(13)C,(13)C]- and [(13)C,(1)H]-TROSY in a triple resonance experiment for ribose-base and intrabase correlations in nucleic acids.

A novel TROSY (transverse relaxation-optimized spectroscopy) element is introduced that exploits cross-correlation effects between (13)C-(13)C dipole-dipole (DD) coupling and (13)C chemical shift anisotropy (CSA) of aromatic ring carbons. Although these (13)C-(13)C effects are smaller than the previously described [(13)C,(1)H]-TROSY effects for aromatic (13)C-(1)H moieties, their constructive use resulted in further transverse relaxation-optimization by up to 15% for the resonances in a 17 kDa protein-DNA complex. As a practical application, two- and three-dimensional versions of the HCN triple resonance experiment for obtaining ribose-base and intrabase correlations in the nucleotides of DNA and RNA (Sklenar, V.; Peterson, R. D.; Rejante, M. R.; Feigon, J. J. Biomol. NMR 1993, 3, 721-727) have been implemented with [(13)C,(1)H]- and [(13)C,(13)C]-TROSY elements to reduce the rate of transverse relaxation during the polarization transfers between ribose (13)C1' and base (15)N1/9 spins, and between (13)C6/8 and N1/9 within the bases. The resulting TROSY-HCN experiment is user-friendly, with a straightforward, robust experimental setup. Compared to the best previous implementations of the HCN experiment, 2-fold and 5-fold sensitivity enhancements have been achieved for ribose-base and intrabase connectivities, respectively, for (13)C,(15)N-labeled nucleotides in structures with molecular weights of 10 and 17 kDa. TROSY-HCN experiments should be applicable also with significantly larger molecular weights. By using modified TROSY-HCN schemes, the origins of the sensitivity gains have been analyzed.     

Insights into the mobility of methyl-bearing side chains in proteins from (3)J(CC) and (3)J(CN) couplings

Side-chain dynamics in proteins can be characterized by the NMR measurement of (13)C and (2)H relaxation rates. Evaluation of the corresponding spectral densities limits the slowest motions that can be studied quantitatively to the time scale on which the overall molecular tumbling takes place. A different measure for the degree of side-chain order about the C(alpha)-C(beta) bond (chi(1) angle) can be derived from (3)J(C)(')(-)(C)(gamma) and (3)J(N)(-)(C)(gamma) couplings. These couplings can be measured at high accuracy, in particular for Thr, Ile, and Val residues. In conjunction with the known backbone structures of ubiquitin and the third IgG-binding domain of protein G, and an extensive set of (13)C-(1)H side-chain dipolar coupling measurements in oriented media, these (3)J couplings were used to parametrize empirical Karplus relationships for (3)J(C)(')(-)(C)(gamma) and (3)J(N)(-)(C)(gamma). These Karplus curves agree well with results from DFT calculations, including an unusual phase shift, which causes the maximum (3)J(CC) and (3)J(CN) couplings to occur for dihedral angles slightly smaller than 180 degrees, particularly noticeable in Thr residues. The new Karplus curves permit determination of rotamer populations for the chi(1) torsion angles. Similar rotamer populations can be derived from side-chain dipolar couplings. Conversion of these rotamer populations into generalized order parameters, S(J)(2) and S(D)(2), provides a view of side-chain dynamics that is complementary to that obtained from (13)C and (2)H relaxation. On average, results agree well with literature values for (2)H-relaxation-derived S(rel)(2) values in ubiquitin and HIV protease, but also identify a fraction of residues for which S(J,D)(2) < S(rel)(2). This indicates that some of the rotameric averaging occurs on a time scale too slow to be observable in traditional relaxation measurements.     

