Anti-Syn Conformational Range of Pyrimidines with Deoxyribofuranose

Abstract

The ability of pyrimidine bases to adopt the syn conformation in DNA has been investigated. The distances between atoms on the sugar and base and the resulting steric energies have been calculated as a function of glycosidic torsion angle for the principal sugar puckers of the deoxyribose of cytosine. The results indicate that pyrimidines can assume both the anti and syn conformations for the ³E, ₄E, ₁E, ²E, ₃E sugar puckers and syn for the ₂E sugar pucker. For these sugar puckers the difference between the minimum energies of the anti and syn conformations is in the range of 0.1–2.0 kcal/mole, with the minimum syn energy being lower in the case of the ₄E, ₁E and ²E sugar puckers. It is particularly significant that cytosine can assume the syn conformation for the ³E sugar pucker commonly observed for the syn nucleotides in Z-DNA with both alternating pyrimidine/purine (APP) and non-APP sequences. The results of this investigation confirm that steric interactions resulting from putting a pyrimidine nucleotide in the syn conformation are not a major factor in the preference for APP base sequences in Z-DNA.     

Syn-anti effects on the spatial configuration of polynucleotide chains

Semiempirical energy calculations have beeb performed on model nucleic acid systems to assess the preferred conformation of the rotation χ about the glycosidic linkage and also the effect of this rotation on the spatial configuration of the sugar-phosphate chain backbone. The rotation angle ?? about bond C_5′–C_4′ in purine polyribonucleotides and 5′-monoribonucleotides is found to depend on whether the conformational range of χ is syn or anti. The preferred conformation of χ in these molecules is also found to depend upon the nature of the attached base. The orientation of χ in poly rA chains is predicted to be predominantly anti, whereas in poly rG the syn conformer is expected to occur in significant proportions. The syn conformer is preferred almost exclusively in certain unusual purine polynucleotides, such as poly 8Br-rA. It is noted that the preferred conformation of x in polynucleotides is not necessarily the same as that calculated for 5′-mononucleotides and nucleosides. On the basis of these calculations, the influence of the orientation and nature of a purine base on the spatial configuration of a polynucleotide chain as a whole has been examined. The random coil dimensions of a syn polynucleotide chain are found to be larger than those of an anti chain as a consequence of the effect of a syn base on the local conformation of the chain skeleton. Finally, it is found that the occurrence of a syn base in an ordered polynucleotide chain may prevent the formation of normal stacking with the preceding base.     

Handedness of duplex DNA: role of the glycosydic torsion

Model building studies on helical structures of polynucleotides indicate the glycosydic torsion, χ, and the sugar pucker to be the most important conformational parameters in determining the handedness of the models. Sugar puckers in two typical regions of the pseudorotational space namely, C2′ endo and C3′ endo and the glycosydic torsion in four ranges, namely, anti, high anti, syn and low anti lead to eight conformational combinations for the model bulding of base-paired double helical structures of polynucleotides. Interestingly, not all combinations of sugar pucker and glycosidic torsion lead to structures with Watson-Crick base-pairing. The stereochemical details of the double stranded Watson-Crick base-paired models and their consistency with X-ray diffraction data are discussed.     

A new anti conformation for N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (AAF-dG) allows Watson-Crick pairing in the Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4)

Primer extension studies have shown that the Y-family DNA polymerase IV (Dpo4) from Sulfolobus solfataricus P2 can preferentially insert C opposite N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (AAF-dG) [F. Boudsocq, S. Iwai, F. Hanaoka and R. Woodgate (2001) Nucleic Acids Res., 29, 4607–4616]. Our goal is to elucidate on a structural level how AAF-dG can be harbored in the Dpo4 active site opposite an incoming dCTP, using molecular modeling and molecular dynamics simulations, since AAF-dG prefers the syn glycosidic torsion. Both anti and syn conformations of the templating AAF-dG in a Dpo4 ternary complex were investigated. All four dNTPs were studied. We found that an anti glycosidic torsion with C1′-exo deoxyribose conformation allows AAF-dG to be Watson–Crick hydrogen-bonded with dCTP with modest polymerase perturbation, but other nucleotides are more distorting. The AAF is situated in the Dpo4 major groove open pocket with fluorenyl rings 3′- and acetyl 5′-directed along the modified strand, irrespective of dNTP. With AAF-dG syn, the fluorenyl rings are in the small minor groove pocket and the active site region is highly distorted. The anti-AAF-dG conformation with C1′-exo sugar pucker can explain the preferential incorporation of dC by Dpo4. Possible relevance of our new major groove structure for AAF-dG to other polymerases, lesion repair and solution conformations are discussed.     

