Rotaxane Probes for the Detection of Hydrogen Peroxide by 129Xe HyperCEST NMR Spectroscopy

The development of sensitive and chemically selective MRI contrast agents is imperative for the early detection and diagnosis of many diseases. Conventional responsive contrast agents used in ¹H MRI are impaired by the high abundance of protons in the body. ¹²⁹Xe hyperCEST NMR/MRI comprises a highly sensitive complement to traditional ¹H MRI because of its ability to report specific chemical environments. To date, the scope of responsive ¹²⁹Xe NMR contrast agents lacks breadth in the specific detection of small molecules, which are often important markers of disease. Herein, we report the synthesis and characterization of a rotaxane‐based ¹²⁹Xe hyperCEST NMR contrast agent that can be turned on in response to H₂O₂, which is upregulated in several disease states. Added H₂O₂ was detected by ¹²⁹Xe hyperCEST NMR spectroscopy in the low micromolar range, as well as H₂O₂ produced by HEK 293T cells activated with tumor necrosis factor.     

Parahydrogen-Induced Polarization of Diethyl Ether Anesthetic

The growing interest in magnetic resonance imaging (MRI) for assessing regional lung function relies on the use of nuclear spin hyperpolarized gas as a contrast agent. The long gas-phase lifetimes of hyperpolarized ¹²⁹Xe make this inhalable contrast agent acceptable for clinical research today despite limitations such as high cost, low throughput of production and challenges of ¹²⁹Xe imaging on clinical MRI scanners, which are normally equipped with proton detection only. We report on low-cost and high-throughput preparation of proton-hyperpolarized diethyl ether, which can be potentially employed for pulmonary imaging with a nontoxic, simple, and sensitive overall strategy using proton detection commonly available on all clinical MRI scanners. Diethyl ether is hyperpolarized by pairwise parahydrogen addition to vinyl ethyl ether and characterized by ¹H NMR spectroscopy. Proton polarization levels exceeding 8 % are achieved at near complete chemical conversion within seconds, causing the activation of radio amplification by stimulated emission radiation (RASER) throughout detection. Although gas-phase T₁ relaxation of hyperpolarized diethyl ether (at partial pressure of 0.5 bar) is very efficient, with T₁ of ca. 1.2 second, we demonstrate that, at low magnetic fields, the use of long-lived singlet states created via pairwise parahydrogen addition extends the relaxation decay by approximately threefold, paving the way to bioimaging applications and beyond.     

Directly Functionalized Cucurbit[7]uril as a Biosensor for the Selective Detection of Protein Interactions by 129Xe hyperCEST NMR

Advancement of hyperpolarized ¹²⁹Xe MRI technology toward clinical settings demonstrates the considerable interest in this modality for diagnostic imaging. The number of contrast agents, termed biosensors, for ¹²⁹Xe MRI that respond to specific biological targets, has grown and diversified. Directly functionalized xenon-carrying macrocycles, such as the large family of cryptophane-based biosensors, are good for localization-based imaging and provide contrast before and after binding events occur. Noncovalently functionalized constructs, such as cucurbituril- and cyclodextrin-based biosensors, benefit from commercial availability and optimal exchange dynamics for CEST imaging. In this work, we report the first directly functionalized cucurbituril used as a xenon biosensor. Biotinylated cucurbit[7]uril (btCB7) gives rise to a ¹²⁹Xe hyperCEST response at the unusual shift of δ=28 ppm when bound to its protein target with substantial CEST contrast. We posit that the observed chemical shift is due to the deformation of btCB7 upon binding to avidin, caused by proximity to the protein surface. Conformational searches and molecular dynamics (MD) simulations support this hypothesis. This construct combines the strengths of both families of biosensors, enables a multitude of biological targets through avidin conjugation, and demonstrates the advantages of functionalized cucurbituril-based biosensors.     

