Double Pulsed Field Gradient Spin Echo Research Articles

Structural analyses of mannose pentasaccharide of high mannose type oligosaccharides by 1D and 2D NMR spectroscopy

NMR spectroscopy is a very important and useful method for the structural analysis of oligosaccharides, despite its low sensitivity. We first applied conventional measuring methods, 2D DQF COSY, (1)H-(13)C HSQC, and (1)H-(13)C HMBC, and also the Double Pulsed Field Gradient Spin Echo (DPFGSE)-TOCSY and DPFGSE-NOESY/ROESY techniques to analyze a branched mannose pentasaccharide as a model of high mannose type N-glycans in natural abundance. The NMR spectra of the model compound are very complex and difficult to analyze owing to overlapping signals. The superior selective irradiation capability of the DPFGSE technique is useful for fine structural and conformational analyses of such complex oligosaccharides. We here introduce a novel technique called DPFGSE-Double-Selective Population Transfer (SPT)-Difference and DPFGSE-NOE/ROE-SPT-Difference spectroscopy. The DPFGSE-Double-SPT-Difference method involves irradiation of two peaks from one proton and the subtraction of higher and lower peaks from each spectrum. The DPFGSE-NOE/ROE-SPT-Difference method involves the transfer of the magnetization polarized by NOE/ROE from the nuclei to the spin-coupled nuclei through scalar spin-spin interaction using the SPT method. Even if the signals in the NMR spectra overlap, each signal can be accurately assigned. In particular, DPFGSE-NOE/ROE-SPT-Difference is very useful for identifying sugar connectivity.

An efficient use of the WATERGATE W5 sequence for observing a ligand binding with a protein receptor

An efficient pulse sequence for observing a ligand binding with a receptor has been developed by incorporating the WATERGATE W5 sequence. In the conventional water ligand observed via gradient spectroscopy (WaterLOGSY) techniques, the water resonance is selectively excited using, e.g. the double-pulsed field gradient spin-echo (DPFGSE) sequence at the initial portion of pulse sequence. In the current version, the modified WATERGATE W5 sequence is incorporated at the initial portion of the pulse sequence, and the resonance at the water frequency can be selectively reserved by the modified WATERGATE W5 sequence. The efficiency of ligand-observed NMR screening techniques has been demonstrated using the human serum albumin (HSA)-tryptophan complex.

The Identification and Location of Succinyl Residues and the Characterization of the Interior Arabinan Region Allow for a Model of the Complete Primary Structure of Mycobacterium tuberculosis Mycolyl Arabinogalactan

The complex cell wall of Mycobacterium tuberculosis is the hallmark of acid fast bacteria and is responsible for much of its physiological characteristics. Hence, much effort has been made to determine its primary structure. Such studies have been hampered by its extreme complexity. Also, its insolubility leads to difficulties determining the presence or absence of base labile groups. We have used an endogenous arabinase to solubilize the arabinan region of the cell wall and have shown using mass spectrometry and NMR that succinyl esters are present on O2 of the inner-branched 1,3,5-alpha-d-arabinofuranosyl residues. In addition, an inner arabinan region of 14 linear alpha-1,5 arabinofuranosyl residues has been identified. These and earlier results now allow the presentation of a model of the entire primary structure of the mycobacterial mycolyl arabinogalactan highlighted by three arabinan chains of 31 residues each.

Bohemamines from a Marine-Derived Streptomyces sp.

Investigation of the culture extracts of a marine-derived Streptomyces sp. led to the isolation of three new bohemamine-type pyrrolizidine alkaloids, bohemamine B (1), bohemamine C (2), and 5-chlorobohemamine C (3). The structures were elucidated using NMR methods, and the relative stereochemistry was determined using double-pulsed-field-gradient spin echo (DPFGSE) NOE studies.