Rapid communication capturing the destabilizing effect of dihydrouridine through molecular simulations

The structural effects of the commonly occurring modified nucleoside dihydrouridine (D) observed experimentally in model oligonucleotides include a strong destabilization of the C3′‐endo sugar conformation of D, the disruption of stacking interactions of neighboring residues with D and a possible destabilization of the C3′‐endo sugar pucker of the 5′‐neighboring nucleoside. Our simulations with a combination of a set of parameters for modified RNA residues with the recently developed AMBER FF99χ force field having reoptimized glycosidic torsion angle parameters for standard nucleosides was found to reproduce the destabilizing effect of dihydrouridine better than with the AMBER FF99 force field for nucleic acids for which the parameters for the modified residues were originally developed. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 985–991, 2014.     

THE SYNTHESIS OF ANTI-FIXED 3-METHYL-3- DEAZA-2′-DEOXYADENOSINE AND OTHER 3H-IMIDAZO[4,5-c]PYRIDINE ANALOGS

ABSTRACT

Rotation of a heterocyclic base around a glycosidic bond allows the formation of syn and anti conformations in nucleosides. The syn conformation has been observed primarily in purine-purine mismatches in DNA duplexes. Such mismatches give rise to false positive oligonucleotide hybridization in DNA-based diagnostics. Here we describe the synthesis of an analog of 2′-deoxyadenosine that retains its Watson-Crick functional groups, but cannot form the syn conformation. In this analog, the N3 atom of 2′-deoxyadenosine is replaced by a C-CH₃ group to give 7-methyl-1-β-D-deoxyribofuranosyl-1H-imidazo[4,5-c]pyridin-4-ylamine or 3-methyl-3-deaza-2′-deoxyadenosine (3mddA). This modification sterically prevents the syn conformation and 3mddA becomes an anti-fixed nucleoside analog of 2′-deoxyadenosine. The synthesis and conformational analysis of 3mddA and several analogs with an 3H-imidazo[4,5-c]pyridine skeleton are described, as well as their potential applications.     

Syn–anti equilibrium of purine bases in dinucleoside monophosphates as studied by proton longitudinal relaxation

The preferential orientations of the purine bases in dinucleoside monophosphates such as ApA, ApG, and GpA in 10^?2M neutral aqueous solutions have been investigated by proton relaxation at 250 MHz. These orientations are deduced from computer simulations of the magnetization recovery curves following a 180° nonselective pulse. The distances between the H(8) proton of a base and the ribose ring protons which are used in these calculations are obtained by minimization as a function of the glycosyl torsion angle ? of the standard deviation between the isotropic reorientation correlation times τ_R derived from the relaxation rates of these protons. The average H(1′) – H(8) distance obtained by this procedure may be readily verified from the reduction of the H(1′) relaxation rate when H(8) is substituted by a deuteron. The limits of validity of the assumption of a single correlation time τ_R governing the proton relaxation have been estimated, taking into account several possible internal motions, e.g., the rotation of the base, of the methylene exocyclic group and the N ? S interconversion of the ribose ring. For 10^?10 < τ_R < 2 × 10^?10 sec, it appears that the influence of these motions on the proton relaxation becomes perceptible when the jump rates among equilibrium positions exceed ca. 10⁹ sec^?1. The whole of the experimental results show that for the ribose ring N conformer, the orientation of the bases is found in the ranges 60° < ? < 80° (syn) and 180° < ? < 210° (anti). For ribose S conformer, it is observed that this orientation is mainly syn with 5° < ? < 90°. The average H(1′) – H(8) distance provides semiquantitative information on the overall syn or anti orientations of the base in each nucleoside moiety. At 298 K the population of the anti conformer is found to increase in the order A- pG < Ap -G ～ Gp -A < Ap -A < A-pA < G-pA . A more detailed analysis of relaxation data shows that the maximum possible fraction of the stacked form of dinucleotides, due to the occurrence of N-anti conformers in both nucleoside moieties, is in the order ApG < GpA < ApA, in agreement with previous works, with however smaller values. Lastly the deuteron linewidth in position 8 of the bases indicates a syn–anti transition rate of the order of 10⁹ sec^?1 at room temperature, without noticeable effects therefore on the proton relaxation.     

