Correlation between the backbone and side chain conformations in 5′-nucleotides. The concept of a ‘rigid’ nucleotide conformation

Conformational energies of the 5′-adenosine monophosphate have been computed as a function of χ and ψ, of the torsion angles about the side-chain glycosyl C(1′)–N(9) and of the main-chain exocyclic C(4′)–C(5′) bonds by considering nonbonded, torsion, and electrostatic interactions. The two primary modes of sugar puckering, namely, C(2′)-endo and C(3′)-endo have been considered. The results indicate that there is a striking correlation between the conformations about the side-chain glyocsyl bond and the backbone C(4′)–C(5′) bond of the nucleotide unit. It is found that the anti and the Gauche–Gauche (gg), conformations about the glycosyl and the C(4′)–C(5′) bonds, respectively, are energetically the most favored conformations for 5′-adenine nucleotide irrespective of whether the puckering of the ribose is C(2′)-endo or C(3′)-endo. Calculations have also shown that the other common 5′-pyrimidine nucleotides will show similar preferences for the glycosyl and C(4′)–C(5′) bond conformations. These results are in remarkable agreement with the concept of the “rigid” nucleotide unit that has been developed from available data on mononucleotides and dinucleoside monophosphates. It is found that the conformational ‘rigidity’ in 5′-nucleotides compared with that of nucleosides is a consequence of, predominantly, the coulombic interactions between the negatively charged phosphate group and the base. The above result permits one to consider polynucleotide conformations in terms of a “rigid” C(2′)-endo or C(3′)-endo nucleotide unit with the major conformational changes being brought about by rotations about the P–O bonds linking the internucleotide phosphorus atom. IT is predicted that the anti and the gg conformations about the glycosyl and the C(4′)–C(5′) bonds would be strongly preferred in the mononucleotide components of different purine and pyrimidine coenzymes and also in the nucleotide phosphates like adenodine di- and triphosphates.     

The A and B conformations of DNA and RNA subunits. Potential energy calculations for dGpdC

In order to obtain a molecular picture of the A and B forms of a DNA subunit, potential energy calculations have been made for dGpdC with C(3′)-endo and C(2′)-endo [or C(3′)-exo] sugar puckerings. These are compared with results for GpC. The global minima for dGpdC and GpC are almost identical. They are like A-form duplex DNA and RNA, respectively, with bases anti, the ω′, ω angle pair near 300°, 280°, and sugar pucker C(3′)-endo. For dGpdC, a B-form helical conformer, with sugar pucker C(2′)-endo and ω′ = 257°, ω = 298°, is found only 0.4 kcal/mol above the global minimum. A second low-energy conformation (2.3 kcal/mol) has ω′ = 263°, ω = 158° and ψ near 180°. This has dihedral angles like the original Watson–Crick model of the double helix. In contrast, for GpC, the C(2′)-endo B form is 6.9 kcal/mol above the global minimum. These theoretical results are consistent with experimental studies on DNA and RNA fibers. DNA fibers exist in both A and B forms, while RNA fibers generally assume only the A form. A low-energy conformation unlike the A or B forms was found for both dGpdC and GpC when the sugars were C(3′)-endo. This conformation—ω′,ω near 20°,80°—was not observed for C(2′)-endo dGpdC. Energy surface maps in the ω′,ω plane showed that C(2′)-endo dGpdC has one low-energy valley. It is in the B-form helical region (ω′ ～ 260°, ω ～ 300). When the sugar pucker is C(3′)-endo, dGpdC has two low-energy regions: the A-form helical region and the region with the minimum at ω′ = 16°, ω = 85°.     