DFT analysis of NMR scalar interactions across the glycosidic bond in DNA

The relationship between the glycosidic torsion angle chi, the three-bond couplings (3)J(C2/4-H1') and (3)J(C6/8-H1'), and the one-bond coupling (1)J(C1'-H1') in deoxyribonucleosides and a number of uracil cyclo-nucleosides has been analyzed using density functional theory. The influence of the sugar pucker and the hydroxymethyl conformation has also been considered. The parameters of the Karplus relationships between the three-bond couplings and chi depend strongly on the aromatic base. (3)J(C2/4-H1') reveals different behavior for deoxyadenosine, deoxyguanosine, and deoxycytidine as compared to deoxythymidine and deoxyuridine. In the case of (3)J(C6/8-H1'), an opposite trans to cis ratio of couplings is obtained for pyrimidine nucleosides in contrast to purine nucleosides. The extremes of the Karplus curves are shifted by ca. 10 degrees with respect to syn and anti-periplanar orientations of the coupled nuclei. The change in the sugar pucker from S to N decreases (3)J(C2/4-H1') and (3)J(C6/8-H1'), while increasing (1)J(C1'-H1') for the syn rotamers, whereas all of the trends are reversed for the anti rotamers. The influence of the sugar pucker on (1)J(C1'-H1') is interpreted in terms of interactions between the n(O4'), sigma*(C1'-H1') orbitals. The (1)J(C1'-H1') are related to chi through a generalized Karplus relationship, which combines cos(chi) and cos(2)(chi) functions with mutually different phase shifts that implicitly accounts for a significant portion of the related sugar pucker effects. Most of theoretical (3)J(C2/4-H1') and (3)J(C6/8-H1') for uracil cyclo-nucleosides compare well with available experimental data. (3)J(C6/8-H1') couplings for all C2-bridged nucleosides are up to 3 Hz smaller than in the genuine nucleosides with the corresponding chi, revealing a nonlocal aspect of the spin-spin interactions across the glycosidic bond. Theoretical (1)J(C1'-H1') are underestimated with respect to the experiment by ca. 10% but reproduce the trends in (1)J(C1'-H1') vs chi.     

Eta(z)/kappa: a transverse relaxation optimized spectroscopy NMR experiment measuring longitudinal relaxation interference

NMR spin relaxation experiments provide a powerful tool for the measurement of global and local biomolecular rotational dynamics at subnanosecond time scales. Technical limitations restrict most spin relaxation studies to biomolecules weighing less than 10 kDa, considerably smaller than the average protein molecular weight of 30 kDa. In particular, experiments measuring eta(z), the longitudinal (1)H(N)-(15)N dipole-dipole (DD)/(15)N chemical shift anisotropy (CSA) cross-correlated relaxation rate, are among those least suitable for use with larger biosystems. This is unfortunate because these experiments yield valuable insight into the variability of the (15)N CSA tensor over the polypeptide backbone, and this knowledge is critical to the correct interpretation of most (15)N-NMR backbone relaxation experiments, including R(2) and R(1). In order to remedy this situation, we present a new (1)H(N)-(15)N transverse relaxation optimized spectroscopy experiment measuring eta(z) suitable for applications with larger proteins (up to at least 30 kDa). The presented experiment also yields kappa, the site-specific rate of longitudinal (1)H(N)-(1)H(') DD cross relaxation. We describe the eta(z)/kappa experiment's performance in protonated human ubiquitin at 30.0 degrees C and in protonated calcium-saturated calmodulin/peptide complex at 20.0 degrees C, and demonstrate preliminary experimental results for a deuterated E. coli DnaK ATPase domain construct at 34 degrees C.     

Measurement of long-range 1H-19F scalar coupling constants and their glycosidic torsion dependence in 5-fluoropyrimidine-substituted RNA

Long-range scalar 5J(H1',F) couplings were observed in 5-fluoropyrimidine-substituted RNA. We developed a novel S3E-19F-alpha,beta-edited NOESY experiment for quantitation of these long-range scalar 5J(H1',F) couplings, where the J-couplings can be extracted from inspection of intraresidual (H1',H6) NOE cross-peaks. Quantum chemical calculations were exploited to investigate the relation between scalar couplings and conformations around the glycosidic bond in oligonucleotides. The theoretical dependence of the observed 5J(H1',F) couplings on the torsion angle chi can be described by a generalized Karplus relationship. The corresponding density functional theory (DFT) analysis is outlined. Additional NMR experiments facilitating the resonance assignments of 5-fluoropyrimidine-substituted RNAs are described, and chemical shift changes due to altered shielding in the presence of fluorine-19 (19F) are presented.     

