Intramolecular hydrogen bonds and structural nonrigidity of pyrimidine nucleosides

Optimal structures and intramolecular web of H-bonds of cytidinc, uridine, thymidine, their deoxyriboanalogues and some 05'-, О У -deuie.ro derivatives were studied by means of MNDO/H semiempirical quantum-chemical method. Effect of the intramolecular H-bonds on the stereochemical structure of nucleosides (particularly, on the stabilization of ant і conformation), on the physico-chemical characteristics of nucleoside molecules (heat of formation, dipole moment, first adiabatic ionization potential and the charge distribution), and on the dynamical characteristics of pyrimidine nucleosides (barriers of the inierconvertion, frequencies of the torsional vibrations) was elucidated. The intramolecular II bonds in polynucleotides and their influence on the nucleic acid architecture and nonlinear dynamic properties are discussed.

Introduction.Nucleoside molecules exhibit many pos sibilities as model objects for biophysical inves tigations [] ].The structure of isolated nucleoside molecule is complicated and nonrigid [2,3 ].Pyri midine nucleosides have in particular a higher barrier of rotation around glycoside bond as purine ones.
While it is possible for pyrimidines to adopt the syri conformations, it is slerically difficult and rarely observed in nature (see [4-6 | and references there) and leads to the preferring of a/z/^-conformation o:f sugar.The optimal conformation of pyrimidine nuc leoside is determined by the dipole-dipole inter actions, torsion rigidity of glycoside Cl'Nl bond and steric hindrances.But there are additional inter actions between the base residue and the sugar moiety contributing in stabilization of the nucleoside molecule in certain conformation.Effect of such interactions was found in some cases.For example, Emerson and Sundaralingam [7 | in their study of dihydrouridine 3'-monophosphate hemihydrate pointed out that puckering of the base could influence the ribose puckering due to inter actions of the C6 methylene proton with the ribose Van Lier, Smits and Buck [4 | in quantum-chemical study of tetrahydrofuryl-l-(5-methylcytosine) explain its unusually high anti-syn transition barrier in terms of an electronic effect of the /;a/Yv-substiluted methyl group on the and C2' methylene fragment through the carbonyl C2 group.
The possibility of participation of CH groups in H-bonds in nucleic acid constituents was supported by the experimental [14,15] and theoretical [12, .16]investigations.It also is known that the existence of С-Н...0 H-bond interactions in the nucleic acid polymers have a strong influence on their molecular structure [12, 16 |.The present study was undertaken to elucidate by means of MNDO/H semiempirical quantum-che mical calculation method, what kinds of intramo lecular H-bonds are in pyrimidine nucleosides and what is the influence of these H-bonds on the parameters of structural пол rigidity and dynamics of the nucleosides Methods.MNDO/H semiempirical quantum-che mical calculation method was shown to be useful for the investigation of the stereochemical nonrigidity of nucleotide bases [17] and in calculation of the parameters of hydrogen bonds [18].It slightly un derestimates the values of geometric and energetical characteristics of nucleotide base nonplanarity effects [1 ], allowing о consider more precisely many of phenomena having been earlier neglecting.
The structure, 1R spectra and some physicochemical properties of pyrimidine nucleosides were calculated with full optimization of all geometric parameters with the gradient norm <0.01.Starting geometries were obtained by the composition of fully optimized structures of components: nucleotide bases [191 and ribose (deoxyribose) molecules.Optimi zation of the structures of all pyrimidine nucleosides were started from anli-conformation of the sugar moiety with respect to base residue.Intramolecular H-bond enthalpies were obtained by comparing of two different heais of formation of the nucleoside mole cule which were calculated in cases «with» and «without» intramolecular H-bonds.For more details of calculations see [17,19].
Results and Discussion.The resulting lowesi energy structures of calculated pyrimidine nucleosides cytidine (Cyd), uridine (Urd), thymidine (Thd) and their deoxynbo-analogues (dCyd, dUrd, dThd res pectively) are shown in Figure .All the pyrimidine nucleoside sugar moieties are in an//-conformation with respect to the base residues and in gauchegauche conformation arcund C4'-C5' bond.This is in accordance with the results of previous molecular modelling |51, so as semiempirical quantum-chemica MNDO [4 1 and CNDO/2 [12] calculations in which the preference of this conformation for pyrimidine nucleosides was shown.In Figure, calculated values of the x (04CJ N1C2 dihedral angle), which charac terizes the art//-conformation, are exhibited.As one can see, x is somewhat higher in ribonucieosides than in deoxyribomjclcosides. Besides, in cytidine nuc leosides x values are obviously higher than in uridine and thymidine ones.
In the structures shown in the Figure the  Our results exhibit the influence of the intra molecular H-bonds on the physico-chemical charac teristics of the nucleosides: heat of formation, dipole moment, first adiabatic ionization potential and the fundamental vibration frequencies -some of these parameters are shown in Table 2.
The data presented in Table 3 demonstrate the most prominent structural peculiarities of the nuc leosides and the effect of the intramolecular H-bonds on the conformation and mutual orientation of their structural fragments.

