Spin state selective coherence transfer: a method for discrimination and complete analyses of the overlapped and unresolved 1H NMR spectra of enantiomers

In general, the proton NMR spectra of chiral molecules aligned in the chiral liquid crystalline media are broad and featureless. The analyses of such intricate NMR spectra and their routine use for spectral discrimination of R and S optical enantiomers are hindered. A method is developed in the present study which involves spin state selective two dimensional correlation of higher quantum coherence to its single quantum coherence of a chemically isolated group of coupled protons. This enables the spin state selective detection of proton single quantum transitions based on the spin states of the passive nuclei. The technique provides the relative signs and magnitudes of the couplings by overcoming the problems of enantiomer discrimination, spectral complexity and poor resolution, permitting the complete analyses of the otherwise broad and featureless spectra. A non-selective 180 degrees pulse in the middle of MQ dimension retains all the remote passive couplings. This accompanied by spin selective MQ-SQ conversion leads to spin state selective coherence transfer. The removal of field inhomogeneity contributes to dramatically enhanced resolution. The difference in the cumulative additive values of chemical shift anisotropies and the passive couplings, between the enantiomers, achieved by detecting Nth quantum coherence of N magnetically equivalent spins provides enhanced separation of enantiomer peaks. The developed methodology has been demonstrated on four different chiral molecules with varied number of interacting spins, each having a chiral centre.     

Binuclear spin state selective detection of 1H single quantum transitions using triple quantum coherence: a novel method for enantiomeric discrimination

In the present work a novel methodology is developed for the unambiguous discrimination of enantiomers aligned in chiral liquid crystalline media and the simultaneous determination of 1H-1H and 13C-1H couplings in a single experiment. An INEPT transfer and back transfer of magnetization to protons retain the 13C edited 1H magnetization which is utilized to generate spin selective homonuclear triple quantum coherence of dipolar coupled methyl protons. Spin selective correlation of triple quantum to single quantum coherence results in spin state selective detection by 13C spin and the remaining passive protons. The difference between the successive transitions in the triple quantum dimension pertains to sum of the passive couplings and results in enhanced resolution by a factor of three. This results in unambiguous chiral visualization. The masked 13C satellite transitions in the single quantum spectrum are extracted for chiral discrimination. The technique retains all the passive homo- and heteronuclear couplings in the triple quantum dimension by the application of non-selective refocusing pulse on 1H as well as on 13C spins. This, however, refocuses the chemical shift evolution in the triple quantum dimension, and also overcomes the problem of field inhomogeneity. The method enables the determination of spectral information which is otherwise not possible to derive from the broad and featureless proton spectra. The elegant experimental technique has been demonstrated on different chiral molecules.     

Band selective small flip angle COSY: a simple experiment for the analyses of 1H NMR spectra of small chiral molecules

The NMR spectroscopic discrimination of enantiomers in the chiral liquid crystalline solvent is more often carried out using ²H detection in its natural abundance. The employment of ¹H detection for such a purpose is severely hampered due to significant loss of resolution in addition to indistinguishable overlap of the spectra from the two enantiomers. This study demonstrates that the band selected small flip angle homonuclear correlation experiment is a simple and robust technique that provides unambiguous discrimination, very high spectral resolution, reduced multiplicity of transitions, relative signs of the couplings and enormous saving of instrument time.     

Selective detection of single-enantiomer spectrum of chiral molecules aligned in the polypeptide liquid crystalline solvent: Transition selective one-dimensional 1H–1H COSY

One-dimensional (1D) proton NMR spectra of enantiomers are generally undecipherable in chiral orienting poly-γ-benzyl-l-glutamate (PBLG)/CDCl₃ solvent. This arises due to large number of couplings, in addition to superposition of spectra from both the enantiomers, severely hindering the ¹H detection. On the other hand in the present study the benefit is derived from the presence of several couplings among the entire network of interacting protons. Transition selective 1D ¹H–¹H correlation experiment (1D-COSY) which utilizes the coupling assisted transfer of magnetization not only for unraveling the overlap but also for the selective detection of enantiopure spectrum is reported. The experiment is simple, easy to implement and provides accurate eanantiomeric excess in addition to the determination of the proton–proton couplings of an enantiomer within a short experimental time (few minutes).     

