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.     

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.     

“Clickable” Hydrosoluble PEGylated Cryptophane as a Universal Platform for 129Xe Magnetic Resonance Imaging Biosensors

We describe the synthesis of a highly water‐soluble cryptophane 1 that can be seen as a universal platform for the construction of ¹²⁹Xe magnetic resonance imaging (MRI)‐based biosensors. Compound 1 is easily functionalized by Huisgen cycloaddition and exhibits excellent xenon‐encapsulation properties. In addition, 1 is nontoxic at the concentrations typically used for hyperpolarized ¹²⁹Xe MRI.     

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.     

Reversible Encapsulation of Xenon and CH2Cl2 in a Solid‐State Molecular Organometallic Framework (Guest@SMOM)

Reversible encapsulation of CH₂Cl₂ or Xe in a non‐porous solid‐state molecular organometallic framework of [Rh(Cy₂PCH₂PCy₂)(NBD)][BAr^F₄] occurs in single‐crystal to single‐crystal transformations. These processes are probed by solid‐state NMR spectroscopy, including ¹²⁹Xe SSNMR. Non‐covalent interactions with the ‐CF₃ groups, and hydrophobic channels formed, of [BAr^F₄]^? anions are shown to be important, and thus have similarity to the transport of substrates and products to and from the active site in metalloenzymes.     

Synthesis and Characterization of [XeOXe]2+ in the Adduct‐Cation Salt, [CH3CN‐ ‐ ‐XeOXe‐ ‐ ‐NCCH3][AsF6]2

Acetonitrile and [FXeOXe‐ ‐ ‐FXeF][AsF₆] react at ?60 °C in anhydrous HF (aHF) to form the CH₃CN adduct of the previously unknown [XeOXe]²⁺ cation. The low‐temperature X‐ray structure of [CH₃CN‐ ‐ ‐XeOXe‐ ‐ ‐NCCH₃][AsF₆]₂ exhibits a well‐isolated adduct‐cation that has among the shortest Xe?N distances obtained for an sp‐hybridized nitrogen base adducted to xenon. The Raman spectrum was fully assigned by comparison with the calculated vibrational frequencies and with the aid of ¹⁸O‐enrichment studies. Natural bond orbital (NBO), atoms in molecules (AIM), electron localization function (ELF), and molecular electrostatic potential surface (MEPS) analyses show that the Xe?O bonds are semi‐ionic whereas the Xe?N bonds may be described as strong electrostatic (σ‐hole) interactions.     

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.     

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.     

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.     

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.     

Nondisruptive Dissolution of Hyperpolarized 129Xe into Viscous Aqueous and Organic Liquid Crystalline Environments

Studies of hyperpolarized xenon‐129 (hp‐¹²⁹Xe) in media such as liquid crystals and cell suspensions are in demand for applications ranging from biomedical imaging to materials engineering but have been hindered by the inability to bubble Xe through the desired media as a result of viscosity or perturbations caused by bubbles. Herein a device is reported that can be reliably used to dissolve hp‐¹²⁹Xe into viscous aqueous and organic samples without bubbling. This method is robust, requires small sample volumes (<60 μL), is compatible with existing NMR hardware, and is made from readily available materials. Experiments show that Xe can be introduced into viscous and aligned media without disrupting molecular order. We detected dissolved xenon in an aqueous liquid crystal that is disrupted by the shear forces of bubbling, and we observed liquid‐crystal phase transitions in (MBBA). This tool allows an entirely new class of samples to be investigated by hyperpolarized‐gas NMR spectroscopy.     

Cucurbit[6]uril Hyperpolarized Chemical Exchange Saturation Transfer Pulse Sequence Parameter Optimization and Detectability Limit Assessment at 3.0T

Molecular imaging is the future of personalized medicine; however, it requires effective contrast agents. Hyperpolarized chemical exchange saturation transfer (HyperCEST) can boost the signal of Hyperpolarized ¹²⁹Xe MRI and render it a molecular imaging modality of high efficiency. Cucurbit[6]uril (CB6) has been successfully employed in vivo as a contrast agent for HyperCEST MRI, however its performance in a clinical MRI scanner has yet to be optimized. In this study, MRI pulse sequence parameter optimization was first performed in CB6 solutions in phosphate-buffered saline (PBS), and subsequently in whole sterile citrated bovine blood. The performance of four different depolarization pulse shapes (sinusoidal, 3-lobe sinc (3LS), rectangular (block), and hyperbolic secant (hypsec) was optimized. The detectability limits of CB6 in a clinical 3.0T MRI scanner was assessed using the optimized pulse sequences. The 3LS depolarization pulses performed best, and demonstrated 24 % depletion in a 25 μM solution of CB6 in PBS. It performed similarly in blood. The CB6 detectability limit was found to be 100 μM in citrated bovine blood with a correspondent HyperCEST depletion of 30 % ±9 %. For the first time, the HP ¹²⁹Xe HyperCEST effect was observed in red blood cells (RBC) and had a similar strength as HyperCEST in plasma.     

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.     

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.     