Intramolecular H-bonds between the base residue and the sugar moiety and nonrigidity of the pyrimidine nucleoside, CI 'H...02=C2
intramolecular H-b о n d.Earlier attention have not been put on the role of СГН group in the hydrogen bonding between base residue and sugar moieties in nucleosides.There were assumptions about the participation of С2=02 in the Fl-bond but they were concerned with 02'H group in pyrimidine nucleosides [4,8,9].
Recently we studied the effect of N1 methylation of pyrimidine nucleotide bases on their structural nonrigidity 122].There the intramolecular H-bond between С2=02 and C1H of methyl group have been found (in agreement with further ab initio inves tigations [23]) which serve us as the model for the СГН...02=С2 interaction in nucleosides.The exist-

Fully optimized by MNDO/H structures of pyrimidine nucleosides with the network of the intramolecular hydrogen bonds {% is the 04'C1'N1C2 dihedral angle)
ence of CTH...02=C2 H-bond can be evidenced by diffuse low frequency band -satellite of fundamental stretching vibration of СГН centered at ~ 2785 cm" 1 in the nucleosides IR spectra [24].The enthalpies of the СГН...02 intramolecular H-bond was shown (Table 1) to be of the same order of value HI uridine and thymidine nucleosides, but

INTRAMOLECULAR HYDROGEN BONDS OF F'YRIMIDJNE NUCLEOSIDES
they are by 0.KH0.16kcal/mol smaller in cytidine ones: 3.47 kcal/mol (Cyd) and 3.57 kcal/mol (Urd and Thd); 3.67 kcal/mol (dCyd) and 3.83 kcal/mol (dUrd and dThd).Besides, one can see that in ribonucleosides СГН...02 H-bonds are by 0.20-^0.26kcal/mol weaker than in their deoxyriboanalogues.These tendencies are in accordance with  From the Table 3 one can conclude, that the neglecting of the intramolecular H-bonds results in the essential changes in the dihedral angle values of nucleosides, especially consisting with glycoside N1C1' bond, that reach 19° for nonhydrogen atoms and 90° for protons.Thus, intramolecular H-bonds involving atoms of the base residue and the sugar moiety (C1H...02, C6H...04' and С6И...05') put the important contribution in the stabilization of anticonformation of pyrimidine nucleosides (and C6H...05' H-bond additionally support also gauchegauche conformation [12]).
The intramolecular H-bonds between the base residue and the sugar moiety also affect the dynamics of pyrimidine nucleosides, particularly the torsion motion.In the Table 2 we have compared the calculated frequencies of torsion (libration) vibrations (wr) in nucleosides in cases «with»(H 0) and «without» (H = 0) intramolecular H-bonds.According to our results, these low frequency torsional vibrations are not only anharmonical but also anisotropic: the direction of decreasing of the НГСГЫ1С2 dihedral angle value of nucleoside is preferred.

