Formation, isolation, spectroscopic properties, and calculated properties of some isomers of C(60)H(36).

Isomers of C(60)H(36) and He@C(60)H(36) have been synthesized by the Birch or dihydroanthracene reduction of C(60) and isolated by preparative high-pressure liquid chromatography. (3)He, (13)C, and (1)H NMR spectroscopic properties were then determined. A comparison of experimental chemical shifts against those computed using density functional theory (B3LYP) with polarized triple- and double-zeta basis sets for He and C,H, respectively, allowed provisional assignment of structure for several isomers to be made. Theoretical calculations have also been carried out to identify low-energy structures. The transfer hydrogenation method using dihydroanthracene gives a major C(60)H(36) isomer and a minor C(60)H(36) isomer with C(3) symmetry as determined by the (13)C NMR spectrum of C(60)H(36) and the (3)He NMR spectrum of the corresponding sample of (3)He@C(60)H(36). In view of the HPLC retention times and the (3)He chemical shifts observed for the Birch and dihydroanthracene reduction products, the two isomers generated by the latter procedure can be only minor isomers of the Birch reduction. A significant energy barrier apparently exists in the dihydroanthracene reduction of C(60) for the conversion of the C(3) and C(1) symmetry isomers of C(60)H(36) to the T symmetry isomer previously predicted by many calculations to be among the most stable C(60)H(36) isomers. Many of the (1)H NMR signals exhibited by C(60)H(36) (and C(60)H(18), previously reported) are unusually deshielded compared to "ordinary" organic compounds, presumably because the unusual structures of C(60)H(36) and C(60)H(18) result in chemical shift tensors with one or more unusual principal values. Calculations clearly show a relationship between exceptionally deshielded protons beta to a benzene ring in C(60)H(18) and C(60)H(36) and relatively long C-C bonds associated with these protons. The additional information obtained from 1D and 2D (1)H NMR spectra obtained at ultrahigh field strengths (up to 900 MHz) will serve as a critical test of chemical shifts to be obtained from future calculations on different C(60)H(36) isomers.

An effort to understand the molecular basis of hypertension through the study of conformational analysis of losartan and sarmesin using a combination of nuclear magnetic resonance spectroscopy and theoretical calculations.

Losartan is the first recently approved drug against hypertension disease that competes with the biological action of angiotensin II (AII) at the AT1 receptor. Its design was based on the mimicry of the C-terminal segment of AII. Due to the biological significance of Losartan, its structure elucidation and conformational properties are reported as determined by NMR spectroscopy and computational analysis. In addition, molecular modeling of the peptide Sarmesin [Sar1Tyr(OMe)4AII], a competitive antagonist of AII, was also developed based on NMR and computational analysis data. Sarmesin's C-terminal was used as a template for superimposition with specific molecular features of interest in the structure of Losartan such as the conformation of biphenyltetrazole, the n-butyl chain, and the orientation of hydroxymethylimidazole relative to the biphenyl template. The major conclusions derived from this study are the following: (a) Sarmesin, like the AII superagonist [Sar1]AII, adopts a conformation which keeps in close proximity the key amino acids Sar1 (or Arg2)-Tyr(OMe)4-His6-Phe8. (b) Losartan favors a low-energy conformation in which imidazole and tetrazole rings are placed in the opposite site relative to the spacer phenyl ring plane; the hydroxymethyl group is placed away from the spacer phenyl ring, the alkyl chain is oriented above the spacer phenyl ring, and the two phenyl rings deviate approximately 60 degrees from being coplanar. (c) Overlay of the C-terminal region of Sarmesin with Losartan using equivalent groups revealed an excellent match. (d) Interestingly, the matching between enantiomeric structures of Losartan was not equivalent, proposing that the chirality of this molecule is significant in order to exert its biological activity. These findings open a new avenue for synthetic chemists to design and synthesize peptidomimetic drugs based on the C-terminal segment of the proposed model of Sarmesin. The new candidate drug molecules are not restricted to structurally resemble Losartan as the design is hitherto focused.

Conformational analysis of a Chlamydia-specific disaccharide alpha-Kdo-(2-->8)-alpha-Kdo-(2-->O)-allyl in aqueous solution and bound to a monoclonal antibody: observation of intermolecular transfer NOEs.

