NMR Tube Research Articles

A mixing microfluidic chip for real-time NMR monitoring of macromolecular reactions

NMR spectroscopy permits real-time monitoring of reactions that involve changes in the spectra of reactants. MICCS (MIcro Channelled Cell for Synthesis monitoring) is a microfluidic chip for such purposes, which is used to rapidly activate reactions by mixing the reactant solutions in the chip inserted into the typical NMR tube. Although it allows monitoring of chemical reactions of small compounds, its simple mixing system dependent on diffusion in the microchannel was not suitable for macromolecules such as proteins with low diffusion rates. Here, we developed a new microfluidic chip based on MICCS by incorporating a mixer of split-and-recombination type within the microchannel. We applied it to monitoring of the protein-folding reaction in a stopped-flow mode. A solution of denaturant-unfolded RNase A was injected from a syringe pump into the microchip set inside the NMR magnet and mixed with a buffer for dilution to reach the folding condition. Immediately after dilution, the reaction was initiated and detected by a series of NMR measurements that were synchronized with activation and inactivation of the pump. The process was repeated for accumulation of the data. By analysing the change of the spectra by factor analysis, a kinetic constant of 0.57 min-1 was obtained.

Reactions of Styrylmalonates with Aromatic Aldehydes: Detailed Synthetic and Mechanistic Studies.

Reactions of styrylmalonates with aromatic aldehydes in the presence of Lewis and Brønsted acids and their regularities have been studied in detail. Approaches to the synthesis of various polysubstituted 5,6-dihydropyran-2-ones, indenes, aryl-containing dienes and trienes, cyclopentadienes, and polycyclic lactones have been developed with chemo-, regio-, stereo-, and diastereoselectivity control. The mechanisms of these reactions were studied in depth using multinuclear NMR experiments, monitoring the reactions in the NMR tube, and 18O isotope labeling.

Clinical-Scale Production of Nearly Pure (>98.5%) Parahydrogen and Quantification by Benchtop NMR Spectroscopy.

Because of the extensive chemical, physical, and biomedical applications of parahydrogen, the need exists for the development of highly enriched parahydrogen in a robust and efficient manner. Herein, we present a parahydrogen enrichment equipment which substantially improves upon the previous generators with its ability to enrich parahydrogen to >98.5% and a production rate of up to 4 standard liters per minute with the added advantage of real-time quantification. Our generator employs a pulsed injection system with a 3/16 in. outside diameter copper spiral tubing filled with iron-oxide catalyst. This tubing is mated to a custom-made copper attachment to provide efficient thermal coupling to the cold head. This device allows for robust operation at high pressures up to 34 atm. Real-time quantification by benchtop NMR spectroscopy is made possible by direct coupling of the p-H2 outlet from the generator to a 1.4 T NMR spectrometer using a regular 5 mm NMR tube that is continuously refilled with the exiting parahydrogen gas at ∼8 atm pressure. The use of high hydrogen gas pressure offers two critical NMR signal detection benefits: increased concentration and line narrowing. Our work presents a comprehensive description of the apparatus for a convenient and robust parahydrogen production, distribution, and quantification system, especially for parahydrogen-based hyperpolarization NMR research.

Low Volume in Vitro Diagnostic Proton NMR Spectroscopy of Human Blood Plasma for Lipoprotein and Metabolite Analysis: Application to SARS-CoV-2 Biomarkers.

The utility of low sample volume in vitro diagnostic (IVDr) proton nuclear magnetic resonance (1H NMR) spectroscopic experiments on blood plasma for information recovery from limited availability or high value samples was exemplified using plasma from patients with SARS-CoV-2 infection and normal controls. 1H NMR spectra were obtained using solvent-suppressed 1D, spin–echo (CPMG), and 2-dimensional J-resolved (JRES) spectroscopy using both 3 mm outer diameter SampleJet NMR tubes (100 μL plasma) and 5 mm SampleJet NMR tubes (300 μL plasma) under in vitro diagnostic conditions. We noted near identical diagnostic models in both standard and low volume IVDr lipoprotein analysis (measuring 112 lipoprotein parameters) with a comparison of the two tubes yielding R2 values ranging between 0.82 and 0.99 for the 40 paired lipoprotein parameters samples. Lipoprotein measurements for the 3 mm tubes were achieved without time penalty over the 5 mm tubes as defined by biomarker recovery for SARS-CoV-2. Overall, biomarker pattern recovery for the lipoproteins was extremely similar, but there were some small positive offsets in the linear equations for several variables due to small shimming artifacts, but there was minimal degradation of the biological information. For the standard untargeted 1D, CPMG, and JRES NMR experiments on the same samples, the reduced signal-to-noise was more constraining and required greater scanning times to achieve similar differential diagnostic performance (15 min per sample per experiment for 3 mm 1D and CPMG, compared to 4 min for the 5 mm tubes). We conclude that the 3 mm IVDr method is fit-for-purpose for quantitative lipoprotein measurements, allowing the preparation of smaller volumes for high value or limited volume samples that is common in clinical studies. If there are no analytical time constraints, the lower volume experiments are equally informative for untargeted profiling.

