HOHAHA Experiments Research Articles

New secoiridoid ester of swertiamarin and secoxyloganic acid with hepatoprotective activity from Centaurium spicatum L.

Context: Centaurium spicatum (L.) Fritsch (Gentianaceae) is an annual herb occurring in Southern Europe and Northern Africa where it is used in traditional medicine for treatment of numerous illness. Aims: To isolate compounds from C. spicatum having hepatoprotective activity. Methods: Structure elucidation of the new compound was established on the basis of 1D and 2D NMR analyses including 1H-, 13C- NMR, DEPT-135 NMR, HMQC, HMBC and H- H HOHAHA experiments in addition to FAB+ and HRFAB+ Mass spectrometry. In vivo transaminases inhibition activity was carried out. Results: A new ester of swertiamarin and secoxyloganic acid (1) was isolated from C. spicatum in addition to its known components secoiridoids swertiamarin (2) and secoxyloganic acid (3). All tested secoiridoids have transaminases inhibition activity with different values for each compound 1, 2, 3 (86, 83, 81%) for SGOT and (96, 93, 91%) for SGPT, respectively. Conclusions: The new ester compound from C. spicatum has hepatoprotective activity over than its components.

Saponin Glycosides from Osteospermum vaillantii

The aerial parts of Osteospermum vaillantii (Decne) T. Norl., collected in Saudi Arabia, yielded four saponin glycosides, one of which was a new natural product. The structures of the isolated compounds were elucidated using MS, 1 H, 13 C NMR, 1 H- 1 H COSY, HMQC, HMBC and HOHAHA experiments.

Proton NMR spectroscopy assignment of d-glucose residues in highly acetylated starch

1H NMR spectroscopy assignments have been obtained for starch acetates using COSY and HOHAHA experiments by comparison with the spectra of amylose triacetate and of peracetylated malto-oligosaccharides (maltotriose, maltotetraose, maltoheptaose). These assignments are valuable for the location and evaluation of the substitution pattern in modified starches. The bulk of the 1H NMR spectra of highly acetylated starch strongly resembles the spectrum of amylose triacetate in which all protons are identified and display distinct chemical shifts. The resolving power of the HOHAHA experiment allowed the distinction of minor spin systems. Beside these strong signals pertaining to an average 2,3,6-tri- O-acetyl- α-(1 → 4) linked d-glucopyranose unit in an infinite chain, the combination of COSY and HOHAHA experiments allowed the identification of these systems to the terminal, n-1, n-2, and to partially acetylated glucopyranosyl units. As an example, two different preparations of starch acetates with degrees of substitution 2.74 and 2.63 were examined. In one case, NMR demonstrates that the defects of acetylation are random on the polymeric chain (with corresponding signals for unacylated secondary hydroxyl positions at δ 3.61 and 3.40) while in another case, these signals are not detectable, probably due to the presence of clusters of non-acetylated residue forming solid-like zones.

Proton Resonance Assignments in Oligosaccharides Containing Multiple Monosaccharide Residues of the Same Type

A technique is described for assisting the resonance assignment process in oligosaccharide proton NMR spectra, where multiple residues of the same type generate extreme resonance overlap in the spectrum. The approach involves the modification of a conventional HOHAHA experiment with constant-time acquisition in t 1, which effectively proton decouples the C-1 protons of residues whose resonances overlap, thus affording a significant increase in effective resolution in that dimension. For a sufficiently long spin-lock time, complete one-dimensional subspectra are obtained essentially free of cross talk from adjacent resonances. Further simplification of the assignment process is illustrated by incorporation of the constant-time modification into a three-dimensional HOHAHA-HOHAHA experiment.

NMR studies and conformational analysis of a DNA four-way junction formed in a linear synthetic oligonucleotide.