Examination by the molecular orbital method of the intrinsic conformation preferences of nucleic acid sequence]

This paper reports a systematic PCILO study of the conformation of the nucleic acid backbone. The authors principally studied the ω′ and ω phosphodiester torsion angles of the disugar triphosphate model as a simultaneous function of (1) the sugar nature, ribose or deoxyribose, (2) the different combinations of the sugar ring puckers C(2′)-endo-C(2′)-endo, C(3′)-endo-C(3′)-endo, C(3′)-endo-C(2′)-endo, and C(2′)-endo-C(3′)-endo, and (3) the different conformations around the ψ(C4′–C5′) exocyclic bond. The dependence of the (ω′,ω) conformational energy maps upon these different factors, is discussed. The results are in very good agreement with the observed structures of ribonucleic (RNA10, RNA11, A′-RNA12, tRNA^Phe) and deoxyribonucleic acids (D-DNA, C-DNA 9.3, B-DNA 10, A-DNA 11). Thus the validity of this model, the disugar triphosphate unit, is ensured. The main conclusions that can be drawn from this systematic study are the following:

• 1 The torsion around P-05′ (angle ω) is, as a general rule, more flexible than the torsion around P-03′ (angle ω′).
• 2 There is no notable difference between the ribose–triphosphate units and the deoxyribose–triphosphate units for the C(3′)-endo–C(3′)-endo and C(3′)-endo–C(2′)-endo sugar puckers.
• 3 The deoxyribose–triphosphate units with C(2′)-endo–C(2′)-endo and C(2′)-endo–C(3′)-endo sugar puckers show much more ω′ flexibility than the ribose–triphosphate units with the same sugar puckers and cis position for the 2′hydroxyl group.
• 4 The preferred values of ω′ are independent of the sugar nature (ribose or deoxyribose) and of ψ values; they are correlated with the sugar pucker of the first sugar-phosphate unit:
  • C(3′)-endo-C(3′)-endo and C(3′)-endo-C(2′)-endo puckers ? ω′ ? 240° (g^? region)
  • C(2′)-endo-C(2′)-endo and C(2′)-endo-C(3′)-endo puckers ? ω′ 180° (t region)
• 5 The preferred values of ω are independent of the nature and the puckering of the sugars; they are correlated with the rotational state of the torsion angle ψ(C4′–C5′): ψ ? 60° (gg) ? ω ? 300° (g^?), ψ ? 180° (gt) or 300° (tg) ? ω ? 60° (g⁺)

     

Molecular Conformation of 2′-Deoxy-2′-methylidene-cytidine: A Potent Antineoplastic Nucleoside

Abstract

2′-Deoxy-2′-methylidenecytidine (DMDC), a potent inhibitor of the growth of tumor cells, was crystallized with two different forms. One is dihydrated (DMDC·2H₂O) and the other is its hydrochloride salt (DMDC·HCLl). Both crystal and molecular structures have been determined by the X-ray diffraction method. In both forms the glycosidic and sugar conformations are anti and C(4′)-exo, respectively, whereas the conformation about the exocyclic bond is trans for DMDC·2H₂O and gauche ⁺ for DMDC·HCl. Proton nuclear magnetic resonance data of DMDC indicate a preference for the anti C(4′)-exo conformation found in the solid state. These molecular conformations were compared with the related pyrimidine nucleosides. When the cytosine bases are brought into coincidence, DMDC displays the exocyclic C(4′)-C(5′) bond located on the very close position to those of pyrimidine nucleosides with typical overall conformations. On the other hand, the hydroxyl O(3′)-H groups are separated by ca. 3 Å in the cases of DMDC and other pyrimidine nucleosides which have the C(2′)-endo sugar conformation. This result may be useful for the implication about the mechanism of the biological activity of DMDC.     

Conformational characteristics of the trinucleoside diphosphate d(ApApA) from energy-minimization studies

Energy-minimization studies were carried out on the trinucleoside diphosphate d(ApApA). The potential energy contributions from nonbonded, electrostatic, hydrogen-bonding, and torsional interactions were minimized by treating the 13 relevant dihedral angles as simultaneous variables. For the C(3′)-endo trimer, 14 low-energy conformations are within 10 kcal/mol above the lowest energy found, compared to only 3 in the case of the C(2′)-endo trimer. This result shows the flexible character of the C(3′)-endo unit. The hairpin-type, loop-promoting conformer with (ω′,ω) = (101°, 59°) was found to be the most favored structure at the 3′-terminus of d(ApApA). The predicted U- and L-type bend conformers were found to lie within 5 kcal/mol, compared to the lowest energy B-DNA structure. The A-DNA and Watson-Crick DNA types of helical conformers also lie within very small energy barriers. The phosphate group at the 5′-end of the nucleotide residue has a definite influence on the base of the corresponding nucleotide, keeping it in the normal anti-region, and hence on the base-stacking property. The results are compared with the presently available experimental data, mainly with the tRNA^Phe crystal.     