Application of the highly sensitive labeling reagent to the structural confirmation of readily isomerizable peptides.

Thioamycolamide A (1) is a biosynthetically unique cytotoxic cyclic microbial lipopeptide that bears a D-configured thiazoline, a thioether bridge, a fatty acid side chain, and a reduced C-terminus. It has gained attention for its unique structure, and very recently we reported the total synthesis of 1 via a biomimetic route. The NMR spectra of synthetic 1 agreed with those of natural 1. However, structural identity between peptidic natural and synthetic compounds is often difficult to confirm by comparison of NMR spectra because their NMR spectra vary depending on the conditions in the NMR tube, which often result in the structural misassignment of peptidic compounds. Especially, our total synthesis based on the putative biomimetic route potentially gives 1 as a diastereomixture at the final step. The problem is that the diastereomers of peptidic mid-sized molecules often exhibit similar properties (such as NMR spectra and bioactivities), and their separation procedures are often laborious. Herein we report the structural confirmation of synthetic 1 by the LC-MS-based chromatographic comparison with the use of our highly sensitive labeling reagent L-FDVDA; the highly sensitive-advanced Marfey's method (HS-advanced Marfey's method). This work demonstrated the utility of our highly sensitive labeling reagent for the structural determination of not only scarce natural products but also readily isomerizable synthetic compounds.

NMR-based metabolomics with enhanced sensitivity.

NMR-based metabolomics, which emerged along with mass spectrometry techniques, is the preferred method for studying metabolites in medical research and food industries. However, NMR techniques suffer from inherently low sensitivity, regardless of their superior reproducibility. To overcome this, we made two beneficial modifications: we detuned the probe to reach a position called “Spin Noise Tuning Optimum” (SNTO), and we replaced the conventional cylindrical 5 mm NMR tube with an electric field component-optimized shaped tube. We found that concerted use of both modifications can increase the sensitivity (signal to noise ratio per unit volume) and detection of metabolites and decrease the measurement time by order of magnitude. In this study, we demonstrate and discuss the achieved signal enhancement of metabolites on model non-human (bovine serum, amino acid standard mixture) and human urine samples.

Full Cell Understanding of Solvents in Battery Systems

Lithium ion batteries are complex systems with multiple components that can be optimized to obtain desired performance capability. These components include electrolyte salts, solvents, and additives, as well as electrode active materials and coatings. Rational optimization of battery components requires an understanding of the fundamental science driving the observed performance. Silatronix® has developed a suite of analytical tools and methods using a combination of full format batteries, lab R&D cells, and model systems to construct a complete understanding of the battery from the cell level performance to the fundamental chemistry. This process includes the recovery and analysis of all parts of a full format pouch cell, including liquid electrolyte, generated gas, and electrode surface layers (Fig. 1).Using this process, Silatronix® has evaluated several battery components, including organosilicon solvents for Li-ion electrolytes, to acquire a comprehensive picture of how these components provide observed performance benefits. For example, OS3®, a novel organosilicon chemistry that merges a silane with a Li+ coordinating nitrile functionality, has provided multiple performance benefits in various advanced Li-ion chemistries. Key experimental results include improved high temperature cycling stability in full format graphite/NMC622 pouch cells with 1%, 2%, or 3% OS3® (Fig. 2a), and using a model system of electrolyte in NMR tubes, we show that enhanced thermal stability of the bulk electrolyte results from HF scavenging and LiPF6 salt stabilization when 2-16% OS3® is present (Fig. 2b). Reduction of gas generation during high temperature testing in pouch cells with a commercial Si-based anode is also shown with 2% OS3® (Fig. 2c), and gas product identification and quantification is determined by GC-TCD for mechanistic analyses. In addition, OS materials change the reactivity of other electrolyte components, particularly at the electrolyte/electrode interfaces that govern critical performance attributes in Li-ion cells such as gas generation and interfacial impedance. These key performance benefits serve to enable several next generation battery chemistries, including a strong synergy with Si-based anodes. Figure 1

