Double-quantum Filter Research Articles

Homonuclear J-couplings and heteronuclear structural constraints

In magic angle spinning (MAS) experiments involving uniformly 13C,15N proteins, 13C–13C and 13C–15N dipolar recoupling experiments are now routinely used to measure direct dipole–dipole couplings that constrain distances and torsion angles and determine molecular structures. When the distances are short (<4 Å), the direct couplings dominate the evolution of the spin system, and the 13C–13C and 13C–15N J-couplings (scalar couplings) are ignored. However, for structurally interesting >4 Å distances, the dipolar and J-couplings are generally of comparable magnitude, and the variation in J must be included in order to optimize the precision of the experiment. This problem is circumvented in cases with well resolved spectra by using frequency-selective dipolar recoupling methods where the effects of J-couplings are refocused. However, for larger molecules with more spectral crowding, the requisite pulse length to achieve selectivity becomes long and leads to unacceptable sensitivity losses during the pulse or the spectral overlap precludes selective excitation. In this paper, we address this problem with two approaches aimed at facilitating higher precision internuclear distance measurements in systems that are not fully resolved. Namely, (1) we describe an approach for high precision measurements of specific J-couplings using the in-phase anti-phase (IPAP) sequence which is integrated into a non-selective dipolar recoupling technique and (2) we utilize the measured J-couplings to implement a double quantum filter experiment capable of providing the resolution necessary for frequency selective dipolar recoupling techniques without resorting to multidimensional spectroscopy. We illustrate these methods using a 7-peptide segment from the amyloidogenic Sup-35p protein, U-13C/15N-GNNQQNY, where we have measured 25 of the 27 possible one bond 13C–13C J-couplings.

Sulfur-containing spiroketals from Breynia disticha and evaluations of their anti-inflammatory effect.

Breynia spp. are a key source of sulfur-containing spiroketal glycosides with potential anti-inflammatory activity. In this study, three new sulfur-containing spiroketals - breynin J (1), epibreynin J (2), and probreynogenin (3) - along with four known compounds - probreynin I (4), phyllaemblic acid (5), breynin B (6), and epibreynin B (7) - were isolated from the roots of Breynia disticha. The structures of compounds 1-7 were elucidated by extensive 1D and 2D NMR spectroscopic analyses, including 1D total correlation spectroscopy (TOCSY), HSQC, HMBC, double quantum-filtered (DQF)-COSY, heteronuclear two-bond correlation (H2BC), and HSQC-TOCSY experiments, as well as high-resolution electrospray ionization HRESIMS analysis, and quantum chemical electronic CD calculations. Furthermore, the absolute configurations of sugar residues were determined by derivatization of the hydrolysates with ʟ-cysteine methyl ester and o-tolyl isothiocyanate followed by HPLC analysis. The anti-inflammatory effects of the isolated compounds were evaluated based on the mRNA levels of proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage cells. Compounds 1, 2, 6, and 7 inhibited the increase in interleukin (IL)-1β and IL-6 mRNA levels stimulated by LPS. Moreover, the most potent compound 7 was found to significantly inhibit the production of IL-1β and IL-6 proteins, as revealed by the analysis of culture supernatants.

Polymer composition optimization approach based on feature extraction of bound and free water using time-domain nuclear magnetic resonance

As global environmental sustainability becomes increasingly emphasized, the development of eco-friendly materials, including solutions to the issue of marine plastics, is thriving. However, the material parameter space is vast, making efficient search a challenge. Time-domain nuclear magnetic resonance offers material property information through the complex T2 relaxation curves resulting from multiple mobilities. In this research, we used the Carr–Purcell–Meiboom–Gill (CPMG) pulse sequence to evaluate the binding state of water (water affinity) in polymers synthesized with various monomer compositions, which were immersed in seawater. We also assessed the T2 relaxation property of the polymers using the magic sandwich echo, double quantum filter, and magic-and-polarization echo filter techniques. We separated the T2 relaxation curves of CPMG into free and bound water for polymers by employing semisupervized nonnegative matrix factorization. By employing the features of separated bound water and polymer properties, a polymer composition optimization method offered crucial factors to monomers through random forests, predicted the components of the polymer using generative topography mapping regression, and determined expected values using Bayesian optimization for polymer composition candidates with the desired high water affinity and high rigidity.