Interaction of molecules with nucleic acids. II. Two pairs of families of intercalation sites, unwinding angles, and the neighbor-exclusion principle

Intercalation-site geometries are generated for a tetramer duplex extracted from B-DNA. Glycosidic angles and puckers of the deoxyribose sugar groups bonded to base pairs BP₁ and BP₄, namely, those at either end of the tetramer duplex, are assumed to be those of B-DNA to insure continuity. All possible geometrical conformations for combinations of C(2′)-endo, C(3′)-endo, C(2′)-exo, and C(3′)-exo sugar puckers are determined for the tetranucleotide backbone. Those with minimum energy are selected as candidates for intercalation sites. Calculations reveal two pairs of physically meaningful families of intercalation sites which occur in two distinct regions, I and II, of helical angles which orient BP₂ relative to BP₃ and with the helical axis disjointed between these base pairs. For each site I and II within BP₂ and BP₃, there are two distinct backbone conformations, A and B, connecting BP₃ to BP₄ or BP₁ to BP₂ which do not disrupt backbone conformations connecting BP₂ to BP₃. Hence two pairs, IA and IB, and IIA and IIB, of intercalation sites exist in which the sugar puckers along the backbone of the tetramer alternate from C(2′)-endo to C(3′)-endo on the backbone (5′p3′) connecting BP₂ to BP₃. The glycosidic angles of the C(3′)-endo sugar χ₃^γ are, coincidentally, 80° ± 2° for both conformations γ = A and B connecting BP₃ to BP₄ along the phosphate backbone (5′p3′). Consistent with the theoretical results, the experimental unwinding angles can be grouped into two categories with absolute values of 18° and 26°. The theoretical unwinding angles for sites IA and IB of 16° and for sites IIA and IIB of 20° occur for a displacement of -0.8 Å in the helical axes of BP₂ and BP₃ and for a 100% G·C composition, with a decrease depending on the amount of A·T base pairs present. Ratios of theoretical unwinding angles of sites I and II, which range from 0.75 to 0.84 for the two principal sites, compare well with the experimental value of 0.71. The theoretical results, in agreement with experimental observation, provide a new interpretation of the nature and conformation of the possible binding sites. Conformations obtained from these studies of intercalation sites in a tetramer duplex are used to rationalize the well-known neighbor-exclusion principle. The possibility of violation of this principle is demonstrated by the existence of two families of physically meaningful conformations. Conformations of unconstrained dimer duplexes are also obtained, one of which corresponds to the experimental crystal structure of ethidium–dinucleoside complexes, but these cannot be joined to the B-DNA structure. Backbone conformations of the tetramer duplex can be constructed until the base-pair separation reaches 8.25 Å, which may limit the molecules that can intercalate.     

Conformational studies on pyrimidine 5'-monophosphates and 3',5'-diphosphates. Effect of the phosphate groups on the backbone conformation of polynucleotides

In continuation of our studies on the effect of the base and the phosphate groups on the glycosyl and the sugar-phosphate backbone conformation, we have carried out semi-empirical potential energy calculations on the common 5′- and 3′5′-ribopyrimidine mono- and diphosphates by considering simultaneous rotations about the glycosyl (χ) and the C(4′)–C(5′) (ψ) bonds. This calculation provides an assessment of the nature and orientation of the base on the sugar–phosphate backbone conformation of nucleotides and polynucleotides. It is found that the attractive inetractions between the 5′-phosphate group and the base mutually stabilize the antiand the gauche-gauche (gg) conformations about χ and ψ, respectively, in 5′-ribopyrimidine nucleotides. The introduction of the 3′-phosphate group as in 3′,5′-ribopyrimidine diphosphates, still leaves the anti-gg as the most favored conformation with the important difference that the probability of occurrence of the anti, gauche-trans (gt) is how substantially increased. This is dependent to a large extent on the sugar conformation and to a lesser extent on the base. Uracil and thymine show a greater probability for the anti-gt than cytosine. The syn conformation is considerably less likely and its occurrence is also dependent on the base type, cytosine showing a lesser tendency than uracil and thymine. For the syn base, the most favourec conformation for ψ is gt, since gg is sterically disallowed and tg is destabilized by electrostatic repulsive interactions between the 3′ and 5′-phosphate groups. Thus, there is a striking correlation between the glycoysl and the backbone C(4′)–C(5′) bond conformations. The rest of the bonds of the backbone are considerable less dependent on the glycosyl conformation. These studies reveal that in poly-ribopyrimidine nucletides the majority of the nucleotide residues are expected to occur in the anti-gg conformation.     

