear form tetramer) groups are involved in H-bonds.Thus, the experimental broad band at 3517.4 cm -1 in the FT-IR spectrum can be specifically assigned to m(N3-H) mode in free groups, in accordance to the scaled value by PSE in the zig-zag form at 3435 cm -1 , or to m(N1-H) mode, if it is considered the value in the linear form at 3434 cm -1 .Similar doubt appears in the assignment of FT-IR band at 3166.4 cm -1 , which can be assigned to the m(N1-H) mode, if it is considered the zig-zag form (scaled at 3194 cm -1 ) or to the m(N3-H) mode at 3178 cm -1 if the linear form is considered.An additional comparison of the calculated in-plane and out-of-plane bending modes of N1-H and N3-H groups with the experimental ones, and with the assignment reported in 5-FOA (Cuellar et al., 2014) resolves this question and the final assignment is included in Table 5.
Contributions of the in-plane bending d(N1-H) (mode 23) appear distributed in different vibrations.The main contribution is predicted in IR by PSE at 1491 cm -1 (zig-zag form) and at 1487 cm -1 (linear form) in accordance to the FTIR band at 1519.3 cm -1 or to the ATR-FTIR band at 1503.1 cm -1 .The Raman lines appearing at 1521.9, 1519.1 and 1521.2 cm -1 in the three recorded spectra, respectively, are also well related to this mode.
In-plane bending d(N3-H) (mode 20) is predicted with weak intensity at 1441 cm -1 in the tetramer linear form, something far of the FTIR experimental value at 1338.7 cm -1 , 1628 2 100 0.17   while in the tetramer zig-zag form it is scaled at 1384 cm -1 , which is more close to the experimental value.This difference (ca.50 cm -1 ) to a lower wavenumber is in agreement with the large shift to a higher value observed in the m(N3-H) mode and its assignment to the IR band at 3517.4 cm -1 , as discussed above.This feature further confirms our assignment.
The out-of-plane bending c(N1-H) (mode 8) appears as an almost pure mode and it is predicted in the tetramer zig-zag form with weak-medium IR intensity at 845 cm -1 in accordance to the FTIR band with medium intensity at 855.8 cm -1 .However, in the tetramer linear form it is predicted with very weak intensity at 637 cm -1 , but no experimental bands appear around this value to relate it.Similar feature also appears in the Raman spectrum, perhaps due to the weak intensity predicted for this mode.
The c(N3-H) bending (mode 9) appears also as an almost pure normal mode and it was scaled in the zig-zag form at 675 cm -1 with weak IR, and at 669 cm -1 with almost null Raman intensity.Because of that, this mode was not related to any experimental band.However, in the tetramer linear form it is predicted at 918 cm -1 in IR and at 896 cm -1 in Raman, also with weak and almost null intensity, respectively, which can be only related to the weak IR band at 889.1 cm -1 .

C2 ¼ O and C4 ¼ O modes. The C ¼ O group vibra-
tions are of great interest because they are involved in Hbonds, in particular in nucleic acid base derivatives.The C ¼ O stretching modes usually appear in the range 1750-1600 cm -1 and their identification is relatively easy (Alcolea Palafox et al., 2021;Rani et al., 2017).In tetramer zig-zag form, the m(C2 ¼ O) stretching (mode 26) appears significantly coupled with the m(N1-C-N3) mode, and it is predicted at 1723 cm -1 by PSE with very strong IR intensity, in good accordance to the very strong and broad FTIR band at 1707.1 cm -1 and to the ATR FTIR band at 1724.2 cm -1 .However, they are far of the very strong IR intensity band predicted at 1768 cm -1 in the tetramer linear form.The Raman lines at 1709.4,1710.2 and 1712.2 cm -1 are also well predicted in the zig-zag form at 1732 cm -1 , but it is poorly predicted in the tetramer linear form at 1773 cm -1 .These features indicate that in the crystal solid state there are intermolecular H-bonds through the C2 ¼ O group, since these bonds only appear in the zig-zag tetramer form Compared to uracil molecule, the wavenumber of this mode appears almost unchanged by the carboxilic and chlorine substitutions of 5-ClOA, This feature could be explained (Alcolea Palafox et al., 2002b) as C2 ¼ O group appears somewhat far of -COOH and C' substituents and is surrounded by the N1-H and N3-H groups, which protect it of interactions.
The m(C4 ¼ O) stretching (mode 25) appears strongly coupled with the m(C2 ¼ O) mode and it is also little affected by the substitution of chlorine atom at position 5, and is calculated in the monomer form at 1771 cm -1 , which is very close to 1768 cm -1 of uracil molecule.In the tetramer zig-zag form it is scaled with strong IR intensity at 1745 cm -1 , but it was not observed experimentally in the IR spectrum because it is involved in the very strong and broad band at 1707.1 cm -1 .However, it was predicted in Raman by LSE at 1723 cm -1 in excellent accordance to the experimental Raman lines at 1723. 5, 1722.7 and 1725.7 cm -1 .This feature further confirms the presence of tetramer zig-zag form in the solid state.
The in-plane d(C2 ¼ O) and d(C4 ¼ O) bending modes appear so strongly coupled with the ring modes, which makes their identification difficult.In addition, their contributions appear distributed in many calculated wavenumbers, in accordance to that observed in 5-FOA and uracil molecules.For this reason, these modes were not discussed here.
The out-of-plane c(C2 ¼ O) bending (mode 11) is clearly identified, although it is strongly coupled to c(NCN) and c(N1-H) modes.It was predicted at 746 cm -1 with weak IR and null Raman intensities, in accordance to its no detection in the experimental spectra.The c(C4 ¼ O) bending corresponds to mode 10 and it is also strongly coupled but with c(N3C4C) mode.It was predicted with very weak IR intensity at 768 cm -1 and very weak Raman intensity at 767 cm -1 , in good accordance to the very weak bands observed experimentally, confirming our tetramer zig-zag form.

