Conformations of cyclic 2,3-nucleotides and 2,3-Cyclic-5-diphosphates. Interrelation between the phase angle of pseudorotation of the sugar and the torsions about the glycosyl C(1)-N and the backbone C(4)-C(5) bonds

Semiempirical potential energy calculations have been carried out for cyclic 2′,3′-nucleotides and their 5′-phosphorylated derivatives, which are the intermediates in the hydrolysis of RNA. Calculations have been performed for both purine and pyrimidine bases for the observed O(1′)-endo, O(1′)-exo and the unpuckered planar sugar ring conformations. It is found that the mode of sugar pucker largely determines the preferred conformations of these molecules. For cyclic 2′,3′-nucleotides themselves, the O(1′)-endo sugars show a preference for the syn glycosyl conformation while the O(1′)-exo sugars exclusively favor the anti conformation regardless of whether the base is a purine or pyrimidine. For the unpuckered planar sugar, the syn conformation is favored for purines and anti for pyrimidines. Both the gauche (+) (60°) and trans (180°) conformations about the C(4′)? C(5′) bond are favored for O(1′)-endo sugars, while the gauche (?) (300°) and trans (180°) are favored for O(1′)-exo sugars. On the contrary, the 5′-phosphorylated cyclic 2′,3′-nucleotides of both purines and pyrimidines show a preference for the anti-gauche (+) conformational combination about the glycosyl and C(4′)? C(5′) bonds for the O(1′)-endo sugars and the anti-trans combination for the O(1′)-exo sugars. The correlation between the phase angle of the sugar ring and the favored torsions about the glycosyl and the backbone C(4′)? C(5′) bonds as one traverses along the pseudorotational pathway of the sugar ring is examined.     

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.     

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.     

Synthesis and Antiviral Activity of Novel Fluorinated 2′,3′‐Dideoxynucleosides

A series of 5‐(trifluoroethoxymethyl)‐2′,3′‐dideoxyuridines and 5‐[bis(trifluoroethoxy)‐methyl]‐2′,3′‐dideoxyuridines have been prepared and screened for antiviral activity. The conformations of these compounds are discussed on the bases of NOE studies and the MO calculations. Modelling and NOE studies suggest both syn‐ and anti conformations for these 5‐(2,2,2‐trifluoroethoxymethyl)‐ and 5‐[bis(2,2,2‐trifluoroethoxy)‐methyl]‐ derivatives. The NOE parameters are also suggested to be more attributable to the nature of the fluorine atom than to structural or conformational changes. Compounds 17, 26 and 30 showed some activity in anti‐HIV‐1 and anti‐HIV‐2 assays, but the compounds were devoid of activity against HSV and human rhinovirus. The compounds tested exhibited low cytotoxicity and were inactive against a bank of cancer cells in vitro.     

Conformation and Antiherpes Activity of 3′- and 5′-Azido and Amino Analogs of 5-Methoxymethyl-2′-Deoxyuridine

Abstract

The molecular conformations of 3′- and 5′-azido and amino derivatives of 5-methoxymethyl-2′-deoxyuridine, 1, were investigated by nmr. The glycosidic conformation of 5-methoxymethyl-5′-amino-2′,5′-dideoxy-uridine, 5 had a considerable population of the syn form. The 5′-derivatives show a preference for the S conformation of the furanose ring as in 1. In contrast, the 3′-derivatives show preference for the N conformation. For 5-methoxymethyl-3′-amino-2′,3′-dideoxyuridine, 3, the shift towards the N state is pH dependent. The preferred conformation for the exocyclic (C4′,C5′) side chain is g⁺ for all compounds except 5 which has a strong preference for the t rotamer (79%). Compounds 1, 3 and 5 inhibited growth of HSV-1 by 50% at 2, 18 and 70 μg/ml respectively, whereas 2 and 4 were not active up to 256 μg/ml (highest concentration tested). The compounds were not cytotoxic up to 3,000 μM.     

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.     

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 2′,5′-Linked Tetraribonucleotide Loops: A Novel RNA Motif

Abstract

We report on the three dimensional structure of an RNA hairpin containing a 2′,5′-linked tetraribonucleotide loop, namely, 5′-rGGAC(UUCG)GUCC-3′ (where UUCG = U_2′p5′U_2′p5′C_2′p5′G_2′p5′). We show that the 2′,5′-linked RNA loop adopts a conformation that is quite different from that previously observed for the native 3′,5′-linked RNA loop. The 2′,5′- RNA loop is stabilized by (a) U:G wobble base pairing, with both bases in the anti conformation, (b) extensive base stacking, and (c) sugar–base contacts, all of which contribute to the extra stability of this hairpin structure.     

NMR study of dideoxynucleotides with anti-human immunodeficiency virus (HIV) activity

The molecular structures of 3′-azido-2′,3′-dideoxyribosylthymine 5′-triphosphate (AZTTP), 2′,3′-dideoxyribosylinosine 5′-triphosphate (ddITP), 3′-azido-2′,3′-dideoxyribosylthymine 5′-monophosphate (AZTMP) and 2′,3′-dideoxyribosyladenine 5′-monophosphate (ddAMP) have been studied by NMR to understand their anti-HIV activity. For ddAMP and ddITP, conformations are almost identical with their nucleoside analogues with sugar ring pucker equilibriating between C3′-endo (∼75%) and C2′-endo (∼25%). AZTMP and AZTTP on the other hand show significant variations in the conformational behaviour compared with 3′-azido-2′,3′-dideoxyribo-sylthymine (AZT). The sugar rings for these nucleotides have a much larger population of C2′-endo (∼75%) conformers, like those observed for natural 2′-deoxynucleosides and nucleotides. The major conformers around C5′-O5′, C4′-C5′ and the glycosidic bonds are the β^t, γ⁺ and anti, respectively.     