Conformational study of trinucleoside tetraphosphate d(pCpGpCp): Transition of right-handed form to left-handed form

Using the semiempirical potential functions, conformational energies of the model compounds DMP^?, d(pCp), d(pGp), and d(pCpGpCp) are calculated, and the B → Z transition is discussed along the pseudorotational path of the sugar ring. For dimethylmonophosphate anion, DMP^?, the energy contour map is presented and the stabilities of the phosphodiester conformations discussed. For the sugar ring without the base attached, the minimum energies for each sugar-puckering form are calculated along the pseudorotational path. The energy barrier of the interconversion between the C(3′)-endo form and the C(2′)-endo form is calculated to be about 2.0 kcal/mol. From the conformational energy calculations of the interconversions of mononucleoside diphosphates, d(pCp) and d(pGp), between the C(2′)-endo conformer and the C(3′)-endo conformer, the purine sugar segment is known to be more convertible than the pyrimidine sugar segment. The results also support the finding that the pseudorotational transition occurred with the O(1′)-endo form more easily than with the O(1′)-exo form. Based on the results of conformational studies of DMP^?, d(pCp), and d(pGp), a topological transition of the handedness of the model compound, d(pCpGpCp), is studied. The left-handed Z-form is found to be less stable by about 8.5 kcal/mol than is the right-handed B-form. The energy barrier of the Z → B transition is calculated to be about 17.4 kcal/mol. The contributions of the electrostatic and nonbonded energies to the energy barrier are discussed in connection with the conformation changes of the model compound, d(pCpGpCp).     

Minimum energy conformations of DNA dimeric subunits: Potential energy calculations for dGpdC,dApdA, dCpdC,dGpdG, and dTpdT

Minimum energy conformations have been calculated for the deoxydinucleoside phosphates dGpdC, dApdA, dCpdC, dGpdG, and dTpdT. In these potential energy calculations the eight diheldral angles and the sugar pucker were flexible parameters. A substantial survey of conformation space was made in which all staggred combination ofthe dihedral angles ω′,ω, and ψ, in conjuction with C(2′)-endo puker, were used as starting conformers for the energy minimization. The most important conformations in the C(3′)-endo-puckering domain have ψ = g⁺; ω′,ω = g^?,g^?(A-form),g⁺, g⁺, and g^?,t. With C(2′)-endo-type puker the most important conformations have ψ = g⁺; ω′,ω =g^_,g^_(B-form) and g⁺,t; and ψ =t; ω′,ω =g^_,t(Watson-Crick from) and t,g⁺ (skewed). Stacked bases are a persistent feature of the low-energy conformations, the g⁺ conformer being an exception. Freeing the suger puker allowed this conformation to become low energy, with C(3′)-exo puker. It also caused other low-energy forms, such and the Waston-Crick conformation, to become more favourable. Conformation flexibility in the sugar puker and in ψ, as well as the ω′,ω angle pair, is indicated for the dimeric subunits of DNA.     

Determination of the molecular conformation of beta-D-O2,2'-cyclouridine in aqueous solution by proton magnetic resonance spectroscopy

The solution conformation of β-D -O²,2′-cyclouridine has been determined at 27 and 88°C in D₂O by proton magnetic resonance spectroscopy. The conformation is described in terms of a fixed syn-like sugar-base torsional angle, a type S furanose ring conformation (similar to 2′-endo), and a temperature-dependent exocyclic C(4)′–C(5′) rotamer population containing approximately 50% of the gauche-gauche form at 27°C. β-D -O²,2′-Cyclouridine 5′-phosphate likewise possesses a type S furanose ring conformation.     

