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Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM Objectives Nuclear magnetic resonance (NMR) spectroscopy provides a holistic snapshot of the metabolome of an organism. There is a dearth of studies till date that had exploited NMR metabolomic platform to study dermatophytes, despite its potential for rapid identification and subsequent application of the knowledge in performing faster antifungal susceptibility of dermatophytes. Here we attempted to study the frequency of various species of dermatophytes in clinically suspected cases of dermatophytosis and perform NMR-based identification of metabolites in the culture suspensions/cell extracts of T. mentagrophyte and T. rubrum. Methods This was a hospital-based prospective study conducted in the isolates obtained from clinically suspected cases of Dermatophytosis in the patients. Skin, nails, and hair samples of patients suspected with superficial fungal infections were processed for dermatophytes using conventional microbiological methods. NMR-based identification of metabolites was carried out in cell extracts prepared from the culture suspensions of T. mentagrophytes and T. rubrum obtained during the study from a subset of the clinical isolates from the samples. Results Dermatophytes were isolated in 85.88% (219/255) cases, with T. mentagrophyte being isolated in 65% (143/219) of isolates, followed by T. rubrum in 31.5% (69/219) isolates. In NMR study was done in the standard ATCC strains (T. mentagrophyte ATCC9533 and T. rubrum ATCC28188) and representative clinical isolates of both the species. Overall, 24 metabolites were identified in T. rubrum and 23 metabolites in T. mentagrophyte amongst which 22 metabolites were common to both fungus, however, ‘4-hydroxyproline’ and ‘acetate’ was found specific to T. rubrum, and ‘allantoin’ was found specific to T. mentagrophyte. Conclusion T. mentagrophyte was the predmominant dermatophyte species in the study. Amongst the number of metabolites detected in T. rubrum and T. mentagrophyte, ‘4-hydroxyproline’ and ‘acetate’ was found specific to T. rubrum, and ‘allantoin’ was found specific to T. mentagrophyte. These specific metabolites could be useful for as early identification of these dermatophytes as well early determination of antifungal susceptibility by using metabolic endpoints, further large-scale study will be helpful in this regard.

A systematic review was carried out to prepare a checklist of the mammalian species of northeastern India. The region is located between two prominent biodiversity hotspots, Himalaya and Indo-Burma. Though it is only 8% of the country, it supports almost half of the country’s total wild flora and fauna. Ongoing developmental activities such as the construction of roads, electrification, and mining in and around the wildlife habitats have threatened the survival of many species of wildlife. A lot of literature has been checked to understand the status and distribution of wildlife in the region and the present manuscript is prepared from existing literature. A total of 267 species representing 11 orders and 38 families from the region have been reported. A state-wise updated list of species along with their status as per IUCN, WPA (Wildlife Protection Act) of India, 1972 and CITES has been provided which is intended to serve as a baseline data for further research in mammalian fauna of the region.

CDCl3 is the most frequently used solvent for the NMR investigation of organic compounds. Busy chemistry labs need to investigate hundreds of compounds daily. While 1H NMR investigation takes a couple of minutes, recording 13C NMR spectra necessitates hours of signal averaging due to the low abundance and low sensitivity of 13C nuclei. The longer acquisition time for 13C NMR results in a loss of precious spectrometer time in a shared multi-user environment. A regular 5 mm o.d. NMR tube is the most commonly used tube for NMR in organic chemistry labs and is also the cheapest option. We show that for analytes soluble in the CDCl3 solvent using a regular 5 mm o.d. NMR tube, the speed of 13C observation can be enhanced by a factor of two by resorting to a sample preparation method that employs a biphasic system made of H2O or D2O at the top of another layer of CDCl3. By using the biphasic system of two immiscible solvents, the analyte can be concentrated in the CDCl3 layer (within the more sensitive volume of the NMR coil), resulting in the improvement of the signal to noise ratio (SNR) by a factor of up to 1.8 for 13C and 2D 1H–13C HSQC spectra, which results in more than two-fold reduction in the experimental time. 1H NMR and other 2D NMR also get a sensitivity boost. The amount of CDCl3 required for sample preparation can also be reduced by 40% using this biphasic system (CDCl3/H2O). Sample preparation in such an immiscible biphasic system is effortless and straightforward. The performance of such biphasic samples is closer to that of Shigemi tubes and better than that of 3 mm o.d. tubes.

