Effect of pH and counterions on the encapsulation properties of xenon in water-soluble cryptophanes

In the ¹²⁹Xe NMR‐based biosensing approach in which the hyperpolarized noble gas is transported to biological receptors for a sensitive molecular imaging, cryptophanes are excellent xenon host systems. However to avoid formation of self‐organized systems, these hydrophobic cage molecules can be rendered water soluble by introduction of ionic groups. We show that the sensitivity of xenon to its local environment and the presence of these ionic functions can lead to interesting properties. For a first water‐soluble cryptophane derivative, we show that a precise monitoring of the local pH can be performed. For a second cryptophane, the presence of ionic groups close to the cryptophane cavity modifies the xenon binding constant and in–out exchange rate. The latter allows the tuning of physical properties of xenon–cryptophane interactions without resorting to a change of the cavity size. These results open new perspectives on the influence of chemical modifications of cryptophanes for optimizing the biosensor properties.     

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.     

Dynamics of xenon binding inside the hydrophobic cavity of pseudo-wild-type bacteriophage T4 lysozyme explored through xenon-based NMR spectroscopy

Wild-type bacteriophage T4 lysozyme contains a hydrophobic cavity with binding properties that have been extensively studied by X-ray crystallography and NMR. In the present study, the monitoring of 1H chemical shift variations under xenon pressure enables the determination of the noble gas binding constant (K = 60.2 M(-1)). Although the interaction site is highly localized, dipolar cross-relaxation effects between laser-polarized xenon and nearby protons (SPINOE) are rather poor. This is explained by the high value of the xenon-proton dipolar correlation time (0.8 ns), much longer than the previously reported values for xenon in medium-size proteins. This indicates that xenon is highly localized within the protein cavity, as confirmed by the large chemical shift difference between free and bound xenon. The exploitation of the xenon line width variation vs xenon pressure and protein concentration allows the extraction of the exchange correlation time between free and bound xenon. Comparison to the exchange experienced by protein protons indicates that the exchange between the open and closed conformations of T4 lysozyme is not required for xenon binding.     

MRI Thermometry Based on Encapsulated Hyperpolarized Xenon

A new approach to MRI thermometry using encapsulated hyperpolarized xenon is demonstrated. The method is based on the temperature dependent chemical shift of hyperpolarized xenon in a cryptophane‐A cage. This shift is linear with a slope of 0.29 ppm °C^?1 which is perceptibly higher than the shift of the proton resonance frequency of water (ca. 0.01 ppm °C^?1) that is currently used for MRI thermometry. Using spectroscopic imaging techniques, we collected temperature maps of a phantom sample that could discriminate by direct NMR detection between temperature differences of 0.1 °C at a sensor concentration of 150 μM . Alternatively, the xenon‐in‐cage chemical shift was determined by indirect detection using saturation transfer techniques (Hyper‐CEST) that allow detection of nanomolar agent concentrations. Thermometry based on hyperpolarized xenon sensors improves the accuracy of currently available MRI thermometry methods, potentially giving rise to biomedical applications of biosensors functionalized for binding to specific target molecules.     

Extreme Confinement of Xenon by Cryptophane‐111 in the Solid State

Solids that sorb, capture and/or store the heavier noble gases are of interest because of their potential for transformative rare gas separation/production, storage, or recovery technologies. Herein, we report the isolation, crystal structures, and thermal stabilities of a series of xenon and krypton clathrates of (±)‐cryptophane‐111 ( 111 ). One trigonal crystal form, Xe@ 111? y(solvent), is exceptionally stable, retaining xenon at temperatures of up to about 300 °C. The high kinetic stability is attributable not only to the high xenon affinity and cage‐like nature of the host, but also to the crystal packing of the clathrate, wherein each window of the molecular container is blocked by the bridges of adjacent containers, effectively imprisoning the noble gas in the solid state. The results highlight the potential of discrete molecule materials exhibiting intrinsic microcavities or zero‐dimensional pores.     