Conformational studies on polynucleotide chains. V. A comparison between the quantum-mechanical and the consistent force field approach

Consistent force field (CFF) calculations were performed for the sugar–phosphate–sugar fragment, taken as a model of the polynucleotide backbone. The potential-energy-function is the sum of four contributions, accounting for bond and angle deformation, torsional motions, and nonbonded interactions. Both deoxyribose and ribose systems, with either C(2′)-endo or C(3′)-endo puckering in the starting geometry of ribose rings, were considered. A fair number of minima of the conformational-energy hypersurface were found. Although the numerical method employed in the CFF context cannot solve the problem of finding the global minimum in a definite way, one of the final conformations has a total energy much more attractive than the others, and may be regarded as the most stable conformation attainable with our potential-energy function. The energy-minimization affects the puckering of the first ribose ring differently from that of the second: in general, for the C(2′)-endo system the second ring retains its starting conformation (Ψ′ = 152°), while in the first the Ψ′ is modified by up to 70°; the opposite occurs for the C(3′)-endo system. This is explained by the different positions of the two rings relative to the phosphate group.     

SYNTHESIS,STRUCTURE, AND CONFORMATION OF ANTI-TUMOR AGENTS IN THE SOLID AND SOLUTION STATES: HYDROXYL DERIVATIVES OF FTORAFUR

ABSTRACT

The pyrimidine antimetabolite Ftorafur [FT; 5-fluoro-1-(tetrahydro-2-furyl)uracil] has shown significant antitumor activity in several adenocarcinomas with a spectrum of activity similar to, but less toxic than, 5-fluorouracil (5-FU). It is considered as a prodrug that acts as a depot form of 5-FU, and hence the two drugs exhibit a similar spectrum ofchemotherapeutic activity. Ftorafur is metabolized in animals and humans when hydroxyl groups are introduced into the tetrahydrofuran moiety. These metabolites are also thought to be as active as ftorafur but less toxic than 5-FU. Hydroxyl derivatives: 2′-hydroxyftorafur (III), 3′-hydroxyftorafur (IV) and 2′,3′-dihydroxyftorafur (II) were synthesized and X-ray and NMR studies of these hydroxyl derivatives were undertaken in our laboratories to study the structural and conformational features of Ftorafur and its metabolites in the solid and solution states. X-ray crystallographic investigations were carried out with data collected on a CAD-4 diffractometer. The structures were solved and refined using the SDP crystallographic package of Enraf-Nonius on PDP 11/34 and Microvax computers. All of the compounds studied had the base in the anti conformation. The glycosidic torsion angles varied from ?20 to 60 degrees. There is an inverse correlation between the glycosyl bond distances and the χ angle. Molecules with a lower χ angle have a larger bond distance and vice versa. The sugar rings show a wide variation of conformations ranging from C2′-endo through C3′-endo to C4′-exo. The crystal structures are stabilized by hydrogen bonds involving the base nitrogen atom N3 and the hydroxyl oxygen atoms of the sugar rings as donors and the keto oxygens O2 and O4 of the base and the hydroxyl oxygen atoms O2′ and O3′ as acceptors. The NMR studies were carried out on Brüker 400 and 600 MHz instruments. Simulated proton spectra were obtained through Laocoon, and pseudorotational parameters were solved by Pseurot. Presence of syn or anti forms was demonstrated with the use of NOE experiments. The glycosyl conformations in solution vary more widely than in the solid state. The conformations of the sugar molecules are in agreement with the values obtained in the solid state. The studies of the structure and conformation in the solid and solution states give a model for the Ftorafur molecule that could be used in structure, function and biological activity correlation studies.     

Structure of guanylyl-3',5'-cytidine monophosphate. II. Description of the molecular and crystal structure of the calcium derivative in space group P2(1).