Dynamic nuclear polarization-enhanced, double-quantum filtered 13C-13C dipolar correlation spectroscopy of natural 13C abundant bone-tissue biomaterial

Here, we describe a method for obtaining a dynamic nuclear polarization (DNP)-enhanced double-quantum filtered (DQF) two-dimensional (2D) dipolar 13C-13C correlation spectra of bone-tissue material at natural 13C abundance. DNP-enhanced DQF 2D dipolar 13C-13C spectra were obtained using a few different mixing times of the dipolar-assisted rotational resonance (DARR) scheme and these spectra were compared to a conventional 2D through-space double-quantum (DQ)-single-quantum (SQ) correlation spectrum. While this scheme can only be used for an assignment purpose to reveal the carbon-carbon connectivity within a residue, the DQF 13C-13C dipolar correlation scheme introduced here can be used to obtain longer distance carbon-carbon constraints. A DQF pulse block is placed before the DARR mixing scheme for removing dominant 13C single-quantum (SQ) signals because these SQ 13C signals are overwhelmingly large compared to those 13C-13C dipolar cross-peaks generated and therefore saturate the dynamic range of the NMR detection. This approach exhibits strong enough 2D cross-peaks in a dipolar 13C-13C correlation spectrum and potentially provides pairwise 13C-13C dipolar constraints because the dipolar truncation effect as well as multi-step signal propagations involving a spin cluster that contains more than two spins can be ignored probabilistically. To obtain fast signal averaging, AsymPolPOK was used to provide a short 1H DNP signal build-up time (1.3 s) and to expedite our MAS DNP NMR acquisitions while still maintaining a satisfactory DNP enhancement factor (ε = 50). Under long DARR mixing, a t1-noise-like artifact was observed at a site that possesses a large chemical shift anisotropy (CSA) and a few different strategies to address this problem were discussed.

3D localized lactate detection in muscle tissue using double-quantum filtered 1 H MRS with adiabatic refocusing pulses at 7T.

Lactate is a key metabolite in skeletal muscle and whole-body physiology. Its MR visibility in muscle is affected by overlapping lipid signals and fiber orientation. Double-quantum filtered (DQF) 1 H MRS selectively detects lactate at 1.3ppm, but at ultra-high field the efficiency of slice-selective 3D-localization with conventional RF pulses is limited by bandwidth. This novel 3D-localized 1 H DQF MRS sequence uses adiabatic refocusing pulses to unambiguously detect lactate in skeletal muscle at 7T. Lactate double-quantum coherences were 3D-localized using slice-selective Shinnar-Le Roux optimized excitation and adiabatic refocusing pulses (similar to semi-LASER). DQF MR spectra were acquired at 7T from lactate phantoms, meat specimens with injected lactate (exploring multiple TEs and fiber orientations), and human gastrocnemius in vivo during and after exercise (without cuff ischemia). Lactate was readily detected, achieving the full potential of 50% signal with a DQF, in solution. The effects of fiber orientation and TE on the lactate doublet (peak splitting, amplitude, and phase) were in good agreement with theory and literature. Exercise-induced lactate accumulation was detected with 30s time resolution. This novel 3D-localized 1 H DQF MRS sequence can dynamically detect glycolytically generated lactate in muscle during exercise and recovery at 7T.

Spectral editing of alanine, serine, and threonine in uniformly labeled proteins based on frequency-selective homonuclear recoupling in solid-state NMR.

Spectral editing is crucial to simplify the crowded solid-state NMR spectra of proteins. New techniques are introduced to edit 13C-13C correlations of uniformly labeled proteins under moderate magic-angle spinning (MAS), based on our recent frequency-selective homonuclear recoupling sequences [Zhang et al., J. Phys. Chem. Lett. 2020, 11, 8077-8083]. The signals of alanine, serine, or threonine residues are selected out by selective 13Cα-13Cβ double-quantum filtering (DQF). The 13Cα-13Cβ correlations of alanine residues are selectively established with efficiency up to ~ 1.8 times that by dipolar-assisted rotational resonance (DARR). The techniques are shown in 2D/3D NCCX experiments and applied to the uniformly 13C, 15N labeled Aquaporin Z (AqpZ) membrane protein, demonstrating their potential to simplify spectral analyses in biological solid-state NMR.

