Proton homonuclear correlated spectroscopy as an assignment tool for hyperfine-shifted resonances in medium-sized paramagnetic proteins: cyanide-ligated yeast cytochrome c peroxidase as an example.

Two types of homonuclear proton COSY experiments are shown to be useful in making resonance assignments in cyanide-ligated cytochrome c peroxidase, a 34 kDa paramagnetic heme protein. Both magnitude COSY and phase-sensitive COSY experiments provide spectra useful for making proton assignments to resonances of strongly relaxed hyperfine-shifted protons. This initial investigation demonstrates that COSY experiments combined with NOESY experiments are feasible for hyperfine-shifted protons of paramagnetic proteins larger than metmyoglobins and ferricytochromes c, for which the nuclear spin-lattice relaxation times are in the range 70-300 ms. Taken together, COSY and NOESY experiments, although not yet widely applied to paramagnetic metalloproteins, provide a reliable protocol for accurately assigning hyperfine-shifted resonances that are part of a metalloenzyme's active site. Specific examples of expected proton homonuclear COSY connectivities that were not observed in these experiments are presented, and utilization of COSY with respect to the proton resonance line widths and apparent nuclear relaxation times is discussed. The COSY experiments presented here provide valuable verification of previously proposed hyperfine resonance assignments for cyanide-ligated cytochrome c peroxidase, which were made by using NOESY experiments alone, and in several instances expand these assignments to additional protons in particular amino acid spin systems.     

Omega 1-decoupled 1H homonuclear shift-correlated nuclear magnetic resonance spectroscopy (COSYDEC) applied to steroids

Problems of cross-peak overlap in two-dimensional 1H homonuclear shift-correlated (COSY) spectra of steroids can often be avoided by use of the omega 1-decoupled COSY (COSYDEC) method. The selection of experimental parameters is discussed, and COSYDEC spectra are illustrated for 17a-oxa-D-homoandrost-4-ene-3,17-dione (testololactone), testosterone, and 17 alpha-hydroxyprogesterone. In a good case, a COSYDEC spectrum obtained at 250 MHz allows cross-peak recognition and assignment with facility comparable to that available only at 500 MHz for normal COSY spectra.     

Identification of glycine spin systems in 1H NMR spectra of proteins using multiple quantum coherences

Double-quantum filtered COSY and triple-quantum filtered COSY techniques have been compared for the tripeptide Gly-Tyr-Gly and for human lysozyme. The insertion of a triple-quantum filter in the COSY experiment leads to dramatic spectral simplification in the fingerprint region of the spectrum and permits the specific identification of glycine spin systems in the complex 1H NMR spectra of proteins. The assignment of these peaks to glycine H alpha can be confirmed using 2D double-quantum correlated spectroscopy.     

Improved spectral resolution in cosy 1H NMR spectra of proteins via double quantum filtering

A double quantum filter is inserted into a two-dimensional correlated (COSY) 1H NMR experiment to obtain phase-sensitive spectra in which both cross peak and diagonal peak multiplets have anti-phase fine structure, and in which the cross peaks and the major contribution to the diagonal peaks have absorption lineshapes in both dimensions. The elimination of the dispersive character of the diagonal peaks in phase-sensitive, double quantum-filtered COSY spectra allows identification of cross peaks lying immediately adjacent to the diagonal, which represents a significant improvement over the conventional COSY experiment.     

Diterpene glucosides from Pieris formosa

Five diterpene glucosides, pierisformosides B-F were isolated from Pieris formosa D. Don (Ericaceae). Their structures were elucidated on the basis of spectral analysis. including 1H-1H COSY, 13C-1H COSY, HMBC and NOESY experiments.     

Application of nonlinear sampling schemes to COSY-type spectra

Summary Nonlinear sampling along the t₁ dimension is applied to COSY-type spectra. The sine dependence of the time domain signals for the cross peaks is matched by a nonlinear sampling scheme that samples most densely around the maximum of the sine function. Data are processed by maximum entropy reconstruction, using a modified implementation of the Cambridge algorithm of Skilling and Bryan. The procedure is demonstrated for P.E.COSY spectra recorded on a cyclic hexapeptide and on a 126-residue domain of the protein villin. The number of t₁ values in the nonlinearly sampled experiments was reduced by a factor of four compared to linear sampling. The sensitivity and resolution of the resulting spectra are comparable to those achieved by conventional methods. The method described can thus significantly reduce the measuring time for COSY-type spectra.Abbreviations COSY two-dimensional correlation spectroscopy - E.COSY exclusive COSY - P.E.COSY primitive E.COSY - TPPI time proportional phase incrementation - FID free induction decay To whom correspondence should be addressed.     