Characterization of CuY zeolites after dehydration,oxidation and reduction with carbon monoxide An adsorption and 129Xe nuclear magnetic resonance spectroscopy study

The adsorption isotherms and ¹²⁹Xe nuclear magnetic resonance (NMR) chemical shifts of xenon and the adsorption isotherms of carbon monoxide of Cu(II)- and Cu(I)-exchanged zeolites NaY were measured. The former zeolites of 53, 75, and 95% exchange degrees were investigated after various pretreatment steps comprising dehydration, oxidation and reduction with CO at 420°C as well as long-term CO reduction at 470°C. The Cu(I)Y zeolite of 70% exchange degree was prepared via a solid-state exchange procedure with CuCl and subjected to dehydration at 420°C. In all cases, except the dehydrated zeolites, almost linear xenon adsorption isotherms and linear ¹²⁹Xe NMR chemical shift _versus xenon concentration curves running parallel to each other are obtained. In contrast, the chemical shift curves for the dehydrated zeolites are non-linear at low xenon concentrations turning towards negative chemical shift values at very low pressures. The whole body of the experimental xenon data can be explained quantitatively with a unifying approach on the basis of a site adsorption model where the sites are (i) two types of cuprous ions of much different adsorption strength and ¹²⁹Xe chemical shift, (ii) Na⁺ cations, (iii) Lewis acid sites generated through autoreduction and reduction of Cu²⁺ by CO, and (iv) framework sites free of cations. These five types of sites are each characterized by Langmuir adsorption isotherm constants and local ¹²⁹Xe NMR chemical shifts. The adsorption site concentrations in the various zeolites are evaluated. The supercage Cu(I) concentration values are in nice agreement with the results deduced from the CO adsorption isotherm measurements.     

A Structural Basis for 129Xe Hyper-CEST Signal in TEM-1 β-Lactamase

Genetically encoded (GE) contrast agents detectable by magnetic resonance imaging (MRI) enable non-invasive visualization of gene expression and cell proliferation at virtually unlimited penetration depths. Using hyperpolarized ¹²⁹Xe in combination with chemical exchange saturation transfer, an MR contrast approach known as hyper-CEST, enables ultrasensitive protein detection and biomolecular imaging. GE MRI contrast agents developed to date include nanoscale proteinaceous gas vesicles as well as the monomeric bacterial proteins TEM-1 β-lactamase (bla) and maltose binding protein (MBP). To improve understanding of hyper-CEST NMR with proteins, structural and computational studies were performed to further characterize the Xe-bla interaction. X-ray crystallography validated the location of a high-occupancy Xe binding site predicted by MD simulations, and mutagenesis experiments confirmed this Xe site as the origin of the observed CEST contrast. Structural studies and MD simulations with representative bla mutants offered additional insight regarding the relationship between local protein structure and CEST contrast.     

Probing polymer colloids by Xe NMR

Model aqueous dispersions of polystyrene, poly(methyl methacrylate), poly(n-butyl acrylate) and a statistical copolymer poly(n-butyl acrylate-co-methyl methacrylate) were studied using xenon NMR spectroscopy. The ¹²⁹Xe NMR spectra of these various latexes reveal qualitative and quantitative differences in the number of peaks and in their line widths and chemical shifts. Above the glass transition temperature, exchange between xenon sorbed in the particle core and free xenon outside the particles is fast on the ¹²⁹Xe spectral time-scale and a single ¹²⁹Xe signal is observed. At temperatures below the glass transition temperature, the exchange between sorbed and free xenon is slow on the ¹²⁹Xe spectral time-scale and two ¹²⁹Xe NMR signals can be observed. If the signal of sorbed ¹²⁹Xe is observed, its chemical shift, line width and integral relative to the integral of free ¹²⁹Xe can be used for the characterization of the particle core. The line width of free ¹²⁹Xe provides the residence time of xenon outside the particles and can be used to determine the rate constant characterizing the kinetics of penetration of xenon in the particles. This rate constant emerges as promising parameter for the characterization of the polymer particle surface.     