Selective J-resolved spectra: A double pulsed field gradient spin-echo approach

A simple method to obtain selective J-resolved spectra is presented, which relies on the refocusing properties of double pulsed field gradient spin-echoes and provides unambiguous assignment of the measured coupling constants. The proposed examples show how this method is of general applicability, and requires no more than a simple optimization strategy to produce artifact-free spectra. Examples of application include the determination of a small, long-range coupling constant (0.7 Hz) in trans-retinal and H α couplings in a tripeptide.

Improved resolution in two‐dimensional 1H NMR spectra of peptides by band‐selective, homonuclear decoupling during both the evolution and acquisition periods: application to characterization of the binding of peptides by heparin

Two-dimensional 1H NMR experiments that achieve band-selective, homonuclear decoupling in both the indirectly detected F1 and directly detected F2 dimensions were used to assign the highly overlapped 1H NMR spectrum of the peptide Ac-SRGKARVRAKVKDQTK-NH2, both free in solution and bound to heparin. Band-selective, homonuclear decoupling during the evolution period was achieved using a double pulsed field gradient spin-echo (DPFGSE) with semi-selective shaped pulses; band-selective, homonuclear decoupling during the acquisition period was achieved by time-shared semi-selective shaped pulse decoupling. Regular TOCSY, ROESY and NOESY spectra and TOCSY, ROESY and NOESY spectra measured with band-selective, homonuclear decoupling in the evolution (F1) dimension (BASHD-TOCSY, ROESY and NOESY spectra) and with band-selective, homonuclear decoupling in both the F1 and F2 dimensions (D-(or Double)-BASHD-TOCSY, ROESY and NOESY spectra) are reported and compared for the peptide and its heparin complex. Complete assignment of the 1H-NMR spectra of the free and heparin-complexed peptide was achieved with the high resolution of the D-BASHD-TOCSY, ROESY and NOESY spectra. Characterization of the heparin-complexed peptide is of interest because of the ability of the peptide to neutralize the anticoagulant activity of heparin.

Solution structures and behavior of trans‐RuH(η1‐BH4) (binap)(1,2‐diamine) complexes

The solution structures of a number of trans-RuH(eta(1)-BH(4))[(S)-tolbinap](1,2-diamine) precatalysts [TolBINAP = 2,2'-bis(di-4-tolylphosphino)-1,1'-binaphthyl; 1,2-diamine==(S,S)- or (R,R)-1,2-diphenylethylenediamine (DPEN), ethylenediamine (EN), and (S)-1,1-di(4-anisyl)-2-isopropylethylenediamine (DAIPEN)] have been determined using 2D NMR ((1)H--(1)H DQF-COSY, (1)H--(13)C HMQC, (1)H--(31)P HSQC, and (1)H--(15)N HSQC), and a double-pulsed field-gradient spin-echo (DPFGSE) NOE technique. All the octahedral Ru complexes adopt a trans configuration with respect to the BH(4) and hydride ligands. Amine protons of trans-RuH(eta(1)-BH(4))[(S)-tolbinap](1,2-diamine) complexes undergo H/D exchange in (CD(3))(2)CDOD. This inherent high acidity, coupled with the lability and chemical properties of the BH(4) ligand, allows for precatalyst activation without the need for an added base, in contrast to trans-RuCl(2)[(S)-tolbinap](1,2-diamine) precatalysts, which require a strong base for generation of a catalytic species. The H/BH(4) complex in a 2-propanol solution is converted to catalytically active [trans-RuH{(S)-tolbinap}{(S,S)-dpen}(ROH)](+) [(RO)(ROH)(n)](-) (R = (CH(3))(2)CH), a loosely associated ion pair of the discrete (solvated) cationic fragment and anionic species.