Comprehensive conformational analysis of the nucleoside analogue 2'-beta-deoxy-6-azacytidine by DFT and MP2 calculations

A comprehensive conformational analysis of isolated 2'-beta-deoxy-6-azacytidine (d6AC), an analogue of therapeutically active 6-azacytidine (6AC), has been performed by means of ab initio calculations at the MP2/6-311++G(2df,pd)//DFT B3LYP/6-31G(d,p) level of theory. Among the 81 conformers located within a 7.83 kcal/mol Gibbs energy range at T = 298.15 K, 38 contain syn-oriented bases with respect to 2'-deoxyribose; the other conformers include anti-oriented bases. Energetic analysis of these conformers shows that conformational equilibrium of isolated d6AC at T = 298.15 K is shifted to syn conformation with a syn/anti ratio estimated as 61.4%:38.6%. As far as the sugar conformation is concerned, 40 conformers contain north (N) (with 0.3 degrees < or = P < or = 40.1 degrees), and the rest possess south (S) (with 157.1 degrees < or = P < or = 207.0 degrees) puckers, where P is the pseudorotational angle of the furanose ring. The S/N occupancy ratio is estimated as 80.2%:19.8% (T = 298.15 K). The two most stable conformers are energetically quasidegenerate and correspond to both C2'-endo/syn conformers differing only by orientation of the O3'H hydroxyl group. They are both stabilized by means of similar intramolecular H-bonds, i.e., O5'H...O2, C2'H2...O2, and C2'H2...O5'. As examined by AIM criteria, from 1 to 3 H-bonds per conformer were identified among 13 possible interactions: O5'H...O2, O5'H...N6, O3'H...O5', O5'H...O3', C1'H...O2, C2'H2...O2, C2'H2...O5', C3'H...O2, C3'H...N6, C5'H1...O2, C5'H2...O2, C5'H1...N6, and C5'H2...N6. The biological effect of d6AC is conceived as an inhibition of replicative DNA polymerase caused by an unusual orientation of the sugar residue against the base in the only A form DNA-like conformer.     

Three-bond sugar-base couplings in purine versus pyrimidine nucleosides: a DFT study of Karplus relationships for (3)J(C2/4-H1') and (3)J(C6/8-H1') in DNA

(3)J(C2/4-H1') and (3)J(C6/8-H1') scalar spin-spin coupling constants have been calculated for deoxyadenosine, deoxyguanosine, deoxycytidine, and deoxythymidine as functions of the glycosidic torsion angle chi by means of density functional theory. Except for deoxythymidine, (3)J(C2/4-H1') depends little on the base type. On the contrary, (3)J(C6/8-H1') follows the usual trans to cis ratio ((3)J(C-H(cis)) < (3)J(C-H(trans))) for purine nucleosides, but reveals the opposite relation ((3)J(C-H(cis)) > (3)J(C-H(trans))) for pyrimidine nucleosides. Our results compare well with the experiment for deoxyguanosine and predict a novel trend in the case of pyrimidine bases for which no NMR results are available in the syn region. A breakdown of the key Fermi contact part of (3)J(C6/8-H1') into MO contributions rationalizes this trend in terms of an unusual coupling mechanism in the syn orientation that is very effective for pyrimidine nucleosides and considerably weaker for purine nucleosides.     