Discerning the degenerate transitions of scalar coupled H NMR spectra: Correlation and resolved techniques at higher quantum

The blend of spin topological filtering and the spin state selective detection of single quantum transitions by the two dimensional multiple quantum-single quantum correlation and higher quantum resolved techniques have been employed for simplifying the complexity of scalar coupled ¹H NMR spectra. The conventional two dimensional COSY and TOCSY experiments, though identify the coupled spin networks, fail to differentiate them due to severe overlap of transitions. Non-selective excitation of homonuclear higher quantum of protons results in filtering of spin systems irrespective of their spin topologies. The spin state selection by passive ¹⁹F spins provides fewer transitions in each cross section of the single quantum dimension simplifying the analyses of the complex spectra. The degenerate single quantum transitions are further discerned by spin selective double and/or triple quantum resolved experiments that mimic simultaneous heteronuclear and selective homonuclear decoupling in the higher quantum dimension. The techniques aided the determination of precise values of spectral parameters and relative signs of the couplings.     

柚皮素7-O-葡萄糖苷非对映异构体的NMR波谱分析

二氢黄酮糖苷化后产生的R与S构型非对映异构体在¹H NMR谱上会呈现一些差别,但文献对其差别描述非常有限.为便于利用¹H NMR谱图判断二氢黄酮糖苷的R与S构型非对映异构体,本文首先在植物药皂荚提取物中分离得到一种二氢黄酮苷-柚皮素7-O-葡萄糖苷R与S构型混合物,分析其氘代二甲亚砜（DMSO-d₆）溶液的¹H NMR、¹³C NMR、¹H-¹H COSY、¹H-¹³C HSQC和¹H-¹³C HMBC谱,对其¹H和¹³C NMR谱峰进行了归属;然后,采用手性色谱柱对该混合物进行分离,结合圆二色光谱（CD）技术确定构型;最后,为鉴别R与S构型柚皮素7-O-葡萄糖苷在¹H NMR谱中特征差别谱峰,避免葡萄糖残基质子对二氢黄酮苷元质子化学位移的影响,采集了R与S构型柚皮素7-O-葡萄糖苷及其混合物氘代乙腈（CD₃CN）溶液的NMR谱,结果显示葡萄糖残基端基质子H-1″化学位移差别最为明显,为9.4 Hz;5-位酚羟基质子化学位移差别为5.8 Hz,C环上3个质子化学位移差也较明显．     

High resolution heteronuclear correlation NMR spectroscopy between quadrupolar nuclei and protons in the solid state

A high resolution two-dimensional solid state NMR experiment is presented that correlates half-integer quadrupolar spins with protons. In this experiment the quadrupolar nuclei evolve during t1 under a split-t1, FAM-enhanced MQMAS pulse scheme. After each t1 period ending at the MQMAS echo position, single quantum magnetization is transferred, via a cross polarization process in the mixing time, from the quadrupolar nuclei to the protons. High-resolution proton signals are then detected in the t2 time domain during wPMLG5* homonuclear decoupling. The experiment has been demonstrated on a powder sample of sodium citrate and 23Na-1H 2D correlation spectra have been obtained. From the HETCOR spectra and the regular MQMAS spectrum, the three crystallographically inequivalent Na+ sites in the asymmetric unit were assigned. This MQMAS-wPMLG HETCOR pulse sequence can be used for spectral editing of half-integer quadrupolar nuclei coupled to protons.     