Practical aspects of ROESY experiments for identification of bound waters in the cyclic tetrasaccharide

ROESY pulse sequences are presented and evaluated to identify bound waters in the cyclic tetrasaccharide. The first experiment incorporated the double-pulsed field gradient spin-echo (DPFGSE) for selective water excitation at the initial portion of the pulse sequence. Although long, shaped pulses were used in DPFGSE to achieve the highly selective excitation of water resonance that is very close to resonances of the cyclic tetrasaccharide, the approach was not effective because of the loss of sensitivity. Concomitant use of long delays and moderate length of shaped pulses in the portion of DPFGSE gained more sensitivity. A simple approach incorporating spin-echo with long delays instead of DPFGSE also afforded a sensitive spectrum. Practical aspects of these ROESY experiments are illustrated using the cyclic tetrasaccharide cyclo-{-->6}-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->).

Application of NMR screening techniques for observing ligand binding with a protein receptor.

Water ligand observed via gradient spectroscopy (WaterLOGSY), saturation transfer difference and NOE pumping NMR techniques were used to identify ligand binding with a receptor. Although these experiments were originally designed to observe ligands in complexes, their application is limited by the affinity of ligands towards target molecules. Here the improved WaterLOGSY pulse sequence was developed by incorporating the double pulsed field gradient spin-echo and gradient-tailored excitation WATERGATE sequences. The efficiency of these ligand-observed NMR screening techniques was investigated using the ribonuclease T1-inhibitor system.

A new selective population transfer experiment using a double pulsed field gradient spin-echo.

A new pulse sequence is proposed for the determination of scalar coupling correlation in small- and medium-sized organic compounds. The method uses a combination of the double pulsed field gradient spin-echo (DPFGSE) and the selective population transfer (SPT) techniques and is shown to be useful in the analysis of complex spectra with many overlapped signals. The usefulness of this method in the structural elucidation of natural substances is demonstrated using strychnine and digitoxin as examples.

Improvement of double pulsed field gradient spin‐echo ROESY experiments for identification of bound waters

AbstractA novel pulse sequence incorporating the double pulsed field gradient spin‐echo (DPFGSE) and the gradient‐tailored excitation WATERGATE techniques is presented that has particular use for identifying bound waters in 15N‐labeled macromolecules. This sequence, DPFGSE–ROESY–HSQC, affords greater spectral sensitivity than the DPFGSE–ROESY–HMQC experiment which was previously presented and is consequently useful for rapidly obtaining reliable information for characterizing macromolecular bound water molecules. A significant enhancement in the sensitivity is achieved by using the gradient‐tailored excitation WATERGATE sequence in the reverse INEPT step as it allows the use of much higher receiver gains. Since coherence selection is not used, the sequence has improved sensitivity together with less spectral artifacts. The advantage of this pulse sequence is illustrated using 15N‐labeled ribonuclease T1. Copyright © 2003 John Wiley & Sons, Ltd.

Homonuclear Selective One-Dimensional Gradient NMR Experiments in the Structure Determination of Natural Diterpene Derivatives

The solution structure of two natural diterpene derivatives, the secondary metabolites esulatin-A and esulatin-B of Euphorbia esula, was investigated by homonuclear NMR experiments. Since the spectral dispersion of the 1H NMR spectra at 500 MHz was sufficient to separate several skeletal protons of the title compounds, they were selectively excited with a double pulsed field gradient spin-echo (DPFGSE) sequence using 180°Gaussian pulses sandwiched between sine shaped gradients. With the use of selective excitation, scalar as well as dipolar interactions of the selected spins were monitored through one-dimensional (1D) COSY, TOCSY, and NOESY experiments. The chemical shifts of the coupling partners could be accurately extracted from the 1D COSY and TOCSY spectra recorded with high digital resolution. The selective TOCSY experiment provided an excellent opportunity to identify spins belonging to the same scalarly coupled spin system. The solution state conformation was investigated by selective gradient enhanced NOESY experiments. Proton–proton distances were evaluated from the cross-relaxation rates obtained from a quantitative analysis of the NOESY spectra recorded with different mixing times. The NMR derived distances were compared to the results of solid state X-ray diffraction measurements.