The disaccharide alpha-Kdo-(2-->8)-alpha-Kdo (Kdo: 3-deoxy-D-manno-oct-2-ulosonic acid) represents a genus-specific epitope of the lipopolysaccharide of the obligate intracellular human pathogen Chlamydia. The conformation of the synthetically derived disaccharide alpha-Kdo-(2-->8)-alpha-Kdo-(2-->O)-allyl was studied in aqueous solution, and complexed to a monoclonal antibody S25-2. Various NMR experiments based on the detection of NOEs (or transfer NOEs) and ROEs (or transfer ROEs) were performed. A major problem was the extensive overlap of almost all 1H NMR signals of alpha-Kdo-(2-->8)-alpha-Kdo-(2-->O)-allyl. To overcome this difficulty, HMQC-NOESY and HMQC-trNOESY experiments were employed. Spin diffusion effects were identified using trROESY experiments, QUIET-trNOESY experiments and MINSY experiments. It was found that protein protons contribute to the observed spin diffusion effects. At 800 MHz, intermolecular trNOEs were observed between ligand protons and aromatic protons in the antibody binding site. From NMR experiments and Metropolis Monte Carlo simulations, it was concluded that alpha-Kdo-(2-->8)-alpha-Kdo-(2-->O)-allyl in aqueous solution exists as a complex conformational mixture. Upon binding to the monoclonal antibody S25-2, only a limited range of conformations is available to alpha-Kdo-(2-->8)-alpha-Kdo-(2-->O)-allyl. These possible bound conformations were derived from a distance geometry analysis using transfer NOEs as experimental constraints. It is clear that a conformation is selected which lies within a part of the conformational space that is highly populated in solution. This conformational space also includes the conformation found in the crystal structure. Our results provide a basis for modeling studies of the antibody-disaccharide complex.

Application of homonuclear 3D NMR experiments and 1D analogs to study the conformation of sialyl Lewis(x) bound to E-selectin.

The conformation of the sialyl Lewis(x) tetrasaccharide bound to E-selectin was previously determined from transfer NOE (trNOE) experiments in conjunction with a distance-geometry analysis. However, the orientation of the tetrasaccharide ligand in the binding site of E-selectin is still unknown. It can be predicted that the accurate quantitative analysis of all trNOEs, including those originating from spin diffusion, is one key to analyze the orientation of sialyl Lewis(x) in the binding pocket of E-selectin. Therefore, we applied homonuclear 3D NMR experiments and 1D analogs to obtain trNOEs that could not unambiguously be assigned from previous 2D trNOESY spectra, due to severe resonance-signal overlap. A 3D TOCSY-trNOESY experiment, a 1D TOCSY-trNOESY experiment, and a 1D trNOESY-TOCSY experiment of the sialyl Lewis(x)/E-selectin complex furnished new interglycosidic trNOEs and provided additional information for the interpretation of trNOEs that have been described before. A 2D trROESY spectrum of the sialyl Lewis(x)/E-selectin complex allowed one to identify the amount of spin-diffusion contributions to trNOEs. Finally, an unambiguous assignment of all trNOEs, and an analysis of spin-diffusion pathways, was obtained, creating a basis for a quantitative analysis of trNOEs in the sialyl Lewis(x)/E-selectin complex.

Determination of (3)J(H (infi) (supN) ,C (infi) (sup') ) coupling constants in proteins with the C'-FIDS method.

We introduce the C'-FIDS-(1)H,(15)N-HSQC experiment, a new method for the determination of (3)J(H (infi) (supN) ,C (infi) (sup') ) coupling constants in proteins, yielding information about the torsional angle ϕ. It relies on the (1)H,(15)N-HSQC or HNCO experiment, two of the the most sensitive heteronuclear correlation experiments for isotopically labeled proteins. A set of three (1)H,(15)N-HSQC or HNCO spectra are recorded: a reference experiment in which the carbonyl spins are decoupled during t(1) and t(2), a second experiment in which they are decoupled exclusively during t(1) and a third one in which they are coupled in t(1) as well as t(2). The last experiment yields an E.COSY-type pattern from which the (2)J(H (infi) (supN) ,C (infi-1) (sup') ) and (1)J(N(i),C (infi-1) (sup') ) coupling constants can be extracted. By comparison of the coupled multiplet (obtained from the second experiment) with the decoupled multiplet (obtained from the first experiment) convoluted with the (2)J(H (infi) (supN) ,C (infi-1) (sup') ) coupling, the (3)J(H (infi) (supN) ,C (infi) (sup') ) coupling can be found in a one-parameter fitting procedure. The method is demonstrated for the protein rhodniin, containing 103 amino acids. Systematic errors due to differential relaxation are small for (n)J(H(N),C') couplings in biomacromolecules of the size currently under NMR spectroscopic investigation.