Hepatic encephalopathy (HE) is a debilitating neuropsychiatric complication associated with acute and chronic liver failure. It is characterized by diverse symptoms with variable severity that includes cognitive and motor deficits. The aim of the study is to assess metabolic alterations in the brain and liver using nuclear magnetic resonance (NMR) spectroscopy and subsequent multivariate analyses to characterize metabolic signatures associated with HE. HE was developed by bile duct ligation (BDL) that resulted in hepatic dysfunctions and cirrhosis as shown by liver function tests. Metabolic profiles from control and BDL rats indicated increased levels of lactate, branched-chain amino acids (BCAAs), glutamate, and choline in the liver, whereas levels of glucose, phenylalanine, and pyridoxine were decreased. In brain, the levels of lactate, acetate, succinate, citrate, and malate were increased, while glucose, creatine, isoleucine, leucine, and proline levels were decreased. Furthermore, neurotransmitters such as glutamate and GABA were increased, whereas choline and myo-inositol were decreased. The alterations in neurotransmitter levels resulted in cognitive and motor defects in BDL rats. A significant correlation was found among alterations in NAA/choline, choline/creatine, and NAA/creatine with behavioral deficits. Thus, the data suggests impairment in metabolic pathways such as the tricarboxylic acid (TCA) cycle, glycolysis, and ketogenesis in the liver and brain of animals with HE. The study highlights that metabolic signatures could be potential markers to monitor HE progression and to assess therapeutic interventions.

Limited chemical shift dispersion and broad multiplet patterns limit resolution in

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NMR is a fast method for obtaining a holistic snapshot of the metabolome and also offers quantitative information without separating the compounds present in a complex mixture. Identification of the metabolites present in a plant extract sample is a crucial step for all plant metabolomics studies. In the present work, we used various two dimensional (2D) NMR methods such as J-resolved NMR, total correlation spectroscopy (TOCSY), and heteronuclear single quantum coherence sensitivity enhanced NMR spectroscopy for the identification of 36 common metabolites present in Coriandrum sativum L. seed extract. The identified metabolites belong to the following classes: organic acids, amino acids, and carbohydrates. 1 H NMR spectra of such complex mixtures in general display tremendous signal overlap due to the presence of a large number of metabolites with closely resonating multiplet signals. This signal overlapping leads to ambiguity in an assignment, and hence, identification of metabolites becomes tedious or impossible in many cases. Therefore, the utility of pure-shift proton spectrum along the indirect (F1 ) dimension of the F1 -PSYCHE-TOCSY spectrum is demonstrated for overcoming ambiguity in assignment of metabolites in crowded spectral regions from Coriandrum sativum L. seed extract sample. Because pure-shift NMR methods yield ultrahigh resolution spectrum (i.e., a singlet peak per chemical site) along one or more dimensions, such spectra provide better identification of metabolites compared with regular 2D TOCSY where signal overlap and peak distortions lead to ambiguity in the assignment. Nine metabolites were unambiguously assigned by pure-shift F1 -PSYCHE-TOCSY spectrum, which was unresolved in regular TOCSY spectrum.

Two dimensional (2D) NMR display better resolution than one-dimensional (1D) 1H NMR. However, 2D NMR does not display a straightforward quantitative aspect due to J-dependent polarization/coherence transfer. 1D 1H NMR is versatile for quantification; however, it displays significant spectral overlap in biological or organic complex mixtures, which forbids quantification of a large number of signals in 1D 1H NMR. The significant variations in 1H13C scalar couplings, T1, T2, and pulse imperfections are the main problems. Although T1, T2 can be suitably chosen to minimize their adverse effect on quantification, the large variations in 1H - 13C couplings lead to variations in cross peak intensity, which is more influenced by the amount of polarization transfer rather than the quantity of metabolites or amount of analytes in a complex mixture. In the present work, we show that spatial encoding of the polarization transfer periods can be executed in 1H13C HSQC using sweep frequency pulses in the presence of a magnetic field gradient. As a result, uniform transfer of polarization from 1H to 13C over a range of 1H - 13C couplings can be performed, subsequently improving the quantitative aspect of HSQC or improve the intensity of cross-peaks, which are mistuned in regular HSQC

This investigation reports the analysis of different microstructures present in poly(methyl methacrylate) (PMMA) using1D and 2D NMR spectroscopy. The PMMA used in this study has been prepared via activators regenerated by electron transfer atom transfer radical polymerization (ARGET-ATRP) in DMF at ambient temperature using methyl 2-chloro propionate (MCP) as initiator, CuBr2 as catalyst in combination with N, N, N´, N´´, N´´- pentamethyldiethylenetriamine (PMDETA) as ligand and ascorbic acid as reducing agent.13C NMR, which provides good information about tacticity of a polymer, has been used extensively along with 1H NMR spectroscopy to extract the information about the different microstructures present in PMMA. Heteronuclear multiple-bond correlation (HMBC), heteronuclear single-quantum coherence (HSQC), and total correlation spectroscopy (TOCSY) were also used to study the position of protons, couplings with carbon etc. in the PMMA.

