Structural Elucidation of Agrochemicals and Related Derivatives Using Infrared Ion Spectroscopy.

Agrochemicals frequently undergo various chemical and metabolic transformation reactions in the environment that often result in a wide range of derivates that must be comprehensively characterized to understand their toxicity profiles and their persistence and outcome in the environment. In the development phase, this typically involves a major effort in qualitatively identifying the correct chemical isomer(s) of these derivatives from the many isomers that could potentially be formed. Liquid chromatography-mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy are often used in attempts to characterize such environment transformation products. However, challenges in confidently correlating chemical structures to detected compounds in mass spectrometry data and sensitivity/selectivity limitations of NMR frequently lead to bottlenecks in identification. In this study, we use an alternative approach, infrared ion spectroscopy, to demonstrate the identification of hydroxylated derivatives of two plant protection compounds (azoxystrobin and benzovindiflupyr) contained at low levels in tomato and spinach matrices. Infrared ion spectroscopy is an orthogonal tandem mass spectrometry technique that combines the sensitivity and selectivity of mass spectrometry with structural information obtained by infrared spectroscopy. Furthermore, IR spectra can be computationally predicted for candidate molecular structures, enabling the tentative identification of agrochemical derivatives and other unknowns in the environment without using physical reference standards.

Recent developments in the use of fluorine NMR in synthesis and characterisation.

A review of developments in fluorine NMR of relevance to synthesis, characterisation and industrial applications of small organic molecules. Developments considered include those in spectrometer technology, computational methods and pulse sequences. The review of 80 references outlines applications in areas of identification, quantitation, mixture analysis, reaction monitoring, environmental studies and fragment-based drug design.

Is perfection ever attainable? Strong coupling effects in the Perfect Echo.

The Perfect Echo sequence, originally proposed in the late 1980s, has recently been popularised with many applications in the field of small-molecule proton NMR spectroscopy. The Perfect Echo refocuses all homonuclear J-couplings for AX spin systems and refocuses magnetization in-phase for more complex weakly coupled spin systems, albeit with some intensity reduction. In contrast to suggestions in previous publications, spectra acquired in our laboratory showed that the Perfect Echo caused intensity distortions in strongly coupled systems where the chemical shift difference between the coupled spins was not large compared to the J-coupling. This paper reports experimental observations and theoretical analysis of strongly coupled spins to confirm the distortions are real and that they originate principally from transfer of magnetization caused by the final inversion pulse of the Perfect Echo. The intensity changes are not large, but because of them, identifications of coupling partners based on resonance intensities ("roofing") can no longer be relied on when the Perfect Echo is used. However, theory and experiment confirm that adding an orthogonal excitation pulse at the end of the Perfect Echo greatly reduces the distortions.

Suppression of Protonated Organic Solvents in NMR Spectroscopy Using a Perfect Echo Low-Pass Filtration Pulse Sequence.

Proton NMR spectra are usually acquired using deuterated solvents, but in many cases it is necessary to obtain spectra on samples in protonated solvents. In these cases, the intense resonances of the protonated solvents need to be suppressed to maximize sensitivity and spectral quality. A wide range of highly effective solvent suppression methods have been developed, but additional measures are needed to suppress the 13C satellites of the solvent. Because the satellites represent 1.1% of the original solvent signal, they remain problematic if unsuppressed. The recently proposed DISPEL pulse sequences suppress 13C satellites extremely effectively, and this Technical Note demonstrates that combining DISPEL and presaturation results in exceptionally effective solvent suppression. An important element in the effectiveness is volume selection, which is inherent within the DISPEL sequence. Spectra acquired in protonated dimethlysulfoxide and tetrahydrofuran show that optimum results are obtained by modifying the phase cycle, cycling the pulse-field gradients, and using broadband 13C inversion pulses to reduce the effects of radiofrequency offset and inhomogeneity.

A qualitative exploration of anesthesia trainees' experiences during transition to a children's hospital.