The structural features of calcium guanosine-3′,5′-cytidine monophosphate (GpC) have been elucidated by X-ray diffraction analysis. The molecule was crystallized in space group P2₁ with cell constants of a = 21.224 Å, b = 34.207 Å, c = 9.327 Å, and β = 90.527°, Z = 8. The hydration of the crystal is 21% by weight with 72 water molecules in the unit cell. The four GpC molecules in the asymmetric unit occur as two Watson-Crick hydrogen-bonded dimers related by a pseudo-C face centering. Each dimer consists of two independent GpC molecules whose bases are hydrogen bonded to each other in the traditional Watson-Crick fashion. Each dimer possesses a pseudo twofold axis broken by a calcium ion and associated solvent. The four molecules are conformationally similar to helical RNA, but are not identical to it or to each other. Instead, values of conformational angles reflect the intrinsic flexibility of the molecule within the range of basic helical conformations. All eight bases are anti, sugars are all C3′-endo, and the C4′-C5′ bond rotations are gauche-gauche. The R factor is 12.6% for 2918 observed reflections at 1.2-Å resolution.     

Theoretical drug design: 6-azauridine-5'-phosphate--its X-ray crystal structure, potential energy maps, and mechanism of inhibition of orotidine-5'-phosphate decarboxylase.

The cytostatic drug 6-azauridine is converted in vivo to 6-azauridine-5′-phosphate (z⁶Urd-5′-P), which blocks the enzyme orotidine-5′-phosphate decarboxylase (Ord-5′-Pdecase) and therefore inhibits the de novo production of uridine-5′-phosphate (Urd-5′-P). In order to relate the structure and function of z⁶Urd-5′-P, it was crystallized as trihydrate, space group P2₁2₁2₁ with a = 20.615 Å, b = 6.265 Å, c = 11.881 Å, and the structure established by Patterson methods. Atomic parameters were refined by full-matrix least-squares methods to R = 0.066 using 1638 counter measured x-ray data. The ribose of z⁶Urd-5′-P is in a twisted C(2′)-exo, C(3′)endo conformation, the heterocycle is in extreme anti position with angle N(6)-N(1)-C(1′)-O(4′) at 86.3°, and the orientation about the C(4′)-C(5′) bond is gauche, trans in contrast to gauche, gauche found for all the other 5′-ribonucleotides. Conformational energy calculations show that z⁶Urd-5′-P may adopt an extreme anti conformation not allowed to Urd-5′-P, and they also predict the same unusual trans, gauche conformation about the C(4′)-C(5′) bond in orotidine-5′-phosphate (Ord-5′-P) and in z⁶Urd-5′-P, which renders the distances O(2)…O(5′) in z⁶Urd-5′-P and O(7)…O(5′) in Ord-5′-P comparable. On this basis the function of z⁶Urd-5′-P as an Ord-5′-Pdecase inhibitor can be explained as being due to its structural similarity with the substrate Ord-5′-P and further clarifies the inhibitory action of 5′-nucleotides bearing the heterocycles oxipurinol, xanthine, or allopurinol [J. A. Fyfe, R. L. Miller, and T. A. Krenitsky, J. Biol. Chem. 248 , 3801 (1973)]. With this in mind, new inhibitors for Ord-5′-Pdecase may be designed.     

Crystal Structure and Conformation of 5‐Fluorouridine: Conformational Preferences for 5‐Fluorinated Pyranosides

Crystals of 5‐fluorouridine (5FUrd) have unit cell dimensions a = 7.716(1), b = 5.861(2), c = 13.041(1)Å, α = γ = 90°, β = 96.70° (1), space group P2₁, Z = 2, ρ_obs = 1.56 gm/c.c and ρ_calc = 1574 gm/c.c The crystal structure was determined with diffractometric data and refined to a final reliability index of 0.042 for the observed 2205 reflections (I ≥ 3σ). The nucleoside has the anti conformation [χ = 53.1(4)°] with the furanose ring in the favorite C2′–endo conformation. The conformation across the sugar exocyclic bond is g⁺, with values of 49.1(4) and ? 69.3(4)° for Φ_θc and Φ_∞ respectively. The pseudorotational amplitude τ_m is 34.5 (2) with a phase angle of 171.6(4)°. The crystal structure is stabilized by a network of N–H…O and O–H…O involving the N3 of the uracil base and the sugar O3′ and O2′ as donors and the O2 and O4 of the uracil base and O3′ oxygen as acceptors respectively. Fluorine is neither involved in the hydrogen bonding nor in the stacking interactions. Our studies of several 5‐fluorinated nucleosides show the following preferred conformational features: 1) the most favored anti conformation for the nucleoside [χ varies from ? 20 to + 60°] 2) an inverse correlation between the glycosyl bond distance and the χ angle 3) a wide variation of conformations of the sugar ranging froni C2′–endo through C3′–endo to C4′–exo 4) the preferred g⁺ across the exocyclic C4′–C5′ bond and 5) no role for the fluorine atom in the hydrogen bonding or base stacking interactions.     