Solution structure of a pentasaccharide representing the repeating unit of the O-antigen polysaccharide from Escherichia coli O142: NMR spectroscopy and molecular simulation studies.

Conformational studies have been performed of a pentasaccharide derived from the O-polysaccharide from Escherichia coli O142. The polymer was selectively degraded by anhydrous hydrogen fluoride and reduced to yield an oligosaccharide model of its repeating unit, which in the branching region consists of four aminosugars. A comparison of (1)H and (13)C chemical shifts between the pentasaccharide and the polymer showed only minor differences, except where the cleavage had taken place, indicating that the oligomer is a good model of the repeating unit. Langevin dynamics and molecular dynamics simulations with explicit water molecules were carried out to sample the conformational space of the pentasaccharide. For the glycosidic linkages between the hexopyranoside residues, small but significant changes were observed between the simulation techniques. One-dimensional (1D) (1)H,(1)H double pulsed field gradient spin echo (DPFGSE) transverse rotating-frame Overhauser effect spectroscopy (T-ROESY) experiments were performed, and homonuclear cross-relaxation rates were obtained. Subsequently, a comparison of interproton distances from NMR experiment and the two simulation approaches showed that in all cases the use of explicit water in the simulations resulted in better agreement. Hydrogen-bond analysis of the trajectories from the molecular dynamics simulation revealed interresidue interactions to be important as a cluster of different hydrogen bonds and as a distinct highly populated hydrogen bond. NMR data are consistent with the presence of hydrogen bonding within the model of the repeating unit.

NMR Spectra of Nitrobenzanthrones Obtained by Using Double-Pulsed Field-Gradient Spin Echo

We have synthesized several nitrobenzanthrones (NBAs) and investigated their structures in solution using NMR spectroscopy. In our study, however, low solubility of NBAs in organic solvents always became a serious problem and led to NMR spectra with low signal-to-noise ratios. To solve this problem, we adopted a new method of conventional coherence transfer pathway selection using pulsed field-gradients. The double-pulsed field-gradient spin echo (DPFGSE) sequence was used for selecting natural-abundance carbon-13 isotopomers by using a bilinear rotation decoupling (BIRD) sequence and a so-called gradient-BIRD pulse for the excitation sculpting technique. We performed NMR measurements on 3,9-dinitro-7 H -benz[ d,e ]anthracene-7-one (compound 1), 3,9,11-trinitro-7 H -benz[ d,e ]anthracene-7-one (compound 2), and 2-nitro-7 H -benz[ d,e ]anthracene-7-one (compound 3) by this method and obtained spectra of high quality. Combining the results from phase-sensitive 2D NOESY, HOHAHA, and DPFGSE-HMQC, DPFGSE-HMQC TOCSY, and phase-sensitive FG-HMBC permitted us to complete NMR assignments. It was confirmed that the present method enables us to improve the sensitivity and to assign the NMR signal readily, even for compounds with low solubility.

NMR Spectra of Nitrobenzanthrones Obtained by Using Double-Pulsed Field-Gradient Spin Echo

NMR Spectra of Nitrobenzanthrones Obtained by Using Double-Pulsed Field-Gradient Spin Echo

Pentacoordinate Siliconate Tetraanionic Molecular Squares. Synthesis, Structure, and Solution Conformation of a Novel Tetraanion−Tetracation Ion Pair