BACKGROUND: The stresses of starting a new job can make anyone feel tired and inefficient. In health care, this may impair the ability to learn at a time when there is most to learn, and increase the risk of error in a context where errors may lead to patient harm. AIM: The aim of this study was to understand issues which influence anesthesia trainees' transition to a pediatric setting. METHODS: This qualitative study utilized in-depth semi-structured interviews to gather data from 31 anesthesia trainees who had commenced work at a tertiary children's hospital between 4 and 6 weeks previously. Data were examined using thematic analysis. RESULTS: Two key themes were identified: feeling ineffective, which appeared to have both a cognitive component (feeling disoriented) and an emotional component (feeling useless), and feeling anxious or afraid. Trainees found the pediatric environment highly unfamiliar, which made them feel disoriented, inefficient, and at times incompetent. Many experienced difficulty identifying a useful role in a highly specialized area of practice, leading to loss of identity as an expert clinician. Many described an ever-present fear of making an anesthetic error or being unable to manage a rapidly evolving clinical situation. Some trainees developed a negative mindset, which was reinforced by subsequent perceived failures. Overall, these experiences impeded trainees' ability to concentrate and learn. CONCLUSIONS: The impact of disorientation and anxiety on anesthesia trainees as they adapt to a highly specialized clinical environment such as a children's hospital should not be underestimated. Study findings illustrate the importance of helping new trainees to feel less afraid, more useful, and more realistic in assessing their own performance during the transition period.

Selective diffusion spectroscopy using excitation sculpting.

Diffusion spectroscopy NMR provides a sensitive and fast way of determining diffusion coefficient. The coefficient is measured by fitting attenuation of resonance intensities to the Stejskal-Tanner equation, but, because it is an exponential equation, this fitting is quite sensitive to experimental artefacts. Intense resonances in NMR spectra, such as solvent signals, are a particular problem because small fractions of intensity of the intense resonances can significantly change the intensities of minor resonances and distort their calculated diffusion coefficients. This problem can be overcome by selective excitation of only the minor resonances, and the method of choice for this is excitation sculpting. This paper shows that the conventional excitation sculpting pulse sequence can be used directly for DOSY spectroscopy with minimal modification. The sequence suppresses intense resonances very effectively, allowing the reproducible measurement of diffusion coefficients of minor solutes in a solvent containing five separate resonances. It is sensitive and robust to convection. Excitation sculpting will allow the application of diffusion spectroscopy to samples which include multiple intense resonances, such as polymeric solvents. Copyright © 2015 John Wiley & Sons, Ltd.

The potential utility of predicted one bond carbon-proton coupling constants in the structure elucidation of small organic molecules by NMR spectroscopy.

NMR spectroscopy is the most popular technique used for structure elucidation of small organic molecules in solution, but incorrect structures are regularly reported. One-bond proton-carbon J-couplings provide additional information about chemical structure because they are determined by different features of molecular structure than are proton and carbon chemical shifts. However, these couplings are not routinely used to validate proposed structures because few software tools exist to predict them. This study assesses the accuracy of Density Functional Theory for predicting them using 396 published experimental observations from a diverse range of small organic molecules. With the B3LYP functional and the TZVP basis set, Density Functional Theory calculations using the open-source software package NWChem can predict one-bond CH J-couplings with good accuracy for most classes of small organic molecule. The root-mean-square deviation after correction is 1.5 Hz for most sp3 CH pairs and 1.9 Hz for sp2 pairs; larger errors are observed for sp3 pairs with multiple electronegative substituents and for sp pairs. These results suggest that prediction of one-bond CH J-couplings by Density Functional Theory is sufficiently accurate for structure validation. This will be of particular use in strained ring systems and heterocycles which have characteristic couplings and which pose challenges for structure elucidation.

Fluorine-proton correlation from isolated trifluoromethyl groups using unresolved J-couplings.

Fluorine-containing compounds are rare in biological systems, so fluorine NMR spectroscopy can selectively detect and quantify fluorinated xenobiotics in crude biological extracts. The high sensitivity of fluorine NMR allows the detection of compounds containing isolated trifluoromethyl groups at nanogramme levels. However, it only provides limited structural information about trifluoromethyl-containing compounds owing to the difficulty of interpreting fluorine chemical shifts and the low sensitivity of HOESY experiments used to correlate fluorine nuclei with protons in the same compound. This paper demonstrates that long-range fluorine-proton J-couplings can be used to correlate isolated trifluoromethyl groups with nearby protons with significantly higher sensitivity than HOESY. Fluorine-observe fluorine-proton HMQC can even give correlations when the fluorine-proton J-couplings are less than the observed fluorine resonance linewidth, so it provides a useful alternative source of structural information about fluorinated xenobiotics.