Ab initio comprehensive conformational analysis of 2'-deoxyuridine, the biologically significant DNA minor nucleoside, and reconstruction of its low-temperature matrix infrared spectrum

A comprehensive conformational analysis of isolated 2'-deoxyuridine (dU), a minor DNA nucleoside, has been performed by means of ab initio calculations at the MP2/6-311++G (d,p)//DFT B3LYP/6-31G (d,p) level of theory. At 298.15 and 420 K, all 94 allowed conformers of dU are within 8.96 and 7.91 kcal/mol Gibbs energy ranges, respectively. Syn orientation for the base and South (S) conformers for the sugar dominate at 298.15 K: syn/anti=62.3%:37.7% and S/N=77.2%:22.8%. At 420 K in the majority of conformers, the base is anti oriented and the population of North (N) sugars increases: syn/anti=39.3%:60.7% and S/N=63.0%:37.0%. Values of all conformational parameters and correlations between them, as well as their correlations with valence bonds, and also correlations between valence bonds and angles were estimated. In general, 14 types of intramolecular H-bonds were detected (1-3 H-bonds per conformer, the total number 175), namely, C1'H...O2 (16 H-bonds), C2'H1...O5' (9), C2'H2...O2 (21), C3'H...O2 (21), C5'H1...O2 (14), C5'H2...O2 (11), C6H...O4' (37), C6H...O5' (22), C3'H...HC6 (4), O5'H...HC6 (2), O3'H...O5' (5), O5'H...O4' (1), O5'H...O3' (4), and O5'H...O2 (8). Geometric, vibrational, structural-topological, and energetic features of the OH...O intramolecular H-bonds in dU conformers were determined. The close similarity between energetic and geometric characteristics of dU and thymidine DNA-like conformers in anti and relevant syn conformations and their transition states of the anti-->syn interconversion implies that mismatch DNA glycosylase discriminates between the two nucleosides, mainly because of the difference in the shapes of their bases. Convolution of calculated IR spectra of all the dU conformers within the limits 3400-3700 cm(-1) appears to be consistent with its low-temperature matrix IR spectrum (Ivanov et al. Spectrochim. Acta, Part A 2003, 59, 1959), wavenumber discrepancy not exceeding 1%. It was concluded that, for a reliable reproduction of the experimental spectrum, the whole set of conformers should be taken into consideration. The suggested method makes reconstruction of the isolated nucleoside IR spectrum at a physiological interval of temperature reasonably possible.     

Sugar pucker modulates the cross-correlated relaxation rates across the glycosidic bond in DNA

The dependence of N1/9 and C1' chemical shielding (CS) tensors on the glycosidic bond orientation (chi) and sugar pucker (P) in the DNA nucleosides 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxycytidine, and 2'-deoxythymidine was studied using the calculation methods of quantum chemistry. The results indicate that these CS-tensors exhibit a significant degree of conformational dependence on chi and P structural parameters. The presented data test underlying assumptions of currently established methods for interpretation of cross-correlated relaxation rates between the N1/9 chemical shielding tensor and C1'-H1' dipole-dipole (Ravindranathan et al. J. Biomol. NMR 2003, 27, 365-75. Duchardt et al. J. Am. Chem. Soc. 2004, 126, 1962-70) and highlight possible limitations of these methods when applied to DNA.     

Effects of ionization, metal cationization and protonation on 2'-deoxyguanosine: changes on sugar puckering and stability of the N-glycosidic bond

The influence of oxidation, protonation, and metal cationization with Cu(+) and Cu(2+) on the strength of the N-glycosidic bond in 2'-deoxyguanosine has been studied by means of quantum chemical calculations. In all cases, the N9-C1' bond distance increases (0.03-0.06 A) upon introducing positive charge in the guanine moiety, the observed variations being more important for the dicationic systems. Binding energies show that the effect of X(n)(+) in guanine hinders the homolytic dissociation, whereas it largely favors the heterolytic process. With respect to the deoxyribose ring, it has been found that metal binding, oxidation, and protonation do not significantly change the values of the phase angle of pseudorotation P. However, the glycosyl torsion angle chi varies considerably (from 242.0 degrees to 189.8 degrees) as a consequence of a stabilizing guanine-sugar (H8-O4') interaction due to the increase of acidity of guanine C8-H8 upon cationization.     