C-C' and C-N modes.
The m(C-C') mode was related to mode 28, m(C5-H), of uracil molecule, which appears noticeable coupled with m(ring) modes and it was predicted in IR at 1084 cm -1 by the zig-zag form and at 1092 cm -1 by the linear form.Both predicted wavenumbers can be well related to the experimental FTIR bands at 1107.3 and 1105.9 cm -1 , and as well as to the experimental Raman line at ca. 1125 cm -1 , which confirm our calculations.
The m(C-N) stretching mode (number 21) appears little affected by the carboxylic and chlorine substituents.i.e. it was calculated in the monomer form at 1411 cm -1 (scaled at 1387 cm -1 ) vs. 1405 cm -1 (scaled at 1382 cm -1 ) in uracil molecule.In the zig-zag form it was scaled at 1411 cm -1 in good accordance to the strong IR band at 1422.6 cm -1 , while the scaled value at 1378 cm -1 by the linear form is somewhat far of the experimental value.This better prediction by the zigzag form was also observed in the Raman spectra, at 1404 cm -1 by this zig-zag form vs. at 1379 cm -1 by the linear form, compared to the experimental Raman bands at 1421.7, 1419.1 and 1419.7 cm -1 .

Vibrations of the carboxyl group
In the solid state, this group appears involved in strong Hbonds with the neighbouring molecules as in carboxylic acid derivatives.The calculated and scaled values in this group are included in Tables 6 and 7, respectively.

O8-H group modes.
The m(O8-H) stretching appears characterized as pure mode in 5-ClOA, and it was well predicted in the zig-zag form by IR at 3037 cm -1 , in good accordance to the very broad and strong FT-IR band at 3016.4 cm -1 .This mode is predicted in Raman with the highest intensity of the spectrum, but experimentally it is found as a weak and broad band.This strong disagreement  (m, cm -1 ) at the B3LYP/6-311þþG(3df,pd) level with the experimental IR and Raman values observed in the solid state of the carboxylic group in 5-ClOA, together with their corresponding absolute errors (cm -1 ).

Groups
perhaps may be due to the strong fluorescence that 5-ClOA exhibits in the solid state.
In-plane bending d(O8-H) mode appears strongly coupled with m(C-O) and d(N1-H) modes, as in 5-FOA.In spite of this, it is well predicted by the tetramer zig-zag form with a strong intensity at 1445 cm -1 in good accordance to the strong FT-IR band at 1437.3 cm -1 .However, in the tetramer linear form it is predicted with weak intensity in contradiction to the strong band observed experimentally.This mode was predicted well in Raman by both tetramer forms.
The out-of-plane c(O8-H) bending was characterized as almost pure mode and scaled in the zig-zag form at 1003 cm -1 with weak intensity in good accordance to the weak FT-IR band at 1014.3 cm -1 and to the weak Raman line at 1011.7 cm -1 .However, in the linear form it is predicted in Raman at 966 cm -1 , somewhat far of any Raman line, confirming again the presence of tetramer zig-zag form in the solid state of 5-ClOA.

COO group modes.
The very strong and broad FT-IR band observed at 1664.5 cm -1 has been assigned to the m(C7 ¼ O) mode, according to the scaled value in the tetramer zig-zag form at 1706 cm -1 by PSE (or at 1688 cm -1 by LSE) or in the linear form at 1694 cm -1 .The large difference with the value in the monomer form at 1740 cm -1 confirms that intermolecular H-bonds through the -COOH group are present in the crystal solid state.However, these H-bonds are noticeably stronger in the crystal than in our tetramer zig-zag model due to the large difference between our prediction at 1706 cm -1 vs. the experimental value at 1664.5 cm - 1 .In Raman it was predicted with very strong intensity at 1647 cm -1 and is well related to the very strong FT-Raman line at 1659.7 cm -1 .
The stretching m(C-O8) mode appears strongly coupled with several ring modes, however, it was clearly identified.Thus, the strong IR band at 1278.6 cm -1 was assigned to this mode in accordance to the scaled value at 1314 cm -1 .The difference between both wavenumbers again confirms that stronger H-bonds are present in the crystal than in our zigzag tetramer model.This large difference is also observed in the Raman spectra, which is predicted with very strong intensity at 1320 cm -1 but in the experimental spectra appear as very weak Raman lines at 1298.1 and 1302.7 cm -1 .This discrepancy can be attributed to other weak intermolecular Hbonds through this group, because Raman lines appearing at 1266.7, 1268.4 and 1263.5 cm -1 can be assigned to this mode.
The in-plane symmetric mode D s (COO) was well predicted with our tetramer models: by the zig-zag form at 748 cm -1 with medium IR intensity and by the linear form at 750 cm -1 , in good accordance to the FT-IR band at 738.3 cm -1 .Similarly, a good agreement was also found in Raman, predicted at 727 cm -1 and experimentally observed at 742.5 and 740.6 cm -1 .