We report, based on biophysical studies and molecular mechanical calculations that curcumin binds DNA hairpin in the minor groove adjacent to the loop region forming a stable complex. UV–Vis and fluorescence spectroscopy indicated interaction of curcumin with DNA hairpin. In this novel binding motif, two ɣ H of curcumin heptadiene chain are closely positioned to the A16-H8 and A17-H8, while G12-H8 is located in the close proximity of curcumin α H. Molecular dynamics (MD) simulations suggest, the complex is stabilized by noncovalent forces including; π-π stacking, H-bonding and hydrophobic interactions. Nuclear magnetic resonance (NMR) spectroscopy in combination with molecular dynamics simulations indicated curcumin is bound in the minor groove, while circular dichroism (CD) spectra suggested minute enhancement in base stacking and a little change in DNA helicity, without significant conformational change of DNA hairpin structure. The DNA:curcumin complex formed with FdU nucleotides rather than Thymidine, demonstrated enhanced cytotoxicity towards oral cancer cells relative to the only FdU substituted hairpin. Fluorescence co-localization demonstrated stability of the complex in biologically relevant conditions, including its cellular uptake. Acridine orange/EtBr staining further confirmed the enhanced cytotoxic effects of the complex, suggesting apoptosis as mode of cell death. Thus, curcumin can be noncovalently complexed to small DNA hairpin for cellular delivery and the complex showed increased cytotoxicity in combination with FdU nucleotides, demonstrating its potential for advanced cancer therapy.

Recently, pure shift NMR methods have delivered ultrahigh resolution broadband proton NMR spectra that display singlet peak per chemical site. BASH/HOBS (band selective homo-decoupling/homonuclear band selective) decoupling is the only method that provides singlet only spectrum for a selected signal or a group of signals with a sensitivity higher than regular proton NMR, and an order of magnitude higher than broadband pure shift techniques. It is this aspect that makes this technique important. In the present work, we show that perfect echo (PE) when combined with band selective homo-decoupling (BASHD) permits increasing the bandwidth of the BASH/HOBS decoupling resulting in reduced experimental time for this class of experiments. Using new PE-BASHD technique two closely resonating coupled signals could be decoupled in a single experiment which otherwise demands two separate BASHD experiments. In polypeptides, it also allows decoupling of Hα and HN signals simultaneously from each other as well as from the side chain protons reducing experimental time. Further, pseudo 2D based PE-BASHD scheme provides spectrum superior to the real-time BASHD spectrum when applied to closely resonating coupled signals. Numerical simulation as well as experimental results indicate that the PE-BASHD sequence display good quality singlet only spectrum even in the presence of moderate strong coupling.

A method is demonstrated for parallel acquisition of several slice selective soft COSY proton spectra. Application of a slice selective mixing pulse in a selective correlation experiment allows slice selective coherence transfer between different coupled spin pairs. During such slice selective coherence transfer, the spin states of the passive spins are undisturbed. In other words, slice selective coherence transfer executes spin-state selective coherence transfer between a given spin and all its coupled neighbours. This results in a final spectrum which contains multiple soft cosy spectra between a given signal(s) and all its coupled signals, significantly reducing experimental time. This provides access to all the couplings for a given proton site and its coupled partners.

A diagonal suppressed F1 decoupled total correlation spectroscopy(TOCSY) experiment is developed for analyses of complex mixtures. In 2D homonuclear correlation, assignment of the cross peaks is crucial for structure elucidation. However, when cross peaks are close to the diagonal peaks in overcrowded spectral regions, their assignment becomes tedious. In complex mixtures, the presence of multiple spectra along with broad and complex proton multiplets owing to homonuclear scalar couplings degrade the resolution to the extent that assignment of various cross peaks becomes tedious or impossible. Herein, a diagonal suppressed total correlation technique with F1 decoupling is presented to improve the resolution of the cross peaks. The resolution of the cross peaks is improved by both diagonal suppression as well as the collapse of the multiplets to singlets. Application of the method to a few mixtures of organic compounds reveals better identification of the cross peaks relative to other TOCSY variants.

Organic chemistry labs routinely perform NMR in a standard 5 mm NMR tube. NMR sample is prepared by filling the lowermost 4 cm length of a regular 5 mm 0.d. tube that holds approximately 0.55 ml of a deuterated solvent. This is actually a sample dilution procedure as the signal mainly comes from the central part, i.e. 1.8 cm length sample that fits the typical coil length of 1.8 cm in regular NMR spectrometers. The diluted top and bottom part of the sample is away from the coil and contributes less signal. This dilution procedure amplifies the requirement of expensive deuterated solvent and lowers sensitivity. The present study explores a new way of sample preparation which involves sandwiching a small amount of the deuterated solvent (D2O) of length 1.8 cm containing the analyte between two non-deuterated solvents (CCl4 at bottom of length ≈ 1 cm and similarly C6H6 at top), which are immiscible with the former using a regular 5 mm o.d. NMR tube in such a way that total length is still 4 cm as demanded by the magnetic field homogeneity considerations. The analyte now being closer to NMR coil dissolved in 1.8 cm solvent, improves sensitivity and reduces deuterated solvent hitherto required. 13C and 15N spectra from such a set-up display two to three-fold higher signal to noise ratio and hence four to eightfold savings in experimental time or faster data collection.