Configurational effects on conformational properties of cyclic nucleotides. I. Theoretical calculations of conformer preferences in α-nucleoside 3′,5′ cyclic monophosphates

A comparative study has been made of the configurational effects on the conformational properties of α- and β-anomers of purine and pyrimidine nucleoside 3′,5′,-cyclic monophosphates and their 2′-arabino epimers. Correlation between orientation of the base and the 2′-hydroxyl group have been studied theoretically using the PCILO (Perturbative Configuration Interaction using Localized Orbitals) method. The effect of change in ribose puckering on the base-hydroxyl interaction has also been studied. The result show that steric repulsions and stabilizing effects of intramolecular hydrogen bonding between the base and the 2′-hydroxyl (OH) group are of major importance in determining configurations of α-anomers and 2′-arabino-β-epimers. For example, hydrogen bonding between the 2′-hydroxyl group and polar centers on the base ring is clearly implicated as a determinant of syn-anti preferences of the purine (adenine) or pyrimidine (uracil) bases in α-nucleoside 3′,5′-cyclic monophosphates. Moreover, barrier heights for interconversion between conformers are sensitive to ribose pucker and 2′-OH orientations. The result clearly show that a change in ribose-ring pucker plays an essential role in relieving repulsive interaction between the base and the 2′-hydroxyl group. Thus a C2′-exo-C3′-endo (₂T³) pucker is favored for α-anomers in contrast with the C4′-exo-C3′-endo (₄T³) from found in β-compounds.     

Conformational analysis of the 2'-deoxyribofuranose ring from proton-proton coupling constants: analysis of a nucleoside-carcinogen adduct formed from 2-acetylaminofluorene utilizing a three-state model

The conformation of the sugar moiety of 8-(N-fluoren-2-ylamino)-2′-deoxyguanosine in solution has been examined as a function of temperature by ¹H-nmr spectroscopy. Analysis of coupling constants shows that lowering the temperature to ?50°C in methanol shifts the conformational equilibrium of the sugar ring resulting in a C2′-endo conformation at a mole fraction of 0.97. The computed phase angle of pseudorotation and amplitude of pucker are 154° and 36°, respectively, with very little discrepancy between the five calculated coupling constants and coupling constants extrapolated from the temperature profiles. A computer program has been written enabling a three-state best-fit analysis. The three-state analysis indicates an equilibrium between C2′-endo, C3′-endo, and 04′-endo conformations. In aqueous solution, the computed mole fraction of the 04′-endo form is 0.18 at 30°C. The conformation associated with the sugar ring and the C4′? C5′ bond is compared to that of 2′-deoxyguanosine.     

Conformational characteristics of dApdA,dApdT, dTpdA,and dTpdT from energy minimization studies