Low-frequency vibrations
The presence of lattice modes indicates a high flexibility in molecular structure of molecules and play an important role in their biological functions, specially, these modes may be important for local recognition because they are very sensitive to changes in the local environment, such as hydration and interaction with other biomolecules.Thus, a theoretical description of the low-lying vibrations below 200 cm -1 was carried out, and the calculated wavenumbers and their assignments were collected in Table 8.The notation used for these modes was in accordance to that reported in 5-FOA (Cuellar et al., 2014) and other molecules (Alcolea Palafox et al., 2002a, 2013).In 5-ClOA, eight different modes were found below this value.This large number and shape agree well with the high conformational flexibility of the 5-ClOA structure and in general of nucleobases,

WC base pairs of 5-ClOA with adenine nucleobase
To check with the possibility to make use of 5-ClOA as an interrupter of DNA/RNA growing, the study of the interaction of this molecule with adenine nucleobase was carried out (Figure 10).Therefore, different WC base pairs with anti conformer of 5-ClOA, rather than with thymine/uracil, were fully optimized at the MP2/6-31G(d,p) level and the molecular structure of the 5-ClOA���adenine system was compared to the canonical WC base pair thymine���adenine, Figure 10b.Four main types of intermolecular H-bonds of 5-ClOA with adenine were analyzed: (a) through N3-H and C4 ¼O groups of 5-ClOA, Figure 10a, in a similar way to the canonical WC pair thymine���adenine.(b) Through the carbonyl oxygen C ¼ O7 of the -COOH group, N1-H and C2 ¼ O, Figure 10c.It is the less stable pair.(c) Through just the carboxylic group and the reverse form of adenine, Figure 10d.Because, the Hbonds through the O-H group are stronger than through the N-H group, this pair is the most stable one, even more stable than with the canonical WC pair thymine-adenine, Figure 10b, and (d) through just the carboxylic group but with the syn conformer of 5-ClOA, Figure 10e.Because, it is in the syn form, it is less stable than that of Figure 10d, but it was included here because of its possible binding to the DNA/RNA helix.Comparing these base pairs, the main changes observed were the following: i.The pair of 5-ClOA with the intermolecular H-bonds through N3-H and C4 ¼ O groups (Figure 10a) is full planar as the canonical WC pair, Figure 10b.However, in the pairs through the -COOH group, the 5-ClOA molecule appears slightly rotated related to adenine molecule, with a value ca. 5 � in Figure 10d and ca. 3 � in Figure 10e.ii.The pair of 5-ClOA through the C ¼ O7 and N1-H intermolecular H-bonds (Figure 10c) appears out-of-planar, as it is expected, and strongly rotated each molecule of the pair to a value of 34 � .In addition, the -COOH group is noticeably rotated by a value of 44.5 � related to the uracil ring.Because of this deformation, this pair is the less stable of those included in Figure 10 and it is not expected to be found in the DNA/RNA helix formation.iii.The intermolecular H-bond lengths of 5-ClOA with adenine through the N3-H and C4 ¼ O H-bonds (Figure 10a) are similar to the canonical WC pair with thymine,  10a) is slightly higher than the canonical WC pair with thymine (Figure 10b), with a deformation energy E def (AB) of 6.567 kJ/mol in the pair with 5-ClOA vs. 5.779 kJ/mol in the pair with thymine, Table 3.In these pairs, E def is very low in adenine, around 2 kJ/mol, and larger in 5-ClOA and thymine.It is because in the canonical WC pair thymine���adenine, the thymine nucleobase is the one, which is mainly deformed (3.910 kJ/ mol) to fit to the WC pair, while adenine is little deformed (1.869 kJ/ mol).This feature is increased in the 5-ClOA pair, where 5-ClOA is more deformed (4.772 kJ/mol) and adenine noticeably less (1.796 kJ/mol).i.e. 5-ClOA seems to be more flexible to fit to partner.vi.The flexibility of 5-ClOA through the -COOH group (Figures 10d-e), or through the -C7 ¼ O7 and -N1-H groups (Figure 10c), is remarkably higher than through N3-H and C4 ¼ O (Figure 10a), with large E def A values and noticeable geometric changes in their pairs formation.This larger flexibility of 5-ClOA could facilitates its H-bond to adenine in the DNA/RNA helix formation.The flexibility of the -COOH group in the pair can also be measured by its rotation related to the uracil ring.In this pair formation, a very slight out-of-plane deformation of this -COOH group is observed.vii.The DE CP interaction energies appear higher in the most stable pairs of Figure 10d-e, as expected, and it is lowest in the least stable pair of Figure 10c.The interaction energy value in our pair of Figure 10a with 5-ClOA appears slightly higher than in the canonical one.Therefore, it can be expected to remain H-bonded to the DNA/RNA helix formation, but with little distortion of it.
In these base pairs, thymine/uracil can be replaced well by 5-ClOA in WC pairs with adenine.This feature broadens the possible applications of 5-ClOA.Though, only the substitution of thymine/uracil for 5-ClOA was considered in the present study, but the results can be transferred to other WC pairs with cytosine and guanine.An example with tautomer 4a and guanine is shown in Figure 9-SUP.

Why our nucleoside proposition?
Most antiviral prodrugs are given to patients in their nucleobase form and, through the cellular biochemical machinery, they are transformed into nucleotides and finally attach to the DNA/RNA helix formation.In this case, the furanose ring is N1-linked to the nucleobase ring.However, other antiviral prodrugs will be given to patients in their nucleoside or nucleotide forms, and in this case, the furanose ring can be N3-linked to the nucleobase ring, or to N1 if it is consider the point of view of Figure 11 in which the standard uracil notation was changed.In the present manuscript, our proposition is as nucleoside analogues, nor as nucleobases, to avoid the cellular biochemical machinery that link the furanose ring to the nucleobase.
The proposed nucleosides I and II (Figure 12) with the furanose ring N3-attached to the nucleobase ring do not have a structural impediment to form stable WC pairs with adenosine, as we have shown in the optimized structures of Figure 12, nor a structural impediment to bind the primer in the helix growing, as we have shown in Figures 13 and 14.Moreover, nucleosides I and II can be seen from another point of view, see Figure 11, in which nitrogen N1 in the standard notation of the uracil ring of uridine, by similarity it can be related to nitrogen N1 of nucleoside I., although in Figure 12 this nitrogen atom appears as N3.Therefore, nucleosides I and II can be considered attached to the furanose ring through N1, but instead of the carbonyl C4 ¼ O of uridine, in nucleosides I and II, the carboxylic COOH group appears, and instead of hydrogen atoms at positions 5 and 6 of uridine, in nucleosides I and II appear the chlorine and oxygen O6 (O4 in Figure 12) atoms, respectively.