Carbon-13 chemical shift anisotropy in DNA bases from field dependence of solution NMR relaxation rates

Knowledge of (13)C chemical shift anisotropy (CSA) in nucleotide bases is important for the interpretation of solution-state NMR relaxation data in terms of local dynamic properties of DNA and RNA. Accurate knowledge of the CSA becomes particularly important at high magnetic fields, prerequisite for adequate spectral resolution in larger oligonucleotides. Measurement of (13)C relaxation rates of protonated carbons in the bases of the so-called Dickerson dodecamer, d(CGCGAATTCGCG)(2), at 500 and 800 MHz (1)H frequency, together with the previously characterized structure and diffusion tensor yields CSA values for C5 in C, C6 in C and T, C8 in A and G, and C2 in A that are closest to values previously reported on the basis of solid-state FIREMAT NMR measurements, and mostly larger than values obtained by in vacuo DFT calculations. Owing to the noncollinearity of dipolar and CSA interactions, interpretation of the NMR relaxation rates is particularly sensitive to anisotropy of rotational diffusion, and use of isotropic diffusion models can result in considerable errors.     

Spectroscopic studies on [(DD18C6H2)(HPA)2](PA)2 and [(DD18C6H2)(DDQ)2](DDQH)2 formed in the reaction of N,N'-dibenzyl-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane with HPA and DDQ

The interaction of the mixed oxygen-nitrogen cyclic base, N,N'-dibenzyl-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane (DD18C6) with pi-acceptors such as picric acid (HPA) and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) has been studied spectrophotometrically in chloroform at 25 degrees C. The results obtained indicate the formation of 1:4 charge-transfer complexes with the general formula (DD18C6)(acceptor)4. The electronic and infrared spectra of charge-transfer complexes along with the (1)H NMR spectra were recorded and discussed. Based on the data obtained, the complexes were formulated as [(DD18C6H2)(HPA)2](PA)2 and [(DD18C6H2)(DDQ)2](DDQH)2. A general mechanism explaining the formation of the DDQ complex has been suggested.     

Determinations of 15N chemical shift anisotropy magnitudes in a uniformly 15N,13C-labeled microcrystalline protein by three-dimensional magic-angle spinning nuclear magnetic resonance spectroscopy

Amide 15N chemical shift anisotropy (CSA) tensors provide quantitative insight into protein structure and dynamics. Experimental determinations of 15N CSA tensors in biologically relevant molecules have typically been performed by NMR relaxation studies in solution, goniometric analysis of single-crystal spectra, or slow magic-angle spinning (MAS) NMR experiments of microcrystalline samples. Here we present measurements of 15N CSA tensor magnitudes in a protein of known structure by three-dimensional MAS solid-state NMR. Isotropic 15N, 13C alpha, and 13C' chemical shifts in two dimensions resolve site-specific backbone amide recoupled CSA line shapes in the third dimension. Application of the experiments to the 56-residue beta1 immunoglobulin binding domain of protein G (GB1) enabled 91 independent determinations of 15N tensors at 51 of the 55 backbone amide sites, for which 15N-13C alpha and/or 15N-13C' cross-peaks were resolved in the two-dimensional experiment. For 37 15N signals, both intra- and interresidue correlations were resolved, enabling direct comparison of two experimental data sets to enhance measurement precision. Systematic variations between beta-sheet and alpha-helix residues are observed; the average value for the anisotropy parameter, delta (delta = delta(zz) - delta(iso)), for alpha-helical residues is 6 ppm greater than that for the beta-sheet residues. The results show a variation in delta of 15N amide backbone sites between -77 and -115 ppm, with an average value of -103.5 ppm. Some sites (e.g., G41) display smaller anisotropy due to backbone dynamics. In contrast, we observe an unusually large 15N tensor for K50, a residue that has an atypical, positive value for the backbone phi torsion angle. To our knowledge, this is the most complete experimental analysis of 15N CSA magnitude to date in a solid protein. The availability of previous high-resolution crystal and solution NMR structures, as well as detailed solid-state NMR studies, will enhance the value of these measurements as a benchmark for the development of ab initio calculations of amide 15N shielding tensor magnitudes.     

