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)A, alpha = gamma = 90 degrees, beta = 96.70 degrees (1), space group P2(1), Z = 2, rho obs = 1.56 gm/c.c and rho 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 > or = 3sigma). The nucleoside has the anti conformation [chi = 53.1(4) degrees] 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) degrees for phi(theta c) and phi (infinity) respectively. The pseudorotational amplitude tau(m) is 34.5 (2) with a phase angle of 171.6(4) degrees. 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 03' and 02' as donors and the 02 and 04 of the uracil base and 03' 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 [chi varies from -20 to + 60 degrees] 2) an inverse correlation between the glycosyl bond distance and the chi 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.     

Determination of the solution conformation of adenosein 2':3'-monophosphate by nuclear magnetic resonance with lanthanide probes.

The aqueous solution conformation of adenosine 2':3'-monophosphate at pH 2.5 has been determined by a nuclear magnetic resonance method utilizing lanthanide ions as shift and relaxation probes. The ribose conformation is best described as a rapid equilibrium of 2'-endo(3'-exo) and 3'-endo(2'-exo) conformations in a ratio of approximately 2 to 1. The orientation of the base relative to ribose is restricted to a narrow range about chiCN=-70 degrees.     

Molecular structures of two new anti-HIV nucleoside analogs: 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl)adenine and 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl)hypoxanthine.

The x-ray crystal structures of two new anti-HIV compounds, 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl)adenine (2'-F-dd-araA) and 9-(2,3-dideoxy-2-fluoro-beta-D-threo- pentofuranosyl)hypoxanthine (2'-F-dd-aral), have been determined at two temperatures. Both crystals are in the space group P2(1)2(1)2(1), and their structures were solved by direct methods. Least-squares refinement produced final R-factors of 0.027 for the 2'-F-dd-araA structure and of 0.044 for the 2'-F-dd-aral structure, respectively. The latter structure contains a two-fold disordered conformation of the sugar moiety. All three conformers (one for 2'-F-dd-araA and two for 2'-F-dd-aral) adopt an anti chi CN glycosyl torsion angle. The sugar in the 2'-F-dd-araA structure has a C2'-endo pucker conformation, whereas the sugar in the 2'-F-dd-aral structure has a mixture of C2'-endo and C3'-endo pucker conformations. When the sugar adopts the C2'-endo conformation, the torsion angle about the C4'-C5' bond is in a transgauche+ conformation. In contrast, when the sugar adopts the C3'-endo conformation, the torsion angle about the C4'-C5' bond is in a gauche(+)-gauche- conformation. The C2'-F bond distance is 1.406(3) A, similar to that found in other aliphatic C-F bonds. The results suggest that the 2'-fluoro-2',3'-dideoxyarabinosyl nucleosides do not have a strong preference for either C2'-endo or C3'-endo sugar pucker.     

A comparison of the conformations adopted by some 5-bromovinyl-2''-deoxyuridines and a correlation with their antiviral properties: an X-ray study.

Crystal structures of (Z)-5-(2-bromovinyl)-2'-deoxyuridine, 3',5'-di-O-acetyl-(E)-5-(2-bromovinyl)-2'-deoxyuridine and 3',5'-di-O-p-chlorobenzoyl-5-(2-dibromovinyl)-2'-deoxyuridine are compared with each other and with that of the most potent antiviral agent (E)-5-(2-bromovinyl)-2'-deoxyuridine (E-BVDU) reported earlier. A comparison of the conformation of 3',5'-di-O-acetyl-pyrimidine nucleoside structures in which intermolecular hydrogen bond network formation is minimized, with those of their parent compounds has shown that the greatest change in rotation about the glycosyl bond and in the sugar ring pucker is exhibited by E-BVDU. Upon acylation this molecule changes from C2'-endo/C3'-exo conformation to C3'-endo/C4'-exo conformation. The relevance of these structures upon the biological activity of the nucleosides and in particular to their ability to be a substrate for thymidine kinase is discussed.     