Acidity-Driven Bidirectional Room-Temperature Spin-State Switch and Fluorescence Modulation of a Mononuclear Fe(II) Complex

Room-temperature switchable materials showing multiple responses toward external stimuli are highly desired. Herein, we report bidirectional spin-state switch and fluorescence modulation of an Fe(I...

An organogel library for solution NMR analysis of nanoparticle suspensions in non-aqueous samples

Surface contrast solution NMR methods (scNMR) are emerging as powerful tools to investigate the adsorption of small molecule ligands to the surface of nanoparticles (NP), returning fundamental insight into the kinetics and thermodynamics of sorption, as well as structural information on the adsorbed species. A prerequisite for the acquisition of high quality solution NMR data is the preparation of homogeneous and stable samples that return consistent NMR spectra and allow extensive signal averaging. Unfortunately, this condition does not apply to NMR samples containing NPs that often show a tendency to sediment and accumulate at the bottom of the NMR tube over the course of the experiment. We have recently shown that preparing NMR samples in an agarose gel matrix inhibits sedimentation and allows the characterization of small molecule-NP interactions by scNMR. Unfortunately, as the agarose gel only forms in aqueous solution, this sample preparation method cannot be used to stabilize NP suspensions in a non-aqueous environment. Here, we introduce a library of 48 organogels, based on low molecular-mass organic gelators (LMOGs), to prepare NMR samples of small molecule/NP systems in a wide range of organic solvents. In addition, we present a simple method that takes advantage of 1H transverse relaxation (1H-R2) measurements to screen the library and identify the best gelator to characterize the small molecule-NP interaction of interest in the solvent of choice. We expect the results of this study will enable the preparation of homogeneous and stable samples of NPs in non-aqueous environments, therefore dramatically increasing the applicability of scNMR to the characterization of heterogeneous interactions and to the investigation of the role played by solvent molecules in regulating the kinetics and thermodynamics of sorption.

Accurate determination of highly volatile chloroethylene by quantitative 1H-NMR

In Japan, chloroethylene is a regulated substance under the soil contamination countermeasures act since 2017, due to the presence of chloroethylene in soil leachate and groundwater. As a consequence, chloroethylene concentrations must be accurately determined to ensure the safety of drinking water, chloroethylene standard solutions with reliable concentrations had not been available because chloroethylene is difficult to handle due to its boiling point of − 13 °C. Here, we developed a quantitative 1H nuclear magnetic resonance (1H-qNMR) method to measure chloroethylene concentration in a standard solution in CD3OD. Sampling temperature must be between − 80 °C and − 21 °C, and the NMR tube headspace volume ratio must be lower than 23% to reduce the volatilization of chloroethylene. We used the signal of CD2HOD present in the standard solution as an internal standard because chloroethylene was volatilized during the addition of another internal standard. The chloroethylene concentration in the standard solution was accurately determined as 117.7 ± 1.4 mg kg−1.

1D 1 H NMR as a Tool for Fecal Metabolomics.