Identification of water compartments in spinal cords by 2 H double quantum filtered NMR.

In 2 H double quantum filtered (DQF) NMR, the various water compartments are characterized by their different residual quadrupolar interactions. The spectral separation between the different signals enables the measurement of the relaxation of each compartment and the magnetization transfer (MT) between them. In the current study, five water compartments were identified in the 2 H DQF spectra of porcine spinal cord. The most prominent signal was the pair of satellites with a quadrupolar splitting of about 550 Hz. 2 H DQF MRI optimized for the 550 Hz quadrupolar splitting indicated that this signal originated mainly from the white matter and it was assigned to the myelin water. This splitting does not change upon changing the orientation of the spinal cord relative to the magnetic field, indicating a liquid crystalline nature. Another site exhibiting splitting of about 1500 Hz was assigned to collagenous connective tissue. The narrow central peak was assigned to a combination of intra- and inter-axonal water. The assignment of the other two sites is not certain and requires further study. The rates of MT between the various sites were recorded.

In vitro singlet state and zero-quantum encoded magnetic resonance spectroscopy: Illustration with N-acetyl-aspartate.

Magnetic resonance spectroscopy (MRS) allows the analysis of biochemical processes non-invasively and in vivo. Still, its application in clinical diagnostics is rare. Routine MRS is limited to spatial, chemical and temporal resolutions of cubic centimetres, mM and minutes. In fact, the signal of many metabolites is strong enough for detection, but the resonances significantly overlap, exacerbating identification and quantification. Besides, the signals of water and lipids are much stronger and dominate the entire spectrum. To suppress the background and isolate selected signals, usually, relaxation times, J-coupling and chemical shifts are used. Here, we propose methods to isolate the signals of selected molecular groups within endogenous metabolites by using long-lived spin states (LLS). We exemplify the method by preparing the LLSs of coupled protons in the endogenous molecules N-acetyl-L-aspartic acid (NAA). First, we store polarization in long-lived, double spin states, followed by saturation pulses before the spin order is converted back to observable magnetization or double quantum filters to suppress background signals. We show that LLS and zero-quantum coherences can be used to selectively prepare and measure the signals of chosen metabolites or drugs in the presence of water, inhomogeneous field and highly concentrated fatty solutions. The strong suppression of unwanted signals achieved allowed us to measure pH as a function of chemical shift difference.

Multiquantum Counting of Trityl Radicals.

We demonstrate a series of multitrityl radical compounds where accurate spin-counting by pulsed electron paramagnetic resonance (EPR) can be achieved at X-band (9 GHz) frequencies, even for molecules with very short and flexible linkers. Multiquantum filter experiments, well-known from NMR, were used to count the number of coupled electron spins in these compounds. The six pulse double quantum filter sequence used in EPR for distance determinations in biradicals was used. Precise phase settings to separate higher quantum coherences were achieved by an arbitrary waveform generator. The trityl radicals have narrow spectral width so that homogeneous excitation of all spins by the pulses is possible. The transversal relaxation times of higher quantum coherences of trityl radicals are sufficiently long to allow their detection. Our results on model compounds show the potential of this approach to determine oligomeric states in protein complexes in their native environment using functionalized trityl spin labels.

NMR techniques in studying water in biotechnological systems.

Different NMR methodologies have been considered in studying water as a part of the structure of heterogeneous biosystems. The current work mostly describes NMR techniques to investigate slow translational dynamics of molecules affecting anisotropic properties of polymers and biomaterials. With these approaches, information about organized structures and their stability could be obtained in conditions when external factors affect biomolecules. Such changes might include rearrangement of macromolecular conformations at fabrication of nano-scaffolds for tissue engineering applications. The changes in water-fiber interactions could be mirrored by the magnetic resonance methods in various relaxations, double-quantum filtered (DQF), 1D and 2D translational diffusion experiments. These findings effectively demonstrate the current state of NMR studies in applying these experiments to the various systems with the anisotropic properties. For fibrous materials, it is shown how NMR correlation experiments with two gradients (orthogonal or collinear) encode diffusion coefficients in anisotropic materials and how to estimate the permeability of cell walls. It is considered how the DQF NMR technique discovers anisotropic water in natural polymers with various cross-links. The findings clarify hydration sites, dynamic properties, and binding of macromolecules discovering the role of specific states in improving scaffold characteristics in tissue engineering processes. Showing the results in developing these NMR tools, this review focuses on the ways of extracting information about biophysical properties of biomaterials from the NMR data obtained.

