Conformation Analysis of Two Anti-HIV Nucleoside Analogues 2′,3′-Dideoxy-3′-fluorocytidine and Its N4-Dimethylaminomethylene Prodrug Derivative

Abstract

Crystal structure analysis of 2′,3′-dideoxy-3′-fluorocytidine (1) and its prodrug derivative, N⁴-dimethylaminomethylene-2′,3′-dideoxy-3′-fluorocytidine (2), active anti-HIV nucleoside analogues, reveals that both structures adopt an anti conformation about the glycosyl bond. The furanose ring is C2′-endo for (2) and C2′-endo/C1′-exo and C2′-endo/C3′-exo for the two independent molecules of (1), respectively.     

NMR SOLUTION STRUCTURE AND CONFORMATIONAL ANALYSIS OF THE CALCIUM RELEASE AGENT CYCLIC ADENOSINE 5′-DIPHOSPHATE RIBOSE (cADPR)

A high-resolution NMR study of the solution structure of the calcium release agent cADPR has been performed. Pseudorotationals analysis reveals that in solution both sugar rings in cADPR adopt predominantly (～75%) South conformations, with the A and N rings adopting approximately ²T₃ (C2′-endo(major)-C3′-exo(minor) and ⁴ ₃T (C3′-exo-C4′-endo) conformations, respectively. The backbone torsion angles β and γ have also been determined. While the minor North conformers were not observed in the crystal structure of cADPR, the solution values of the major South conformers compare well to those found in crystal structure.     

DNA-RNA hybrid secondary structures

DNA-RNA and DNA-DNA duplexes are even more polymorphic than observed previously. DNA-RNA hybrids can have secondary structures like A-DNA or A-RNA, but double helices of the synthetic DNA-RNA hybrids poly(dA) X poly(rU) and poly(dI) X poly(rC), respectively, form 11-fold and 10-fold double-helical structures in which the two chains have quite different conformations. Extensive X-ray fiber diffraction analyses show that in both structures the DNA chains have C-2'-endo-puckered furanose rings, while the anti-parallel RNA chains have C-3'-endo-puckered rings. The bidirectional properties of such duplexes may be important in the transfer of biological information from nucleic acids.     

Structures for polyinosinic acid and polyguanylic acid

X-ray-diffraction analysis of oriented, partially crystalline fibres of polyinosinic acid has resulted in a new molecular model. This model consists of four identical polynucleotide chains related to one another by a fourfold rotation axis. The coaxial helices are righthanded (screw symmetry 23(2)) and have an axial translation per residue h=0.341nm and a rotation per residue t=31.3 degrees . Incorporated in the model are standard bond lengths, bond angles and C-2-endo furanose rings. The nucleotide conformation angles, determined by linked-atom least-squares methods, are orthodox and the fit with the X-ray intensities is good. Each hypoxanthine base is linked to two others by hydrogen bonds involving O-6 and N-1. Further stability may arise from intrachain hydrogen bonds between each ribose hydroxyl group and the phosphate oxygen O-3. If guanine were to be substituted for hypoxanthine in an isogeometrical molecular structure, additional hydrogen bonds could be made between every N-2 and N-7.     

A method for the determination of furanose ring coordinates in its pseudorotation circuit for different amplitudes of pucker

Interconversion between energetically favored molecular conformations must proceed through less favored intermediate states. Thus, a knowledge of the nucleotide furanose ring conformations, other than the crystallographically well-determined ones, are of interest in investigating nucleotide conformational energies and dynamics. The sugar ring flexibility affects the conformation and dynamics of the monomer and determines the range of feasible nucleic acid secondary and tertiary structures. We have generated furanose geometries for varying amplitudes of pucker over its entire range of pseudorotation by making use of a ring closure procedure and the empirical dependence of endocyclic bond lengths and bond angles on sugar pucker. Atomic coordinates are tabulated here for the furanose ring at pseudorotation phase angle intervals of 9 degrees for the average amplitude (tau m) of pucker of 39 degrees as well as for decreased (20 degrees and 30 degrees) and increased (44 degrees) values of tau m. However, the coordinates for any values of P and tau m can be readily calculated.     

Synthesis of novel 2'-deoxy type trans-3',4'-bridged nucleic acid

We newly designed and synthesized a 2'-deoxy type trans-3',4'-bridged nucleic acid (trans-3',4'-BNA) analogues bearing a 4,7-dioxabicyclo[4.3.0]nonane structure. The synthesis of the trans-3',4'-BNA was carried out successfully from thymidine over 21 steps. The structure of trans-3',4'-BNA was confirmed by x-ray crystallographic analysis, indicating that the furanose ring has a typical S-type conformation with C(3')-exo puckering.     