Molecular and electronic structures of iron complexes containing N,S-coordinated,open-shell o-iminothionebenzosemiquinonate(1-) pi radicals

The reaction of the dinuclear species (mu-NH,NH)[Fe(III)(L(IP))(L(AP))](2) dissolved in CH(2)Cl(2) with dioxygen affords black microcrystals of diamagnetic (mu-S,S)[Fe(III)(L(IP))(L(ISQ))](2).n-hexane (6) upon the addition of n-hexane, where (L(IP))(2)(-) represents the dianion of 4,6-di-tert-butyl-2-aminothiophenol, (L(AP))(-) is the corresponding monoanion, and (L(ISQ))(-) is the corresponding o-iminothionebenzosemiquinonate(1-) pi radical monoanion; similarly, the dianion ('H(2)N(2)S(2)')(2)(-) is derived from 1,2-ethanediamine-N,N'-bis(2-benzenethiol), and ('N(2)S(2)(*)')(3)(-) is its monoradical trianion. The above reaction in a CH(2)Cl(2)/CH(3)OH (1:1) mixture yields the diamagnetic isomer (mu-NH,NH)[Fe(III)(L(IP))(L(ISQ))](2).5CH(3)OH (7), whereas air oxidation of (mu-S,S)[Fe(II)('H(2)N(2)S(2)')](2) in CH(3)CN yields diamagnetic (mu-S,S)[Fe(III)('N(2)S(2)(*)')](2) (8). Complexes 6 and 8 were shown to undergo addition reactions with phosphines, phosphites, or cyanide affording the following complexes: trans-[Fe(II)(L(ISQ))(2)(P(OPh)(3))] (9; S(t) = 0) and [N(n-Bu)(4)][Fe(II)(L(ISQ))(2)(CN)] (S(t) = 0). Oxidation of 6 in CH(2)Cl(2) with iodine, bromine, and chlorine respectively yields black microcrystals of [Fe(III)(L(ISQ))(2)X] (X = I, Br, or Cl) with S(t) = (1)/(2). The structures of complexes 6-9 have been determined by X-ray crystallography at 100 K. The oxidation level of the ligands and iron ions in all complexes has been unequivocally established, as indicated by crystallography; electron paramagnetic resonance, UV-vis, and M?ssbauer spectroscopies; and magnetic-susceptibility measurements. The N,S-coordinated o-iminothionebenzosemiquinonate(1-) pi radicals have been identified in all new complexes. The electronic structures of the new complexes have been determined, and it is shown that no evidence for iron oxidation states >III is found in this chemistry.     

含咔唑和偶氮苯的乙炔衍生物的合成

采用Sonagashira偶联反应和N-烷基化反应合成了含有咔唑和偶氮苯的乙炔衍生物:3-乙炔基-9-(4-[4-(硝基)苯基偶氮苯]氧)亚丁基咔唑3.其结构通过IR,1H NMR,13C NMR,元素分析和X-射线单晶衍射法测定.标题化合物属单斜晶系,P21/c空间群;a=9.238(3),b=28.240(8),c...     