Lactate concentration in breast cancer using advanced magnetic resonance spectroscopy

BackgroundPrecision medicine in breast cancer demands markers sensitive to early treatment response. Aerobic glycolysis (AG) upregulates lactate dehydrogenase A (LDH-A) with elevated lactate production; however, existing approaches for lactate quantification are either invasive or impractical clinically.MethodsThirty female patients (age 39–78 years, 15 grade II and 15 grade III) with invasive ductal carcinoma were enrolled. Lactate concentration was quantified from freshly excised whole tumours with double quantum filtered (DQF) magnetic resonance spectroscopy (MRS), and Nottingham Prognostic Index (NPI), LDH-A and proliferative marker Ki-67 were assessed histologically.ResultsThere was a significantly higher lactate concentration (t = 2.2224, p = 0.0349) in grade III (7.7 ± 2.9 mM) than in grade II (5.5 ± 2.4 mM). Lactate concentration was correlated with NPI (ρ = 0.3618, p = 0.0495), but not with Ki-67 (ρ = 0.3041, p = 0.1023) or tumour size (r = 0.1716, p = 0.3645). Lactate concentration was negatively correlated with LDH-A (ρ = −0.3734, p = 0.0421).ConclusionOur results showed that lactate concentration in whole breast tumour from DQF MRS is sensitive to tumour grades and patient prognosis.

New insight into the organization of myelin water using deuterium NMR.

Myelin water is commonly characterized by its short proton T2 relaxation time, suggesting strong association with the polar head groups of the bilayer constituents. Deuterium NMR of water in ordered structures exhibits splittings as a result of quadrupolar interactions that are observable using the double-quantum filter. The purpose of the current study was to identify and characterize the water populations. The 2 H double-quantum-filtered spectroscopic experiments were conducted at 62MHz (9.4 T) on a sample of reconstituted myelin from ovine spinal cord after exchange of native water with D2 O. Signals passing the double-quantum filter were attributed to 2 water pools: 1 consisting of a doublet of 650-Hz splitting, and a second unsplit signal. Similar signals were observed in the sciatic and optic nerves and in the spinal cord. Further, data suggest that diffusion of water molecules in these 2 pools (Dapp ≤5×10-7 cm2 /s) is either hindered or restricted. An estimate of exchange lifetime of 10-15ms between water pertaining to the single peak and that of the split peaks suggests exchange occurs in a slow-intermediate rate regime. Further distinction between the 2 pools was obtained from T1 measurements. Deuterons belonging to the doublet resonance were found to have short T1 , estimated to be on the order of 10-20ms, whereas those corresponding to the single peak were close to that of bulk D2 O. The results suggest that myelin extract water consists of 2 hindered populations with distinct degrees of anisotropic motion that can be studied by 2 H double-quantum-filtered NMR.

Magnetization transfer in liposome and proteoliposome samples that mimic the protein and lipid composition of myelin.

Although magnetization transfer (MT) has been widely used in brain MRI, for example in brain inflammation and multiple sclerosis, the detailed molecular origin of MT effects and the role that proteins play in MT remain unclear. In this work, a proteoliposome model system was used to mimic the myelin environment and to examine the roles of protein, cholesterol, brain cerebrosides, and sphingomyelin embedded in the liposome matrix. Exchange parameters were determined using a double-quantum filter experiment. The goal was to determine the relative contributions to exchange and MT of cerebrosides, sphingomyelin, cholesterol, and proteins in 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayers. The main finding was that cerebrosides produced the strongest exchange effects, and that these were even more pronounced than those found for proteins. Sphingomyelin (which also has exchangeable groups at the head of the fatty acid chains, albeit closer to the lipid acyl chains) and cholesterol showed only minimal transfer. Overall, the extracted exchange rates appeared much smaller than commonly assumed for -OH and -NH groups.

DQF J-RES NMR: Suppressing the singlet signals for improving the J-RES spectra from complex mixtures.

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.

Determining Cholesterol Binding to Membrane Proteins by Cholesterol 13C Labeling in Yeast and Dynamic Nuclear Polarization NMR.