A Method for the Determination of Furanose Ring Coordinates in its Pseudorotation Circuit for Different Amplitudes of Pucker

Abstract

Interconversion between energetically favored molecular conformations must proceed through less favored intermediate states. Thus, a knowledge of the nucleotide furanose ring conformations, other than the crystallographically well-determined ones, are of interest in investigating nucleotide conformational energies and dynamics. The sugar ring flexibility affects the conformation and dynamics of the monomer and determines the range of feasible nucleic acid secondary and tertiary structures. We have generated furanose geometries for varying amplitudes of pucker over its entire range of pseudorotation by making use of a ring closure procedure and the empirical dependence of endocyclic bond lengths and bond angles on sugar pucker. Atomic coordinates are tabulated here for the furanose ring at pseudorotation phase angle intervals of 9° for the average amplitude (τ_m) of pucker of 39° as well as for decreased (20° and 30°) and increased (44°) values of τ_m. However, the coordinates for any values of P and τ_m can be readily calculated.     

The crystal and molecular structure of 2'-deoxy-2'-fluoroinosine monohydrate.

The structure of the hydrate of 2'-deoxy-2'-fluoroinosine has been determined by single-crystal x-ray diffraction. The nucleoside crystallizes in space group P2(1)2(1)2(1) with unit cell dimensions, a = 33.291, b = 10. 871, c = 6.897A. There are two nucleosides and two water molecules in the asymmetric unit. The structure was solved by direct methods and refined to a residual R = 0.095. The two independent nucleosides in the asymmetric unit show different conformations about the glycosidic bond, while other structural details are similar. The base orientation to the sugar is syn in molecule A, whereas anti in molecule B. The exocyclic C(4')-C(5') bond conformation defined with respect to C(3')-C(4')-C(5')-O(5') is gauche+ in both molecules A and B. The sugar ring pucker defined by the pseudorotation phase angle P is a twisted conformation in both, C(3')-endo-C(4')-exo with P = 29 degrees in molecule A and C(4')-exo-C(3')-endo with P = 41 degrees in molecule B. It is shown by comparison with x-ray results of other 2'-fluoronucleosides and unmodified nucleosides including inosines that, in addition to a strong preference of the C(3')-endo type pucker, twisted conformations involving C(4')-exo puckering may be one of characteristic features of 2'-fluoronucleosides.     

Conformation of the common purine (beta) ribosides in solution: further evidence for a correlation between N-S state of the ribose moiety and syn-anti equilibrium.

The solution conformations of adenosine, guanosine and inosine in liquid ND3 have been determined by NMR. Comparison of the Karplus analysis of the proton HR spectra of the ribose moiety obtained in this solvent with the data from aqueous solutions of A and I proves that the conformations of the nucleosides are very similar in both liquids. From the analysis of the vicinal coupling constants of the ring protons it has been deduced that the S state C(2')-endo is slightly preferred. The mole fraction in S approximates 0.6 for all three nucleosides. C-13 relaxation measurements have been applied in the determination of the correlation times for rotational diffusion. Only at temperatures below - 40 degrees C is the pseudo-rotation of the furanoside ring slowed down sufficiently for it not to contribute to the measured relaxation rates. From NOE studies and T1 measurements on the individual protons it is derived that the N, C(3')-endo, form of the ribose is correlated with an anti conformation of the base (Y approximately 210 degrees to 220 degrees) and the S, C(2')-endo, form of the ribose with a syn conformation of the base (Y approximately 30 degrees to 50 degrees). The glycosyl torsion angles derived for the two conformations of A, G, and I are equal within the limits of accuracy.     

Conformational Flexibility of the Furanose Ring in DNA and its Dipole Moment

Abstract

The influence of conformational rearrangement of the furanose ring in DNA on its dipole moment is studied. The dipole moment of the deoxyribose molecule as a function of its puckered state is calculated by the quantum-mechanical method using the MINDO/3 approximation. The values of the dipole moment and its components are obtained at various magnitudes of the pseudorotation phase angle. The C3′-endo = C2′-endo conformational transition of deoxyribose is shown to be accompanied by the change in the dipole moment up to 3D. The results obtained are used to explain the structural properties of the DNA hydration shell.     

1H-, 13C-, 31P-NMR studies and conformational analysis of NADP+, NADPH coenzymes and of dimers from electrochemical reduction of NADP+.