Background-Free Detection of Spin-Exchange Dynamics at Ultra-Low Magnetic Field

Ultra-low field nuclear magnetic resonance spectroscopy (NMR) and imaging (MRI) inherently suffer from a low signal-to-noise ratio due to the small thermal polarization of nuclear spins. Transfer of polarization from a pre-polarized spin system to a thermally polarized spin system via the Spin Polarization Induced Nuclear Overhauser Effect (SPINOE) could potentially be used to overcome this limitation. SPINOE is particularly advantageous at ultra-low magnetic field, where the transferred polarization can be several orders of magnitude higher than thermal polarization. Here we demonstrate direct detection of polarization transfer from highly polarized ¹²⁹Xe gas spins to ¹H spins in solution via SPINOE. At ultra-low field, where thermal nuclear spin polarization is close to background noise levels and where different nuclei can be simultaneously detected in a single spectrum, the dynamics of the polarization transfer can be observed in real time. We show that by simply bubbling hyperpolarized ¹²⁹Xe into solution, we can enhance ¹H polarization levels by a factor of up to 151-fold. While our protocol leads to lower enhancements than those previously reported under extreme Xe gas pressures, the methodology is easily repeatable and allows for on-demand enhanced spectroscopy. SPINOE at ultra-low magnetic field could also be employed to study ¹²⁹Xe interactions in solutions.     

129Xe and 131Xe nuclear magnetic dipole moments from gas phase NMR spectra

³He, ¹²⁹Xe and ¹³¹Xe NMR measurements of resonance frequencies in the magnetic field B₀ = 11.7586 T in different gas phase mixtures have been reported. Precise radiofrequency values were extrapolated to the zero gas pressure limit. These results combined with new quantum chemical values of helium and xenon nuclear magnetic shielding constants were used to determine new accurate nuclear magnetic moments of ¹²⁹Xe and ¹³¹Xe in terms of that of the ³He nucleus. They are as follows: μ(¹²⁹Xe) = ?0.7779607(158)μ_N and μ(¹³¹Xe) = +0.6918451(70)μ_N. By this means, the new ‘helium method’ for estimations of nuclear dipole moments was successfully tested. Gas phase NMR spectra demonstrate the weak intermolecular interactions observed on the ³He and ¹²⁹Xe and ¹³¹Xe shielding in the gaseous mixtures with Xe, CO₂ and SF₆. Copyright © 2015 John Wiley & Sons, Ltd.     

Molecular Sensing with Hyperpolarized 129Xe Using Switchable Chemical Exchange Relaxation Transfer

An approach for hyperpolarized ¹²⁹Xe molecular sensors is explored using paramagnetic relaxation agents that can be deactivated upon chemical or enzymatic reaction with an analyte. Cryptophane encapsulated ¹²⁹Xe within the vicinity of the paramagnetic center experiences fast relaxation that, through chemical exchange of xenon atoms between cage and solvent pool, causes accelerated hyperpolarized ¹²⁹Xe signal decay in the dissolved phase. In this proof‐of‐concept work, the relaxivity of Gadolinium ^III‐DOTA on ¹²⁹Xe in the solvent was increased eightfold through tethering of the paramagnetic molecule to a cryptophane cage. This potent relaxation agent can be ′turned off′ specifically for ¹²⁹Xe through chemical reactions that spatially separate the Gd^III centre from the attached cryptophane cage. Unlike ¹²⁹Xe chemical shift based sensors, the new concept does not require high spectral resolution and may lead to a new generation of responsive contrast agents for molecular MRI.     