The conformational characteristics of the deoxydinucleoside monophosphates with adenine and thymine bases in all possible sequences, namely, dApdA, dApdT, dTpdA, and dTpdT have been studied using an improved set of energy parameters to calculate the total potential energy and an improved set of energy parameters to calculate the total potential energy and an improved version of the minimization technique to minimize the total energy by allowing all seven dihedral angles of the molecular fragment to vary simultaneously. The results reveal that the most preferred conformation in all these units usually corresponds to one of the four helical conformations, namely, the A-DNA, B-DNA, C-DNA, and Watson-Crick DNA models. These helical conformations differ in energies by about 3 kcal/mol with respect to one another. The conformations which could promote a loop or bend in the backbone are, in general, less stable by about 3.5 kcal/mol with respect to the respective lowest-energy helical conformation. The results indicate that there is a definite influence of bases and their actual sequences on the preferred conformations of the deoxydinucleoside monophosphates. The lowest-energy structure, although corresponding to one of the four helical conformations, differ with the type of the deoxydinucleoside monophosphate. Good or reasonable base stacking is noted in dApdA and dTpdA with both C(3′)-endo and C(2′)-endo sugars and in dApdT and dTpdT with only C(3′)-endo sugar. The inversion of the base sequence in deoxydinucleoside monophosphates alters the order of preference of low-energy conformations as well as the base-stacking property of the unit. The paths linking the starting and final states in the (ω′, ω) plane show interesting features with regard to the energy spread, thus providing insight into the path of conformational movement ofthe molecule under slight perturbation. The stabilities of the A and B forms, including the internal energies of the C(3′)-endo ans C(2′)-endo sugar systems, indicate that for dTpdT the B → A transition is less probable. For dApdA, dApdT, and dTpdA this transition is probable in the same order of preference. We propose that the T-A sequence in the polynucleotide chain might serve as the site accessible for B ? A transitions. The theoretical predictions are in good agreement with the experimental observations.     

Cytldlne Cyclic (3′, 5′) Monophosphate: Cyclic Nucleotide With a Non-Characteristic Ribose Ring Conformation

Abstract

Cytidine 3′,-5′-cyclic phosphate (cCMP) occurs in nature and has growth stimulatory activity on L-1210 cells. The initiation of cell growth by cCMP, under conditions where CAMP, cGMP and cUMP delay the onset of proliferation suggests that cCMP may play a regulatory role in the cell metabolism. It has been reported that in 3′,5′-cyclic nucleotides, the phosphate ring fused to the furanose ring resuicts the conformation of the furanose ring to the twist form C(3′) endo C(4′) exo (³T₄), in contrast to the C(2′) endo C(3′) endo (²T₃) and C(3′) endo C(2′) exo (³T₂) twist forms normally found in nucleotides and nucleosides. We have carried out an accurate crystal structure of cCMP and found that the furanose ring in cCMP has the C(3′) endo C(2′) exo conformation (³T₂), with a pseudo rotation amplitude (P) of 44° and phase angle τ_m of 12°. cCMP is in low anti conformation (X_CN = 15.4°) and O(5′) has the fixed g conformation. The phosphate ring is constrained to the chair conformation, as in other cyclic nucleotides. The two exocyclic P-O bond distances are short (1.489, 1.476Å) and the ring angle at N(3) is large (125.2°) suggesting that the molecule in the solid state is a zwitterion with a plus charge on N(3). The crystals are hydrated and highly unstable. The three water molecules are highly disordered in ten locations. The crystals of cCMP 3H₂O are hexagonal, a = 16.294(3), b = c = 11.099(4)Å, space group P6₁, final R value is 0.067 for 1620 reflections 230.     

Conformational study of ribonucleotides, 2′-deoxyribonucleotides,and arabinonucleotides by carbon-13 nuclear magnetic resonance

The geminal and vicinal ¹³C-³¹P coupling constants have been monitored, as a function of pH, for a series of uracil and cytosine 3′- and 5′-nucleotides with a ribose, arabinose, or 2′-deoxyribose sugar. Data were also obtained for two 3′,5′-diphosphates in the ribose and arabinose series. The geminal J(C5′-P5′) and J(C3′-P3′) couplings show only a small dependence on the ionization state of the phosphate, decreasing by < 0.5 Hz in the pH 5–7 range. For the ribose and arabinose 3′-nucleotides, the vicinal J(C4′-P3′) increase (up to 1.5 Hz) on secondary phosphate ionization in the pH 5–7 range, whereas their J(C2′-P3′) couplings decrease (up to 1.5 Hz) over the same pH range. In contrast for the 2′-deoxyribose molecules, both couplings decrease (～0.5 Hz) on phosphate ionization. The titration curves provide information about the influence of the sugar on the conformation about the C3′? O3′ bond. Some conformational trends could be rationalized by consideration of the sugar-puckerdependent contact interactions between the 3′-phosphate and the substituents on the furanose ring.