HNCCH-TOCSY, a triple resonance experiment for the correlation of backbone 13C alpha and 15N resonances with aliphatic side-chain proton resonances and for measuring vicinal 13CO,1H beta coupling constants in proteins.

A 3D triple resonance experiment has been designed to provide intraresidual and sequential correlations between amide nitrogens and alpha-carbons in uniformly 13C/15N-labeled proteins. In-phase 13C alpha magnetization is transferred to the aliphatic side-chain protons via the side-chain carbons using a CC-TOCSY mixing sequence. Thus, the experiment alleviates the resonance assignment process by providing information about the amino acid type as well as establishing sequential connectivities. Leaving the carbonyl spins untouched throughout the transfer from 13C alpha to 1H beta leads to E.COSY-type cross peaks, from which the 3JH beta CO coupling constants can be evaluated. The pulse sequence is applied to oxidized Desulfovibrio vulgaris flavodoxin.

Backbone dynamics of ribonuclease T1 and its complex with 2'GMP studied by two-dimensional heteronuclear NMR spectroscopy.

The backbone dynamics of free ribonuclease T1 and its complex with the competitive inhibitor 2'GMP have been studied by (15)N longitudinal and transverse relaxation experiments, combined with {(1)H, (15)H} NOE measurements. The intensity decay of individual amide cross peaks in a series of ((1)H, (15)N)-HSQC spectra with appropriate relaxation periods (Kay, L.E. et al. (1989) Biochemistry, 28, 8972-8979; Kay, L.E. et al. (1992) J. Magn. Reson., 97, 359-375) was fitted to a single exponential by using a simplex algorithm in order to obtain (15)N T(1) and T(2) relaxation times. These experimentally obtained values were analysed in terms of the 'model-free' approach introduced by Lipari and Szabo (Lipari, G. and Szabo, A. (1982) J. Am. Chem. Soc., 104, 4546-4559; 4559-4570). The microdyramical parameters accessible by this approach clearly indicate a correlation between the structural flexibility and the tertiary structure of ribonuclease T1, as well as restricted mobility of certain regions of the protein backbone upon binding of the inhibitor. The results obtained by NMR are compared to X-ray crystallographic data and to observations made in molecular dynamics simulations.

Determination of H(N),H (α) and H (N),C' coupling constants in (13)C, (15)N-labeled proteins.

Sensitive three-dimensional NMR experiments, based on the E.COSY principle, are presented for the measurement of the (3)J(H(N),H(α)) and (3)J(H(N),C') coupling constants in uniformly (13)C- and (15)N-labeled proteins. They employ gradient coherence selection in combination with the sensitivity enhancement method in HSQC-type spectra (Cavanagh et al., 1991; Palmer et al., 1991). In most cases, the two measured coupling constants unambiguously define the ϕ-angle for protein structure determination. The method is applied to uniformly (13)C, (15)N-labeled ribonuclease T(1).

3D triple-resonance NMR techniques for the sequential assignment of NH and 15N resonances in 15N- and 13C-labelled proteins.

Two new 3D 1H-15N-13C triple-resonance experiments are presented which provide sequential cross peaks between the amide proton of one residue and the amide nitrogen of the preceding and succeeding residues or the amide proton of one residue and the amide proton of the preceding and succeeding residues, respectively. These experiments, which we term 3D-HN(CA)NNH and 3D-H(NCA)NNH, utilize an optimized magnetization transfer via the 2JNC alpha coupling to establish the sequential assignment of backbone NH and 15N resonances. In contrast to NH-NH connectivities observable in homonuclear NOESY spectra, the assignments from the 3D-H(NCA)NNH experiment are conformation independent to a first-order approximation. Thus the assignments obtained from these experiments can be used as either confirmation of assignments obtained from a conventional homonuclear approach or as an initial step in the analysis of backbone resonances according to Ikura et al. (1990) [Biochemistry, 29, 4659-4667]. Both techniques were applied to uniformly 15N- and 13C-labelled ribonuclease T1.