Conformation of 3'CMP bound to RNase A using TrNOESY

The conditions for accurately determining distance constraints from TrNOESY data on a small ligand (3'CMP) bound to a small protein (RNase A, <14 kDa) are described. For small proteins, normal TrNOESY conditions of 10:1 ligand:protein or greater can lead to inaccurate structures for the ligand-bound conformation due to the contribution of the free ligand to the TrNOESY signals. By using two ligand:protein ratios (2:1 and 5:1), which give the same distance constraints, a conformation of 3'CMP bound to RNase A was determined (glycosidic torsion angle, chi=-166 degrees ; pseudorotational phase angle, 0 degrees < or = P < or =36 degrees ). Ligand-protein NOESY cross peaks were also observed and used to dock 3'CMP into the binding pocket of the apo-protein (7rsa). After energy minimization, the conformation of the 3'CMP:RNase A complex was similar to the X-ray structure (1 rpf) except that a C3'-endo conformation for the ribose ring (rather than C2'-exo conformation) was found in the TrNOESY structure.     

Computer modeling studies of ribonuclease T1-guanosine monophosphate complexes

The three-dimensional structures of ribonuclease (RNase) T1 complexes with the inhibitors 2'-guanylic acid (2'-GMP), 3'-guanylic acid (3'-GMP), and 5'-guanylic acid (5'-GMP) were predicted by energy minimization studies. It is shown that these inhibitors can bind to RNase T1 in either of the ribose puckered conformations (C2'-endo and C3'-endo) in solid state and exist in significant amounts in both forms in solution. These studies are in agreement with the x-ray crystallographic studies of the 2'-GMP-Lys25-RNase T1 complex, where the inhibitor binds in C2'-endo puckered conformation. These results are also in good agreement with the available 1H-nmr results of Inagaki et al. [(1985) Biochemistry 24, 1013-1020], but differ from their conclusions where the authors favor only the C3'-endo ribose conformation for all the three inhibitors. The calculations explain the apparent discrepancies in the conclusions drawn by x-ray crystallographic and spectroscopic studies. An extensive hydrogen-bonding scheme was predicted in all the three complexes. The hydrogen-bonding scheme predicted for the 2'-GMP (C2'-endo)-RNase T1 complex agrees well with those reported from x-ray crystallographic studies. In all three complexes the base and the phosphate bind in nearly identical sites independent of the position of the phosphate or the ribose pucker. The glycosyl torsion angle favors a value in the +syn range in the 2'-GMP (C2'-endo)-RNase T1, 3'-GMP (C2'-endo)-RNase T1, and 3'-GMP (C3'-endo)-RNase T1 complexes; in the high-syn range in the 2'-GMP (C3'-endo)-RNase T1 complex; and in the -syn range in the 5'-GMP (C2'-endo)-RNase T1 and 5'-GMP (C3'-endo)-RNase T1 complexes. These results are in agreement with experimental studies showing that the inhibitory power decreases in the order 2'-GMP greater than 3'-GMP greater than 5'-GMP, and they also explain the high pKa value observed for Glu58 in the 2'-GMP-RNase T1 complex.     

Secondary structure of the hybrid poly(rA).poly(dT) in solution. Studies involving NOE at 500 MHz and stereochemical modelling within the constraints of NOE data