Tuning the Magnetization Dynamic Properties of Nd⋅⋅⋅Fe and Nd⋅⋅⋅Co Single‐Molecular Magnets by Introducing 3 d–4 f Magnetic Interactions

By using paramagnetic [Fe(CN)₆]^3? anions in place of diamagnetic [Co(CN)₆]^3? anions, two field‐induced mononuclear single‐molecular magnets, [Nd(18‐crown‐6)(H₂O)₄][Co(CN)₆] ? 2 H₂O ( 1 ) and [Nd(18‐crown‐6)(H₂O)₄][Fe(CN)₆] ? 2 H₂O ( 2 ), have been synthesized and characterized. Single‐crystal X‐ray diffraction analysis revealed that compounds 1 and 2 were ionic complexes. The Nd^III ions were located inside the cavities of the 18‐crown‐6 ligands and were each bound by four water molecules on either side of the crown ether. Magnetic investigations showed that these compounds were both field‐induced single‐molecular magnets. By comparing the slow relaxation behaviors of compounds 1 and 2 , we found significant differences between the direct and Raman processes for these two complexes, with a stronger direct process in compound 2 at low temperatures. Complete active space self‐consistent field (CASSCF) calculations were also performed on two [Nd(18‐crown‐6)(H₂O)₄]³⁺ fragments of compounds 1 and 2 . Ab initio calculations showed that the magnetic anisotropies of the Nd^III centers in complexes 1 and 2 were similar to each other, which indicated that the difference in relaxation behavior was not owing to the magnetic anisotropy of Nd^III. Our analysis showed that the magnetic interaction between the Nd^III ion and the low‐spin Fe^III ion in complex 2 played an important role in enhancing the direct process and suppressing the Raman process of the single‐molecular magnet.     

Covalent‐Bond Formation in the Course of the Inhibition of δ‐Chymotrypsin with trans‐Decalin‐Type Organophosphates: 31P‐NMR Evidence

Earlier investigations have shown that the irreversible inhibition of δ‐chymotrypsin with the axially substituted trans‐3‐(2,4‐dinitrophenoxy)‐2,4‐dioxa‐3λ⁵‐phosphabicyclo[4.4.0]decan‐3‐one (=2‐(2,4‐dinitrophenoxy)hexahydro‐4H‐1,3,2‐benzodioxaphosphorin 2‐oxide) proceeds under inversion of the configuration at the P‐atom. Since this assignment is based on the comparison of the respective chemical shifts with model compounds, the covalent nature of the binding interaction between enzyme and inhibitor was formulated in analogy. To prove this assumption, inhibition experiments were performed with the deuterated inhibitor (±)‐trans‐3‐(2,4‐dinitrophenoxy)‐2,4‐dioxa‐3λ⁵‐phospha(1,5,5‐²H₃)bicyclo[4.4.0]decan‐3‐one ((±)‐ 6a ). ³¹P{²H}‐NMR‐Spectroscopic monitoring of the reaction of stoichiometric amounts of the enzyme with (±)‐ 6a at pH 7.8 yielded the diastereoisomeric adducts 9 (−3.88 ppm) and 9′ (−3.96 ppm). Comparing the ³¹P chemical shifts of the corresponding deuterated covalent phosphoserine model compounds 8a/8a′ (−6.70 ppm, axial) and 8b/8b′ (−4.11/−4.13 ppm, equatorial) confirmed the inversion of the configuration at the P‐atom. ¹H‐Correlated ³¹P{²H}‐NMR spectra revealed a cross peak of the Ser¹⁹⁵‐H₂ (4.45 ppm) with the P‐atom of the inhibitor at −3.88/−3.96 ppm, thus establishing the covalent nature of the Ser¹⁹⁵−O−P bond.     

<Superscript>129</Superscript>Xe NMR spectroscopy of adsorbed xenon: Possibilites for exploration of microporous carbon materials

Despite the extensive use of ¹²⁹Xe NMR for characterization of high surface-to-volume porous solids, particularly zeolites, this method has not been widely used to explore the properties of microporous carbon materials. In this study, commercial amorphous carbons of different origin (produced from different precursors) and a series of activated carbons obtained by successive cyclic air oxidation/pyrolysis treatments of a single precursor were examined. Models of ¹²⁹Xe chemical shift as a function of local Xe density, mean pore size, and temperature are discussed. The virial coefficient arising from binary xenon collisions, σ_Xe-Xe, varied linearly with the mean pore size given by N₂ adsorption analysis; σ_Xe-Xe appeared to be a better probe of the mean pore size than the chemical shift extrapolated to zero pressure, σ_S.     

Controlling Gas Selectivity in Molecular Porous Liquids by Tuning the Cage Window Size

Control of pore window size is the standard approach for tuning gas selectivity in porous solids. Here, we present the first example where this is translated into a molecular porous liquid formed from organic cage molecules. Reduction of the cage window size by chemical synthesis switches the selectivity from Xe‐selective to CH₄‐selective, which is understood using ¹²⁹Xe, ¹H, and pulsed‐field gradient NMR spectroscopy.     

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.     

ATRP of MMA under 60Co γ‐irradiation at room temperature

In this work, atom transfer radical polymerization (ATRP) of methyl methacrylate (MMA) was successfully carried out at room temperature (25 °C) under ⁶⁰Co γ‐irradiation environment. The polymerization proceeded smoothly with high conversion (>90%) within 7 h. The polymerizations kept the features of controlled radical polymerization: first‐order kinetics, well‐predetermined number‐average molecular weights (M_n,GPC), and narrow molecular weight distributions (M_w/M_n < 1.25). ¹H NMR spectroscope and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry confirmed that poly(methyl methacrylate) (PMMA) chain was end‐capped by the initiator moieties. The Cu(II) concentration could reduce to 20 ppm level while keeping good control over molecular weights. This is the first successful example for the ATRP of MMA under ⁶⁰Co γ‐irradiation at room temperature. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011