Boosting the Resolution of 1H NMR Spectra by Homonuclear Broadband Decoupling

Broadband homonuclear decoupling of proton spectra, that is, the collapse of all multiplets into singlets, has the potential of boosting the resolution of ¹H NMR spectra. Several methods have been described in the last 40 years to achieve this goal. Most of them can only be applied in the indirect dimension of multi‐dimensional NMR spectra or special data processing is necessary to yield decoupled 1D proton spectra. Recently, complete decoupling of proton spectra during acquisition has been introduced; this not only significantly reduced the experimental time to record these spectra, but also removed the need for any sophisticated processing schemes. Here we present an introduction and overview of the techniques and applications of broadband proton‐decoupled proton experiments.     

HOBS methods for enhancing resolution and sensitivity in small DNA oligonucleotide NMR studies

¹H NMR spectra from biopolymers give chemical shifts classified according to proton type and often suffer from signal degeneracy. Data from nucleic acids are particularly prone to this failing. Recent developments in proton broadband decoupling techniques with the promise of enhanced resolution at full sensitivity have allowed us to investigate the application of homonuclear band‐selective (HOBS) decoupling to the study of small synthetic DNA molecules and to compare these with results from classical and pure shift techniques. Improved signal resolution at full sensitivity in both HOBS‐1D ¹H and HOBS‐2D [¹H, ¹H] NOESY NMR data is reported for three example small DNA molecules. Comparisons of ¹H T₁ and integrals of signals from HOBS‐1D ¹H and HOBS‐2D [¹H, ¹H] NOESY NMR data with those of standard data collection methods are also reported. The results show that homonuclear HOBS‐NOESY data are useful for data assignment purposes and have some merit for quantification purposes. In general, we show that resolution and sensitivity enhancement of ¹H NMR data for small DNA samples may be achieved without recourse to higher magnetic field strength at full sensitivity in a band‐selected manner. Copyright © 2014 John Wiley & Sons, Ltd.     

青蒿素的二维核磁共振研究

报导了采用二维核磁共振技术研究青蒿素的核磁氢谱和碳谱的谱线归属,以今后为^1^3C中间体的生物转化产物提供鉴定的基础.     

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.     

J‐Edited Pure Shift NMR for the Facile Measurement of nJHH for Specific Protons

We report a novel 1D J‐edited pure shift NMR experiment (J‐PSHIFT) that was constructed from a pseudo 2D experiment for the direct measurement of proton–proton scalar couplings. The experiment gives homonuclear broad‐band ¹H‐decoupled ¹H NMR spectra, which provide a single peak for chemically distinct protons, and only retain the homonuclear‐scalar‐coupled doublet pattern at the chemical‐shift positions of the protons in the coupled network of a specific proton. This permits the direct and unambiguous measurement of the magnitudes of the couplings. The incorporation of a 1D selective correlation spectroscopy (COSY)/ total correlation spectroscopy (TOCSY) block in lieu of the initial selective pulse, results in the exclusive detection of the correlated spectrum of a specific proton.     

A method for dynamical characterization and high resolution 1H-NMR in dipolar coupled systems: application to liquid crystals

We study the variation of 13C spectra as function of off-resonances in protons during decoupling, for continuous wave (cw) and small phase incremental alternation with 64-step (SPINAL-64) schemes in the liquid crystals 4-n-octyl-4'-cyanobiphenyl (8CB) and 4-n-pentyl-4'-cyanobiphenyl (5CB). The self-decoupling mechanism induced by the strong homonuclear dipolar interactions provides a method to study the dynamics of the proton system through the 13C spectra. In the n-cyanobiphenyl (nCB) liquid crystals each nonquaternary carbon is coupled through dipolar interactions to more than one proton constituting a SI(N) group (with N> or =2). We extend the analytical treatment of the variation of the 13C spectrum with the off-resonance, described for SI groups, to SI(N) under cw decoupling. The dependence of the maxima of the 13C spectra as a function of proton off-resonance follows a Lorentzian line that depends on the rate of exchange among proton spin states. From the fitting parameters of this curve and the heteronuclear interaction measured in cross-polarization experiments, we extract dynamical information of the intramolecular 1H-1H interactions. In the case of SPINAL-64 we experimentally observe the same behavior. Under both kinds of decouplings, we characterize the chemical shift of the protons through the NMR spectra of carbons. The resulting values are in very good agreement with those obtained by other methods.     