One-dimensional nuclear Overhauser effect (NOE) in nuclear magnetic resonance spectroscopy along with stereochemically sound model building was employed to derive the structure of the hybrid poly(rA).poly(dT) in solution. Extremely strong NOE was observed at AH2' when AH8 was presaturated; strong NOEs were observed at TH2'TH2' when TH6 was presaturated; in addition the observed NOEs at TH2' and TH2' were nearly equal when TH6 was presaturated. There was no NOE transfer to AH3' from AH8 ruling out the possibility of (C-3'-endo, low anti chi approximately equal to 200 degrees to 220 degrees) conformation for the A residues. The observed NOE data suggest that the nucleotidyl units in both rA and dT strands have equivalent conformations: C-2'-endo/C-1'-exo, anti chi approximately equal to 240 degrees to 260 degrees. Such a nucleotide geometry for rA/dT is consistent with a right-handed B-DNA model for poly(rA).poly(dT) in solution in which the rA and dT strands are conformationally equivalent. Molecular models were generated for poly(rA).poly(dT) in the B-form based upon the geometrical constraints as obtained from the NOE data. Incorporation of (C-2'-endo pucker, chi congruent to 240 degrees to 260 degrees) into the classical B-form resulted in severe close contacts in the rA chain. By introducing base-displacement, tilt and twist along with concomitant changes in the backbone torsion angles, we were able to generate a B-form for the hybrid poly(rA).poly(dT) fully consistent with the observed NOE data. In the derived model the sugar pucker is C-1'-exo, a minor variant of C-2'-endo and the sugar base torsion is 243 degrees, the remaining torsion angles being: epsilon = 198 degrees, xi = 260 degrees, alpha = 286 degrees, beta = 161 degrees and gamma = 72 degrees; this structure is free of any steric compression and indicates that it is not necessary to switch to C-3'-endo pucker for rA residues in order to accommodate the 2'-OH group. The structure that we have proposed for the polynucleotide RNA-DNA hybrid in solution is in complete agreement with that proposed for a hexamer hybrid in solution from NOE data and is inconsistent with the heteronomous model proposed for the fibrous state.     

Stereochemical studies on nucleic acid analogues. I. Conformations of alpha-nucleosides and alpha-nucleotides: interconversion of sugar puckers via O4'-exo.

The preferred conformations of ribo and deoxyribo alpha-nucleosides and alpha-nucleotides, the stereoisomers of the naturally occurring beta-isomers, are worked out by minimizing the conformational energy as a function of all the major parameters including the sugar ring conformations along the pseudorotation path. The results of the studies bring out certain distinct conformational features that are at variance with their beta counterparts. The range of glycosyl conformations are found to be not only quite restricted here but favor predominantly the anti conformation. The syn glycosyl conformation for the entire region of P values are found to be energetically less favorable, with the barrier to anti in equilibrium with syn interconversion being higher especially in alpha-ribonucleosides. The energetically preferred range of pseudorotation phase angles (P) is also considerably restricted and P values corresponding to the C1'-exo range of sugars are highly unfavorable for alpha-nucleosides, in sharp contrast to the broad range of sugar ring conformations favored by beta-isomers. While both trans congruent to 180 degrees and skew congruent to 270 degrees conformations around the C3'-O3' (phi') bond are favored for alpha-3'-nucleotides with deoxyribose sugars, ribose sugars seem to favor only the skew values of phi'. Most interestingly and in sharp contrast to beta-stereoisomers, an energy barrier is encountered at P values corresponding to O4'-endo sugars. This suggests that the possible sugar pucker interconversion between C2'-endo/C3'-exo and C3'-endo/C2'-exo in alpha-anomers could take place only through the O4'-exo region. Likewise the possible path of anti in equilibrium with syn interconversion in alpha-nucleosides is not via high anti, in sharp contrast to beta-nucleosides. These observations should be borne in mind while assigning the sugar ring conformers in alpha-nucleosides and those containing them from nmr investigations. Comparison of the results with beta-anomers seem to suggest on the whole a lack of conformational variability or the restricted nature of alpha-stereoisomers. This could be one of the reasons for its nonselection in the naturally occurring nucleic acids.     

Modified bases in tRNA: the structures of 5-carbamoylmethyl- and 5-carboxymethyl uridine.