All H,H, H,P and several C,P coupling constants, including those between C-4' and the vicinal phosphorus atom, have been determined for NADP+, NADPH coenzymes and for a 4,4-dimer obtained from one-electron electrochemical reduction of NADP+. From these data the preferred conformation of the ribose, that of the 1,4-dihydronicotinamide rings, and the conformation about bonds C(4')-C(5') and C(5')-O(5') were deduced. The preferred form of the 1,4- and 1,6-dihydropyridine rings and the conformation about the ring-ring junction were also obtained for all the other 4,4- and 4,6-dimers formed in the same reduction. All the dimers show a puckered structure, i.e., a boat form for the 1,4- and a twist-boat for the 1,6-dihydronicotinamide ring; both protons at the ring-ring junctions are equatorial and have preferred gauche orientation. On the contrary, the reduced coenzyme NADPH displays a planar or highly flexible conformation, rapidly flipping between two limiting boat structures. The conformation of the ribose rings, already suggested for the NADP coenzymes to be an equilibrium mixture of C(2')-endo (S-type) and C(3')-endo (N-type) puckering modes, has been reexamined by using the Altona procedure and the relative proportion of the two modes has been obtained. The S and N families of conformers have almost equal population for the adenine-ribose, whereas for the nicotinamide-ribose rings the S-type reaches the 90%. The rotation about the ester bond C(5')-O(5') and about C(4')-C(5'), defined by torsion angles beta and gamma respectively, displays a constant high preference for the trans conformer beta t (75-80%), whereas the rotamers gamma are spread out in a range of different populations. The values are distributed between the gauche gamma + (48-69%) and the trans gamma t forms (28-73%). The gamma + conformer reaches a 90% value in the case of NADP+ and NMN+. The conformations of the mononucleotides 5'-AMP, NMN+ and NMNH were also calculated from the experimental coupling constant values of the literature.     

Theoretical studies of cis-Pt(II)-diammine binding to duplex DNA

The binding of cis-Pt(II) diammine (cis-DP) to double-stranded DNA was studied with several kinked conformations that can accommodate the formation of a square planar complex. Molecular mechanics (MM) calculations were performed to optimize the molecular fit. These results were combined with quantum mechanical (QM) calculations to ascertain the relative energetics of ligand binding through water vs direct binding of the phosphate to the ammine and platinum, and to guide the selection of DNA conformations to model complex formation. Based on QM and MM calculations, models are proposed that may be characterized by several general features. A structure involving hydrogen bonding between each ammine and distinct adjacent phosphate groups, referred to as closed conformation (CC), has already been reported. This is also found in the crystal structure of small dimers. We report alternative conformations that may be important in platination of duplex DNA. They are characterized by an intermediate conformation (IC), involving hydrogen bonding between one ammine and phosphate group, and an open conformation (OC), without ammine phosphate hydrogen bonding. The IC and OC can be stabilized by water bridges in the space between the ammine and the phosphate groups. Sugar puckers alternate from the type C(2')-endo or C(1')-exo (S), to the type C(3')-endo or C(2')-exo (N), with intermediate types near O(1')-endo (O). In general, the sugar puckers alternate from S to N to S through the platinated region (3'-TpG*pG*p-5'), with the complexed strand exhibiting, (3')-S*-N*-S-(5') alternation, while the complementary strand shows either (3')-S*-N*-S-(5') or (3')-S*-N*-O-(5') alternation. In both the OC and IC, a hydrogen bond is found between the ammine and O4(T) on thymine (T) at the (3') end, adjacent to the complex site. There is a continuous range of backbone conformations through the platinated region which relate the OC to the IC. The models presented suggest that the dynamics of the binding of the cis-Pt(II)-diammines to adjacent N7(G) in double-stranded DNA may encompass several conformational possibilities, and that water bridges may play a roll in supporting open and intermediate conformations. Proton-proton distances are reported to assist in the experimental determination of conformations.     

Conformational studies of proline-, thiaproline- and dimethylsilaproline-containing diketopiperazines.

As proline plays an important role in biologically active peptides, many analogues of this residue have been developed to modulate the proportion of cis and trans conformers. A correlation between the pyrrolidine ring shape and structural properties of proline has been established. Diketopiperazine (DKP) is the model of choice to study the influence of the proline ring modification. In this contribution, cyclo(Gly-Pro) and two analogues cyclo(Sip-Pro) and cyclo(Thz-Pro) have been studied with proton NMR. We showed that both analogues with heteroatoms in gamma position, silicon and sulfur respectively, display a more rigid five-member ring. The usual flexibility of proline ring is restrained in both cases and only the two C(beta)-exo and C(beta)-endo conformations are observed.     

Why ribose was selected as the sugar component of nucleic acids

During evolution ribose was selected as the exclusive sugar component of nucleic acids. The selection is explained by using molecular models and by eliminating most of the other common sugars by looking at their chemical structure and envisioning how they would fit in a nucleic acid model. Comparisons of sugar pucker conformations and configurations of pentoses indicate that ribose was not randomly selected but the only choice, since beta-D-ribose fits best into the structure of physiological forms of nucleic acids. In other nucleotides containing arabinose, xylose, or lyxose, the C(2)'-OH and/or the C(3)'-OH are above the furanose ring, causing steric interference with the bulky base and the C(5)'-OH group.     