We present a general strategy for determining the cholesterol-binding site of eukaryotic membrane proteins in native-like lipid membranes by NMR spectroscopy. The strategy combines yeast biosynthetic 13C enrichment of cholesterol with detection of protein-cholesterol 13C-13C cross peaks in 2D correlation NMR spectra under the dynamic nuclear polarization (DNP) condition. Low-temperature DNP not only allows high-sensitivity detection of weak protein-cholesterol cross peaks in 2D spectra but also immobilizes cholesterol and protein to enable intermolecular distance measurements. We demonstrate this approach on the influenza M2 protein, which utilizes cholesterol to conduct membrane scission in the last step of virus budding and release from the host cell plasma membrane. A 13C-13C double-quantum filter was employed to significantly simplify the 2D 13C-13C correlation spectra and facilitate the identification of protein-cholesterol cross peaks. A number of cross peaks between the M2 transmembrane residues' side chains and the cholesterol sterol group were detected, which complement recently measured protein contacts to the isooctyl tail of cholesterol to define an extended binding interface. These results provide atomic-level evidence of M2-cholesterol interaction to cause membrane curvature and scission, and the approach is generally applicable to other eukaryotic membrane proteins for understanding the influence of cholesterol on membrane protein function.

Only Para-Hydrogen Spectroscopy (OPSY) Revisited: In-Phase Spectra for Chemical Analysis and Imaging.

We revisited only para-hydrogen spectroscopy (OPSY) for the analysis of para-hydrogen-enhanced NMR spectra at high magnetic fields. We found that the sign of the gradients and interpulse delays are pivotal for the performance of the sequence: the variant of double-quantum filter OPSY, where the second time interval is twice as long as the first one (OPSYd-12) converts the antiphase spectrum to in-phase and efficiently suppresses the background signal in a single scan better than the other variants. OPSYd-12 strongly facilitates the analysis of para-hydrogen-derived NMR spectra in homogeneous and inhomogeneous magnetic fields. Furthermore, the net magnetization produced is essential for subsequent applications such as imaging, e.g., in a reaction chamber or in vivo.

Biological effects of Salvia Officinalis leaf extract on murine myeloma cells

Background: In the Mediterranean region, Salvia officinalis plant is widely used and routinely added to food for a traditional culinary utilization, and moreover, from long time, it is recognized to possess pharmacological activities. Objectives: In the present study, we investigated the biological properties of S. officinalis leaf ethanol extracts and a thin layer chromatography-isolated spot on murine myeloma cells. Materials and Methods: 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide tests were carried out to determinate the EC50 of both the leaf extracts and the isolated spot; the isolated spot was also investigated by liquid chromatography–mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). Electron microscopy and immunofluorescence were used for morphological studies on treated P3X murine myeloma cells. Results: LC-MS and NMR techniques identified methyl carnosate, a methyl derivative of carnosic acid, as the main component of spot B. Moreover, scanning electron microscopy, transmission electron microscopy, and immunofluorescence investigations carried out on murine myeloma cells treated for 20 h with EC50 values of spot B revealed some changes both in the cellular morphology and in the microtubular array. Conclusions: The present studies indicate that S. officinalis extracts have biological effects on murine myeloma cells and identify methyl carnosate as an interesting molecule for further investigations on human cell lines and possibly on cancer prevention. Abbreviations used: EC50: Effective concentration 50, LC-MS: Liquid chromatography–mass spectrometry, NMR: Nuclear magnetic resonance, TLC: Thyn layer chromatography, MTT: 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide, RF: Retention factor, MS/Ms: Tandem mass spectrometry, dqf-COSY: double quantum filter COSY

Dynamic Disorder and Electronic Structures of Electron-Precise Dianionic Diboranes: Insights from Solid-State Multinuclear Magnetic Resonance Spectroscopy.