The crystal structures of two nucleosides, 5-carbamoylmethyluridine (1) and 5-carboxymethyluridine (2), were determined from three-dimensional x-ray diffraction data, and refined to R = 0.036 and R = 0.047, respectively. Compound 1 is in the C3'-endo conformation with chi +5.2 degrees (anti), psiinfinity = +63.4 degrees and psialpha = +180.0 degrees (tt); 2 is in the C2'endo conformation with chi +49.4 degrees (anti), psiinfinity -60.5 degrees and psialpha +60.0 degrees (gg). For each derivative, the plane of the side chain substituent is skewed with respect to the plane of the nucleobase; for 1, the carboxamide group is on the same side of the uracil plane vis a vis the ribose ring; for 2, the carboxyl group is on the opposite side of this plane. No base pairing is observed for either structure. Incorporation of structure 1 into a 3'-stacked tRNA anticodon appears to place 08 within hydrogen bonding distance of the 02' hydroxyl of ribose 33, which may limit the ability of such a molecule of tRNA to "wobble".     

Structure and conformation of 1-beta-D-ribo furanosyl pyridin-2-one-5-carboxamide: an anti-inflammatory agent

The pyrimidine nucleoside, 1-beta-D-ribofuranosyl pyridine-2-one-5-carboxamide, is an anti inflammatory agent used in the treatment of adjuvant-induced arthritis. It is the 2-one isomer of 1-beta-D-ribofuranosyl pyridine-4-one 5-carboxamide, an unusual nucleoside isolated from the urine of patients with chronic myelogenic leukemia and an important cancer marker. Crystals of 1-beta-D-ribofuranosyl pyridine-2-one-5-carboxamide are monoclinic, space group C2, with the cell dimensions a = 31.7920(13), b = 4.6872 (3), c = 16.1838(11), beta = 93.071(3) degrees , V = 2408.2(2) A(3), D(calc) = 1.496 mg/m(3) and Z = 8 (two molecules in the asymmetric unit). The structure was obtained by the application of direct methods to diffractometric data and refined to a final R value of 0.050 for 1669 reflections with I >or= 3sigma. The nucleoside exhibits an anti conformation across the glycosidic bond (chi(CN) = -15.5 degrees , -18.9 degrees ), a C3 '-endo C2 '-exo [(3)(2)T] ribose pucker and g(+) across the C(4 ')-C(5 ') exocyclic bond. The amino group of the carboxamide group is distal from the 2-one and lacks the intramolecular hydrogen bonding found in the related 2-one molecule. Nuclear magnetic resonance studies shows also an anti conformation across the glycosidic bond but the solution conformation of the furanose ring is not the same as that found in the solid state.     

NMR conformational analysis of p-tolyl furanopyrimidine 2'-deoxyribonucleoside and crystal structure of its 3',5'-di-O-acetyl derivative

The conformation of a representative molecule of a new, potent class of antiviral-active modified nucleosides is determined. A bicyclic nucleoside, 3-(2'-deoxy-beta-D-ribofuranosyl)-6-(4-methylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one, shows C2'-endo and C3'-endo ribose conformations in solution (63:37, 37 degrees C; DMSO-d6), as determined by 1H NMR studies. The crystal structure of a 3',5'-di-O-acetyl-protected derivative (monoclinic, P21, a/b/c= 6.666(1)/12.225(1)/24.676(2) A, beta=90.24(1) degrees , Z=4) shows exclusively C2'-endo deoxyribose puckering. The base is found in the anti position both in solution and in crystalline form.     