Resolution enhancement in spectra of natural products dissolved in weakly orienting media with the help of 1H homonuclear dipolar decoupling during acquisition: application to 1H-13C dipolar couplings measurements

In weakly orienting media such as poly-gamma-benzyl-L-glutamate (PBLG) a polymer that forms a chiral liquid crystal in organic solvents, the spectral resolution for embedded molecules is usually poor because of numerous (1)H, (1)H dipolar couplings that generally broaden proton spectra. Therefore (1)H, (13)C dipolar couplings are difficult or impossible to measure. Here, we incorporate Flip-Flop decoupling during detection into an HSQC experiment. Flip-Flop removes the (1)H, (1)H dipolar couplings and scales the chemical shifts of the protons as well as the (1)H, (13)C dipolar couplings during detection. A resolution gain by a factor 1.5-4.2 and improved signal intensity by an average factor of 1.6-1.7 have been obtained. This technique is demonstrated on (+)-menthol dissolved in a PBLG/CDCl(3) phase.     

Application of z-COSY experiment and its variant for accurate chiral discrimination by 1H NMR

We report the application of z-COSY experiment and a band selected version of it by employing a selective 90° pulse entitled BASE-z-COSY for precise chiral discrimination, quantification of enantiomeric excess and the analyses of the ¹H NMR spectra of chiral molecules aligned in the chiral liquid crystalline solvent poly-γ-benzyl-l-glutamate (PBLG). We have demonstrated their applicability for obtaining very high resolution in the ¹H NMR spectra of small organic molecules. It is well known that the commonly employed z-COSY experiment disentangles the spectral complexity, provides pure phase spectra with high resolution, aids in the complete spectral analyses, in addition to yielding information on relative signs of the couplings. The BASE-z-COSY experiment possesses all these properties, permits the measure of enantiomeric excess, in addition to large saving of instrument time.     

Visualisation of enantiomers via insertion of a BIRD module in X-H correlation experiments in chiral liquid crystal solvent

Several 13C-1H NMR techniques are derived simplifying the visualisation of enantiomers in chiral ordering solvents. They proceed through various heteronuclear 2D experiments where a bilinear rotation decoupling sequence (BIRD) is inserted in the middle of the t1 evolution period. In this way, the small couplings are refocused while the large couplings are preserved. The methods allow extracting precise values of one-bond carbon-proton residual dipolar couplings for each enantiomer out of unresolved proton-coupled 13C or carbon-coupled 1H spectra. Illustrative examples are analysed and discussed using various pulse sequences.     

Dipolar interactions in molecules aligned by strong AC electric fields

We observed magnetization transfer and spectroscopic splittings due to dipolar couplings in the solution NMR spectra of neat nitrobenzene aligned using AC electric fields. Weak dipolar splittings have been previously observed for nitrobenzene in a DC electric field (T. M. Plantenga, et al., Chem. Phys. 66, 1-9, 1982); the use of homogeneous pulsed AC fields has allowed us to establish stable experimental conditions, which were more tolerable to sample impurities and required no sample purification, and to carry out multidimensional experiments. A pulse sequence is discussed in which the electric field is present only for the indirect dimension: this sequence records the dipolar splittings for each proton in the indirect dimension; the direct dimension presents the isotropic chemical shift. Another pulse sequence is discussed that uses the applied electric field only in the mixing period to produce cross peaks between dipolar coupled pairs and correlate their isotropic chemical shifts. The order parameter describing molecular alignment was in good agreement with that previously determined from deuterium quadrupolar measurements of deuterated nitrobenzene in a similar range of electric fields: S(mol) approximately 0.025% for a field strength of 7.0 MV/m (rms). The dipolar splittings for ortho-meta, meta-para, and ortho-para protons were in qualitative agreement with the known geometry. Copyright 2000 Academic Press.     