Radio frequency-driven recoupling at high magic-angle spinning frequencies: homonuclear recoupling sans heteronuclear decoupling

We describe solid-state NMR homonuclear recoupling experiments at high magic-angle spinning (MAS) frequencies using the radio frequency-driven recoupling (RFDR) scheme. The effect of heteronuclear decoupling interference during RFDR recoupling at high spinning frequencies is investigated experimentally and via numerical simulations, resulting in the identification of optimal decoupling conditions. The effects of MAS frequency, RF field amplitude, bandwidth, and chemical shift offsets are examined. Most significantly, it is shown that broadband homonuclear correlation spectra can be efficiently obtained using RFDR without decoupling during the mixing period in fully protonated samples, thus considerably reducing the rf power requirements for acquisition of (13)C-(13)C correlation spectra. The utility of RFDR sans decoupling is demonstrated with broadband correlation spectra of a peptide and a model protein at high MAS frequencies and high magnetic field.     

Mixed IR/Vis Two‐Dimensional Spectroscopy: Chemical Exchange beyond the Vibrational Lifetime and Sub‐ensemble Selective Photochemistry

Two‐dimensional exchange spectroscopy (2D EXSY) is a powerful method to study the interconversion (chemical exchange) of molecular species in equilibrium. This method has recently been realized in femtosecond 2D‐IR spectroscopy, dramatically increasing the time resolution. However, current implementations allow the EXSY signal (and therefore the chemical process of interest) only to be tracked during the lifetime (T₁) of the observed spectroscopic transition. This is a severe limitation, as typical vibrational T₁ are only a few ps. An IR/Vis pulse sequence is presented that overcomes this limit and makes the EXSY signal independent of T₁. The same pulse sequence allows to collect time‐resolved IR spectra after electronic excitation of a particular chemical species in a mixture of species with strongly overlapping UV/Vis spectra. Different photoreaction pathways and dynamics of coexisting isomers or of species involved in different intermolecular interactions can thus be revealed, even if the species cannot be isolated because they are in rapid equilibrium.     

Supercycled homonuclear dipolar decoupling in solid-state NMR: toward cleaner 1H spectrum and higher spinning rates

A homonuclear dipolar decoupling scheme based on windowed phase-modulated Lee-Goldburg (wPMLG) pulse sequences that causes a"z-rotation" of the spins for high-resolution proton NMR spectroscopy of solids is described and analyzed. This supercycled scheme suppresses the effect of pulse imperfections on the spectra and significantly relaxes the off-resonance dependence of the line-narrowing efficiency and scale factor. This leads to a broad spectral window that is free of artifacts such as zero lines, image peaks, and localized rotor-radio-frequency resonances. High-resolution (1)H spectra and two-dimensional homonuclear (1)H-(1)H correlation spectra of standard amino acids, obtained by a combination of this supercycled scheme with magic angle spinning frequencies up to 25 kHz, are demonstrated.     

High resolution 1H detected 1H,13C correlation spectra in MAS solid-state NMR using deuterated proteins with selective 1H,2H isotopic labeling of methyl groups

MAS solid-state NMR experiments applied to biological solids are still hampered by low sensitivity and resolution. In this work, we employ a deuteration scheme in which individual methyl groups are selectively protonated. This labeling scheme allows the acquisition of proton carbon correlation spectra with a resolution comparable to that in solution-state NMR experiments. We observe an increase in resolution by a factor of 10-15 compared to standard heteronuclear correlation experiments using PMLG for 1H,1H dipolar decoupling in the indirect dimension. At the same time, the full sensitivity of the proton-based experiment is retained. In comparison to the heteronuclear detected version of the experiment, a gain in sensitivity of a factor of approximately 4.7 is achieved.     