Intramolecular H-bonds and structural nonrigidity of the base residues.
In the optimized structures of Cyd, Urd, Thd and their deoxyribo-analogues the intramolecular H-bonds between the base residue atoms are observed: N4H'...N3 H-bond in cytosine and N3H...02, N3H...04 in uracil and thymine bases (Figure, Table 1 1) -it must be due to the sugar moiety presence and its H-bonding with the base, but the mechanism of this effect is rather complicated.
The base residues of Cyd, Urd and Thd nucleo sides and their deoxyribo-analogues all have slightly puckering base rings, in accordance with the data of the previous quantum-chemical structural investiga tions of free nucleotide bases [19].Maximal deviations from planarity are observed for the N1C1 glycoside bond region of the pyrimidine nuc leoside base rings with dihedral angle values not more than 2,6°, 3.7°, 3.6° for Cyd, Urd, Thd and 3.7 C , 5.9% 6.1° for dCyd, dUrd and dThd respectively.It would be noted that the neglecting of the intramolecular H-bonds in the base residues results in increasing of the base ring puckering: corresponding dihedral angle reach the values 11.7% 9.6°, 9.3° for Cyd, Urd, Thd and 8.0°, 6.5°, 7.1° for dCyd, dUrd, dThd respec tively.Such behavior of the base rings upon the influence of intramolecular H-bonds shows that in the nucleosides the bases are also stereochemically nonrigid fragments 119].
The cytosine base in the nucleoside has the asymmetrically pyramidal aminogroup, so as Cyt in the isolated state [19,30].The N4H4..N3 intramo lecular H-bond affects the parameters of > CNH, fragment pyramidality (Table 3).The character of potential energy hypersurface which determines ste reochemical nonrigidity of Cyt base remains almost unchanged [31 j.The base residues with aminogroups in nucleosides are usually considered as planar rigid structures [3,7,[9][10][11][12].In contrast with this appro ach, our data evidenced that the planar inversion barrier of > CNH 2 fragment of Cyt residue in nucle oside is LI (Cyd) -1.2 (dCyd) times higher than in free Cyt (0.15 kcal/mol [32]) and consist 0.17 and 0.18 kcal/mol respectively.These data exhibit parti cularly that the р/г-соп jugation of lone electron pair of N4 with ^-electron system of the base ring reduce upon the transition from nucleotide base to nuc leoside.
The internal rotation barriers of the > CNH : , fragment mainly decrease in nucleosides in com parison with free Cyt and consist 3.37, 10.56 kcal/mol for Cyd and 2.33, 10.46 kcal/mol for dCyd (3.72, 10.47 kcal/mol for Cyt [17]) for rotation to the N3 atom and C5H bond respectively.The intermediate state with two aminoprotons turned to the N3 atom (with planar base ring and plane symmetrical location of protons) is stabilized by the bifurcated pair of intramolecular N4H'...N3 and N4H"...N3 H-bonds with the resulting enthalpy 3.59 and 3.55 kcal/mol for Cyd and dCyd respectively.The internal rotation of aminogroup is the dipole active process, transition dipole moment (~ 0.8 D) lies in the ring plane in 90° with respect to C4N4 bond.

Intramolecular H-bonds and nonrigidity of the sugar moiety.
Analysis of a number of crystal struc tures of nucleosides shows that furanose ring is usually nonplanar conformationally nonrigid molecule [2].Theoretical studies and several NMR experi ments show for furanose two preferred ring puckering conformations, C2'-endo and СЗ'-endo, with almost equal energy and 2-5 kcal/mol barrier of interconversion through 04'-endo intermediate confor mation (see [5,6] and bibliography there).
According to our data, all energy differences between C2'-endo, СЗ'-endo and 04'-endo confor mations of furanose ring in the pyrimidine nucleo sides are find to be not more than 0.95 kcal/mol.This fact is in agreement with the results of the Levitt and Warshel 133 ] on the force field calculation of ener getic profile of furanose ring in nucleosides, where such energy differences including repuckering barrier consist not more than 0.60 kcal/mol.
In pyrimidine nucleosides with either C2'-endo or СЗ'-endo sugar puckering modes the ribose atoms are involved into ОЗН...02' H-bonds, while the 2'-deoxyribose atoms form C4'H. .03H-bonds, which are more than 1.5 times weaker than the 03H...02' in ribose (Figure , Table 1).In spite the fact that in crystal structures of some nucleoside derivatives [27] 02'H...03' intramolecular H-bond occurs more frequently than 03'H...02\ in free pyrimidine nucleosides the fully optimized confor mation with 02Н...ОЗ' H-bond is 4.00-^5.95kcal/mol higher in energy than confor mation shown in Figure .The intramolecular H-bonds involving the sugar moiety atoms reduce the furanose ring puckering parameters (Table 3) and cause the essentially free interconvertion at room temperatures.Increase of dihedral angle values, which characterized the fura nose ring puckering, is observed when the amplitude of torsion motions around glycoside Cl'Nl bond increase.This is due to the weakness of the base residue-sugar H-bonds caused by the torsion motions.Other studies using AMI and PM3 semiempirical quantum-chemical methods [11], in which the full optimization process involve calculation of hydrogen bonding interactions between all accessible atoms, also show the unusually small values of the sugar puckering parameters.Influence of intramolecular H-bonds results in decrease of the interconvertion barrier of furanose ring in pyrimidine nucleosides.