Off-angle correlation spectroscopy applied to spin-1/2 and quadrupolar nuclei

A two-dimensional correlation experiment is described, in which homonuclear dipolar couplings are used to realize through-space magnetization exchange on spin-1/2 (³¹P) and on quadrupolar nuclei (²³Na and ¹¹B). In the detection period, Magic Angle Spinning is applied to enhance resolution, and the dipole couplings are re-introduced in the mixing period by spinning off the Magic Angle. The dependency of the exchange rates on the mixing time and the spinning angle is investigated. The influence of strong spin-locking during mixing is discussed, and shown in the spin-1/2 case to remove the dependence on chemical shift offset effects. For quadrupolar spins, the experiment yields information on the relative tensor orientations of the coupled quadrupoles. Applications to crystalline sodium aluminum diphosphate, sodium sulphite, and potassium borate glasses are shown.     

Application of biselective refocusing soft pulses to the simplification of heteronuclear correlation spectra

The biselective spin echo technique allows the signals of coupled proton pairs to be extracted from crowded liquid state proton NMR spectra. Its use as a preparation sequence in heteronuclear chemical shift correlation experiments requires the removal of the heteronuclear coupling interaction during the biselective echo time. The discrimination between coupled and uncoupled protons signals is achieved by double quantum filtration, which delivers antiphase magnetization states. The latter are not directly compatible with the design of an HSQC-like pulse sequence. The conversion of antiphase to in-phase magnetization states by a second biselective echo sequence solves this problem. The optimization of spin echo delays is also discussed. Lastly, the article presents modified HSQC and HMBC pulses sequences in which information is obtained solely for the biselectively selected proton pairs. A peracetylated trisaccharide was used as a test molecule.     

Selection rules for multiple quantum NMR excitation in solids: derivation from time-reversal symmetry and comparison with simulations and (13)C NMR experiments.

New derivations of selection rules for excitation and detection of multiple quantum coherences in coupled spin-1/2 systems are presented. The selection rules apply to experiments in which the effective coupling Hamiltonian used for multiple quantum excitation is both time-reversal invariant and time-reversible by a phase shift of the radiofrequency pulse sequence that generates the effective couplings. The selection rules are shown to be consequences of time-reversal invariance and time-reversibility and otherwise independent of the specific form of the effective coupling Hamiltonian. Numerical simulations of multiple quantum NMR signal amplitudes and experimental multiple quantum excitation spectra are presented for the case of a multiply (13)C-labeled helical polypeptide. The simulations and experiments confirm the selection rules and demonstrate their impact on multiple quantum (13)C NMR spectra in this biochemically relevant case.     

Selection Rules for Multiple Quantum NMR Excitation in Solids: Derivation from Time-Reversal Symmetry and Comparison with Simulations and C NMR Experiments

New derivations of selection rules for excitation and detection of multiple quantum coherences in coupled spin-1/2 systems are presented. The selection rules apply to experiments in which the effective coupling Hamiltonian used for multiple quantum excitation is both time-reversal invariant and time-reversible by a phase shift of the radiofrequency pulse sequence that generates the effective couplings. The selection rules are shown to be consequences of time-reversal invariance and time-reversibility and otherwise independent of the specific form of the effective coupling Hamiltonian. Numerical simulations of multiple quantum NMR signal amplitudes and experimental multiple quantum excitation spectra are presented for the case of a multiply ¹³C-labeled helical polypeptide. The simulations and experiments confirm the selection rules and demonstrate their impact on multiple quantum ¹³C NMR spectra in this biochemically relevant case.     

Residual 2H quadrupolar couplings in weakly aligned carbohydrates.

We report a novel two-dimensional NMR pulse scheme for the 1H-detected observation of 2H in isotopically 13C, 2H-enriched carbohydrates. This scheme is used for the indirect observation of residual quadrupolar couplings in 13C, 2H-enriched methyl-beta-D-glucopyranoside weakly aligned in a dilute lyotropic liquid-crystalline medium comprising 20% (w/v) dihexanoyl-phosphatidylcholine/dimyristoyl-phosphatidylcholine (1:3 mol/mol) in D2O. The observed residual quadrupolar couplings are substantially larger than residual dipolar one-bond 13C-1H couplings under the same experimental conditions. These quadrupolar couplings are thus a useful alternative to dipolar couplings for the structural analysis of small molecules that align very weakly in dilute liquid-crystalline media. Moreover, since the quadrupolar coupling constant is very uniform throughout endocyclic deuterons of the carbohydrate, these data suggest that adoption of a single average value of this parameter in 2H relaxation studies on the glycan moieties of glycoproteins and glycopeptides is a valid assumption.     