A linear DNA oligomer (M(r) 14,000, 46 nucleotides) was especially designed, chemically synthesized, and studied by means of 1H NMR spectroscopy. The design of the oligomer was guided by the idea that incorporation of three short palindromic sequences, each interspersed by 5'-CTTG-3' motifs at predetermined positions in the oligomer, would give rise to the formation of three stable minihairpin loops [Ippel, J. H., et al. (1992) J. Biomol. Struct. Dyn. 9, 1-16], which in turn were expected to encourage further folding of the strand into a stable four-way junction containing three "hairpin" arms and an open-ended duplex stem as the fourth arm. Linear DNA four-way junctions constructed according to this concept can be more compact and are therefore expected to be more suitable as model compounds for conformational studies compared to junctions that are built from two or more separate strands. A stable cruciform conformation was substantiated for the 46-mer in aqueous solution in the presence of Mg2+. Complete sequential 1H NMR assignments of the nonexchangeable protons (except H4', H5', and H5") were obtained with the aid of NOESY and HOHAHA experiments. The NMR data gave evidence for the expected existence of minihairpin-loop structures at the three 5'-CTTG-3' motifs in the sequence. The complementary stem domains adopt a regular B-DNA form. Watson-Crick type base pairing is preserved for all residues in the stem domains, including the residues at the center of the junction. A systematic investigation of the interresidual NOEs observed between the protons of the eight central residues revealed the complete stacking pattern of the residues at the branch point.

Magnetization transfer via isotropic mixing: An introduction to the HOHAHA experiment

AbstractThe theory of the cross‐polarization two‐dimensional homonuclear Hartmann‐Hahn experiment is introduced. The product operator formalism is used to develop expressions to explain the transfer of magnetization under the influence of the isotropic Hamiltonian. The various modifications of the HOHAHA/TOCSY experiment are presented. The MLEV‐17 pulse cycle for generation of the spin‐lock pulse is discussed in detail. The intensity of magnetization transfer is shown both mathematically and graphically to be dependent on the relationship mixing time 1/(2JAX). The HOHAHA experiment is also discussed in relation to the other spin‐lock experiments, ROESY and CAMELSPIN.

1H-NMR study of Ca(2+)-and Mg(2+)-dependent interaction between troponin C and troponin I inhibitory peptide (96-116).

The Ca(2+)-and Mg(2+)-dependence of the interaction between rabbit skeletal muscle troponin C (TnC) and a 21 residue peptide corresponding to 96-116 of troponin I (denoted as CN4) was examined by means of 1H-NMR spectroscopy. The spectral changes of TnC with 4 mol of Ca2+ (Ca4TnC) and TnC with 4 mol of Mg2+ (Mg4TnC) were observed as a function of CN4 concentration. As CN4 was added to Ca4TnC, resonances of the following residues changed in chemical shift: Tyr10, Phe23, Phe72, Ala106, Gly108, Tyr109, Ile110, His125, Gly144, Ile146, Phe102 or Phe151, and Phe148 located in the N- and C-domains of Ca4TnC. Such CN4-induced change was also observed for resonances of Phe19, 26, and 75 in the N-domain of Ca4TnC by means of NOESY and HOHAHA experiments. The presence of CN4 increased the native-to-unfolded transition temperature of the N-domain of Ca4TnC. On the basis of these results, we conclude that CN4 binds to both the C- and N-domains of Ca4TnC ([CN4]:[TnC] = 1:1) and stabilizes the structure of the N-domain. The CN4-binding constant was estimated to be 1.1 x 10(5) M-1. As CN4 was added to Mg4TnC, chemical shift change was observed for resonances of Phe99, Tyr109, and Ile110 in the C-domain, while no change was observed for resonances arising from the N-domain. The presence of CN4 did not change the thermal stability of the N- and C-domains of Mg4TnC. The CN4-binding constant of Mg4TnC was obtained as 0.9 x 10(4) M-1, which is one-tenth of that of Ca4TnC.(ABSTRACT TRUNCATED AT 250 WORDS)

Dihydrofolate reductase; sequential resonance assignments using 2D and 3D NMR and secondary structure determination in solution