Proposed nucleosides based on 5-ClOA structure and WC pairs with the canonical ones
Two types of nucleosides are proposed with the furanose ring started in the 3 0 -endo orientation: (i) binding to N3 nitrogen of the 5-ClOA molecule, nucleosides I and II, Figure 12a and b, respectively, or (ii) binding to N1 nitrogen of the 5-ClOA molecule, nucleoside III, Figure 12c.These RNA nucleosides were optimized and the global minimum obtained plotted in Figure 12.The main characteristic parameters of these optimized nucleosides in the global minimum are collected in Table 9. WC pairs of these nucleosides with adenosine were also optimized.The calculated total energy was included in the bottom of each figure.The glycosidic bond length between the furanose ring and the 5-ClOA molecule has almost the same value whether it is N3-C1 0 or N1-C1 0 , 1.466 Å, and it is almost but slightly shorter than in uridine, 1.479 Å.The value of the C2-N-C1 0 angle is also closely between our predicted nucleosides and the uridine molecule.These features indicate that in the RNA helix, our modified nucleosides with 5-ClOA (Figure 12) can substitute uridine in the WC-pair with adenosine.Nucleosides with the furanose ring in the 2 0 -endo orientation and in the 2 0 -deoxy form (DNA) can also replace uridine in the WC-pair, and for this reason they were also optimized but for simplicity, they were not included in the manuscript.
Nucleoside I is building with conformer anti and it corresponds to the base pair of Figure 10e, while nucleoside II is formed with conformer syn and corresponds to base pair of Figure 10c.Both WC pair of these nucleosides with adenosine are through the -COOH group, which leads to stronger intermolecular H-bonds than those with the canonical ones through N3-H and C4 ¼ O bonds.Only the pair with nucleoside III is through N3-H and C4 ¼ O bonds, in analogy to the natural WC pair uridine-adenosine (U�A), Figure 12d.Although conformer anti of 5-ClOA is less stable than conformer syn, and its nucleoside form I less stable than nucleoside II, however, its WC pair with adenosine is more stable than that with nucleoside II.Moreover, the ring plane between the nucleobases is full planar in the pair with nucleoside I, while it appears rotated 47.7 � with nucleoside II, similarly as it is observed in the base pair with nucleobases (44.5 � ), Figure 10 c.
In nucleoside III the -COOH group appears rotated 64.9 � related to the uracil ring to form two intramolecular H-bonds with the hydroxyl O5 0 -H group, which stabilizes the structure.Because of these intramolecular H-bonds in nucleoside III, its WC pair is more stable than with nucleosides I and II.This large rotation changes the orientation of the -COOH group to a conformer closely to the anti form of 5-ClOA, Figure 1.This orientation is expected to noticeable deform the helix formation when attached.The base pair with nucleoside III appears full planar as in the natural WC pair, Figure 12d.
Nucleosides I and II appears with an orientation of the hydroxyl hydrogen H5 0 (b ¼ À 171.8 � and 170.1 � , respectively) closely to uridine molecule (174.9 � ) and appropriated for the phosphorylation process, which should be close to 180 � .This orientation little change in their WC pairs.Therefore, these nucleosides can bind to ATP in the enzymatic monophosphorylation step, and after other intermediate steps finally bind to the helix growing.Nucleoside I appears with stronger H-bonded through the -COOH group to adenosine than uridine nucleoside, which is expected that difficult the replication process of the helix.In addition, the intermolecular H-bonds to adenosine through this carboxylic group will require slightly more space when it will be inserted into the helix, i.e. an increase in the helix diameter, which will increase its deformation.However, nucleoside II appears with similar but slightly weaker H-bonds to adenosine than uridine nucleoside and therefore can replace it well.Thus, both nucleosides I and II are expected to exhibit mutagenic activity.
Because the intramolecular H-bonds in nucleoside III, the orientation of the hydroxyl hydrogen H5 0 (b ¼ 87.9 � ) is not the appropriated for phosphorylation.However, these intramolecular H-bonds are expected to be broken in a water environment and therefore, the O-H5 0 group can be free to move toward the appropriated orientation that facilitates its binding to ATP, and finally to the helix.
The main geometric difference of our proposed nucleosides with the natural uridine molecule appears in the v angle: in nucleosides I and II with a long rotation of the nucleobase related to the furanose ring to avoid repulsion between O4 and O5 0 oxygen atoms, and in nucleoside III due to intramolecular H-bonds.The pseudorotation phase angle P value is also larger in our nucleosides than in uridine molecule, which leads to a remarkable change in the sugar conformation, as it is shown in Table 9.Therefore, the furanose ring appear in uridine molecule at C3 0 -endo orientation, but it changes to C4 0 -exo form 4 E in nucleoside I, and to symmetrical twist of half-chair C4 0 -exo-C3 0 -endo form 3 4 T in nucleoside II.This large change is due to the noticeable effect of the O4 atom placed close to the furanose ring.The effect is remarkable greater in nucleoside III, with the carboxylic group placed on the furanose ring, Figure 12c, which leads to a change of the furanose ring to an O4 0 -endo orientation.The large difference in the furanose puckering ring among the nucleosides leads to a variation in the exocyclic torsional angles c, d and e, especially in the latter (e) due to the high flexibility in the rotation of the 3 0 -OH group.
These features with nucleosides I to III can lead when they are bonded to the terminal nucleotide of a primer DNA/RNA growing to a deformation or an interruption of this viral helix.Finally, the dipole moment value is noticeable lower in our proposed nucleosides than in uridine molecule, which slightly difficult its water solubility.This expected lower solubility may reduce the effectiveness of the cellular biochemical machinery for its incorporation into the helix.