A model for the single stranded random coil form of polydeoxyadenylic acid from minimum energy conformations of the dimeric subunit.

The minimum energy conformations of dApdA have been examined for their suitability as buildings blocks of the single stranded coil form of polynucleotides. Calculations of the characteristic ratio C difference = less than ro greater than 2/n liter2 were made for a polymer generated from all the low energy conformers, as well as for selected combinations. A polymer composed of a conformer with omega', omega = t*,g+,(skewed) psi = t, C-(2)-endo type pucker, in combination with the 'B' form, has a C difference equal to that observed in coils of apurinic acid (6) when the fraction of 'B' form conformers is approximately 25% and approximately 91%. The t*,g+ conformer is the second lowest energy form in the C-(2)-endo puckering domain, following the 'B' form.     

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.     

8-Chloroguanosine: solid-state and solution conformations and their biological implications

The three-dimensional structure of 8-chloroguanosine dihydrate was determined by X-ray crystallography. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), and the cell dimensions are a = 4.871 (1) A, b = 12.040 (1) A, and c = 24.506 (1) A. The structure was determined by direct methods, and least-squares refinement, which included all hydrogen atoms, converged at R = 0.031 for 1599 observed reflections. The conformation about the glycosidic bond is syn with chi CN = -131.1 degrees. The ribose ring has a C(2')-endo/C-(1')-exo (2T1) pucker, and the gauche+ conformation of the -CH2OH side chain is stabilized by an intramolecular O-(5')-H...N(3) hydrogen bond. Conformational analysis by means of 1H NMR spectroscopy showed that, in dimethyl sulfoxide, the sugar ring exhibits a marked preference for the C(2')-endo conformation (approximately 70%) and a conformation about the glycosidic bond predominantly syn (approximately 90%), hence similar to that in the solid state. However, the conformation of the exocyclic 5'-CH2OH group exhibits only a moderate preference for the gauche+ rotamer (approximately 40%), presumably due to the inability to form the intramolecular hydrogen bond to N(3) in a polar medium. The conformational features are examined in relation to the behavior of 8-substituted purine nucleosides in several enzymatic systems, with due account taken of the steric bulk and electronegativities of the 8-substituents.     

Conformation of the common purine (β) ribosides in solution: further evidence for a correlation between N-S state of the ribose moiety and syn-anti equilibrium

The solution conformations of adenosine, guanosine and inosine in liquid ND₃ have been determined by NMR. Comparison of the Karplus analysis of the proton HR spectra of the ribose moiety obtained in this solvent with the data from aqueous solutions of A and I proves that the conformations of the nucleosides are very similar in both liquids. From the analysis of the vicinal coupling constants of the ring protons it has been deduced that the S state C(2′)-endo is slightly preferred. The mole fraction in S approximates 0.6 for all three nucleosides. C-13 relaxation measurements have been applied in the determination of the correlation times for rotational diffusion. Only at temperatures below −40‡ C is the pseudorotation of the furanoside ring slowed down sufficiently for it not to contribute to the measured relaxation rates. From NOE studies and T¹ measurements on the individual protons it is derived that the N, C(3′)-endo, form of the ribose is correlated with an anti conformation of the base (Y≈210‡ to 220‡) and the S, C(2′)-endo, form of the ribose with a syn conformation of the base (Y≈30‡ to 50‡). The glycosyl torsion angles derived for the two conformations of A, G, and I are equal within the limits of accuracy.     

Conformational studies of per-O-trimethylsilyl derivatives of D-fructoses and oligosaccharides containing β-D-fructofuranose residues by 220- and 300-MHz P.M.R. spectroscopy

The interpretation of 220- and 300-MHz P.M.R. spectra and the accurate chemical shifts and coupling constants of a number of per-O-trimethylsilyl-(TMS-) D-fructose derivatives and TMS-oligosaccharides containing β-D-fructofuranose residues are presented. On the basis of calculations with an adapted Karplus equation it is concluded that TMS-α- and -β-D-fructopyranose occur in the ²C₅(D) chair conformation whereas the D-glucopyranose rings in the oligosaccharides adopt the usual ⁴C₁(D) chair conformation. The structure of the latter units is very similar to that of TMS-α-_D-glucopyranose. The ⁴E(D) envelope and ⁴T₅(D) twist are the principal conformations of the D-fructofuranose rings. The conformation of the furanose ring depends on the number and kind of monosaccharide units attached thereto. The calculated, preferred conformation of the C-5-CH₂OTMS group of the D-fructofuranose moieties correlates with the time-averaged displacement of C-4 above the plane of C-2, C-3, and O-5.