Capture and manipulation of magnetically aligned Pf1 with an aqueous polymer gel

The magnetic alignment of the Pseudomonas bacteriophage Pf1 is captured indefinitely in a gel of the aqueous triblock copolymer Pluronic F-127. In addition to preserving high-resolution liquids NMR spectra for dissolved solutes, the gel prevents the reorientation of the phage allowing mechanical manipulation of the angle between the axis of the phage alignment and the static magnetic field. The residual 2H quadrupolar couplings for several solutes dissolved in this material as a function of the angle Theta between the non-spinning sample tube and the static magnetic field are consistent with the value of P(2)(cosTheta)=(3cos(2)Theta-1)/2. The variable-angle correlation spectrum for these solutes is shown to separate residual quadrupolar effects from isotropic chemical shifts. Finally, the compatibility of Pluronic F-127 with NMR studies of aqueous biological macromolecules is demonstrated in a measurement of residual dipolar couplings in an 15N-labeled nucleic acid.     

Evidence for a dipolar-coupled AM system in carnosine in human calf muscle from in vivo 1H NMR spectroscopy

Spin systems with residual dipolar couplings such as creatine, taurine, and lactate in skeletal muscle tissue exhibit first-order spectra in in vivo 1H NMR spectroscopy at 1.5 T because the coupled protons are represented by (nearly) symmetrized eigenfunctions. The imidazole ring protons (H2, H4) of carnosine are suspected to form also a coupled system. The ring's stiffness could enable a connectivity between these anisochronous protons with the consequence of second-order spectra at low field strength. Our purpose was to study whether this deviation from the Paschen-Back condition can be used to detect the H2-H4 coupling in localized 1D 1H NMR spectra obtained at 1.5 T (64 MHz) from the human calf in a conventional whole-body scanner. As for the hydrogen hyperfine interaction, a Breit-Rabi equation was derived to describe the transition from Zeeman to Paschen-Back regime for two dipolar-coupled protons. The ratio of the measurable coupling strength (Sk) and the difference in resonance frequencies of the coupled spins (Deltaomega) induces quantum-state mixing of various degree upon definition of an appropriate eigenbase of the coupled spin system. The corresponding Clebsch-Gordan coefficients manifest in characteristic energy corrections in the Breit-Rabi formula. These additional terms were used to define an asymmetry parameter of the line positions as a function of Sk and Deltaomega. The observed frequency shifts of the resonances were found to be consistent with this parameter within the accuracy achievable in in vivo NMR spectroscopy. Thus it was possible to identify the origin of satellite peaks of H2, H4 and to describe this so far not investigated type of residual dipolar coupling in vivo.     

A simulation algorithm based on Bloch equations and product operator matrix: application to dipolar and scalar couplings

A product operator matrix is proposed to describe scalar couplings in liquid NMR. Combination of the product operator matrix and non-linear Bloch equations is employed to describe effects of chemical shift, translational diffusion, dipolar field, radiation damping, and relaxation in multiple spin systems with both scalar and dipolar couplings. A new simulation algorithm based on this approach is used to simulate NMR signals from dipolar field effects in the presence of scalar couplings. Several typical coupled spin systems with both intra-molecular scalar couplings and inter-molecular dipolar couplings are simulated. Monte Carlo methods are incorporated into simulations as well to analyze diffusion process in these complicated spin systems. The simulated results of diffusion and relaxation parameters and 2D NMR spectra are coincident with the experimental measurements, and agree with theoretical predictions as well. The simulation algorithm presented herein therefore provides a convenient means for designing pulse sequences and quantifying experimental results in complex coupled spin systems.