Structural characterization of an anhydrous polymorph of paclitaxel by solid-state NMR

The three-dimensional structure of a unique polymorph of the anticancer drug paclitaxel (Taxol) is established using solid state NMR (SSNMR) tensor ((13)C & (15)N) and heteronuclear correlation ((1)H-(13)C) data. The polymorph has two molecules per asymmetric unit (Z' = 2) and is thus the first conformational characterization with Z' > 1 established solely by SSNMR. Experimental data are correlated with structure through a series of computational models that extensively sample all conformations. For each computational model, corresponding tensor values are computed to supply comparisons with experimental information which, in turn, establishes paclitaxel's structure. Heteronuclear correlation data at thirteen key positions provide shift assignments to the asymmetric unit for each comparison. The two distinct molecules of the asymmetric unit possess nearly identical baccatin III moieties with matching conformations of the C10 acetyl moiety and, specifically, the torsion angle formed by C30-O-C10-C9. Additionally, both are found to exhibit an extended conformation of the phenylisoserine sidechain at C13 with notable differences in the dihedral angles centered around the rotation axes of O-C13, C2'-C1' and C3'-C2'.     

Cross-correlated relaxation between H1' chemical shift anisotropy and H1'-H2' dipolar relaxation mechanisms in ribonucleosides: application to the characterization of their anomeric configuration

Cross-correlated nuclear spin relaxation between 1H chemical shift anisotropy (CSA) and 1H-1H dipolar relaxation mechanisms in ribonucleosides in solution phase are observed and used to identify their anomeric configuration. Only alpha-ribonucleosides showed the presence of cross-correlated spin relaxation through differential spin-lattice relaxation (T1) of the H1' doublet. Dependence of the magnitude and the orientation of the H1' CSA tensor values on the glycosidic torsion angle and the fast time-scale internal motions present in the ribose moiety play a significant role in the characterization of the anomeric configuration of the nucleosides via cross-correlated relaxation.     

Novel synthesis and oxidizing ability of tropylium ions annulated with two 2,4-dimethylfuro[2,3-d]pyrimidine-1(2H),3(4H)-diones

Convenient preparation of novel tropylium ions annulated with two 2,4-dimethylfuro[2,3-d]pyrimidine-1(2H),3(4H)-diones, 12a(+).BF(4)(-) and 12b(+)().BF(4)(-), consists of a reaction of 2-methoxytropone with dimethylbarbituric acid to give 7,9-dimethyl-3-[1',3'-dimethyl-2'(1'H),4'(3'H),6'(5'H)-trioxopyrimidin-5'-ylidene]cyclohepta[b]pyrimido[5,4-d]furan-8(7H),10(9H)-dione 8 and the following oxidative cyclization by using DDQ or photoirradiation under aerobic conditions. On the basis of the MO calculations, the selectivity of two types of oxidative cyclization reactions of 8 was rationalized. X-ray crystal analyses and MO calculations were carried out to clarify the structural characteristics of 12a(+). BF(4)(-) and 12b(+).BF(4)(-). The stability of cations 12a(+) and 12b(+) is expressed by the pK(R) + values which were determined spectrophotometrically as 8.8 and 8.6. The electrochemical reduction of 12a(+) and 12b(+) exhibited reduction potential at -0.63 and -0.62 (V vs Ag/AgNO(3)), respectively. Reactions of 12a(+)().BF(4)(-) and 12b(+)().BF(4)(-) with some nucleophiles, hydride and diethylamine, were carried out to clarify that the reactivity of 12a(+)().BF(4)(-) and 12b(+).BF(4)(-) was substantially dependent on the annulating position. The oxidizing ability of 12a(+).BF(4)(-) and 12b(+).BF(4)(-) toward alcohols and amines in the autorecycling process was demonstrated as well.     

Unprecedented head-to-head conformers of d(GpG) bound to the antitumor active compound tetrakis (mu-carboxylato)dirhodium(II,II)