Two-dimensional J-RESolved spectroscopy (J-RES) finds routine use in metabolomics for reducing signal overlap as it separates chemical shift and multiplet information along two frequency axes. However, only magnitude mode of the experiment is practical which prevents exploitation of its full resolving power. Tailing from high-intensity metabolite peaks often obscure nearby low-intensity metabolite peaks which leads to ambiguity in assignment of metabolites. Absorptive mode J-RES spectroscopy offers better-resolving power but comes at the cost of either sensitivity or complicated post-processing. Quite often for certain complex mixtures such as bio-fluids some components of the mixture display intense singlet signals which dominate the whole spectrum resulting in less reliable detection of weaker metabolite signals. Multi-frequency presaturation could suppress these intense singlets but will also remove the useful weaker multiplet peaks which are either totally eclipsed with the intense singlets or very close in frequency. We show that by using a double quantum filter (DQF) in magnitude mode J-RES technique, the intensity of the strong singlet metabolite peaks can be reduced relative to the intensity of the sparsely present multiplet metabolite signals. This approach leads to the identification of many weak intensity multiplet peaks which are otherwise undetected due to their overlap with intense singlet peaks in regular J-RES as well as 1D 1H spectra. Although the improved intensity of most of the weaker peaks relative to the strong singlet peaks is observed, some multiplets can disappear due to the delay-dependent modulation of the signals by the DQF. A few DQF J-RES spectra recorded with different DQF delays, therefore, produce better assignment when analyzed together. The technique is demonstrated on a mixture of eight compounds, human urine, and plant extract samples.

Homonuclear correlated spectroscopy such as COSY and TOCSY provides crucial structural information. In all homonuclear correlation, the most intense peaks are represented by the diagonal. As a result, the useful cross peaks close to the diagonal get obscured by the huge tails of diagonal peaks. Herein, we show that by editing the proton magnetization by a 13C nucleus in natural abundance, it is possible to eliminate the inphase coherence or untransferred magnetization that leads to the diagonal peak while retaining the antiphase coherence or transferred magnetization required for creation of cross peak. After the coherence transfer step, the untransferred magnetization directly attached to 13C evolves under one bond heteronuclear coupling while the transferred transverse magnetization directly attached to remote 12C does not. As a result, the untransferred magnetization directly attached to 13C can be converted to an unobservable heteronuclear multiple quantum coherence leading to a diagonal free correlated spectrum with a sensitivity penalty of two orders of magnitude but comparable to HSQC kind of experiments at natural abundance. The method demonstrated for COSY and TOCSY allows all proton-proton correlations to be observed except the geminal proton-proton correlations. Further, protons directly attached to heteronuclei other than 13C must be scalar coupled to protons directly attached to 13C to have a detectable cross peak.

Enantiodiscrimination and their quantification using nuclear magnetic resonance (NMR) spectroscopy has always been a subject of great interest. Proton is the nucleus of choice for enantiodiscrimination due to its high sensitivity and ubiquitous presence in nature. Despite its advantages, enantiodiscrimination suffers from extensive signal splitting by the proton-proton scalar couplings, which give complex multiplets that spread over a frequency range of some tens of hertz. These multiplets often overlap, further complicating interpretation of the spectra and quantifications. In the present review, we discuss some of the recent developments in the pure shift 1 H NMR based methods for enantiomer resolution and enantiodiscrimination. We also compare various pure shift methods used for enantiodiscrimination and measurement of enantiomeric excess, considering the fact that conventional 1 H NMR fails to provide any detailed insight.

A method is demonstrated for parallel acquisition of several slice selective soft COSY proton spectra. Application of a slice selective mixing pulse in a selective correlation experiment allows slice selective coherence transfer between different coupled spin pairs. During such slice selective coherence transfer, the spin states of the passive spins are undisturbed. In other words, slice selective coherence transfer executes spin-state selective coherence transfer between a given spin and all its coupled neighbours. This results in a final spectrum which contains multiple soft cosy spectra between a given signal(s) and all its coupled signals, significantly reducing experimental time. This provides access to all the couplings for a given proton site and its coupled partners.

A diagonal suppressed F

The heteronuclear multiple-quantum coherence in the indirect dimension of the two-dimensional HMQC experiment evolves under the passive 1H-1H J-couplings leading to multiplet structures in the F1 dimension. Besides, 1H-1H J-multiplets appear in the direct dimension as well. Thus, multiplets along both dimensions lower the resolution and sensitivity of this technique, when high resolution is required along both dimensions. An efficient broadband homodecoupling scheme along the F1 dimension of the HMQC experiment has not been realized to date. We have implemented broadband homonuclear decoupling using bilinear rotation decoupling (BIRD) by adding a 1H SQ evolution period followed by BIRD before the 1H-13C multiple-quantum evolution period in the HMQC. In the direct time domain, BIRD is implemented using a real-time or single-scan scheme, which further improves resolution and sensitivity of this technique. The resulting pure shift HMQC provides singlet peak per chemical site along F1 as well as F2 axes and, hence, better resolution and sensitivity than conventional HMQC spectrum for all peaks except diastereotopic methylene protons. Due to the incorporation of the BIRD, the indirect time domain becomes double in length compared to the conventional HMQC. However, slow relaxation of small molecules favors better sensitivity for ps-HMQC relative to conventional HMQC under all conditions. We also found that the sensitivity of ps-HMQC is only slightly less than ps-HSQC for small molecules.