Three-dimensional (3D) heteronuclear NMR techniques have been used to make sequential 1H and 15N resonance assignments for most of the residues of Lactobacillus casei dihydrofolate reductase (DHFR), a monomeric protein of molecular mass 18,300 Da. A uniformly 15N-labeled sample of the protein was prepared and its complex with methotrexate (MTX) studied by 3D 15N/1H nuclear Overhauser-heteronuclear multiple quantum coherence (NOESY-HMQC), Hartmann-Hahn-heteronuclear multiple quantum coherence (HOHAHA-HMQC), and HMQC-NOESY-HMQC experiments. These experiments overcame most of the spectral overlap problems caused by chemical shift degeneracies in 2D spectra and allowed the 1H-1H through-space and through-bond connectivities to be identified unambiguously, leading to the resonance assignments. The novel HMQC-NOESY-HMQC experiment allows NOE cross peaks to be detected between NH protons even when their 1H chemical shifts are degenerate as long as the amide 15N chemical shifts are nondegenerate. The 3D experiments, in combination with conventional 2D NOESY, COSY, and HOHAHA experiments on unlabelled and selectively deuterated DHFR, provide backbone assignments for 146 of the 162 residues and side-chain assignments for 104 residues of the protein. Data from the NOE-based experiments and identification of the slowly exchanging amide protons provide detailed information about the secondary structure of the binary complex of the protein with methotrexate. Sequential NHi-NHi+1 NOEs define four regions with helical structure. Two of these regions, residues 44-49 and 79-89, correspond to within one amino acid to helices C and E in the crystal structure of the DHFR.methotrexate.NADPH complex [Bolin et al. (1982) J. Biol. Chem. 257, 13650-13662], while the NMR-determined helix formed by residues 26-35 is about one turn shorter at the N-terminus than helix B in the crystal structure, which spans residues 23-34. Similarly, the NMR-determined helical region comprising residues 102-110 is somewhat offset from the crystal structure's helix F, which encompasses residues 97-107. Regions of beta-sheet structure were characterized in the binary complex by strong alpha CHi-NHi+1 NOEs and by slowly exchanging amide protons. In addition, several long-range NOEs were identified linking together these stretches to form a beta-sheet. These elements align perfectly with corresponding elements in the crystal structure of the DHFR.methotrexate.NADPH complex, which contains an eight-stranded beta-sheet, indicating that the main body of the beta-sheet is preserved in the binary complex in solution.

Application de la résonance magnétique nucléaire à la détermination de la structure des protéines en solution

Knowledge of three-dimensional structure is a key factor in protein engineering. It is useful, for example, in predicting and understanding the functional consequences of specific substitution of one or more amino acids of the polypeptide chain. It is also necessary for the design of new effectors or analogs of the substrates of enzymes and receptors. X-ray diffraction by crystals of the biomolecule was for a long time the only method of determining three-dimensional structures. In the last 5 years, it has been joined by a new technique, two-dimensional nuclear magnetic resonance (2D NMR), which can resolve the structure of middle-sized proteins (less than 10 kilodaltons). The technique is applied on solutions whose pH, ionic strength, and temperature can be chosen and changed. The two basic measurements, COSY and NOESY, detect respectively the systems of hydrogen nuclei, or protons, coupled through covalent bonds, and those in which the interproton distances are less than 0.5 nm. A systematic strategy leads from resonance assignments of the two-dimensional spectrum to molecular modeling with constraints and finally to the determination of the molecular structure in the solution. Much sophistication is needed even today for the first task, the assignment of the resonances. Each of the COSY and NOESY spectra is a two-dimensional map, where the diagonal line is the one-dimensional spectrum, and the off-diagonal peaks indicate connectives between protons. Peak assignment to a specific type of amino acid is based on the pattern of scalar couplings observed in the COSY spectrum. Next, the amino acids are positioned in the primary sequence, using the spatial proximities of polypeptide chain protons, as observed in the NOESY spectrum. The principal secondary structures (alpha helix, beta sheets, etc.) are then identified by their specific connectivities. The tertiary structure is detected by NOESY connectivities between protons of different amino acids which are far apart in the primary sequence. The distance constraints from the NOESY connectivities also provide the starting point for modeling the tertiary structure. This is then refined using distance geometry and molecular dynamics algorithms. The resolution of the structures obtained with the help of recent algorithmic developments may be comparable to that provided by X-ray diffraction. The COSY measurement can be completed or substituted by other measurements, useful albeit more complex. For example, the HOHAHA experiment, currently in wide use, gives the correlations through multiple covalent bonds. Multiquanta experiments, which select systems of a given number of coupled spins, provide spectral simplification.(ABSTRACT TRUNCATED AT 400 WORDS)

A lupine alkaloid from Leontice leontopetalum

Abstract Leontice leontopetalum from Jordan has yielded the new lupine alkaloid, 3α-hydroxylupanine. Its structure was confirmed by the use of mono- and bi-dimensional NMR techniques (GASPE, DEPT, homonuclear spindecoupling experiments, 1 H/ 1 H-COSY, 1 H/ 13 C-COSY), linked scan measurements and by chemical transformation. The isolation of 13α-hydroxylupanine and isolupanine are also described and their 1 H and 13 C NMR spectral data have been reinvestigated employing GASPE, DEPT, HMQC and HOHAHA experiments.