Inclusion Complex Formation of Thiacalix[4]arene and Xe in Aqueous Solution Studied by Hyperpolarized 129Xe NMR

The inclusion complex formation of 4-sulfothiacalix[4]arene sodium salt (STCAS) and Xe has been investigated by using hyperpolarized ¹²⁹Xe NMR spectroscopy. Our new continuous-flow type hyperpolarizing system has advantageous capabilities that can supply hyperpolarized gases continuously and directly to a sample solution in a NMR tube. Consequently saturated Xe concentration in the aqueous solution of STCAS is maintained during the NMR experiment, and ¹²⁹Xe NMR spectra can be obtained in remarkably short time. STCAS concentration dependence of ¹²⁹Xe chemical shift has been analyzed in an elaborated way by a computer method as well as a simple graphic method that we have proposed. The association constant K:13.6±0.8 M⁻¹ at 25 °C was obtained, and further analysis of the temperature dependence has successfully given thermodynamic parameters of enthalpy (ΔH) and entropy (ΔS) for the inclusion complex formation: ΔH = −11.9±1.9 kJ mol⁻¹ and ΔS = −17.4±5.8 JK⁻¹ mol⁻¹. The energetic aspects of complex formation are discussed from the size effect and from the molecular theory of standard entropy, and a release of definite number of water molecules from STCAS cavity is suggested in the inclusion complex formation with Xe.     

129Xe NMR chemical shift in Xe@C60 calculated at experimental conditions: Essential role of the relativity,dynamics, and explicit solvent

The isotropic ¹²⁹Xe nuclear magnetic resonance (NMR) chemical shift (CS) in Xe@C₆₀ dissolved in liquid benzene was calculated by piecewise approximation to faithfully simulate the experimental conditions and to evaluate the role of different physical factors influencing the ¹²⁹Xe NMR CS. The ¹²⁹Xe shielding constant was obtained by averaging the ¹²⁹Xe nuclear magnetic shieldings calculated for snapshots obtained from the molecular dynamics trajectory of the Xe@C₆₀ system embedded in a periodic box of benzene molecules. Relativistic corrections were added at the Breit–Pauli perturbation theory (BPPT) level, included the solvent, and were dynamically averaged. It is demonstrated that the contribution of internal dynamics of the Xe@C₆₀ system represents about 8% of the total nonrelativistic NMR CS, whereas the effects of dynamical solvent add another 8%. The dynamically averaged relativistic effects contribute by 9% to the total calculated ¹²⁹Xe NMR CS. The final theoretical value of 172.7 ppm corresponds well to the experimental ¹²⁹Xe CS of 179.2 ppm and lies within the estimated errors of the model. The presented computational protocol serves as a prototype for calculations of ¹²⁹Xe NMR parameters in different Xe atom guest–host systems. © 2013 Wiley Periodicals, Inc.     

129Xe NMR study of the localization of PdCl2 supported on carbon nanotubes

Localization of PdCl₂ clusters supported on multi-wall carbon nanotubes (MWCNT) has been investigated using ¹²⁹Xe NMR of adsorbed xenon. As-made MWCNTs with channels initially inaccessible for adsorption and ball-milled MWCNTs with the totally accessible internal surface were used as supports. The observed ¹²⁹Xe NMR spectra were determined by the dynamics of xenon exchange between the aggregate pores and nanotube channels. No considerable changes of the ¹²⁹Xe NMR spectrum with the concentration of supported PdCl₂ were observed for the as-made MWCNT, while an additional resonance appeared for the ball-milled nanotubes. The ¹²⁹Xe NMR experiments evidenced the supported species to be localized on the internal surface of the ball-milled MWCNT.     

High Exchange Rate Complexes of 129Xe with Water-Soluble Pillar[5]arenes for Adjustable Magnetization Transfer MRI

Macrocyclic host structures for generating transiently bound ¹²⁹Xe have been used in various ultra-sensitive NMR and MRI applications for molecular sensing of biochemical analytes. They are based on hyperpolarized nuclei chemical exchange saturation transfer (Hyper-CEST). Here, we tested a set of water-soluble pillar[5]arenes with different counterions in order to compare their potential contrast agent abilities with that of cryptophane-A (CrA), the most widely used host for such purposes. The exchange of Xe with such compounds was found to be sensitive to the type of ions present in solution and can be used for switchable magnetization transfer (MT) contrast that arises from off-resonant pre-saturation. We demonstrate that the adjustable MT magnitude depends on the interplay of saturation parameters and found that the optimum MT contrast surpasses the CrA CEST performance at moderate saturation power. Since modification of such water-soluble pillar[5]arenes is straightforward, these compounds can be considered a promising platform for designing various sensors that may complement the field of Xe HyperCEST-based biosensing MRI.     