Metabolomic studies allow a deeper understanding of the processes of a given ecological community than nucleic acid-based surveys alone. In the case of the gut microbiota, a metabolic profile of, for example, a fecal sample provides details about the function and interactions within the distal region of the gastrointestinal tract, and such a profile can be generated in a number of different ways. This unit elaborates on the use of 1D 1 H NMR spectroscopy as a commonly used method to characterize small-molecule metabolites of the fecal metabonome (meta-metabolome). We describe a set of protocols for the preparation of fecal water extraction, storage, scanning, measurement of pH, and spectral processing and analysis. We also compare the effects of various sample storage conditions for processed and unprocessed samples to provide a framework for comprehensive analysis of small molecules from stool-derived samples. © 2020 Wiley Periodicals LLC Basic Protocol 1: Extracting fecal water from crude fecal samples Alternate Protocol 1: Extracting fecal water from small crude fecal samples Basic Protocol 2: Acquiring NMR spectra of metabolite samples Alternate Protocol 2: Acquiring NMR spectra of metabolite samples using Bruker spectrometer running TopSpin 3.x Alternate Protocol 3: Acquiring NMR spectra of metabolite samples by semiautomated process Basic Protocol 3: Measuring sample pH Support Protocol 1: Cleaning NMR tubes Basic Protocol 4: Processing raw spectra data Basic Protocol 5: Profiling spectra Support Protocol 2: Spectral profiling of sugars and other complex metabolites.

Chemical Reaction Monitoring using Zero-Field Nuclear Magnetic Resonance Enables Study of Heterogeneous Samples in Metal Containers.

We demonstrate that heterogeneous/biphasic chemical reactions can be monitored with high spectroscopic resolution using zero‐field nuclear magnetic resonance spectroscopy. This is possible because magnetic susceptibility broadening is negligible at ultralow magnetic fields. We show the two‐step hydrogenation of dimethyl acetylenedicarboxylate with para‐enriched hydrogen gas in conventional glass NMR tubes, as well as in a titanium tube. The low frequency zero‐field NMR signals ensure that there is no significant signal attenuation arising from shielding by the electrically conductive sample container. This method paves the way for in situ monitoring of reactions in complex heterogeneous multiphase systems and in reactors made of conductive materials while maintaining resolution and chemical specificity.

2 H SOLCOR: A novel tool for reducing volume variation as a source of error in external standard quantitative NMR.

Tube to tube volume difference presents a challenge in obtaining correct external standard quantitative NMR (esqNMR) results. Deuterium (2 H) NMR is easily observable, intrinsically quantitative, present in all samples, free of interfering signals, and insensitive to probe tune/match and sample saltiness. These properties make 2 H peak integral an ideal parameter in esqNMR for correcting volume differences between the reference standard and analyte. We demonstrate a novel and practical technique abbreviated as "2 H SOLCOR" (2 H SOLvent CORrected), where the 2 H peak integral from the solvent is used as a universal internal standard to correct volume variations in NMR tubes, thereby improving accuracy and precision of esqNMR method. Herein, this simple yet effective technique is described, and practical considerations for successful implementation are presented. 2 H SOLCOR can be applied anywhere esqNMR is used, including where precious samples need to be accurately quantified for qualification as an authentic analytical standard.

A Fast and Reliable Screening Setup for Homogeneous Catalysis with Gaseous Reactants at Extreme Temperatures and Pressures

Modern catalysis research strongly focusses on optimizing multiple reaction parameters en route to find optimal conditions. However, a very limited number of fundamental parameter combinations, such as inert conditions, gaseous reactants, variable temperature, and elevated pressure, can be screened in a convenient and straightforward manner. We report a ready-to-use reaction setup that is designed for screening reactions with gaseous reactants under inert conditions in standard NMR tubes (0.5 mL) using temperatures between −40 and +100 °C and extreme pressures. Proof-of-concept reaction examples are discussed, and the coupling to modern analytic techniques including in situ IR spectroscopy is possible.