Twisting of glycosidic bonds by hydrolases

Patterns of scissile bond twisting have been found in crystal structures of glycoside hydrolases (GHs) that are complexed with substrates and inhibitors. To estimate the increased potential energy in the substrates that results from this twisting, we have plotted torsion angles for the scissile bonds on hybrid Quantum Mechanics::Molecular Mechanics energy surfaces. Eight such maps were constructed, including one for α-maltose and three for different forms of methyl α-acarviosinide to provide energies for twisting of α-(1,4) glycosidic bonds. Maps were also made for β-thiocellobiose and for three β-cellobiose conformers having different glycon ring shapes to model distortions of β-(1,4) glycosidic bonds. Different GH families twist scissile glycosidic bonds differently, increasing their potential energies from 0.5 to 9.5 kcal/mol. In general, the direction of twisting of the glycosidic bond away from the conformation of lowest intramolecular energy correlates with the position (syn or anti) of the proton donor with respect to the glycon’s ring oxygen atom. This correlation suggests that glycosidic bond distortion is important for the optimal orientation of one of the glycosidic oxygen lone pairs toward the enzyme’s proton donor.     

The interaction of the 7,8-dihydrodiol-9, 10-oxides of benzo(a) pyrene with bacteriophages R17 and T7

Dose-survival curves for bacteriophages R17 and T7 treated with the syn- and anti-isomers for 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene in 0.02 M phosphate buffer, pH 7.0, have been determined. In both cases the anti-isomer proved to be the more toxic: mean lethal dose for R17; syn- 3 μg/ml, anti- 2 μg/ml: and for T7, syn- 3 μg/ml, anti- 0.3 μg/ml. With both reagents reaction with bacteriophage or loss by solvolysis were complete within minutes. Physico-chemical studies of the RNA failed to detect any degradation 1 and 24 h after the addition of the reagents to the bacteriophage and no change in survival of the bacteriophage occurred during this period. In experiments with bacteriophage T7 and T7-DNA reaction did not, in the first hour, introduce any significant number of alkali-labile sites in the nucleic acid. These results suggest that no reaction occurs with the phosphate groups of the nucleic acids. Following the initial loss of infectivity when bacteriophage T7 was treated with the syn-isomer there was a further, progressive loss of biological activity over 4 days which was associated with the development of alkali labile lesions. It seems probable that these latter effects are due to the loss of alkylated bases from the DNA, a process similar to the depurination reactions observed following the reaction of DNA with e.g. methylating agents.     

Glycosidic Bond Conformation Preference Plays a Pivotal Role in Catalysis of RNA Pseudouridylation: A Combined Simulation and Structural Study

The most abundant chemical modification on RNA is isomerization of uridine (or pseudouridylation) catalyzed by pseudouridine synthases. The catalytic mechanism of this essential process remains largely speculative, partly due to lack of knowledge of the pre-reactive state that is important to the identification of reactive chemical moieties. In the present study, we showed, using orthogonal space random-walk free-energy simulation, that the pre-reactive states of uridine and its reactive derivative 5-fluorouridine, bound to a ribonucleoprotein particle pseudouridine synthase, strongly prefer the syn glycosidic bond conformation, while that of the nonreactive 5-bromouridine-containing substrate is largely populated in the anti conformation state. A high-resolution crystal structure of the 5-bromouridine-containing substrate bound to the ribonucleoprotein particle pseudouridine synthase and enzyme activity assay confirmed the anti nonreactive conformation and provided the molecular basis for its confinement. The observed preference for the syn pre-reactive state by the enzyme-bound uridine may help to distinguish among currently proposed mechanisms.     