Blood loss and replacement for paediatric cranioplasty in Australia - a prospective national audit.

We prospectively audited blood loss and blood replacement in every child less than 24 months of age undergoing cranioplasty for craniosynostosis in Australia during 2008, in order to obtain more accurate data for the discussion of perioperative transfusion risk. A total of 127 cases were performed at seven centres. There were no directed or autologous blood donations. No patient received preoperative erythropoietin. A total of 233 units of homologous red blood cells were transfused. Overall, 83% of patients received a blood transfusion. This included 100% of patients undergoing cranial vault reconstruction (CVR) and 98% of patients undergoing fronto-orbital advancement (FOA), but only 32% of spring cranioplasty patients. Exposure to no more than one donor was achieved in 60% of FOA patients and 36% of CVR patients. Estimated blood volume loss was more than one blood volume in 36% of CVR and 36% of FOA, but only 12% of spring cranioplasty, and more than two blood volumes in 4% of CVR and 11% of FOA. Differences in surgical technique and volume of surgery between different centres appeared to affect transfusion rates. Children with recognised craniofacial syndromes and those undergoing repeat surgery appeared to have greater blood loss and blood product exposure. There were two cases of sudden massive haemorrhage secondary to dural venous sinus tear, but no death or perioperative cardiac arrest. These findings indicate that blood loss requiring blood product replacement is common in patients <24 months of age undergoing cranioplasty for craniosynostosis, particularly in patients undergoing FOA and CVR.

High resolution 13C DOSY: the DEPTSE experiment.

High Resolution Diffusion-ordered Spectroscopy (HR-DOSY) is a valuable tool for mixture analysis by NMR. It separates the signals from different components according to their diffusion behavior, and can provide exquisite diffusion resolution when there is no signal overlap. In HR-DOSY experiments on (1)H (by far the most common nucleus used for DOSY) there is frequent signal overlap that confuses interpretation. In contrast, a (13)C spectrum usually has little overlap, and is in this respect a much better option for a DOSY experiment. The low signal-to-noise ratio is a critical limiting factor, but with recent technical advances such as cryogenic probes this problem is now less acute. The most widely-used pulse sequences for (13)C DOSY perform diffusion encoding with (1)H, using a stimulated echo in which half of the signal is lost. This signal loss can be avoided by encoding diffusion with (13)C in a spin echo experiment such as the DEPTSE pulse sequence described here.

Application of NMR-based metabolomics to the investigation of salt stress in maize (Zea mays).

INTRODUCTION: High salinity, caused by either natural (e.g. climatic changes) or anthropic factors (e.g. agriculture), is a widespread environmental stressor that can affect development and growth of salt-sensitive plants, leading to water deficit, the inhibition of intake of essential ions and metabolic disorders. OBJECTIVE: The application of an NMR-based metabolic profiling approach to the investigation of saline-induced stress in Maize plants is presented. METHODOLOGY: Zea Maize seedlings were grown in either 0, 50 or 150 mM saline solution. Plants were harvested after 2, 4 and 6 days (n = 5 per class and time point) and (1) H NMR spectroscopy was performed separately on shoot and root extracts. Spectral data were analysed and interpreted using multivariate statistical analyses. RESULTS: A distinct effect of time/growth was observed for the control group with relatively higher concentrations of acetoacetate at day 2 and increased levels of alanine at days 4 and 6 in root extracts, whereas concentration of alanine was positively correlated with the shoot extracts harvested at day 2 and trans-aconitic acid increased at days 4 and 6. A clear dose-dependent effect, superimposed on the growth effect, was observed for saline treated shoot and root extracts. This was correlated with increased levels of alanine, glutamate, asparagine, glycine-betaine and sucrose and decreased levels of malic acid, trans-aconitic acid and glucose in shoots. Correlation with salt-load shown in roots included elevated levels of alanine, γ-amino-N-butyric acid, malic acid, succinate and sucrose and depleted levels of acetoacetate and glucose. CONCLUSIONS: The metabolic effect of high salinity was predominantly consistent with osmotic stress as reported for other plant species and was found to be stronger in the shoots than the roots. Using multivariate data analysis it is possible to investigate the effects of more than one environmental stressor simultaneously.