DNA: DNA and RNA: RNA microhelices with three nucleotide base pairs
The effect of the three predicted 5-ClOA nucleosides on the helical parameters of several microhelices with three nucleoside base pair was analyzed.Our proposed nucleoside analogues have strong effect on the helix because they can deform it through the chlorine atom, the carboxylic group and in nucleosides I and II also through the O4 oxygen.Only the 5 0 -TTT-3 0 (B-type), 5 0 -UAU-3 0 (A-type), Figure 13, and the 5 0 -UUU-3 0 microhelices, Figure 14, were considered.The notation used for the microhelices was that reported in previous publications (Alcolea Palafox et al., 2020;Palafox et al., 2022).The notation in full was also included in these figures.In these microhelices the uracil/thymine nucleobases were replaced by 5-ClOA as follows: i.The syn conformer of 5-ClOA (nucleoside III, Figure 12c) was inserted in the central fragment (WC pair of plane n) of strand I (pyrimidine strand) in the DNA:DNA microhelix 5 0 -TTT-3 0 of B-type, Figure 13a.The notation for the planes is indicated in Figure 13a.ii.The anti conformer of 5-ClOA (nucleoside II, Figure 12b) was inserted in the plane n þ 1 of strand I in the RNA:RNA microhelix 5 0 -UAU-3 0 of A-type, Figure 13b.iii.The anti conformer of 5-ClOA (nucleoside II, Figure 12b) was inserted in the plane n þ 1 of strand I in the RNA:RNA microhelix 5 0 -UUU-3 0 of A-type, Figure 14a.
iv.The syn conformer of 5-ClOA (nucleoside I, Figure 12a) was inserted in the plane n þ 1 of strand I in the RNA:RNA microhelix 5 0 -UUU-3 0 of A-type, Figure 14b.
This A-type and B-type helix notation was in accordance to the corresponding A-type and B-type found in polynucleotide double-helices (Saenger, 1984), and where A-types are with the furanose ring in the C3 0 -endo orientation and B-type in the C 2 0 -endo.This notation was also used by us in related microhelices (Alcolea Palafox, 2019;Alcolea Palafox et al., 2019).The furanose orientation controls the helix shape, which is mostly of B-type in DNA helices, while it is typically of A-type in RNA:RNA helices and in DNA:RNA hybrid-helices (Kozlov & Orgel, 2000).
In the first microhelix of Figure 13a, the base pair of 5-ClOA with adenine was established through N3-H and C4 ¼ O, according to the WC pair of nucleoside III (Figure 12c), and inserted in the 5 0 -TTT-3 0 microhelix of Btype.In this microhelix, the carboxylic group was initially placed in-plane to the uracil ring, as well as in perpendicular form to the uracil ring.In both cases, the interaction of this carboxylic group with the oxygens of phosphate broke the helix formation (for simplicity its plot is not included).This feature indicates that our proposed modified nucleoside III can replace thymidine and therefore, it can be H-bonded to deoxyadenosine, and this binding breaks the viral DNA helix.
In the microhelix of Figure 13b the base pair of 5-ClOA nucleoside with adenosine was established through N1-H and the carboxylic oxygen O7, according to the WC pair of nucleoside II, and inserted in the plane n þ 1 of the 5 0 -UAU-3 0 microhelix of A-type.The optimization of this microhelix also leads breaking of the RNA helix formation.This feature indicates that the proposed nucleoside II can be used to interrupt the viral RNA helix formation.A new optimization with this nucleoside II was also carried out but inserted into the 5 0 -UUU-3 0 microhelix of A-type, Figure 14a.As it is observed, the carboxylic hydrogen H8 interacts with the phosphate oxygen atoms, establishing intramolecular H-bonds that remarkably deform the helix formation.This feature confirms that nucleoside II can have antiviral activity by binding to viral RNA.
Finally, a new microhelix was optimized by using nucleoside I, Figure 14b.In this case, the base pair was established with the carboxylic group only, with its O7 and H8 atoms.The effect of this nucleoside is breaking of the intermolecular H-bonds corresponding to the WC base pairs U���A of n and n-1 planes, as observed in Figure 14b.
In these microhelices, it is noted that the carboxylic group is mainly responsible to deform and break the DNA/RNA helix formation.The effect of the chlorine atom is almost null in the helix deformation and it only slightly changes the structure and base pair interactions of the nucleobase.The great flexibility of our proposed nucleosides allows them to be easily adapted to form intermolecular H-bonds with the complementary adenosine nucleoside of the helix.However, the great reactivity of the carbonyl and hydroxyl groups not involved in H-bonds try to interact with other atoms in the b The maximum torsional angle m max ¼ m2 cos P (Saenger, 1984).helix, and they are the responsible of the noticeable helix deformation or its breakage.
A further improvement of the helix model was carried out with the inclusion of four sodium atoms around the oxygen atoms of the phosphate group together with 16 water molecules around the sodium and phosphate groups, Figure 15.Optimization of this system was only performed with the microhelix of Figure 14b.The final result obtained indicated an increase in the helix deformation through the 5-ClOA nucleobase, with the -COOH group being almost perpendicular to the uracil ring of 5-ClOA, and this one appears oriented to interact with the uracil ring of upper plane n.This type of deformation clearly leads to a break of helix formation.Although this improvement of the helix model was only carried out with the microhelix of Figure 14b, it is expected similar behavior with the remaining helices, Figure 13a and b  and 14a.