Conformational features of benzo‐homologated yDNA duplexes by molecular dynamics simulation

yDNA is a base‐modified nucleic acid duplex containing size‐expanded nucleobases. Base‐modified nucleic acids could expand the genetic alphabet and thereby enhance the functional potential of DNA. Unrestrained 100 ns MD simulations were performed in explicit solvent on the yDNA NMR sequence [5′(yA T yA yA T yA T T yA T)₂] and two modeled yDNA duplexes, [5′(yC yC G yC yC G G yC G G)₂] and [(yT5′ G yT A yC yG C yA yG T3′)?(yA5′ C T C yG C G yT A yC A3′)]. The force field parameters for the yDNA bases were derived in consistent with the well‐established AMBER force field. Our results show that DNA backbone can withstand the stretched size of the bases retaining the Watson‐Crick base pairing in the duplexes. The duplexes retained their double helical structure throughout the simulations accommodating the strain due to expanded bases in the backbone torsion angles, sugar pucker and helical parameters. The effect of the benzo‐expansion is clearly reflected in the extended C1′‐C1′ distances and enlarged groove widths. The size expanded base modification leads to reduction in base pair twist resulting in larger overlapping area between the stacked bases, enhancing inter and intra strand stacking interactions in yDNA in comparison with BDNA. This geometry could favour enhanced interactions with the groove binders and DNA binding proteins., 2016. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 55–64, 2016     

Conformations of cyclic 3',5'-nucleotides. Effect of the base on the synanti conformer distribution

The furanose and the phosphate rings of cyclic 3′,5′-nucleotides are locked in the ₄T³ and chair conformations respectively. The only variable which shows major conformational flexibility in these molecules is the rotation about the glycosyl bond which describes the orientation of the base relative to the sugar-phosphate bicyclic system. The glycosyl torsion angle has been analyzed for cyclic nucleotides with different purine and pyrimidine bases by use of conformational energy calculations. The results indicate that all the pyrimidine bases, U, T and C show a very strong energetic preference for the anti range of conformations. The calculations predict that among cyclic 3′,5′-purine nucleotides cyclic GMP and cyclic IMP favor the syn conformation to the anti by 95:5 and 70:30 respectively, while cyclic AMP shows a preference for the anti conformation to syn by 70:30. Thus the purines show a greater probability for the syn conformation than the pyrimidines in cyclic 3′,5′-nucleotides.     

Interpretation of DNA vibration modes: I-The guanosine and cytidine residues involved in poly(dG-dC)·poly(dG-dC) and d(CG)3·d(CG)3

Abstract

A normal coordinate analysis has been carried out on guanosine and cytidine residues appearing in oligo and polynucleotides by using a simplified valence force field that allows the vibrational spectra of 5′-dGMP and 2′-deoxycytidine molecules to be reproduced. The role of both C2′-endo and C3′-endo conformations on sugar pucker, as well as that of glycosidic torsion angle (χ), on several characteristic vibration modes of these residues have been studied. The present calculations based on a non-redundant set of internal coordinates preserving the harmonic approximation of the potential field, allows us to explain quite satisfactorily the modifications of the vibrational spectra in the 1550-1250 cm^?1 and 785-500 cm^?1 regions, when the right → left-handed conformational transition occurs.     