Proton line narrowing in solid-state nuclear magnetic resonance: new insights from windowed phase-modulated Lee-Goldburg sequence

We present here a bimodal Floquet analysis of the windowed phase-modulated Lee-Goldburg (wPMLG) sequence for homonuclear dipolar decoupling. One of the main criteria for an efficient homonuclear dipolar decoupling scheme is an effective z-rotation condition. This is brought about by the presence of radio-frequency imperfections in the pulse sequence together with a systematic manipulation of the wPMLG pulses. Additional improvement in the (1)H spectral resolution was obtained by a proper understanding of the off-resonance dependence of the wPMLG irradiation scheme based on bimodal Floquet theory. Numerical investigations further corroborate both theoretical and experimental findings. Theoretical analysis points to accidental degeneracies between the cycle time of the wPMLG sequence and the rotor period leading to the experimentally observed off-resonance dependence of the resolution. Two-dimensional (1)H-(1)H homonuclear single-quantum correlation spectra of model amino acids are also presented, highlighting the improved spectral resolution of wPMLG sequences.     

Heteronuclear chemical shift correlation and J-resolved MAS NMR spectroscopy of lipid membranes

Direct observation of J-couplings remains a challenge in high-resolution solid-state NMR. In some cases, it is possible to use Lee-Goldburg (LG) homonuclear decoupling during rare spin observation in MAS NMR correlation spectroscopy of lipid membranes to obtain J-resolved spectra in the direct dimension. In one simple implementation, a wide line separation-type (13)C-(1)H HETCOR can provide high-resolution (1)H/(13)C spectra, which are J-resolved in both dimensions. Coupling constants, (1)J(HC), obtained from (1)H doublets, can be compared with scaled (1)J(θ)(CH)-values obtained from the (13)C multiplets to assess the LG efficiency and scaling factor. The use of homonuclear decoupling during proton evolution, LG-HETCOR-LG, can provide J-values, at least in the rare spin dimension, and allows measurements in less mobile membrane environments. The LG-decoupled spectroscopic approach is demonstrated on pure dioleoylphosphatidylcholine (DOPC) membranes and used to investigate lipid mixtures of DOPC/cholesterol and DOPC/cholesterol/sphingomyelin.     

Application of a 1H δ‐resolved 2D NMR experiment to the visualization of enantiomers in chiral environment,using sample spatial encoding and selective echoes

We present the application of a 2D broadband homodecoupled proton NMR experiment to the visualization of enantiomers. In a chiral environment, the existence of diastereoisomeric intermolecular interactions can yield—generally slight—variations of proton chemical shifts from one enantiomer to another. We show that this approach, which relies on a spatial encoding of the NMR sample, is particularly well suited to the analysis of enantiomeric mixtures, since it allows, within one single 2D experiment, to detect subtle chemical shift differences between enantiomers, even in the presence of several couplings. This sequence, which uses semiselective radio‐frequency (rf) pulses combined to a z‐field gradient pulse, produces different selective echoes in various parts of the sample. The resulting homonuclear decoupling provides an original δ‐resolved spectrum along the diagonal of the 2D map where it becomes possible to probe the chiral differentiation process through every proton site where the resulting variation in the chemical shift is detectable. We discuss the advantages and drawbacks of this approach, regarding other experiments which provide homodecoupled proton spectra. This methodology is applied to the observation of enantiomers of (1) ( ± )2‐methyl‐isoborneol coordinated to europium (III) tris[3‐(trifluoromethyl‐hydroxymethylene)‐(+)‐camphorate] in isotropic solution, and (2) ( ± )3‐butyn‐2‐ol dissolved in a chiral liquid‐crystal solvent, in order to show the robustness of this pulse sequence for a wide range of chiral samples. Copyright © 2009 John Wiley & Sons, Ltd.     