SO015URINE NMR-BASED METABOLOMICS FOR EARLY DETECTION OF POST-PROCEDURAL CONTRAST NEPHROPATHY

Abstract Background and Aims Contrast induced nephropathy (CIN) has been reported to be the third foremost cause of acute renal failure. Metabolomics is a robust technique that has been used to identify potential biomarkers for early detection of renal damage after procedures with using contrast media. We aim to analyze the serum and urine metabolites changes, after using contrast for coronary angiography, to determine if metabolomics can use as a tool for early detection of CIN. Method Sixty-six patients with positive primary non-invasive diagnostic tests for coronary artery disease (CAD) who were candidate of elective coronary angiography recruited. Spot urine samples collected in the morning before angiography and 4 hours after angiography. Patients with > 0.5 mg/dL creatinine rise compared to baseline were considered as case (CIN group). Urine samples were centrifuged at 3000 rcf for 20 minutes at 4°C to remove the cell debris and after addition of sodium azide to prevent bacterial growth, were stored at -80 degree Celsius in aliquots until required. The mixtures were then transferred to a standard 5 mm NMR tube for analysis. 1H-NMR spectra were acquired at 300 K on a Bruker DRX 500 MHz spectrometer by using Carr–Pucell–Meiboom–Gill (CPMG) technique. For each spectrum, 154 scans were collected into 32K data points using a spectral width of 8389.26 Hz during the relaxation time of 2.5 s. Results Structure and outliers of the dataset composed of patient with CIN (n = 10) before angiography and after angiography were evaluated by PCA. A model with two principal components (PC1 and PC2) with R2X = 0.775 and Q2(cum) = 0.487 was obtained .A supervised OPLS-DA model was built to identify discriminative variables between metabolite profiles before and after angiography in patients with CIN. The high level of AUC 0.95 that was obtained from 10-fold cross validation besides decreased R2 (0.0, 0.415) and Q2 (0.0, -0.454) intercepts of 999 random permutations reflects the good validity of this diagnostic model. According to this valid OPLS-DA model, 15 chemical shifts were significant based on VIP > 1 and FC > 1.2. To check these suggested chemical shifts if their changes are due to kidney injury and not caused by contrast agent, a decoy OPLS-DA model was built for non-CIN patients before and after angiography .Two common significant chemical shifts (2.42 and 2.78 ppm) were found in comparison of these two models (i.e. before vs. after angiography in CIN group in compared with before vs. after angiography in non-CIN group) and were excluded from the results. Metabolites corresponding with the remaining list of 12 significant chemical shifts were identified and suggested as early detection biomarker candidates for CIN (Fig 1). The AUC value of a panel of four biomarker candidates were higher than single biomarkers that reflects the value of simultaneous measurement of these four metabolite candidates than single candidates. Figure 2 shows the list of diagnostic metabolite candidates with p < 0.05 . Pathway characterization was used to better understanding of pathophysiology of CIN. As the input data was small list of metabolites, only “Histidine_ lysine_ phenylalanine, tyrosine, proline and tryptophan catabolism” pathway (p < 0.05) was significant and suggested as the most important disturbed pathway in CIN. Conclusion Early detection of CIN as early as only 4 hours after using contrast can help better management of these patients. In this study, only after 4 hours passed from using contrast a panel of metabolites could be found in urine of patients who develop CIN, which facilitates early detection of CIN. This is the first study to investigate urine metabolic profile using NMR-based metabolomics for early detection of CIN after coronary angiography. The use of this suggested panel might significantly improve clinical consequences of this harmful complication.

An NMR Protocol for In Vitro Paclitaxel Release from an Albumin-Bound Nanoparticle Formulation.

The paclitaxel protein-bound particles for injectable suspension (marketed under the brand name Abraxane®) contains nanosized complexes of paclitaxel and albumin. The molecular interaction between paclitaxel and albumin within the higher-order nanostructure is analytically challenging to assess, as is any correlation of differences to differences in therapeutic effect. However, because the higher-order nanostructures may affect the paclitaxel release, a suitable in vitro assay to detect potential differences in paclitaxel release between comparator lots and products is desirable. Herein, solution NMR spectroscopy with a T2-filtering technique was developed to detect paclitaxel signal while suppressing albumin signals to follow the released paclitaxel in the NMR tube upon dilution. The non-invasive nature of NMR allows for precise measurement of a full range of dilution-induced drug release percentage from 14 to 92% without any sample extraction. The critical concentration of the drug product (DP) at 50% of release was 0.63 ± 0.04mg/mL in PBS buffer. In addition, 2D diffusion ordered NMR spectroscopy (DOSY) results revealed that the released paclitaxel experiencing slightly slowed diffusion rates than free paclitaxel, which was attributed to paclitaxel in equilibrium with albumin-bound states. Collectively, the dilution-based NMR method offered an analytical approach to investigate physicochemical attributes of complex injectable products with minimal needed sample preparation and perturbation to nanoparticle formulation.