A “perfect echo” based INEPT experiment that demonstrates more efficient heteronuclear polarization transfer over conventional INEPT has been developed. This scheme refocuses the effect of homonuclear 1 H– 1 H J -evolution and simultaneously allows heteronuclear 13 C– 1 H J -evolution to continue during INEPT. This improves one bond heteronuclear polarization transfer efficiency at longer INEPT transfer delays and also enhances the sensitivity of long range INEPT. The refocusing of homonuclear 1 H– 1 H J -coupling could be achieved by doubling the INEPT transfer period leading to a doubling of T 2 losses. Therefore, the sensitivity gain is observed when loss of magnetization due to homonuclear 1 H– 1 H J -modulation is more severe than that of T 2 decay. However, in general, INEPT transfer period is rather short compared to the longer T 2 observed in small molecules. The long range PE-INEPT transfer to carbonyl carbon in beta-butyrolactone, showed much faster build up of C-13 signal than conventional long range INEPT as the long range heteronuclear J -coupling is comparable in magnitude to homonuclear 1 H- 1 H J -coupling in this case. For one bond heteronuclear polarization transfer at shorter delay, PE-INEPT and conventional INEPT displays equal transfer efficiency. Efficient polarization transfer is observed for small molecules dissolved in isotropic as well as weakly aligned media. Further, simulation results obtained using the full propagator and product operator analysis agree well with the experimental observations.

Traumatic brain injury (TBI) has been shown to affect hippocampus-associated learning, memory and higher cognitive functions, which may be a consequence of metabolic alterations. Hippocampus-associated disorders may vary depending on the severity of injury [mild TBI (miTBI) and moderate TBI (moTBI)] and time since injury. The underlying hippocampal metabolic irregularities may provide an insight into the pathological process following TBI. In this study, in vivo and in vitro proton magnetic resonance spectroscopy (

The small chemical shift dispersion and complex multiplicity pattern in proton NMR limit quantifications, for instance the determination of enantiomeric excess (ee) for an enantiomeric mixture. Herein, we present a simple proton-proton correlation experiment with band selective homonuclear (BASH) decoupling in both F

International audience; In a preliminary communication (B. Baishya, T. F. Segawa, G. Bodenhausen, J. Am. Chem. Soc. 2009, 131, 17538-17539), we recently demonstrated that it is possible to obtain clean echo decays of protons in biomolecules despite the presence of homonuclear scalar couplings. These unmodulated decays allow one to determine apparent transverse relaxation rates R(2) (app) of individual protons. Herein, we report the observation of R(2) (app) for three methyl protons, four amide H(N) protons, and all 11 backbone H(α) protons in cyclosporin A. If the proton resonances overlap, their R(2) (app) rates can be measured by transferring their magnetization to neighboring (13)C nuclei, which are less prone to overlap. The R(2) (app) rates of protons attached to (13)C are faster than those attached to (12)C because of (13)C-(1)H dipolar interactions. The differences of these rates allow the determination of local correlation functions. Backbone H(N) and H(α) protons that have fast decay rates R(2) (app) also feature fast longitudinal relaxation rates R(1) and intense NOESY cross peaks that are typical of crowded environments. Variations of R(2) (app) rates of backbone H(α) protons in similar amino acids reflect differences in local environments.

The routine use of proton NMR for the visualization of enantiomers, aligned in the chiral liquid crystal solvent poly-gamma-benzyl-l-glutamate (PBLG), is restricted due to severe loss of resolution arising from large number of pair wise interaction of nuclear spins. In the present study, we have designed two experimental techniques for their visualization utilizing the natural abundance (13)C edited selective refocusing of single quantum (CH-SERF) and double quantum (CH-DQSERF) coherences. The methods achieve chiral discrimination and aid in the simultaneous determination of homonuclear couplings between active and passive spins and heteronuclear couplings between the excited protons and the participating (13)C spin. The CH-SERF also overcomes the problem of overlap of central transitions of the methyl selective refocusing (SERF) experiment resulting in better chiral discrimination. Theoretical description of the evolution of magnetization in both the sequences has been discussed using polarization operator formalism.

NMR spectroscopic discrimination of optical enantiomers is most often carried out using $^2H$ and $^{13}C$ spectra of chiral molecules aligned in a chiral liquid crystalline solvent. The use of proton NMR for such a purpose is severely hindered due to the spectral complexity and the significant loss of resolution arising from numerous short- and long-distance couplings and the indistinguishable overlap of spectra from both R and S enantiomers.The determination of all the spectral parameters by the analyses of such intricate NMR spectra poses challenges,such as, unraveling of the resonances for each enantiomer, spectral resolution, and simplification of the multiplet pattern. The present study exploits the spin state selection achieved by the two-dimensional $^1H$ NMR correlation of selectively excited isolated coupled spins (Soft-COSY) of the molecules to overcome these problems. The experiment provides the relative signs and magnitudes of all of the proton-proton couplings, which are otherwise not possible to determine from the broad and featureless one-dimensional $^1H$ spectra. The utilization of the method for quantification of enantiomeric excess has been demonstrated. The studies on different chiral molecules, each having a chiral center, whose spectral complexity increases with the increasing number of interacting spins, and the advantages and limitations of the method over SERF and DQ-SERF experiments have been reported in this work.