Heteronuclear two-bond correlation: suppressing heteronuclear three-bond or higher NMR correlations while enhancing two-bond correlations even for vanishing 2J(CH)

A novel two-dimensional NMR pulse sequence, H2BC, for long-range correlation of 1H and 13C nuclei is presented. The experiment has several attractive features compared to the widely used HMBC experiment, for example, (a) typically strong enhancement of correlations over two bonds while suppressing those over more bonds, that is, resolving ambiguities in standard HMBC spectra and showing two-bond correlations not present in HMBC spectra, (b) independence of long-range 1H-13C coupling constants, (c) full homo- and heteronuclear decoupling in the indirect dimension and heteronuclear decoupling in the acquisition dimension, (d) pure 2D absorption peak shapes, and (e) a pulse sequence duration significantly shorter than that of HMBC. The experiment is quite complementary to HMBC and does not effect correlations to quaternary carbons that must be obtained by HMBC.     

Real‐Time Band‐Selective Homonuclear Proton Decoupling for Improving Sensitivity and Resolution in Phase‐Sensitive J‐Resolved Spectroscopy

Real‐time band‐selective homonuclear ¹H decoupling during data acquisition of z‐filtered J‐resolved spectroscopy produces ¹H‐decoupled ¹H NMR spectra and leads to sensitivity enhancement and improved resolution, and thus aids the measurement of J couplings and residual dipolar couplings in crowded regions of ¹H NMR spectrum. High quality spectra from peptides, organic molecules, and also from enantiomers dissolved in weakly aligned chiral media are reported.     

Proton-selective 17O-1H distance measurements in fast magic-angle-spinning solid-state NMR spectroscopy for the determination of hydrogen bond lengths

We present a proton-selective method to determine 17O-1H distances in organic, biological, and biomimetic materials by fast magic-angle-spinning solid-state NMR spectroscopy. This method allows the determination of internuclear distances between specific (17O, 1H) spin pairs selectively. It enables the estimation of medium-range 17O...1H distances across hydrogen bonds in the presence of short-range 17O-1H contacts sharing the same 17O site. The method employs the newly developed symmetry-based radiofrequency pulse sequence SR%@mt;sys@%4%@sx@%1%@be@%2%@sxx@%%@mx@% applied to the protons to achieve heteronuclear dipolar recoupling, while simultaneously decoupling the homonuclear proton dipolar interactions. Fast MAS (50 kHz) and high static magnetic fields (18.8 T) achieve the required proton spectral resolution.     

Real‐time homonuclear broadband and band‐selective decoupled pure‐shift ROESY

Unambiguous spectral assignments in ¹H solution‐state NMR are central, for accurate structural elucidation of complex molecules, which is often hampered by signal overlap, primarily because of scalar coupling multiplets, even at typical high magnetic fields. The recent advances in homodecoupling methods have shown powerful means of achieving high resolution pure‐shift ¹H spectra in 1D and also in 2D J‐correlated experiments, by effectively collapsing the multiplet structures. The present work extends these decoupling strategies to through‐space correlation experiments as well and describes two new pure‐shift ROESY pulse schemes with homodecoupling during acquisition, viz., homodecoupled broadband (HOBB)‐ROESY and homodecoupled band‐selective (HOBS)‐ROESY. Furthermore, the ROESY blocks suppress the undesired interferences of TOCSY cross peaks and other offsets. Despite the reduced signal sensitivity and prolonged experimental times, the HOBB‐ROESY is particularly useful for molecules that exhibit an extensive scalar coupling network spread over the entire ¹H chemical shift range, such as natural/synthetic organic molecules. On the other hand, the HOBS‐ROESY is useful for molecules that exhibit well‐separated chemical shift regions such as peptides (NH, Hα and side‐chain protons). The HOBS‐ROESY sensitivities are comparable with the conventional ROESY, thereby saves the experimental time significantly. The power of these pure‐shift ROESY sequences is demonstrated for two different organic molecules, wherein complex conventional ROE cross peaks are greatly simplified with high resolution and sensitivity. The enhanced resolution allows deriving possibly more numbers of ROEs with better accuracy, thereby facilitating superior means of structural characterization of medium‐size molecules. Copyright © 2014 John Wiley & Sons, Ltd.