1H NMR study of the sugar pucker of 2',3'-dideoxynucleosides with anti-human immunodeficiency virus (HIV) activity

The sugar ring conformations of 2',3'-dideoxyribosyladenine (ddA), 2',3'-dideoxyribosylcytosine (ddC), 2',3'-dideoxyribosylguanine (ddG), 2',3'-dideoxyribosylhypoxanthine (ddI), 3'-azido-2',3'-dideoxyribosylthymine (AZT), 3'-azido-2',3'-dideoxyribosyluracil (AZU) and 3'-fluoro-2',3'-dideoxyribosylthymine (FddT) have been investigated by 1H NMR spectroscopy. While the sugar ring in FddT exists almost totally in C2'-endo geometry, other nucleosides show equilibrium between sugar puckers of C3'-endo family (N-type) and C2'-endo family (S-type). For unsubstituted dideoxynucleosides C3'-endo conformer is favoured (congruent to 75%), whereas for AZT and AZU both the conformers have almost equal populations. Unlike X-ray diffraction studies, the NMR results do not support the suggestion that C3'-exo sugar puckers are desirable for the anti-HIV activity of these nucleosides.     

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.     

On the conformational dependence of the proton chemical shifts in nucleosides and nucleotides. II. Proton shifts in the ribose ring of purine nucleosides as a function of the torsion angle about the glycosyl bond

The variations of the ring current, the local diamagnetic susceptibility anisotropy and the polarization contributions to the chemical shift of the non exchangeable protons of the ribose ring of purine nucleosides are computed as a function of the torsion angle about the glycosyl bond, χ_CN. The results show that the ring current effect is relatively more important in the purines than in the pyrimidines. In addition, N₃ of purines has a local magnetic anisotropy effect similar to the one of the carbonyl group C₂O₂ of pyrimidine nucleosides. The experimental differences between the chemical shift of the ribose protons of purine nucleosides and of 8 substituted derivatives are discussed in relation to the theoretical variations.     

Three-state models of furanose pseudorotation

A general procedure is described to treat the pseudorotation of the furanose ring in terms of a three-state conformational equilibrium. In addition to the principal n (C3'-endo) and s (C2'-endo) puckering domains, the unusual e (01'-endo) intermediate is included in the analysis. Each of these three conformational categories is represented by a blend of five closely related puckered forms rather than by a single rotational isomeric state. Using this model together with experimentally measured nmr coupling constants, the puckering populations of various nucleic acid analogs are estimated. The conventional two-state n/s equilibria is confirmed in ordinary ribose and deoxyribose systems. The e domain, however, is found to be of major importance in several chemically modified furanoses including certain pyrimidine deoxynucleosides damaged by radiation and various nucleosides and nucleotides forced by bulky substituents on the base into unusual syn glycosyl arrangements. The "free" pseudorotation of these modified systems is not detected by conventional two-state puckering analyses.     

Refined X-ray structure of the low pH form of ribonuclease T1-2'-guanylic acid complex at 1.9 A resolution

The three-dimensional X-ray structure of the RNase T1[EC 3.1.27.3]-2'GMP complex crystallized at low pH value (4.0) was determined, and refined to 1.9 A resolution to give a final R value of 0.203. The refined model includes 781 protein atoms, 24 inhibitor atoms, and 43 solvent molecules. The imidazole rings of His27 and His40 interact with the carboxyl side chains of Glu82 and Glu58, respectively, whereas that of His92 is in contact with the main chain carbonyl oxygen of Ala75. In the complex, the ribose ring of the 2'GMP molecule adopts a C2'-endo puckering, and the exocyclic conformation is gauche(-)-gauche(+). The glycosyl torsion angle is in the syn range with an intramolecular hydrogen bond between N3 and O5', and the 2'-phosphate orientation is trans-gauche(-). The guanine base of the inhibitor is tightly bound to the base recognition site with five hydrogen bonds (N1--Glu46O epsilon 2, N2---Asn98O,O6---Asn44N, and N7 ---Asn43N delta 2/Asn43N) and is sandwiched between the phenolic ring portions of Tyr42 and Tyr45 by stacking interactions. The 2'-phosphate group interacts with Arg77N eta 2, Glu58O episilon 2, and Tyr 38O eta but not with any of the histidine residues. Arg77N eta 2 also interacts with Tyr38O eta. There is no interaction between the ribose moiety of the inhibitor and the enzyme.     