The differential values of NMR spectral parameters like chemical shift anisotropies, dipolar couplings and quadrupolar couplings of enantiomers in chiral liquid crystalline media are employed not only for their visualization but also for their quantification. Large differences in chemical shift anisotropies and the quadrupolar couplings between the enantiomers enable the use of 13C and extensive 2H NMR detection for such a purpose. In spite of high magnetic moment, high sensitivity and abundant presence of protons in all the chiral molecules, 1H detection is not routinely employed due to severe overlap of unresolved transitions arising from short and long distance couplings. Furthermore, the doubling of the spectra from two enantiomers and their indistinguishable overlap due to negligible difference in chemical shift anisotropies hampers their discrimination. The present study demonstrates the use of proton chemical shift anisotropy as an exclusive parameter for such a discrimination. The method employs the non-selective excitation of homonuclear Nth quantum coherence of N coupled protons. The simultaneous flipping of all the coupled spins results in a single transition in the multiple quantum dimension at the cumulative sum of their anisotropic chemical shifts for each enantiomer, with the measurable difference between them, resulting in their complete unraveling.

NMR spectra of molecules oriented in liquid-crystalline matrix provide information on the structure and orientation of the molecules. Thermotropic liquid crystals used as an orienting media result in the spectra of spins that are generally strongly coupled. The number of allowed transitions increases rapidly with the increase in the number of interacting spins. Furthermore, the number of single quantum transitions required for analysis is highly redundant. In the present study, we have demonstrated that it is possible to separate the subspectra of a homonuclear dipolar coupled spin system on the basis of the spin states of the coupled heteronuclei by multiple quantum (MQ)-single quantum (SQ) correlation experiments. This significantly reduces the number of redundant transitions, thereby simplifying the analysis of the complex spectrum. The methodology has been demonstrated on the doubly 13C labeled acetonitrile aligned in the liquid-crystal matrix and has been applied to analyze the complex spectrum of an oriented six spin system.

“Pure shift” NMR spectra display singlet peak per chemical site. Thus, high resolution is offered at the cost of valuable J-coupling information. In the present work, real-time BIRD (BIlinear Rotation Decoupling) is applied to the absorptive-mode 2D J-spectroscopy to provide pure shift spectrum in the direct dimension and J-coupling information in the indirect dimension. Quite often in metabolomics, proton NMR spectra from complex bio-fluids display tremendous signal overlap. Although conventional J-spectroscopy in principle overcomes this problem by separating the multiplet information from chemical shift information, however, only magnitude mode of the experiment is practical, sacrificing much of the potential high resolution that could be achieved. Few J-spectroscopy methods have been reported so far that produce high-resolution pure shift spectrum along with J-coupling information for crowded spectral regions. In the present work, high-quality J-resolved spectrum from important metabolomic mixture such as tissue extract from rat cortex is demonstrated. Many low-intensity metabolite peaks which are obscured by the broad dispersive tails from high-intensity metabolite peaks in regular magnitude mode J-spectrum can be clearly identified in real-time BIRD J-resolved spectrum. The general practice of removing such spectral overlap is tedious and time-consuming as it involves repeated sample preparation to change the pH of the tissue extract sample and subsequent spectra recording.

Real-time band-selective homonuclear (1) H decoupling during data acquisition of z-filtered J-resolved spectroscopy produces (1) H-decoupled (1) H 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 (1) H NMR spectrum. High quality spectra from peptides, organic molecules, and also from enantiomers dissolved in weakly aligned chiral media are reported.

Traumatic brain injury (TBI) has been shown to affect hippocampus-associated learning, memory and higher cognitive functions, which may be a consequence of metabolic alterations. Hippocampus-associated disorders may vary depending on the severity of injury [mild TBI (miTBI) and moderate TBI (moTBI)] and time since injury. The underlying hippocampal metabolic irregularities may provide an insight into the pathological process following TBI. In this study, in vivo and in vitro proton magnetic resonance spectroscopy (1 H-MRS) data were acquired from the hippocampus region of controls and TBI groups (miTBI and moTBI) at D0 (pre-injury), 4 h, Day 1 and Day 5 post-injury (PI). In vitro MRS results indicated trauma-induced changes in both miTBI and moTBI; however, in vivo MRS showed metabolic alterations in moTBI only. miTBI and moTBI showed elevated levels of osmolytes indicating injury-induced edema. Altered levels of citric acid cycle intermediates, glutamine/glutamate and amino acid metabolism indicated injury-induced aberrant bioenergetics, excitotoxicity and oxidative stress. An overall similar pattern of pathological process was observed in both miTBI and moTBI, with the distinction of depleted N-acetylaspartate levels (indicating neuronal loss) at 4 h and Day 1 and enhanced lactate production (indicating heightened energy depletion leading to the commencement of the anaerobic pathway) at Day 5 in moTBI. To the best of our knowledge, this is the first study to investigate the hippocampus metabolic profile in miTBI and moTBI simultaneously using in vivo and in vitro MRS.