A tetraanionic molecular square containing four pentacovalent siliconates is produced in high yield by the condensation of phenyltriethoxysilane and spirocatechol 1. A single isomer, meso 2, is produced from this diastereoselective condensation reaction. The steps leading up to the formation of meso-2 are not known, but the reaction appears to be driven by the insolubility of the tetrakistriethylammonium salt 2a. The tetraanionic macrocycle is a stable salt that undergoes ion-exchange reactions. An ion pair composed of the tetraanionic siliconate macrocycle and cyclobis(paraquat-p-phenylene) tetracation (2e) has been synthesized by ion exchange. Following complete assignment of all proton resonances in this 4 × 4 complex, the solution conformation was analyzed by an intermolecular double pulsed field gradient spin−echo (DPFGSE) NOE study. These results, aided by a molecular mechanics modeling study, have led to a proposed dynamic solution structure that features the cyclic bisparaquat tetracation selectively intercalating around a phenyl ring of the C2 face of the macrocyclic tetraanion which in turn is engulfed by the interior of the tetraanion (Figure 10). This novel host−host complexation calls attention to the conformational flexibility of the macrocyclic tetrasiliconates and, in all probability, all macrocycles be they charged or neutral.

Stabilization of a C7 equatorial gamma turn in DMSO-d6 by a ditryptophan crosslink

Covalent crosslinks can control local peptide conformation. In tripeptide sequences of the general formula Cys-Xxx-Cys, cysteine disulfides have been previously shown to enforce a C7 equatorial γ-turn conformation (also referred to as an inverse γ-turn). Much less is known about the effects of dityrosine and ditryptophan crosslinks on local peptide structure. In a series of tripeptides, ditryptophan crosslinks were formed using the two-step process of acid-promoted Mannich dimerization followed by oxidative aromatization. In these peptides, with the general formula Trp-Xxx-Trp (Xxx≠Gly), ditryptophan crosslinks were found to stabilize a C7 equatorial γ-turn conformation in DMSO-d6. Rigorous support for a C7 equatorial conformation in the crosslinked sequence Trp-Pro-Trp came from a variety of 1H NMR experiments and molecular modelling. Interproton distances were derived from NOE buildups that were determined through a series of double pulsed field gradient spin echo (DPFGSE) experiments. In addition, the small temperature dependence of the i+2 NH chemical shifts (Δδ/ΔT<2ppm/°C) provided further support for the intramolecular hydrogen bond which defines a γ-turn.

An ω1-band-selective, ω1-homonuclear decoupled ROESY experiment: application to the assignment of1H NMR spectra of difficult-to-assign peptide sequences

Application of an ω1-band-selective, ω1-homonuclear decoupled ROESY (BASHD-ROESY) experiment to the assignment of 1H NMR spectra of peptides is demonstrated. Band selection in the ω1 dimension is achieved with the double pulsed field gradient spin echo (DPFGSE) technique; homonuclear decoupling in the ω1 dimension is achieved by placing a non-selective 180° pulse together with the first half of the DPFGSE in the middle of the evolution period. Application of the BASHD-ROESY experiment is demonstrated with the complete assignment of the proton resonances of the synthetic 19 amino acid peptide N-Ac–Ala–Glu–Ala–Ala– Ala–Arg–Ala–Ala–Ala–Arg–Arg–Ala–Ala– Arg–Arg–Ala–Ala–Ala–Arg–NH2. Critical to making the assignments was the significantly increased resolution in the CαH–NH region of the ROESY spectrum measured with the BASHD-ROESY pulse sequence with band selection and homonuclear decoupling in the CαH region. NOEs observed for the peptide indicate it has a helical secondary structure in solution. © 1998 John Wiley & Sons, Ltd.

Structural assignment of a molybdenum-containing silsesquioxane which catalyzes the metathesis of olefins: DPFGSE-NOE and x-ray diffraction studies

The reaction of [(c-C6H11)7Si7O9(OSiMe3)(OTl)2] with [Mo(CHCMe2Ph)(NAr)(OSO2CF3)2(dme)] (Ar=2,6-diisopropylphenyl) affords 4a as the predominant (>99%) molybdenum-containing product. Assignment of 4a as the syn-isomer with juxtaposed arylimido and Me3Si groups was made on the basis of double pulsed field gradient spin echo (DPFGSE) NOE experiments and confirmed by a single-crystal x-ray diffraction study. © 1997 John Wiley & Sons, Ltd.