05-and 03'-deprotonated nucleoside molecules.
We have modelled the charge situation in nucleotides and polynucleotides (polyanions) by deprotonalion of Cyd and dCyd molecules at 05' and 03 sites.This was done in order to elucidate the effect of negative charged phosphate groups on the stereochemical non rigidity and intramolecular H-bond web parameters of nucleosides.
It was found that the change of the charge state of nucleoside molecule by the deprotonation of its sugar moiety does nol destroy the intramolecular H-bond web.The deprotonation results in essential perturbation of the balance of intramolecular H-bonds in nucleoside (Table 4) and increasing of their cooperativity (Л = -2.1043.64 kcal/mol).In the nuc leosides deprotonated at the 05' site C6H...05' H-bond becomes much stronger, that results in the weakness of СГН...02 and other H-bonds (Table 4).This is in accordance with the predicted in [12] strengthening of C6H...05' H-bond when 05' belongs to P0 4 ~.In the case of 03' deprotonation site C6H...05' becomes weaker (Table 4) because of the turning of 05 proton to C6H (Table 5), which reduces the interaction of 05' atom with C6H group.The data in Table 5 describe the changes of most prominent structural parameters -dihedral angles in case of neglecting of intramolecular H-bonds in the anions under discussion.
The structural perturbation of cytidine nucleo sides under the deprotonation can be considered particularly by comparison of % values of anions: 134.6° (Cyd C)5 /~), 133.6° (dCyd 05 ), 140.1° (Cyd 0 ,/) and 121.2° (dCyd 03 ) with corresponding angles in Cyd and dCyd (Figure).The deprotonation of the sugar moiety affects also the stereochemical nonrigidity of the base residue in nucleoside molecule.In Cyd" and dCyd~ the aminogroups become more pyra midal, the base ring puckering parameters decrease (Table 5), their planar inversion barriers are 1.7 (Cyd -) -1.8 (dCyd") times higher and the internal anisotropic rotation barriers are reduced by 20-40 %.These effects can be explained particularly by the reducing of ря-conjugation in the base residue upon the influence of the surplus negative charge in the sugar moiety.The reverse is also true: possible mtermolecular interactions which change the struc tural and dynamic properties of the base, especially in the vicinity of < CNH 2 fragment attachment, cause the changes in charge distribution in the sugar moiety.
Thus the local change of the charge state of polynucleotide, for example, in the protein-nucleic acid recognition processes, can affect its structural and dynamical properties, particularly the local curva ture |341, through the disturbance of p7r-conjugatior of base residue.
Indeed, ii is known that poly(dT) and poly(dU) don't form single strand helical structure and on the same conditions that poly(rT) poly(rU) does (see [6 j and bibliography there).And study of CD spectra leads 'TVо 113 I to the conclusion that poly(rC) single strands have more secondary structure (stacking) than poly (dC) in the same conditions.Hydrogen bonding of 02'H groups with 3'phosphate and neig hbour furanose can successfully explain such relative stability of RNA strands with respect to DNA ones.
Conclusions.The structural and dynamical pro perties of pyrimidine nucleosides are shown at first to be formed with participation of the web of cooperative intramolecular H-bonds with enthalpies ~ 2-4 kcal/mol.Involving of any site of the nucleoside into intermolecular hydrogen bonding (Watson-Crick pairing, specific solvent, crystal packing and so on) can perturb its intramolecular H-bond web that results in changes of structural and dynamical para meters of nucleoside.
Pyrimidine nucleosides, their base residues and sugar moieties are stereochemically nonrigid struc tures.The aminogroup pyramidality and plane inver sion barrier increase in nucleoside in comparison with the nucleotide base and these parameters also depend on the charge situation on the sugar moiety.The intramolecular H-bonds effect the stereochemical structure of nucleosides, conformation and mutual orientation of their fragments, particularly, intra molecular H-bonds involving atoms of the base resi due and sugar moiety put the important contribution in the stabilization of a/i/z-conformation of pyrimidine nucleosides.
Intramolecular H-bonds affect the physico-che mical characteristics of pyrimidine nucleosides (heat of formation, dipole moment, first adiabatic ionization potential and the charge distribution), and they also can change the dynamical characteristics of nucleo side: barriers of the base residue and the sugar moiety interconvertion, frequencies of the torsional vibrations in nucleoside Intramolecular H-bonds in polynucleotides take part in the formation of the nucleic acid architecture and nonlinear dynamic properties.Local change of the charge distribution in the sugar region of polynuc leotide, for example, in the protein-nucleic acid recognition processes, can affect its structural and dynamical properties through the disturbance of prr conjugation of base residue.
Taking into ассошії of the intramolecular Hbonds is important for the NMR and IR spectra interpretation, for molecular modelling, for the eluci dation of mechanisms of structural and dynamic changes under molecular recognition processes.

Intramolecular hydrogen bonds and structural nonrigidity of pyrimidine nucleosides Yanina R. Mishchuk, Dmytro M. Hovorun Institute of Molecular Biology and Genetics of Ukrainian National Academy of Sciences 150 vul. Zabolotnoho, 252143 Kyiv, Ukraine
are mutually weakened by 0.29, 0.60 and 0.80 kcal/mol for Cyd, Urd and Thd respectively, and these values are much higher for dCyd, dUrd and dThd: 1Л0, 2.10 and 2.16 kcal/mol respectively.Resulting enthalpy of all intramolecular H-bonds are 18.07 and 14.32 kcal/mol for Cyd and dCyd, 19.21 and 15.64 kcal/mol for Urd and dUrd, 18.97 and 15.41 kcal/mol for Thd and dThd.