Special characteristics of the proposed nucleosides
Nucleoside analogues must be phosphorylated by cellular kinase enzymes to give successively the corresponding nucleoside 5 0 -mono-, di-and triphosphates (Hsu et al., 2007).The effectiveness of this process is in general extremely low (El-Sayed et al., 2015), and usually the first phosphorylation step, i.e. the synthesis of nucleoside 5 0 -monophosphates (NMP), is the crucial step for the nucleoside triphosphate (NTP) formation.However, this problem does not appear with any of the natural nucleosides, which always contain the 3 0 -OH group.We have explained it by a proton-transfer mechanism in which the 3 0 -hydroxyl group plays an important role (Alcolea Palafox, 2014).Our proposed modified nucleosides have molecular structures similar to the canonical ones with some changes, but including the 3 0 -OH group.The addition or removal of some groups to facilitate their activity as anticancer/antivirus drugs is discussed below.The main points are as follows: i.The presence in the sugar ring of the 3 0 -OH substituent for DNA helices is necessary, or both, the 3 0 -OH and 2 0 -OH substituents in RNA helices, as in the canonical nucleosides.This presence will guarantee that our proposed nucleosides will be well identified by the viral reverse transcriptase (RT) and therefore, they will not be rejected when they will move into the active center of this RT.Canonical 2 0 -deoxynucleosides (dN) have not this problem either.However, prodrug nucleosides without these 2 0 -OH and 3 0 -OH substituents, it means 2 0 ,3 0dideoxynucleosides (ddN), have serious problems by their side effects and by drug-resistant virus variants.It was explained by the capacity of RT to differentiate ddN drugs from canonical dN, and therefore avoiding the incorporation of ddN drugs into the helix formation (Ami & Ohrui, 2021).We think that the main problem appears due to the lack of the 3 0 -OH substituent in a prodrug, which makes difficult the first phosphorylation step of this prodrug, which is the crucial step in its bonding to the growing helix.This lack of the 3 0 -OH substituent impedes the pincer formation with the water molecules and the 5 0 -OH group (Alcolea Palafox, 2014;Alcolea Palafox et al., 2023).ii.The presence of the 5 0 -OH group in our proposed prodrug is also necessary for its phosphorylation by adenosine triphosphate (ATP) molecule.The absence of this group impedes its binding to the DNA/RNA helix.In addition, it is necessary that the H5 0 proton of this 5 0 -OH group has the appropriate orientation (b � 180 � , Table 9) (Alcolea Palafox, 2014) for its transfer to an oxygen atom of ATP, and for further bonding of O5 0 to the terminal phosphate group of ATP (Alcolea Palafox, 2014).A final hydrolysis in the ATP kinase cavity leads to the mononucleotide.This proton transfer between the proposed prodrug and ATP appears performed by means of the neighbouring water molecules, as reported by us (Alcolea Palafox, 2014).The presence of -OH groups at both 3 0 and 5 0 positions (Saenger, 1984) of furanose ring appears as a requisite (Alcolea Palafox et al., 2023) to make the first phosphorylation step possible, which is a crucial step.Thus, our proposed prodrugs with 5-ClOA possess both -OH groups.The pronucleotide approach (El-Sayed et al., 2015) with 5 0 -monophosphates of nucleoside analogues was not used, because it leads to dianions under physiological conditions, which cannot cross negatively charged cell membranes.iii.The presence of other substituents in positions 5 and 6 on the pyrimidine ring appears also essential for antiviral activity.In our proposed prodrugs, these substituents were the chlorine and the carboxylic -COOH groups.It is necessary at least two new substituents (or two chemical modifications) in the pyrimidine ring (or furanose ring) for a better viral activity but with lower toxicity, which is our case with the proposed nucleoside analogues.This feature was explained by the fact that with only one single substitution on the canonical nucleoside, it has high antiviral activity (Ami Ohrui, 2021), but it has also high toxicity for clinical use.It is because of human DNA polymerases cannot distinguish it from canonical nucleosides, which leads to healthy cells introduce these prodrugs into their DNA, disrupting their function and ultimately killing them (it means high toxicity).However, with more than one substitution on the canonical nucleoside this toxicity is drastically reduced (Ami & Ohrui, 2021) because the human DNA polymerases do not recognize these prodrugs (in its triphosphate form) as their substrates, but the viral DNA polymerases do, which take them for their lower selectivity.It means, the prodrugs with two or more substituents (as in our case) can kill better the virus disrupting its DNA/RNA growing, but they do not affect healthy cells.
Our proposed prodrugs from 5-ClOA with two new substituents on the pyrimidine ring are expected their phosphorylation by the viral DNA polymerases, and inserted into the DNA viral due to they present WC pairs with canonical nucleobases and nucleosides.Because they also show strong deformation of the viral helix, therefore it is expected that they may disrupt its growing, and kill it.Our proposed prodrugs are also expected to present little toxicity due to which they can be distinguished by the human DNA polymerases and therefore they will not be inserted into the human DNA formation.