Spectroscopic studies on the structure of poly(8-bromoadenylic acid): Effect of glycosidic torsion angle on the conformation and flexibility in polyribonucleotides

The helix–coil transition and conformational structure of poly(8-bromoadenylic acid) [poly(8BrA)] have been investigated using ¹H- and ¹³C-nmr, CD, and ir spectroscopy. The results have been compared with the structure of the related 5′-mono- and polynucleotides. The chemical shifts of H(2′), H(3′), C(2′), and C(3′) nmr signals show an interesting correlation with both the puckering of ribose ring and glycosidic bond torsion angle. Poly(8BrA) shows an upfield shift of the C(3′) signal and a downfield shift of the H(3′) signal compared to the chemical shifts in poly(A). These shifts are consistent with a C(3′) endo-syn conformation for poly(8BrA). A similar effect has been reported previously and is also observed here on the C(2′) and H(2′) signals when the preferred conformation is C(2′)endo-syn (e.g., in 5′-8BrAMP). The chemical-shift parameters thus act as a probe for studying syn ? anti and N ? S equilibria in solutions. The three-bond ¹H-′¹³C coupling constants between H(1′) and C(8) and C(4) have been measured in poly(8BrA) and 5′-8BrAMP and their structural implications have been discussed. The observed preference of a C(3′)endo-syn conformation for poly(8BrA), coupled with other evidence, throws doubt on the validity of a correlation previously reported whereby a syn conformation is associated with a C(2′)endo ribose pucker. The backbone conformation of randomly coiled poly(8BrA) is very similar to the structures found in polyribonucleotides: poly(A) and poly(U). All three polymers show strong preferences for the backbone angles found in RNA helices. The CD spectrum of poly(8BrA) has a striking relationship to that of poly(A). The signs of all extrema are inverted, and the magnitudes are related by a constant factor. We suggest that these differences result from a change in the angle between coupled transition moment vectors in the two polymers. Infrared spectra of poly(8BrA) in H₂O and D₂O solution are reported for the frequency range below 1400 cm^?1. The antisymmetric >PO stretching vibration is observed at an unusually low frequency in the helix (1214 cm^?1). The symmetric >PO stretch occurs at ～1095 cm^?1 but is not resolved from a ring vibration near this frequency. A conformationally sensitive band, characteristic of helical RNA structures, is observed at 817 cm^?1 and disappears when the helix is melted. This observation confirms the conclusion that ordered poly(8BrA) has a regular helical structure with an RNA backbone conformation. A stereochemical explanation is provided for the failure of poly(8BrA) (or other syn polymers) to form double helices with anti-polyribonucleotides.     

A Modified Version of the Cornell et al. Force Field with Improved Sugar Pucker Phases and Helical Repeat

Abstract

We have examined some subtle parameter modifications to the Cornell et al. force field, which has proven quite successful in reproducing nucleic acid properties, but whose C2′-endo sugar pucker phase and helical repeat for B DNA appear to be somewhat underestimated. Encouragingly, the addition of a single V₂ term involving the atoms C(sp³)-O-(sp³)-C(sp³)- N(sp²), which can be nicely rationalized because of the anomeric effect (lone pairs on oxygen are preferentially oriented relative to the electron withdrawing N), brings the sugar pucker phase of C2′-endo sugars to near perfect agreement with ab initio calculations (W near 162°). Secondly, the use of high level ab initio calculations on entire nucleosides (in contrast to smaller model systems necessitated in 1994–95 by computer limitations) lets one improve the % torsional potential for nucleic acids. Finally, the O(sp³)-C(sp³)- C(sp³)-O(sp³) V₂ torsional potential has been empirically adjusted to reproduce the ab initio calculated relative energy of C2′- endo and C3′-endo nucleosides. These modifications are tested in molecular dynamics simulations of mononucleosides (to assess sugar pucker percentages) and double helices of DNA and RNA (to assess helical and sequence specific structural properties). In both areas, the modified force field leads to improved agreement with experimental data.     

Conformationally rigid nucleoside probes help understand the role of sugar pucker and nucleobase orientation in the thrombin-binding aptamer