129Xe NMR as a probe of polymer blends

The miscibility of two-component polymer blends has been investigated using xenon-129 (¹²⁹Xe) nuclear magnetic resonance (NMR) to probe the phase morphology. The chemical shift of ¹²⁹Xe dissolved in a given polymer is unique, thus heterogeneous blends with large domain sizes exhibit two ¹²⁹Xe NMR lines. When a single resonance is obtained, the data are consistent with miscibility, yielding an upper bound on the domain size. The temperature dependence of the relative solubilities and chemical shifts of ¹²⁹Xe dissolved in the pure components may allow a determination of the phase morphology in blends exhibiting a single resonance. The method is used to demonstrate that polychloroprene and 25% epoxidized 1,4-polyisoprene form a miscible blend.     

Simulations of 129Xe NMR chemical shift of atomic xenon dissolved in liquid benzene

The isotropic ¹²⁹Xe NMR chemical shift of atomic Xe dissolved in liquid benzene was simulated by combining classical molecular dynamics and quantum chemical calculations of ¹²⁹Xe nuclear magnetic shielding. Snapshots from the molecular dynamics trajectory of xenon atom in a periodic box of benzene molecules were used for the quantum chemical calculations of isotropic ¹²⁹Xe chemical shift using nonrelativistic density functional theory as well as relativistic Breit?CPauli perturbation corrections. Thus, the correlation and relativistic effects as well as the temperature and dynamics effects could be included in the calculations. Theoretical results are in a very good agreement with the experimental data. The most of the experimentally observed isotropic ¹²⁹Xe shift was recovered in the nonrelativistic dynamical region, while the relativistic effects explain of about 8% of the total ¹²⁹Xe chemical shift.     

Biomembrane Interactions of Functionalized Cryptophane‐A: Combined Fluorescence and 129Xe NMR Studies of a Bimodal Contrast Agent

Fluorescent derivatives of the ¹²⁹Xe NMR contrast agent cryptophane‐A were obtained by functionalization with near infrared fluorescent dyes DY680 and DY682. The resulting conjugates were spectrally characterized, and their interaction with giant and large unilamellar vesicles of varying phospholipid composition was analyzed by fluorescence and NMR spectroscopy. In the latter, a chemical exchange saturation transfer with hyperpolarized ¹²⁹Xe (Hyper‐CEST) was used to obtain sufficient sensitivity. To determine the partitioning coefficients, we developed a method based on fluorescence resonance energy transfer from Nile Red to the membrane‐bound conjugates. This indicated that not only the hydrophobicity of the conjugates, but also the phospholipid composition, largely determines the membrane incorporation. Thereby, partitioning into the liquid‐crystalline phase of 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine was most efficient. Fluorescence depth quenching and flip‐flop assays suggest a perpendicular orientation of the conjugates to the membrane surface with negligible transversal diffusion, and that the fluorescent dyes reside in the interfacial area. The results serve as a basis to differentiate biomembranes by analyzing the Hyper‐CEST signatures that are related to membrane fluidity, and pave the way for dissecting different contributions to the Hyper‐CEST signal.     

Programming A Molecular Relay for Ultrasensitive Biodetection through 129Xe NMR

A supramolecular strategy for detecting specific proteins in complex media by using hyperpolarized ¹²⁹Xe NMR is reported. A cucurbit[6]uril (CB[6])‐based molecular relay was programmed for three sequential equilibrium conditions by designing a two‐faced guest (TFG) that initially binds CB[6] and blocks the CB[6]–Xe interaction. The protein analyte recruits the TFG and frees CB[6] for Xe binding. TFGs containing CB[6]‐ and carbonic anhydrase II (CAII)‐binding domains were synthesized in one or two steps. X‐ray crystallography confirmed TFG binding to Zn²⁺ in the deep CAII active‐site cleft, which precludes simultaneous CB[6] binding. The molecular relay was reprogrammed to detect avidin by using a different TFG. Finally, Xe binding by CB[6] was detected in buffer and in E. coli cultures expressing CAII through ultrasensitive ¹²⁹Xe NMR spectroscopy.     