The J(11B,11B) coupling constants of various salts of the electron-precise hexacyanodiborane(6) dianion, [B2(CN)6]2-, were obtained using 11B double-quantum-filtered (DQF) J-resolved solid-state nuclear magnetic resonance (SSNMR) spectroscopy. Our results show that the magnitude of the DQF J splitting is influenced by both the crystallographic symmetry of the system and the presence of dynamics. The splittings are amplified by a factor of 3 as compared to the corresponding theoretical J coupling constants for cases where (1) there is an absence of dynamics but the boron pairs are crystallographically equivalent or (2) the boron pairs are crystallographically inequivalent but are rendered magnetically equivalent on the time scale of the experiment due to dynamic disorder, which was identified by 11B and 13C SSNMR experiments. Consequently, molecular motions need to be taken into consideration when interpreting the results of DQF J-resolved experiments, and conversely, these experiments may be used to identify dynamic disorder. Variable-temperature NMR data support the notion of three different motional processes with correlation times ranging from 102 to 106 s-1 over the temperature range of 248-306 K. When molecular motion and crystallographic symmetry are both accounted for, the J(11B,11B) coupling constants for various [B2(CN)6]2- salts were measured to range from 29.4 to 35.8 Hz, and their electronic origins were determined using natural localized molecular orbital and natural bond orbital analyses. The coupling constants were found to strongly correlate to the hybridization states of the boron orbitals that form the B-B bonds and to the strength of the B-B bonds. This study provides a novel tool to study dynamics in ordered and disordered solids and provides new perspectives on electron-precise dianionic diboranes featuring two-center-two-electron bonds in the context of related compounds featuring multiply and singly bonded boron spin pairs.

1H–1H Double Quantum NMR Investigation of Proton Dynamics in Solid Acids

Currently, the most popular proton exchange membrane (PEM) for fuel cell applications is Nafion. However, Nafion does not retain its high conductivity at high temperatures due to its dependence on water for proton transport. Because operational temperatures higher than the evaporation point of water are desirable, a family of solid acids was investigated. Cations known to transport protons were paired with anions to make acidic salts. Solid acids discussed here include imidazole paired with trifluoromethanesulfuric acid as well as imidazole, benzimidazole and adenine paired with methanesulfonic acid. Solid-state NMR was utilized to show the relative mobility of protons through double-quantum filter (DQF) experiments. The POST-C7 homonuclear dipolar-recoupling scheme was paired with DUMBO homonuclear decoupling to produce 1H double-quantum coherence buildup curves for the hydrogen-bonded protons of interest. Experimental buildup curves, which reflect both local structure as well as dynamics, are compared to theoretical curves of the static system. The SPINEVOLUTION-simulated curves utilized up to eight pairs of homonuclear dipolar couplings within a sphere of 7 Å diameter centered on the proton of interest. Steep buildup of the DQ curve and maxima at short recoupling times in the buildup curves indicate strong dipole–dipole coupling and are interpreted to indicate limited dynamics of the H-bonded protons. In contrast, shallower buildup curves and maxima at longer recoupling times imply that H-bonded protons (in an otherwise similar structure) are associated with local mobility, which reduces their local dipolar coupling and may facilitate proton transport. Bulk proton conductivities measured via electrochemical impedance spectroscopy were compared to DQF measurements to understand proton conduction within these materials.

Quantitative Detection of PEGylated Biomacromolecules in Biological Fluids by NMR.

The accumulation, biodistribution, and clearance profiles of therapeutic agents are key factors relevant to their efficacy. Determining these properties constitutes an ongoing experimental challenge. Many such therapeutics, including small molecules, peptides, proteins, tissue scaffolds, and drug delivery vehicles, are conjugated to poly(ethylene glycol) (PEG) as this improves their bioavailability and in vivo stability. We demonstrate here that (1)H NMR spectroscopy can be used to quantify PEGylated species in complex biological fluids directly, rapidly, and with minimal sample preparation. PEG bears a large number of spectroscopically equivalent protons exhibiting a narrow NMR line width while resonating at a (1)H NMR frequency distinct from most other biochemical signals. We demonstrate that PEG provides a robust signal allowing detection of concentrations as low as 10 μg/mL in blood. This PEG detection limit is lowered by another order of magnitude when background proton signals are minimized using (13)C-enriched PEG in combination with a double quantum filter to remove (1)H signals from non-(13)C-labeled species. Quantitative detection of PEG via these methods is shown in pig blood and goat serum as examples of complex biological fluids. More practically, we quantify the blood clearance of (13)C-PEG and PEGylated-BSA (bovine serum albumin) following their intravenous injection in live rats. Given the relative insensitivity of line width to PEG size, we anticipate that the biodistribution and clearance profiles of virtually any PEGylated biomacromolecule from biological fluid samples can be routinely measured by (1)H NMR without any filtering or treatment steps.