Zero-Quantum artifacts seriously degrade the performance of 2D NOESY. Homonuclear J-evolution during t(1) period generates Zero-Quantum and other higher quantum coherences which represent the magnetization loss and the artifacts created. We demonstrate that creation of such artifacts itself can be prevented for shorter t1 period by a perfect echo based decoupling technique during t1 period in a single scan. This is in contrast to existing methods that create unwanted coherence, and subsequently suppress that to produce a clean spectrum with a sensitivity penalty. Although decoupling performance of the present scheme remains robust for echo time 2τ short compared to 1/2J, we show that even a partial decoupling effect for extended t(1) (=2τ) period up to 100 ms along with a Zero-Quantum filter generates NOE spectrum from Cyclosporine A, in which majority of the cross peaks displayed partial sensitivity enhancement with few exceptions. However, in crowded proton spin systems like menthol, the enhancements were not observed and perfect echo NOESY displays similar performance as Zero-Quantum filtered NOESY.

Recent studies described an "ultrafast" scanning method based on spatiotemporal (SPEN) principles. SPEN demonstrates numerous potential advantages over EPI-based alternatives, at no additional expense in experimental complexity. An important aspect that SPEN still needs to achieve for providing a competitive ultrafast MRI acquisition alternative, entails exploiting parallel imaging algorithms without compromising its proven capabilities. The present work introduces a combination of multi-band frequency-swept pulses simultaneously encoding multiple, partial fields-of-view, together with a new algorithm merging a Super-Resolved SPEN image reconstruction and SENSE multiple-receiving methods. This approach enables one to reduce both the excitation and acquisition times of sub-second SPEN acquisitions by the customary acceleration factor R, without compromises in either the method's spatial resolution, SAR deposition, or capability to operate in multi-slice mode. The performance of these new single-shot imaging sequences and their ancillary algorithms were explored and corroborated on phantoms and human volunteers at 3 T. The gains of the parallelized approach were particularly evident when dealing with heterogeneous systems subject to major T2/T2* effects, as is the case upon single-scan imaging near tissue/air interfaces.

Homonuclear 1 H– 1 H J -modulation leads to J -multiplets in F 1 dimension of 2D 1 H– 13 C HMQC spectra. This hampers unambiguous signal assignment for overcrowded 13 C spectra. Broadband homonuclear decoupling has been achieved in the indirect t 1 evolution period by incorporating blocks of perfect echo. This method of perfect echo HMQC demonstrates better resolution and sensitivity than conventional HMQC spectra. The results on Cyclosporine demonstrate that the method is very efficient for refocusing geminal couplings in weakly coupled – 13 CH 2 groups. Partial refocusing of vicinal couplings is also observed for – 13 CH and – 13 CH 3 groups. Interpretation of the result based on product operator formalism is also given. Comparison of pe-HMQC, HMQC and HSQC reveals that the F 1 linewidth of pe-HMQC is much narrower than HMQC and very close to that of HSQC for CH 2 groups.

The transverse relaxation rates R 2 = 1/T 2 of protons can be determined by spin-echo sequences with multiple refocusing pulses using moderate radio-frequency field strengths and properly chosen inter-pulse delays so as to suppress echo modulations due to homonuclear scalar couplings. Combination with 2D heteronuclear correlation spectroscopy (HSQC) allows one to measure R 2 of arbitrary protons attached to nitrogen-15 or carbon-13 nuclei. Decays of six amide protons in the protein Ubiquitin that is nitrogen-15 enriched (but not deuterated) were measured at different temperatures.

International audience ; Trains of 2π or 4π pulses fail to refocus offsets but can suppress the effects of bilinear interactions such as homonuclear scalar couplings.

The 1 H NMR spectra of partially aligned molecules become rapidly complex with the increase in the number of interacting spins and the decrease in the symmetry of the molecules. In the strongly orienting thermotropic liquid crystals, the analyses of the complex spectra are very challenging due to their second-order character. The numerical iterative calculations that are generally employed for such analyses are tedious and time consuming. On the other hand, in weakly ordering media such as bicelles or in chiral liquid crystal solvent poly-γ-benzyl- l -glutamate (PBLG), the spin systems are weakly coupled and the first-order analysis is generally possible. However, for the chiral molecules aligned in the chiral liquid crystal medium, the 1 H NMR spectra are not only complex, but also broad and featureless due to large number of pair-wise interactions of nuclear spins resulting in degenerate or near-degenerate transitions, in addition to an indistinguishable overlap of the spectra of enantiomers. This enormous loss of resolution severely hampers the analyses of proton spectra, even for spin systems with five to six interacting protons, thereby restricting its routine application. In this chapter, we report the diverse methods available to circumvent the difficulties in the analyses of such spectra, in both strongly and weakly orienting media. The discussion is devoted to recent methodological developments in the context of spectral simplification, chiral discrimination, and the discerning of the degenerate transitions.