Resume and conclusions
Three proposed antiviral modified nucleosides based on 5-ClOA molecule are presented.A spectroscopic study of 5-ClOA and their possible nucleoside forms was carried out first.The main findings of the present study are as follows: 1.The structural and conformational analysis was carried out on 5-ClOA molecule for the first time by using the B3LYP and M06-2X DFT methods and by ab initio MP2 method, with different basis sets.The anti form was the most stable one, although the small difference found in the energies indicated that both anti and syn forms are possible in the solid and gas phase.However, the slight higher dipole moment in the syn form indicates that it may be the most stable in water environments.2. A tautomeric study was also carried out from the most stable anti form of 5-ClOA.A total of 11 tautomers were optimized taking into account the spatial arrangement of the H-atoms.The keto T1 form was the most stable one.The next most stable is the enol T3a, and closely T2a and T4a tautomers.3. The possible crystal unit cell of the solid state was reproduced by several tetramer forms, which were built and optimized with the keto T1 tautomer, as well as with the dienols T4a and T4d tautomers.The linear tetramer form with the keto T1 tautomer was the most stable one by EþZPE, but the zig-zag form was the most stable according to the Gibbs energy.A spectroscopy study confirms the zig-zag form as the expected arrangement present in the solid state.Because of the noticeable lower stability with the dienol tautomers in the tetramer form, and as well as due to the out-of-planarity of the carboxylic group, these forms are not expected to be in the crystal.The CP-corrected energy DE CP AB was also higher in the zig-zag form.4. The effect of the chlorine atom on the uracil ring structure and on the NBO charges is slightly higher than that produced by the -COOH substituent, but lower than that produced by the fluorine substituent.5.The calculated wavenumbers were improved using two best scaling procedures: the linear-scaling equation (LSE) and the polynomic scaling equation (PSE).With these procedures a good reproduction of the experimental wavenumbers was obtained with errors very small for majority of bands.6.The FT-IR and FT-Raman spectra were recorded at different radiation powers for the first time.In a detailed analysis, these spectra were compared with those optimized in the monomer, dimer and several tetramer forms with both, the keto T1 and di-enol T4a tautomers.The wavenumbers obtained in the zig-zag tetramer with the keto T1 tautomer appear the most close to the experimental ones.The tetramer form spectra with tautomer T4a shows several remarkable fails, which means that in the solid state only the keto form appears. 7. 5-ClOA can take the place of thymine to form WC pair with adenine.Four pairs with 5-ClOA were optimized.The flexibility with 5-ClOA through the N3-H and C4 ¼ O bonds is slightly higher than in the canonical pair thymi-ne���adenine, and it is remarkably higher through the -COOH group.The interaction energy with 5-ClOA is close to that of the canonical ones.8. Three new RNA nucleoside analogues with 5-ClOA, and sugar ring in 3 0 -endo form were studied and their base pairs with adenosine were optimized.Pairs through the -COOH group were also analyzed because they can be found in the helix formation.In general, geometric parameters and molecular structure of our nucleoside analogues appear close to those of canonical thymidine and therefore, they can be proposed as antiviral prodrugs.
9. Four microhelices DNA:DNA and RNA:RNA with three nucleotide base pairs were optimized, in which thymidine was replaced in different positions by the three types of nucleosides formed with 5-ClOA.The carboxylic group in these microhelices is the mainly responsible to deform and break the helix formation.
In the present study, our proposed prodrugs can have antiviral activity by binding to viral DNA/RNA helix formation, because: (i) their slightly higher flexibility through N3-H and C4 ¼ O groups (and through -COOH) to adenosine than the canonical one thymidine���adenosine.(ii) The good orientation of its O5 0 H group that favours the phosphorylation by viral DNA polymerases.(iii) Their base pair formation with canonical nucleosides and with similar interaction energy, and (iv) their lower toxicity due to the human DNA polymerases do not recognize these prodrugs as their substrates.
Because of these reasons and the necessity of new antiviruses with high activity, in the present study, we offer a possibility to have new prodrugs based on 5-ClOA.

Figure 1 .
Figure 1.Labeling of the atoms in the syn and anti-conformations of the isolated state of 5-ClOA by rotation of the -COOH group.

Figure 2 .
Figure 2. Optimised structure of the main tautomers in the most stable anti conformer of 5-ClOA.The calculated dipole moments at the B3LYP/6-311þþG(3df,pd) level are in debyes.
-H. Due to the orientation of the N1-H and -COOH groups, they do not interact to each other.As it is expected, these intermolecular H-bonds lead to a lengthening of the C2 ¼ O and C4 ¼ O bonds and therefore, the N3-C4 and C4-C5 bond lengths are shortened by 0.02 and 0.004 Å, respectively.

Figure 4 .
Figure 4. Optimized structures corresponding to the simulation of the crystal unit cell of 5-ClOA in the solid state in a tetramer system: (a) two views of the linear form with the C ¼ O���H-O and N3-H���O4 ¼ C H-bonds in alternative way at the B3LYP/6-311þþG(3df,pd) level, (b) in zig-zag form with the C ¼ O���H-O and N1-H���O2 ¼ C H-bonds in alternative way at the B3LYP/6-311þþG(3df,pd) level, (c) with one of the tetramer molecules in staking form calculated at the M06-2X/6-311þþG(3df,pd) level.

Figure 6 .
Figure 6.Theoretical scaled IR spectrum in the 3700-2700 cm -1 range of 5-ClOA using the scale equation procedure in the monomer and tetramer optimizations, and comparison with the experimental FTIR ones.� in groups not H-bonded.

Figure 7 .
Figure 7. Theoretical scaled IR spectrum in the 1800-550 cm -1 range of 5-ClOA using the scale equation procedure in the monomer and tetramer optimizations, and comparison with the experimental FTIR ones.� in groups not H-bonded.

Figure 8 .
Figure 8. Theoretical scaled Raman spectrum in the 3700-2700 cm -1 range of 5-ClOA using the scale equation procedure in the monomer and tetramer optimizations, and comparison with the experimental FT-Raman ones.