Structure elucidation of RP 63834 a new macrocyclic lactone antibiotic

The structure of RP 63834, a new macrocyclic lactone antibiotic, was determined using homo and heteronuclear 2D NMR spectroscopy. The molecular formula C 63H 111O 20N 3 agrees with IR, FAB-MS and NMR measurements. The skeletal structure was inferred from the concerted compilation of the results of COSY, HETCOR, HMBC and HOHAHA experiments using a matrix approach. This approach allows consideration of all possible correlations consistent with the experimental data.

Improvements for the 1 D HOHAHA experiment

Improvements for the 1 D HOHAHA experiment

Relayed HOHAHA, a useful method for extracting subspectra of individual components of sugar chains

The ma in difficulty encountered in the analysis of NMR spectra of sugar chains is the overlap of most sugar proton resonances except for reporter groups such as anomerit proton resonances ( 1). Therefore, NMR methods for extracting the proton resonances of each sugar component from an overlapping region are very useful. In previous studies, we applied mu ltiple relayed COSY (2-5) and homonuclear Hartmann-Hahn spectroscopy (HOHAHA) (6, 7) to extract the subspectrum of each sugar component of glycolipids (8, 9)) utilizing magnetization transfer from wellresolved anomeric proton resonances. In contrast to homonuclear mu ltiple relay experiments, the HOHAHA experiment does not generate large amounts of mu ltiplequantum coherence during the m ixing period and it redistributes the entire integrated intensity of one proton among all N protons in the same spin system. Hence, besides relaxation during the m ixing time, the sensitivity of a ID HOHAHA spectrum is reduced only by a factor N relative to a conventional one-dimensional ‘H spectrum. Magnetization transfer in the HOHAHA experiment is especially efficient when all couplings are of a similar order of magn itude. For example, for sugar residues such as G lc and G lcNAc which have couplings of 6-9 Hz all a round the ring, complete subspectra can be obtained very efficiently. If one of the couplings around the sugar ring is very small, it essentially blocks the Hartmann-Hahn flow of magnetization. For example, in Ga l, Ga lNAc, and Fuc residues, magnetization is rapidly transferred among protons HI through H4, but because of the typically very small coupling constant between H4 and H5 protons ( l1.5 Hz), HOHAHA transfer to H5 is not very efficient. Moreover, any magnetization transferred to H5 is rapidly “diluted” because of further HOHAHA transfer to the usually fast relaxing H6 protons. Here we demonstrate that a combination of HOHAHA and the conventional ‘H‘H relay mechanism can be used effectively to circumvent this problem. The pulse sequence of 1 D-relayed HOHAHA is shown in F ig. 1, where magnetization is first transferred from Hl to H4 via H2 and H3 by HOHAHA using MLEV-

8] Homonuclear Hartmann-Hahn experiments

This chapter discusses the HOHAHA or TOCSY, and with particular emphasis on the experimental details required for recording optimal spectra on biological samples. This chapter presents methods for recording the HOHAHA spectra in H2O solution are especially useful for connecting the side-chain protons with the usually better resolved amide resonances. At presence, most commercial spectrometers require some special hardware for producing the rf fields that have to be generated during the mixing period. Two-dimensional HOHAHA experiments are particularly useful for delineating individual spin systems in proteins. The method provides high-sensitivity, high-resolution spectra that contain a wealth of relayed connectivity information. There are two reasons HOHAHA spectroscopy may be more sensitive than conventional COSY-type experiments. First, because of the in-phase nature of the transfer of magnetization, cancellation of absorptive components within a cross-multiplet does not occur. Second, no magnetization is lost to the generation of spurious multiple-quantum coherence, a major source of sensitivity loss in conventional RELAY experiments. Resolution in the fingerprint region of double-quantum-filtered COSY and HOHAHA spectra is quite similar; although in the experience, the sensitivity of the HOHAHA spectra is typically much higher. For studying spectral regions close to the diagonal, the HOHAHA spectrum usually offers the best appearance because of the relatively low intensity of the diagonal.