Monitoring anthracycline binding to nucleotides by two dimensional cosy NMR experiments.

The nucleotide binding ability of the novel anthracycline drug, 3-fluoro-4-demethoxydaunomycin, has been studied by two dimensional 1H NMR correlated spectroscopy (COSY). In the COSY spectrum of the nucleotide mini-helix d(CTGCAG)2 cross-peaks are observed from the spin-coupled H6 and H5 protons of the cytidine bases. Additional cytidine H6/H5 cross-peaks are observed in the COSY spectrum of the anthracycline-d(CTGCAG)2 complex. These additional cytidine cross-peaks enable the identification of the anthracycline binding sites and the determination of the relative kinetic stability of the bound drug at each binding site.     

Study of Sesquiterpene Alkaloids from Tripterygium wilfordii (IV)

Two sesquiterpene alkaloids,wilforine and wilfordsuine were isolated from Tripterygium wilfordii Hook. f. Wilfordsuine was a new alkaloid. The structures of wilforine and wilfordsuine were elucidated on the basis of spectral data,especially 2D-NMR techniques　(H'-'H COSY,H'-13C COSY, NOESY, COLOC methods).     

2D COSY 1H NMR: a new tool for studying in situ brain metabolism in the living animal

2D COSY 1H NMR with surface coil has been used to resolve and assign cerebral metabolites which had previously been detected but could not be resolved or assigned in situ in the living animal by conventional 1D 1H NMR. A wide range of cerebral metabolites, including alanine, N-acetyl aspartate, aspartate, choline derivatives, creatine/phosphocreatine pool, GABA, glucose, glutamate/glutamine pool, inositol, lactate and taurine were simultaneously resolved and assigned in situ in the whole animal using the 2D COSY correlation graphs. Global irreversible ischemia caused the appearance and the disappearance of cross-peaks in the 2D COSY 1H NMR map, corresponding to increases in alanine, GABA and lactate and glucose depletion.     

Abruquinone A,B,D,E,F and G from the Root of Abrus precatorius

Eight flavonoids have been isolated from the root of Abrus precatorius L. Among them, six isoflavanquinones, designated (3R)-abruquinone A, B, D and E, (3S)-abruquinone F and G, are characterized by chemical and spectral means including 1H-1H COSY, 1H-13C COSY and CD methods.     

A Stilbene derivative from Gnetum parvifolium

Gnetifolin F, a novel stilbene derivative, was isolated from the lianas of Gnetum parvifolium. The structure was deduced mainly by the use of ¹H-¹H COSY, ¹³C-¹H COSY, ¹³C-¹H COLOC and NOE difference spectrum, and verified with X-ray crystallographic analysis.     

Comparative structural connectivity spectra analysis (CoSCoSA) models of steroids binding to the aromatase enzyme

A method that combines NMR spectral and structural information into a constructed three-dimensional (3D)-connectivity matrix is developed for modeling biological binding activity of small molecules. The 3D-connectivity matrix for a molecule is defined by associating the distances between all possible carbon-to-carbon connections with their assigned carbon NMR chemical shifts. In this project we selected from the total 3D-connectivity matrix a subset, the two-dimensional (2D) (13)C-(13)C COSY and a theoretical long range 2D (13)C-(13)C distance connectivity spectral plane. Patterns of (13)C chemical shifts observed at these two relative distances for 50 steroids were used to produce a mathematical relationship for the steroids' relative binding affinity (pK(i)) to the aromatase enzyme. We call this technique comparative structural connectivity spectra analysis (CoSCoSA) modeling. Using combinations of the 2D COSY and 2D long-range distance spectra as modeling parameters, we built four CoSCoSA models. One model was made from the 2D COSY spectra alone and another was developed using only the 2D long-range distance spectra. Then the COSY and long-distance spectra were combined in two different ways: starting with the combined principal components (PCs) from the separately calculated COSY and distance spectra or using the combined raw spectra (3D). The best CoSCoSA model was based on the combined PCs from COSY and distance spectra. This model had an r(2) of 0.96 and a leave-one-out cross-validation (q(2)) of 0.92. In general CoSCoSA modeling combines the quantum mechanical information inherent in NMR chemical shifts with internal molecular atom-to-atom distances to give a reliable and straightforward basis for predictive modeling. The technique has the flexibility and accuracy to outperform not only the cross-validated variance q(2) of previously published quantitative structure-activity relationships (QSAR) but also those obtained by related quantitative spectral data-activity relationships (QSDARs) lacking connectivity dimensions.     