Figure 9 .
Figure 9. Theoretical scaled Raman spectrum in the 1800-550 cm -1 range of 5-ClOA using the scale equation procedure in the monomer and tetramer optimizations, and comparison with the experimental FT-Raman ones.
)(69) þ d as (COO)(19) þ d(C-C')(12)Notes: In bold and in italic forms is shown the highest IR and Raman intensity, respectively, and in the tetramer form it is only marked when the vibration is on the rings II and III. a Normalized to the highest value.bThe number corresponds to the normal mode of the uracil structure according to ref.(Alcolea & Rastogi, 2002).c Substituent mode in 5-ClOA.
relative IR (A) and Raman (S) calculated intensities in the zig-zag tetramer form are also listed.The experimental wavenumbers of the IR and Raman bands observed in the solid state and corresponding to the uracil normal modes are included with their main assignments.a With the LSE procedure from uracil molecule, equation from benzoic acid (Alcolea Palafox et al., 2002a), m scal from benzoic acid (Alcolea Palafox et al., 2002a).c With the specific scale factors of the previous column.d From Figure 5-SUP.e From Figure 8-SUP-a.f From Figure 8-SUP-b.

Figure 10b :
The slight shortening (0.034 Å) of the central H-bond N-H���N(adenine) with 5-ClOA is similar to the slight lengthening (0.027 Å) of its C ¼ O���H-N(adenine) H-bond.However, the H-bond lengths of 5-ClOA through the -COOH group (Figure10d and e) are noticeably shorter than with the canonical WC pair (Figure10b), especially the O8-H��N(adenine) H-bond.This means that a stronger H-bond of 5-ClOA in Figures10d-ecan cause it to continue binding to adenine in the DNA/RNA helix replication, which can hinder this replication.iv.The calculated dipole moment of 5-ClOA in the pair of Figure 10a was noticeably lower (0.648 D) than in the canonical WC pair of Figure 10b, 1.999 D. However, it is remarkably higher in the other pairs with 5-ClOA through the -COOH group: 6.948 D in Figure 10c, 9.004 D in Figure 10d and 9.199 D in Figure 10e, which facilitates its stability in water environments, although these values are larger than those found in DNA/RNA helices.E.g. 5-ClOA stabilizes helices with more water molecules around them, which is the purpose of 5-ClOA as possible antiviral drug.v.The flexibility of 5-ClOA pair with adenine through N3-H and C4 ¼ O bonds (Figure

Figure 10 .
Figure 10.Comparison at the MP2/6-31G(d,p) level of four optimized WC base pairs of conformer anti of 5-ClOA with adenine vs. the natural WC pair T�A.These base pairs were through the followings H-bonds: (a) N3-H���N A and C4 ¼ O���H-N A , (c) N1-H���N A and C7 ¼ O���H-N A of the -COOH group, (d) C7 ¼ O���H-N A and O8-H���N A of the -COOH group, (e) C7 ¼ O���H-N A and O8-H���N A of the -COOH group but in conformer syn of 5-ClOA.

Figure 11 .
Figure 11.Comparison of the canonical uridine nucleoside with our proposed nucleoside I in which the uracil ring notation was modified.

Figure 12 .
Figure 12.Comparison of the optimized structure of the proposed RNA modified nucleosides with 5-ClOA molecule at the M06-2X/6-31G(d,p) level, and the possible WC pairs established, with the natural WC pair U�A.The total energy obtained is included in the bottom of each figure.The base pairs were through the H-bonds: (a) of the -COOH group in conformer syn of 5-ClOA, (b) of the N1-H and ¼ O7 in conformer anti of 5-ClOA, (c) of the N3-H and C4 ¼ O groups in conformer syn of 5-ClOA.(d) of the N3-H and C4 ¼ O groups in the natural WC pair.

Figure 13 .
Figure 13.Two of the microhelices studied with three nucleoside base pair: (a) with 5-ClOA in the central fragment (WC pair of plane n, ref. (Alcolea Palafox, 2019)) of strand I in the DNA:DNA microhelix 5 0 -TTT-3 0 of B-type.(b) with 5-ClOA in the plane n þ 1 of strand I in the RNA:RNA microhelix 5 0 -UAU-3 0 of A-type.

Figure 15 .
Figure 15.Microhelix 5 0 -UUU-3 0 RNA:RNA of A-type with nucleoside I of 5-ClOA in the plane n þ 1 of strand I, which was optimized with four sodium atoms and 16 water molecules around the phosphate groups.

Table 1 .
Calculated energies þ ZPE in u.a. and several parameters with the B3LYP and MP2 methods and with two different basis set in the anti and syn (in parentheses) monomers and dimer forms of 5-ClOA molecule.DE ¼ E(anti form) -E(syn form).

Table 5 .
Scaled wavenumbers (m, cm -1 ) with the LSE and PSE procedures on the uracil ring wavenumbers of 5-ClOA obtained at the B3LYP/6-311þþG(3df,pd) level in the monomer and tetramer forms.

Table 7 .
Comparison of the scaled wavenumbers

Table 6 .
Calculated theoretical harmonic wavenumbers (m, cm -1 ), relative infrared intensities (A, %), relative Raman intensities (S, %), and Raman depolarization ratios for plane (P) and unpolarized (UP) incident light obtained at the B3LYP/6-311þþG(3df,pd) level in the carboxylic group of the dimer and tetramer forms of 5-ClOA.The highest IR intensity is shown in bold type while the highest Raman intensity is printed in italic type.
a Normalized to the highest value.

Table 9 .
Main characteristic parameters determined at the M06-2X/6-31G(d,p) level of our proposed nucleosides prodrugs I, II and III compared to those of the natural uridine nucleoside with the C3 0 -endo orientation.
a Pseudorotation phase angle, tg P ¼