Compensating for variation in 1J(CH) coupling constants in HSQC spectra acquired on small organic molecules.

The HSQC sequence provides a sensitive way of determining the (13)C chemical shift of protonated carbons. It uses INEPT elements for magnetization transfer, which can only be optimized for one value of (1)J(CH), but small organic molecules contain a wide range of (1)J(CH) values. One popular method of compensating for (1)J(CH) variation is to incorporate adiabatic pulses into the INEPT elements. This article shows that this method fails for a significant subset of functional groups. It also shows that the effects of this failure can be reduced by avoiding refocusing delays and by using a J-compensated excitation element.

Indirect determination of chemical shift by coupling evolution during adiabatic pulses.

The use of adiabatic 180 degrees X-pulses within INEPT refocusing periods results in chemical shift-dependent evolution of J-couplings. This has been viewed as a disadvantage and several methods of overcoming it have been suggested. This article shows that there is the potential to use this chemical shift dependence to determine heteronuclear chemical shift without a heteronuclear evolution time. In this way, it possible to estimate heteronuclear chemical shift indirectly from a single one-dimensional proton-observe spectrum and determine it with high accuracy from a extensively-folded two-dimensional proton-observe spectrum.

Removal of zero-quantum peaks from 1D selective TOCSY and NOESY spectra.

Selective 1D TOCSY and NOESY experiments are widely used for structure determination. However, they often give distorted peak shapes owing to coherence transfer though zero-quantum coherence (ZQ) which cannot be suppressed by conventional phase cycling or pulse-field gradients. This paper demonstrates that ZQ contributions can be removed from selective 1D spectra by introducing a ZQ evolution time, as previously demonstrated for 2D NOESY spectra by Wang et al. [A three-dimensional method for the separation of zero-quantum coherence and NOE in NOESY spectra, J. Magn. Reson. A 102 (1993) 116-121]. This approach is simple to implement and robust, and is not demanding of spectrometer hardware. Using a new approach to phase cycling described here, spectra can be acquired in a similar time to spectra without a ZQ evolution time.

Isolation and characterisation of a class of carbohydrate oxidases from higher plants, with a role in active defence.

In a search for novel plant-derived antimicrobial proteins, we screened extracts from salicylic acid (SA)-treated lettuce and sunflower leaves. These extracts displayed very potent antimicrobial activity against a set of phytopathogens. Characterisation of these extracts revealed that in both extracts, proteins of approximately 60 kDa were responsible for the antimicrobial activity. Further characterisation of these proteins and cloning of the respective cDNAs revealed close homology to a range of (plant) oxidases. Dissection of the enzymatic activity of both proteins revealed them to be carbohydrate oxidases (Helianthus annuus carbohydrate oxidase (Ha-CHOX) and Lactuca sativa carbohydrate oxidase (Ls-CHOX)) with broad substrate specificity and with hydrogen peroxide (H(2)O(2)) as one of the reaction products. The sunflower transcript, in addition to being SA inducible, was also inducible by fungal pathogens but not by ethylene and jasmonate. To determine whether Ha-CHOX plays a role in pathogen defence, it was transformed into tobacco and the effect of resistance to Pectobacterium carotovorum ssp. carotovorum was examined. Transgenic plants overexpressing Ha-CHOX displayed enhanced resistance to infection by this pathogen, and the resistance level was proportional to enzyme expression.

A straight-forward method of optimising protein solubility for NMR.

Maximising solubility is a key step in applying solution-state NMR to proteins. The 'microbatch' crystallisation screening method can be adapted to screen for protein solubility. In this approach, drops of test solutions are placed under paraffin oil in 96-well screening plates. This requires very small amounts of protein, is easy to set up and is readily automatable.