Interactions between an anthracycline antibiotic and DNA: molecular structure of daunomycin complexed to d(CpGpTpApCpG) at 1.2-A resolution

The crystal structure of a daunomycin-d(CGTACG) complex has been solved by X-ray diffraction analysis and refined to a final R factor of 0.175 at 1.2-A resolution. The crystals are in a tetragonal crystal system with space group P4(1)2(1)2 and cell dimensions of a = b = 27.86 A and c = 52.72 A. The self-complementary DNA forms a six base pair right-handed double helix with two daunomycin molecules intercalated in the d(CpG) sequences at either end of the helix. Daunomycin in the complex has a conformation different from that of daunomycin alone. The daunomycin aglycon chromophore is oriented at right angles to the long dimension of the DNA base pairs, and the cyclohexene ring A rests in the minor groove of the double helix. Substituents on this ring have hydrogen-bonding interactions to the base pairs above and below the intercalation site. O9 hydroxyl group of the daunomycin forms two hydrogen bonds with N3 and N2 of an adjacent guanine base. Two bridging water molecules between the drug and DNA stabilize the complex in the minor groove. In the major groove, a hydrated sodium ion is coordinated to N7 of the terminal guanine and the O4 and O5 of daunomycin with a distorted octahedral geometry. The amino sugar lies in the minor groove without bonding to the DNA. The DNA double helix is distorted with an asymmetrical rearrangement of the backbone conformation surrounding the intercalator drug. The sugar puckers are C1,C2'-endo, G2,C1'-endo, C11,C1'-endo, and G12,C3'-exo. Only the C1 residue has a normal anti-glycosyl torsion angle (chi = -154 degrees), while the other three residues are all in the high anti range (average chi = -86 degrees). This structure allows us to identify three principal functional components of anthracycline antibiotics: the intercalator (rings B-D), the anchoring functions associated with ring A, and the amino sugar. The structure-function relationships of daunomycin binding to DNA as well as other related anticancer drugs are discussed.     

Synthesis,structure, and conformation of anti-tumor agents in the solid and solution states: hydroxyl derivatives of Ftorafur

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 of chemotherapeutic 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 chi angle. Molecules with a lower chi 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.     

1H-NMR studies on nucleotide binding to the sarcoplasmic reticulum Ca2+ ATPase. Determination of the conformations of bound nucleotides by the measurement of proton-proton transferred nuclear Overhauser enhancements

The glycosidic bond torsion angles and the conformations of the ribose of Mg2+ATP, Mg2+ADP and Mg2+AdoPP[NH]P (magnesium adenosine 5'-[beta, gamma-imido]triphosphate) bound to Ca2+ATPase, both native and modified with fluorescein isothiocyanate (FITC), in intact sarcoplasmic reticulum have been determined by the measurement of proton-proton transferred nuclear Overhauser enhancements by 1H-NMR spectroscopy. This method shows clearly the existence of a low-affinity ATP binding site after modification of the high-affinity site with FITC. For all three nucleotides bound to both the high-affinity (catalytic) site and the low-affinity site, we find that the conformation about the glycosidic bond is anti, the conformation of the ribose 3'-endo of the N type and the conformation about the ribose C4'-C5' bond either gauche-trans or trans-gauche. The values for the glycosidic bond torsion angles chi (O4'-C1'-N9-C4) for Mg2+ATP, Mg2+ADP and Mg2+AdoPP[NH]P bound to the low-affinity site of FITC-modified Ca2+ATPase are approximately equal to 270 degrees, approximately equal to 260 degrees and approximately equal to 240 degrees respectively. In the case of the nucleotides bound to the high-affinity (catalytic) site of native Ca2+ATPase, chi lies in the range 240-280 degrees.     

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 (approximately 75%) South conformations, with the A and N rings adopting approximately 2T3 (C2'-endo(major)-C3'-exo(minor) and 4(3)T (C3'-exo-C4'-endo) conformations, respectively. The backbone torsion angles beta and gamma 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.