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.     

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.     

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.     

An isotopic assay of cyclic 3',5'-nucleotide phosphodiesterase with aluminum oxide columns

A method is described for separating purine bases and nucleosides from cyclic 3′,5′-nucleotides on aluminum oxide columns. Purine bases and nucleosides were found to pass through columns equilibrated with ammonium acetate buffer at pH 4.0 while the cyclic nucleotides were retarded enough to permit separation. Optimal conditions and factors affecting separation are described. The method was shown to be applicable in the isotopic assay of cyclic 3′,5′-nucleotide phosphodiesterase activity over a broad range of substrate concentrations. The advantages of this method and its possible use in a simultaneous cyclase-phosphodiesterase assay are described.     

Conformational Analysis of 6-Substituted Uridine Analogues: Crystal Structures of Uridine-6-Thiocarboxamide and 6-Cyanouridine

Abstract

Crystal structure analyses of uridine-6-thiocarboxamide (I) and 6-cyanouridine (II) show that both structures adopt a syn conformation about the glycosyl bond. The conformation of I is similar to that of orotidine (III). The furanose ring conformation of I is C4′-exo, unusual for syn conformers, and is C3′-endo in II. These results have a bearing on the inhibition of orotidylate decarboxylase by the 5′-phosphate of I.     

Structural basis of error‐prone replication and stalling at a thymine base by human DNA polymerase ι

Human DNA polymerase ι (polι) is a unique member of Y‐family polymerases, which preferentially misincorporates nucleotides opposite thymines (T) and halts replication at T bases. The structural basis of the high error rates remains elusive. We present three crystal structures of polι complexed with DNA containing a thymine base, paired with correct or incorrect incoming nucleotides. A narrowed active site supports a pyrimidine to pyrimidine mismatch and excludes Watson–Crick base pairing by polι. The template thymine remains in an anti conformation irrespective of incoming nucleotides. Incoming ddATP adopts a syn conformation with reduced base stacking, whereas incorrect dGTP and dTTP maintain anti conformations with normal base stacking. Further stabilization of dGTP by H‐bonding with Gln59 of the finger domain explains the preferential T to G mismatch. A template ‘U‐turn’ is stabilized by polι and the methyl group of the thymine template, revealing the structural basis of T stalling. Our structural and domain‐swapping experiments indicate that the finger domain is responsible for polι's high error rates on pyrimidines and determines the incorporation specificity.     

The Crystal and Molecular Structure of the Complex of 2′3′-Didehydro-2′3′-Dideoxyguanosine with Pyridine

Abstract

The structure of 2′,3′-didehydro-2′,3′-dideoxyguanosine was determined by X-ray crystallographic analysis of the complex with pyridine. The two independent nucleoside molecules have similar, commonly observed glycosyl link (x = -102.3° and -94.2°) and 5′-hydroxyl (y = 54.0° and 47.6°) conformations. The five-membered rings are very planar with r.m.s. deviations from planarity of less than 0.015 A. 2′,3′-Didehydro-2′,3′-dideoxyadenosine has a similar glycosyl link conformation but a different 5′-hydroxyl group orientation and a slightly less planar 5-membered ring.     

Synthesis of Pyrazolo[3, 4-d]Pyrimidine Ribonucleoside 3′, 5′-Cyclic Phosphates Related to cAMP,cIMP and cGMP1

Abstract

The synthesis of pyrazolo[3,4-d]pyrimidine ribonucleoside 3′, 5′-cyclic phosphates related to cAMP, cIMP and cGMP has been achieved for the first time. Phosphorylation of 4-amino-6-methylthio-1-β-D-ribo-furanosylpyrazolo[3,4-d]pyrimidine (1) with POCl₃ in trimethyl phosphate gave the corresponding 5′-phosphate (2a). DCC mediated intramolecular cyclization of 2a gave the corresponding 3′, 5′-cyclic phosphate (3a), which on subsequent dethiation provided the cAMP analog 4-amino-1-β-D-ribofuranosylpyrazolo[3, 4-d]pyrimidine 3′, 5′-cyclic phosphate (3b). A similar phosphorylation of 6-methylthio-1-β-D-ribofuranosylpyrazolo[3, 4-d]pyrimidin-4(5H)-one (5), followed by cyclization with DCC gave the 3′, 5′-cyclic phosphate of 5 (9a). Dethiation of 9a with Raney nickel gave the cIMP analog 1-β-D-ribofuranosylpyrazolo[3, 4-d]pyrimidin-4(5H)-one 3′, 5′-cyclic phosphate (9b). Oxidation of 9a with m-chloroperoxy benzoic acid, followed by ammonolysis provided the cGMP analog 6-amino-1-β-D-ribofuranosylpyrazolo [3, 4-d] pyrimidin-4(5H)-one 3′, 5′-cyclic phosphate (7). The structural assignment of these cyclic nucleotides was made by UV and H NMR spectroscopic studies.     

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 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.     

Conformational Studies on Nucleosides with Furanose Ring Modifications. 1.

Abstract

Conformational energy calculations have been presented on adenine and thymine nucleosides in which the furanose ring is replaced by 2′,3′-dideoxy-2′,3′-didehydrofuran using molecular mechanics and conformational analysis. Conformational energies have been evaluated using the MM2 and AMBER94 force field parameters at two different dielectric constants. The results are presented in terms of isoenergy contours in the conformational space of the glycosidic (χ) and C4′-C5′ (γ) bonds torsions. In general, the χ-γ interrelationships exhibit similarities with the corresponding plots for unmodified nucleosides and nucleotides, reported previously. Consistency of the calculated preferred conformations with the X-ray data is sensitive to the force field employed.