Modified thrombin-binding aptamers carrying 2′-deoxyguanine (dG) residues with locked North- or South-bicyclo[3.1.0]hexane pseudosugars were synthesized. Individual 2′-deoxyguanosines at positions dG5, dG10, dG14 and dG15 of the aptamer were replaced by these analogues where the North/anti and South/syn conformational states were confined. It was found that the global structure of the DNA aptamer was, for the most part, very accommodating. The substitution at positions 5, 10 and 14 with a locked South/syn-dG nucleoside produced aptamers with the same stability and global structure as the innate, unmodified one. Replacing position 15 with the same South/syn-dG nucleoside induced a strong destabilization of the aptamer, while the antipodal North/anti-dG nucleoside was less destabilizing. Remarkably, the insertion of a North/anti-dG nucleoside at position 14, where both pseudosugar conformation and glycosyl torsion angle are opposite with respect to the native structure, led to the complete disruption of the G-tetraplex structure as detected by NMR and confirmed by extensive molecular dynamics simulations. We conclude that conformationally locked bicyclo[3.1.0]hexane nucleosides appear to be excellent tools for studying the role of key conformational parameters that are critical for the formation of a stable, antiparallel G-tetrad DNA structures.     

Conformational Analysis of Nucleosides Constructed on a Bicyclo[3.1.0]hexane Template. Structure-Antiviral Activity Analysis for the Northern and Southern Hemispheres of the Pseudorotational Cycle

Abstract

A conformational analysis of carbocyclic nucleosides built on a rigid bicyclo[3.1.0]hexane template (1–4, Northern and 5–8 Southern) showed that the Northern conformation prefers an anti glycosyl torsion angle whereas the Southern conformation favors the syn range. Antiviral activity was mostly associated with the Northern conformers.     

Substituent effects on the puckering mode of the cyclobutane ring and the glycosyl bond of cis–syn photodimers

The cyclobutane ring (CB) puckering of a cis–syn DNA photodimer (cis–syn d-T[p]T) differs from that of a cis–syn RNA photodimer (cis–syn r-U [p] U) [J.-K. Kim and J. L. Alderfer (1992) Journal of Biomolecular Structure and Dynamics, Vol. 9 , p. 1705]. In cis–syn d-T [p] T, interconversion of the CB ring between CB⁺ and CB^? is observed, while in cis–syn r-U [p] U only CB^? is observed. In the CB⁺ conformation, the two thymine rings of the dimer are twisted in a right-handed fashion, as are the bases in B-form DNA. In case of CB^? they are twisted in a left-handed fashion. The C5 (base) and/or C2′ (sugar) substituents apparently affect the CB ring flexibility in cis–syn d-T [p] T and cis–syn r-U [p] U. To study the effects of the C5 substituent on CB ring flexibility, two-dimensional nuclear Overhauser effect (NOE) and ³¹P-nmr experiments were performed on cis–syn d-T [p] U, cis–syn d-U [p] T, and cis–syn d-U [p] U photodimers to investigate the CB puckering mode and overall molecular conformation and dynamics. The NOE results indicate the 5-methyl group in the photodimer induces conformational flexibility of the CB ring. In cis–syn d-T [p] U and cis–syn d-U [p] T, both CB⁺ and CB^? puckering modes are observed. This indicates interconversion between two modes takes place as observed in cis–syn d-T [p] T. In the case of cis–syn d-U [p] U, only the puckering CB^? mode is observed. All three DNA-type dimers—cis–syn d-T [p] U, cis–syn d-U [p] T, cis–syn d-U [p] U—show a characteristic flexibility of glycosidic bonds at the 5′ residue; cis–syn d-T [p] T demonstrates syn–anti interconversion for both the 3′ and 5′ sides, while the others are exclusively anti on the 3′ side. In contrast, the ribophotodimer, cis–syn r-U [p] U, lacking the C5 methyls and having a C2′-OH, demonstrates no conformational flexibility in the CB ring or in either of the glycosidic bonds. Differential flexibility of the three DNA-type dimers (cis–syn d-T [p] U, cis–syn d-U [p] T, cis–syn d-U [p] U) and the RNA dimer (cis–syn r-U [p] U) in the sugar-phosphate backbone region is also apparent from the temperature dependence of the ³¹P chemical shifts of these photodimers compared to their normal dimer analogues. Over the temperature range 18-63°C, the chemical shift change is reduced 22–42% in three DNA-type dimers, while it is reduced 71% in cis–syn r-U [p] U, suggesting the RNA-type dimer is more rigid. © 1993 John Wiley & Sons, Inc.