Synthesis and Use of a Nickel Oxidation Catalyst Using Glove Box Methods

The ability to perform air-free chemistry and catalytic reactions on an NMR tube scale are valuable skills in chemistry and not often included in undergraduate chemistry curriculum. This laboratory experiment provides students experience in synthesizing the metal complex, Ni(dcpe)(CH3CN)2(BF4)2 (dcpe = 1,2-bis(dicyclohexylphosphino)ethane), inside an inert atmosphere glovebox, performing a catalytic alcohol oxidation reaction on a microscale, and monitoring the reaction progress by 1H NMR spectroscopy. This activity has been employed in the Advanced Inorganic Chemistry course at Wabash College for three years. The reactions have proven to be reliable, and the students have gained valuable experience using advanced techniques common in inorganic chemistry research laboratories.

Full Cell Understanding of Solvents in Battery Systems

Lithium ion batteries are complex systems with multiple components that can be optimized to obtain desired performance capability. These components include electrolyte salts, solvents, and additives, as well as electrode active materials and coatings. Rational optimization of battery components requires an understanding of the fundamental science driving the observed performance. Silatronix® has developed a suite of analytical tools and methods using a combination of full format batteries, lab R&D cells, and model systems to construct a complete understanding of the battery from the cell level performance to the fundamental chemistry. This process includes the recovery and analysis of all parts of a full format pouch cell, including liquid electrolyte, generated gas, and electrode surface layers (Fig. 1).Using this process, Silatronix® has evaluated several battery components, including organosilicon solvents for Li-ion electrolytes, to acquire a comprehensive picture of how these components provide observed performance benefits. For example, OS3®, a novel organosilicon chemistry that merges a silane with a Li+ coordinating nitrile functionality, has provided multiple performance benefits in various advanced Li-ion chemistries. Key experimental results include improved high temperature cycling stability in full format graphite/NMC622 pouch cells with 1%, 2%, or 3% OS3® (Fig. 2a), and using a model system of electrolyte in NMR tubes, we show that enhanced thermal stability of the bulk electrolyte results from HF scavenging and LiPF6 salt stabilization when 2-16% OS3® is present (Fig. 2b). Reduction of gas generation during high temperature testing in pouch cells with a commercial Si-based anode is also shown with 2% OS3® (Fig. 2c), and gas product identification and quantification is determined by GC-TCD for mechanistic analyses. In addition, OS materials change the reactivity of other electrolyte components, particularly at the electrolyte/electrode interfaces that govern critical performance attributes in Li-ion cells such as gas generation and interfacial impedance. These key performance benefits serve to enable several next generation battery chemistries, including a strong synergy with Si-based anodes. Figure 1

Chan‐Evans‐Lam C−N Coupling Promoted by a Dinuclear Positively Charged Cu(II) Complex. Catalytic Performance and Some Evidence for the Mechanism of CEL Reaction Obviating Cu(III)/Cu(I) Catalytic Cycle

AbstractIn the present study, we report the synthesis of a series of copper(II) complexes with a wide range of ligands and their testing in the copper catalyzed Chan‐Evans‐Lam (CEL) coupling of aniline and phenylboronic acid. The efficiency of the coupling was directly connected with the ease of the reduction of Cu(II) to Cu(I) of the complexes. The most efficient catalyst was derived from 4‐t‐butyl‐2,5‐bis[(quinolinylimino)methyl]phenolate and two Cu(II) ions. Depending on the counter‐anion nature and the concentration of the reaction mixture, the reaction can be directed to predominant C−N‐bond formation. Forty‐three derivatives of diphenylamine were prepared under the optimized conditions. The proposed mechanism of the catalysis was based on the reduction potential of a series of complexes, molecular weight measurements of the catalytic complex in MeOH and the kinetic studies of aniline and phenylboronic acid coupling. In addition, an 1H NMR experiment in a sealed NMR tube, without external oxygen supply available, proved that no complete Cu(II) to Cu(I) conversion was observed under the condition, ruling out the usually accepted mechanism of the C−N coupling, which included the oxygenation of the intermediately formed Cu(I) complexes after the key step of C−N conversion had already been completed. Instead, a mechanism was proposed, involving an oxygen molecule coordinated to two copper ions in the key C−N bond formation without any detectable conversion of the Cu(II) complexes to Cu(I).

From Ag2S to luminescent Ag–In–S nanocrystals via an ultrasonic method – an in situ synthesis study in an NMR tube

Taking advantage of “mild” preparation conditions we have been able for the first time to perform the synthesis of Ag2S and luminescent AgInS2 nanocrystals in an NMR tube.