The proton NMR spectra of fluorine-substituted benzamides are very complex (Figure 1) due to severe overlaP of ${^1}H$ resonances from the two aromatic rings, in addition to several short and long-range scalar couplingS experienced by each proton. With no detectable scalar couplings between the inter-ring spins, the ${^1}H$ NMR spectra can be construed as an overlap of spectra from two independent phenyl rings. In the present study we demonstrate that it is possible to separate the individual spectrum for each aromatic ring by spin system filtering employing the multiple-quantum-single-quantum correlation methodology. Furthermore, the two spin states of fluorine are utilized to simplify the spectrum corresponding to each phenyl ring by the spinstate selection. The demonstrated technique reduces spectral complexity by a factor of 4, in addition to permitting the determination of long-range couplings of less than 0.2 Hz and the relative signs of heteronuclear couplings. The technique also aids the judicious choice of the spin-selective double-quantum-Single-quantum J-resolved experiment to determine the long-range homonuclear couplings of smaller magnitudes. filtering employing the multiple-quantum-single-quantum correlation methodology. Furthermore, the two spin states of fluorine are utilized to simplify the spectrum corresponding to each phenyl ring by the spinstate selection. The demonstrated technique reduces spectral complexity by a factor of 4, in addition to permitting the determination of long-range couplings of less than 0.2 Hz and the relative signs of heteronuclear couplings. The technique also aids the judicious choice of the spin-selective double-quantum-single-quantum J-resolved experiment to determine the long-range homonuclear couplings of smaller magnitudes.

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.

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 $^{13}C-^1H$ couplings in a single experiment. An INEPT transfer and back transfer of magnetization to protons retain the $^{13}C$ 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 $^{13}C$ 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 $^{13}C$ 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 $^{13}C$ 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.

The differences in chemical shift anisotropies, dipolar couplings, and quadrupolar couplings of two enantiomers in the chiral liquid crystalline media are employed to visualize enantiomers. In spite of the fact that proton has high magnetic moment and is abundantly present in all the chiral molecules, $^1H$ NMR is not exploited to its full potential because of severe overlap of unresolved transitions arising from long- and short-distance couplings. Furthermore, the two spectra from R and S enantiomers result in doubling of the number of observable transitions. The present study demonstrates the application of the selectively excited homonuclear double quantum (DQ) coherence correlated to its single quantum coherence of an isolated methyl group in a chiral molecule. The DQ dimension retains only the passive couplings within the protons of the methyl group while the long-distance passive couplings are refocused, removing the overlap of central transitions, and each enantiomer displays a doublet instead of a triplet unlike in regular selective refocusing experiment. The doublet separation being different for each enantiomer results in their discrimination. The cross section taken along the single quantum dimension pertaining to each transition in the DQ dimension provides the one-dimensional spectra for each individual enantiomer with the complete removal of the overlapped transitions from the other enantiomer. The experiment is robust, the pulse sequence is easy to implement, and the methodology has been demonstrated on different chiral molecules.

The spectra of molecules oriented in liquid crystalline media are dominated by partially averaged dipolar couplings. In the $^{13}C-^1H$ HSQC, due to the inefficient hetero-nuclear dipolar decoupling in the indirect dimension, normally carried out by using a \pi pulse, there is a considerable loss of resolution. Furthermore, in such strongly orienting media the $^1H-^1H$ and $^{13}C-^1H$ dipolar couplings leads to fast dephasing of transverse magnetization causing inefficient polarization transfer and hence the loss of sensitivity in the indirect dimension. In this study we have carried out $^{13}C-^1H$ HSQC experiment with efficient polarization transfer from $^1H$ to $^{13}C$ for molecules aligned in liquid crystalline media. The homonuclear dipolar decoupling using FFLG during the INEPT transfer delays and also during evolution period combined with the \pi pulse heteronuclear decoupling in the $t_1$ period has been applied. The studies showed a significant reduction in partially averaged dipolar couplings and thereby enhancement in the resolution and sensitivity in the indirect dimension. This has been demonstrated on pyridazine and pyrimidine oriented in the liquid crystal. The two closely resonating carbons in pyrimidine are better resolved in the present study compared to the earlier work [H.S. Vinay Deepak, Anu Joy, N. Suryaprakash, Determination of natural abundance $^{15}N-^1H$ and $^{13}C-^1H$ dipolar couplings of molecules in a strongly orienting media using two-dimensional inverse experiments, Magn. Reson. Chem. 44(2006) 553–565].

International audience ; The modulation of spin echoes by homonuclear scalar couplings render the determination of transverse relaxation rates of individual spins difficult, in particular for molecules that are isotopically enriched in (13)C or (15)N, and for all molecules with scalar-coupled protons. To avoid echo modulations, most studies using refocusing pulses have so far been restricted to isolated (1)H, (13)C, or (15)N spins. We report measurements of apparent (1)H transverse relaxation rates of backbone and side-chain protons in Cyclosporin A (CsA) determined by quenching the echo modulations that arise from homonuclear scalar couplings between protons.