A dimeric ArC2 compound from Peperomia pellucida

Pellucidin A, a novel dimeric ArC2 compound, together with dill-apiol have been isolated from the aerial parts of Peperomia pellucida. The structure of pellucidin A was established by 1D and 2D NMR spectroscopy (1H-1H COSY; 1H-13C COSY; DEPT; NOESY and double irradiation) and other spectroscopic techniques. The biogenesis of pellucidin A is also briefly discussed.     

A New Diterpene Glycoside in Aster ageratoides Turcz.

A new natural diterpene glycoside was isolated from Aster ageratoides Turcz. Its structure was elucidated by means of IR, MS (FAB, EI), NMR (1H-, 13C-, DEPT), 2D-NMR (1H-1H COSY, 13C-1 H COSY, J-resolved, CoLoC) spectra and chemical method as 16β, 17-dihydroxy- (—) -kauran- 19-oic acid-β-D-glucopyanosyl ester.     

The conversion of 3-C-beta-D-galactopyranosyl phloroacetophenone to a spiroketal derivative

Treatment of 3-C-beta-D-galactopyranosylphloroacetophenone in hot water with a catalytic amount of p-toluenesulfonic acid afforded a spiroketal compound as the main product. The chirality of the spiro carbon of the product was R, which is the opposite of the spiroketal obtained by the conversion of 3-C-beta-D-glucopyranosyl phloroacetophenone under identical conditions. The structure was determined by 1H-1H COSY, 1H-13C COSY, NOESY and HMBC spectroscopy.     

A triterpenoid saponin from Phytolacca esculenta.

A new triterpenoid saponin, 3-O-[beta-D-glucopyranosyl(1----4)-beta-D- xylopyranosyl]-28-O-beta-D-glucopyranosyl-30-methyloleanate-9(11), 12-dien- 2,3,23-trihydroxyl-28-oic acid, was isolated from the roots of Phytolacca esculenta. The structure was assigned by chemical methods and spectral analysis (1H, 13C, DEPT NMR, EIMS and FABMS) including 1H-1H COSY, 1H-13C COSY and 1H-1H NOESY.     

Combined use of COSY and double quantum two-dimensional NMR spectroscopy for elucidation of spin systems in polymyxin B

Two-dimensional single quantum correlation NMR spectroscopy (COSY) and two-dimensional double quantum NMR spectroscopy (2QT) are used to study spin systems in the 1H NMR spectrum of polymyxin B. Because of different frequency relationships, the two types of two-dimensional NMR experiments are found to be highly complementary. This is demonstrated by combined use of COSY and 2QT spectroscopy to obtain a complete analysis of the complicated spectral overlap which occurs in the 1H NMR spectrum of polymyxin B.     

Assignment of the non-exchangeable proton resonances of d(C-G-C-G-A-A-T-T-C-G-C-G) using two-dimensional nuclear magnetic resonance methods

A general method of assigning the non-exchangeable protons in the nuclear magnetic resonance spectra of small DNA molecules has been developed based upon two-dimensional autocorrelated (COSY) and nuclear Overhauser (NOESY) spectra in 2H2O solutions. Groups of protons in specific sugars or bases are identified by their scalar couplings (COSY), then connected spatially in a sequential fashion using the Overhauser effect (NOESY). The method appears to be generally applicable to moderate-sized DNA duplexes with structures close to B DNA. The self-complementary DNA sequence d(C-G-C-G-A-A-T-T-C-G-C-G) has been synthesized by the solid-phase phosphite triester technique and studied by this method. Analysis of the COSY spectrum and the NOESY spectrum leads to the unambiguous assignment of all protons in the molecule except the poorly resolved H5' and H5" resonances. The observed NOEs indicate qualitatively that, in solution, the d(C-G-C-G-A-A-T-T-C-G-C-G) helix is right-handed and close to the B DNA form with a structure similar to that determined by crystallography.     

Two Novel Diterpenoid Alkaloids Isolated from the Roots of Aconitum brevicalcaratum

Two new diterpenoid alkaloids were isolated from the roots of Aconitum brevicalcaratum Diels. They were acobretine D ( Ⅰ )and acobretine E ( Ⅱ ), and the structures of which were identified on the basis of spectroscopic evidences (IR, MS, 1 H and 13C-NMR) and confirmed by chemical transformations. The 1 H and 13C chemical shifts of the hydrochloride of Ⅰ were assigned in relation to of 1 H-1 H COSY and 13C-1 H COSY.