The N7/O6 equatorial binding interactions of the antitumor active complex Rh(2)(OAc)(4)(H(2)O)(2) (OAc(-) = CH(3)CO(2)(-)) with the DNA fragment d(GpG) have been unambiguously determined by NMR spectroscopy. Previous X-ray crystallographic determinations of the head-to-head (HH) and head-to-tail (HT) adducts of dirhodium tetraacetate with 9-ethylguanine (9-EtGH) revealed unprecedented bridging N7/O6 guanine nucleobases that span the Rh-Rh bond. The absence of N7 protonation at low pH and the notable increase in the acidity of N1-H (pK(a) approximately 5.7 as compared to 8.5 for N7 only bound platinum adducts), suggested by the pH dependence titrations of the purine H8 (1)H NMR resonances for Rh(2)(OAc)(2)(9-EtG)(2) and Rh(2)(OAc)(2-)[d(GpG)],are consistent with bidentate N7/O6 binding of the guanine nucleobases. The pK(a) values estimated for N1-H (de)protonation, from the pH dependence studies of the C6 and C2 (13)C NMR resonances for the Rh(2)(OAc)(2)(9-EtG)(2) isomers, concur with those derived from the H8 (1)H NMR resonance titrations. Comparison of the (13)C NMR resonances of C6 and C2 for the dirhodium adducts Rh(2)(OAc)(2)(9-EtG)(2) and Rh(2)(OAc)(2)[d(GpG)] with the corresponding resonances of the unbound ligands [at pH 7.0 for 9-EtGH and pH 8.0 for d(GpG)], shows substantial downfield shifts of Deltadelta approximately 11.0 and 6.0 ppm for C6 and C2, respectively; the latter shifts reflect the effect of O6 binding to the dirhodium centers and the ensuing enhancement in the acidity of N1-H. Intense H8/H8 ROE cross-peaks in the 2D ROESY NMR spectrum of Rh(2)(OAc)(2)[d(GpG)] indicate head-to-head arrangement of the guanine bases. The Rh(2)(OAc)(2)[d(GpG)] adduct exhibits two major right-handed conformers, HH1 R and HH2 R, with HH1 R being three times more abundant than the unusual HH2 R. Complete characterization of both adducts revealed repuckering of the 5'-G sugar rings to C3'-endo (N-type), retention of C2'-endo (S-type) conformation for the 3'-G sugar rings, and anti orientation with respect to the glycosyl bonds. The structural features obtained for Rh(2)(OAc)(2))[d(GpG)] by means of NMR spectroscopy are very similar to those for cis-[Pt(NH(3))(2))[d(GpG)]] and corroborate molecular modeling studies.     

Chemical shift tensors of protonated base carbons in helical RNA and DNA from NMR relaxation and liquid crystal measurements

Knowledge of (13)C chemical shift anisotropy (CSA) tensors in nucleotide bases is important for interpretation of NMR relaxation data in terms of local dynamic properties of nucleic acids and for analysis of residual chemical shift anisotropy (RCSA) resulting from weak alignment. CSA tensors for protonated nucleic acid base carbons have been derived from measurements on a uniformly (13)C-enriched helical A-form RNA segment and a helical B-form DNA dodecamer at natural (13)C abundance. The magnitudes of the derived CSA principal values are tightly restricted by the magnetic field dependencies of the (13)C transverse relaxation rates, whereas the tensor orientation and asymmetry follow from quantitative measurements of interference between (13)C-{(1)H} dipolar and (13)C CSA relaxation mechanisms. Changes in the chemical shift between the isotropic and aligned states, Deltadelta, complement these measurements and permit cross-validation. The CSA tensors are determined from the experimental Deltadelta values and relaxation rates, under the assumption that the CSA tensor of any specific carbon in a given type of base is independent of the base position in either the RNA or DNA helix. However, the experimental data indicate that for pyrimidine C(6) carbons in A-form RNA the CSA magnitude is considerably larger than in B-form DNA. This result is supported by quantum chemical calculations and is attributed in part to the close proximity between intranucleotide C(6)H and O(5)' atoms in RNA. The magnitudes of the measured CSA tensors, on average, agree better with previous solid-state NMR results obtained on powdered nucleosides than with prior results from quantum chemical calculations on isolated bases, which depend rather strongly on the level of theory at which the calculations are carried out. In contrast, previously computed orientations of the chemical shift tensors agree well with the present experimental results and exhibit less dependence on the level of theory at which the computations are performed.