129Xe NMR Studies of Pecan Shell-Based Biochar and Structure-Process Correlations

Pecan shell-based biochar is utilized as a filtration medium, sequestrant for metallic ions, soil conditioner, and other applications. One process for creating the biochar involves the use of phosphoric acid at high temperature in a partial oxygen atmosphere to produce a highly porous carbonaceous material. In this work, we found ¹²⁹Xe NMR to be an excellent technique to study micropores in biochar. Thus, the ¹²⁹Xe chemical shift in biochar was found to vary linearly with the xenon pressure; from the data an estimate of about 8–9 Å could be proposed for the average pore diameter in pecan shell-based biochar. Through saturation recovery and 2-D NMR exchange experiments, information on the exchange between free versus bound xenon was obtained. Furthermore, correlations of ¹²⁹Xe NMR data with the carbonization process conditions were made.     

A Genetically Encoded β‐Lactamase Reporter for Ultrasensitive 129Xe NMR in Mammalian Cells

Molecular imaging holds considerable promise for elucidating biological processes in normal physiology as well as disease states, but requires noninvasive methods for identifying analytes at sub‐micromolar concentrations. Particularly useful are genetically encoded, single‐protein reporters that harness the power of molecular biology to visualize specific molecular processes, but such reporters have been conspicuously lacking for in vivo magnetic resonance imaging (MRI). Herein, we report TEM‐1 β‐lactamase (bla) as a single‐protein reporter for hyperpolarized (HP) ¹²⁹Xe NMR, with significant saturation contrast at 0.1 μm . Xenon chemical exchange saturation transfer (CEST) interactions with the primary allosteric site in bla give rise to a unique saturation peak at 255 ppm, well removed (≈60 ppm downfield) from the ¹²⁹Xe‐H₂O peak. Useful saturation contrast was also observed for bla expressed in bacterial cells and mammalian cells.     

Enantiopure cryptophane-129Xe nuclear magnetic resonance biosensors targeting carbonic anhydrase

The (+) and ( ? ) enantiomers for a cryptophane-7-bond-linker-benzenesulfonamide biosensor (C7B) were synthesised and their chirality was confirmed by electronic circular dichroism spectroscopy. Biosensor binding to carbonic anhydrase II (CAII) was characterised for both enantiomers by hyperpolarised (HP) ¹²⁹Xe NMR spectroscopy. Our previous study of the racemic ( ± ) C7B biosensor–CAII complex [Chambers, J.M.; Hill, P.A.; Aaron, J.A.; Han, Z.H.; Christianson, D.W.; Kuzma, N.N.; Dmochowski, I.J. J. Am. Chem. Soc.2009, 131, 563–569] identified two ‘bound’ ¹²⁹Xe@C7B peaks by HP ¹²⁹Xe NMR (at 71 and 67 ppm, relative to ‘free’ biosensor at 64 ppm), which led to the initial hypothesis that (+) and ( ? ) enantiomers produce diastereomeric peaks when coordinated to Zn²⁺ at the chiral CAII active site. Unexpectedly, the single enantiomers complexed with CAII also identified two ‘bound’ ¹²⁹Xe@C7B peaks: (+) 72, 68 ppm and ( ? ) 68, 67 ppm. These results are consistent with X-ray crystallographic evidence for benzenesulfonamide inhibitors occupying a second site near the CAII surface. As illustrated by our studies of this model protein–ligand interaction, HP ¹²⁹Xe NMR spectroscopy can be useful for identifying supramolecular assemblies in solution.