ChemInform Abstract: Generation of Pure Phase NMR Subspectra for Measurement of Homonuclear Coupling Constants

Recently it has been demonstrated that dramatic spectral simplification can be obtained by editing a regular ‘H NMR spectrum into a set of subspectra by means of the one-dimensional HOHAHA experiment (I). As pointed out earlier, scalar couplings are difficult to measure from such a spectrum because of phase and intensity distortions of the muhiplet components in the various subspectra. Here, it is demonstrated that pure phase subspectra can be obtained readily by combination of the 1D HOHAHA experiment with the z-filtering technique (2). Guidelines for minimizing the number of T averages, needed for the z filter, are also presented. If the Zeeman term from the Hamiltonian is eliminated, the spin system will evolve solely under the influence of scalar couplings. Braunschweiler and Ernst (3) lirst utilized this concept in isotropic liquids for generating total coherence transfer (TOCSY) 2D spectra of spin systems of coupled protons. A number of more efficient mixing schemes based on the same mechanism have since been reported (1, 4, 5). We prefer to use the so-called MLEV- 17 sequence (4) for mixing because it covers a substantial bandwidth (kO.3~) and because it is relatively insensitive to exact phase shifting or pulse width adjustment. Moreover, this mixing scheme lengthens the apparent Tip (4, 5) so that less signal is lost during this mixing period relative to other mixing schemes. To record pure phase spectra, Braunschweiler and Ernst proposed cc&ding a number of spectra, recorded with different mixing times. This procedure works satisfactorily when few spins are involved in a J-coupled network. For larger spin systems however, this procedure is not perfect because the magnetization transfer appears to lose its oscillatory character, i.e., the oscillation is overdamped. Consequently, the spectra obtained are not 100% absorptive and measurement of scalar couplings may lead to errors. Moreover, in a large network of coupled spins (alkaloids, steroids, etc.) averaging over a number of mixing times, some of which would have to be quite long, gives a subspectrum of nearly the entire molecule and therefore defeats the purpose of the experiment. As demonstrated here, the z-filtering technique, proposed by Slarensen

Generation of pure phase NMR subspectra for measurement of homonuclear coupling constants

Recently it has been demonstrated that dramatic spectral simplification can be obtained by editing a regular ‘H NMR spectrum into a set of subspectra by means of the one-dimensional HOHAHA experiment (I). As pointed out earlier, scalar couplings are difficult to measure from such a spectrum because of phase and intensity distortions of the muhiplet components in the various subspectra. Here, it is demonstrated that pure phase subspectra can be obtained readily by combination of the 1D HOHAHA experiment with the z-filtering technique (2). Guidelines for minimizing the number of T averages, needed for the z filter, are also presented. If the Zeeman term from the Hamiltonian is eliminated, the spin system will evolve solely under the influence of scalar couplings. Braunschweiler and Ernst (3) lirst utilized this concept in isotropic liquids for generating total coherence transfer (TOCSY) 2D spectra of spin systems of coupled protons. A number of more efficient mixing schemes based on the same mechanism have since been reported (1, 4, 5). We prefer to use the so-called MLEV- 17 sequence (4) for mixing because it covers a substantial bandwidth (kO.3~) and because it is relatively insensitive to exact phase shifting or pulse width adjustment. Moreover, this mixing scheme lengthens the apparent Tip (4, 5) so that less signal is lost during this mixing period relative to other mixing schemes. To record pure phase spectra, Braunschweiler and Ernst proposed cc&ding a number of spectra, recorded with different mixing times. This procedure works satisfactorily when few spins are involved in a J-coupled network. For larger spin systems however, this procedure is not perfect because the magnetization transfer appears to lose its oscillatory character, i.e., the oscillation is overdamped. Consequently, the spectra obtained are not 100% absorptive and measurement of scalar couplings may lead to errors. Moreover, in a large network of coupled spins (alkaloids, steroids, etc.) averaging over a number of mixing times, some of which would have to be quite long, gives a subspectrum of nearly the entire molecule and therefore defeats the purpose of the experiment. As demonstrated here, the z-filtering technique, proposed by Slarensen