Development of a functionalized xenon biosensor

NMR-based biosensors that utilize laser-polarized xenon offer potential advantages beyond current sensing technologies. These advantages include the capacity to simultaneously detect multiple analytes, the applicability to in vivo spectroscopy and imaging, and the possibility of "remote" amplified detection. Here, we present a detailed NMR characterization of the binding of a biotin-derivatized caged-xenon sensor to avidin. Binding of "functionalized" xenon to avidin leads to a change in the chemical shift of the encapsulated xenon in addition to a broadening of the resonance, both of which serve as NMR markers of ligand-target interaction. A control experiment in which the biotin-binding site of avidin was blocked with native biotin showed no such spectral changes, confirming that only specific binding, rather than nonspecific contact, between avidin and functionalized xenon leads to the effects on the xenon NMR spectrum. The exchange rate of xenon (between solution and cage) and the xenon spin-lattice relaxation rate were not changed significantly upon binding. We describe two methods for enhancing the signal from functionalized xenon by exploiting the laser-polarized xenon magnetization reservoir. We also show that the xenon chemical shifts are distinct for xenon encapsulated in different diastereomeric cage molecules. This demonstrates the potential for tuning the encapsulated xenon chemical shift, which is a key requirement for being able to multiplex the biosensor.     

Investigation of chloromethane complexes of cryptophane‐A analogue with butoxy groups using 13C NMR in the solid state and solution along with single crystal X‐ray diffraction

Host‐guest complexes between cryptophane‐A analogue with butoxy groups (cryptophane‐But) and chloromethanes (chloroform, dichloromethane) were investigated in the solid state by means of magic‐angle spinning ¹³C NMR spectroscopy. The separated local fields method with ¹³C‐¹H dipolar recoupling was used to determine the residual dipolar coupling for the guest molecules encaged in the host cavity. In the case of chloroform guest, the residual dipolar interaction was estimated to be about 19 kHz, consistent with a strongly restricted mobility of the guest in the cavity, while no residual interaction was observed for encaged dichloromethane. In order to rationalize this unexpected result, we performed single crystal X‐ray diffraction studies, which confirmed that both guest molecules indeed were present inside the cryptophane cavity, with a certain level of disorder. To improve the insight in the dynamics, we performed a ¹³C NMR spin‐lattice relaxation study for the dichloromethane guest in solution. The system was characterized by chemical exchange, which was slow on the chemical shift time scale but fast with respect to the relaxation rates. Despite these disadvantageous conditions, we demonstrated that the data could be analyzed and that the results were consistent with an isotropic reorientation of dichloromethane within the cryptophane cavity. Copyright © 2015 The Authors. Magnetic Resonance in Chemistry published by John Wiley & Sons Ltd.     

Investigation of DOTA–Metal Chelation Effects on the Chemical Shift of 129Xe

Recent work has shown that xenon chemical shifts in cryptophane‐cage sensors are affected when tethered chelators bind to metals. Here, we explore the xenon shifts in response to a wide range of metal ions binding to diastereomeric forms of 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid (DOTA) linked to cryptophane‐A. The shifts induced by the binding of Ca²⁺, Cu²⁺, Ce³⁺, Zn²⁺, Cd²⁺, Ni²⁺, Co²⁺, Cr²⁺, Fe³⁺, and Hg²⁺ are distinct. In addition, the different responses of the diastereomers for the same metal ion indicate that shifts are affected by partial folding with a correlation between the expected coordination number of the metal in the DOTA complex and the chemical shift of ¹²⁹Xe. These sensors may be used to detect and quantify many important metal ions, and a better understanding of the basis for the induced shifts could enhance future designs.     

NMR spectroscopic study of noble gas binding into the engineered cavity of HPr(I14A) from Staphylococcus carnosus

Xenon binding into preexisting cavities in proteins is a well-known phenomenon. Here we investigate the interaction of helium, neon, and argon with hydrophobic cavities in proteins by NMR spectroscopy. 1H and 15N chemical shifts of the I14A mutant of the histidine-containing phosphocarrier protein (HPr(I14A)) from Staphylococcus carnosus are analyzed by chemical shift mapping. Total noble gas induced chemical shifts, Delta, are calculated and compared with the corresponding values obtained using xenon as a probe atom. This comparison reveals that the same cavity is detected with both argon and xenon. Measurements using the smaller noble gases helium and neon as probe atoms do not result in comparable effects. The dependence of amide proton and nitrogen chemical shifts on the argon concentration is investigated in the range from 10 mM up to 158 mM. The average dissociation constant for argon binding into the engineered cavity is determined to be about 90 mM.     

Probing the hydrophobic cavity of lipid transfer protein from Nicotiana tabacum through xenon-based NMR spectroscopy

The hydrophobic cavity of Lipid Transfer Protein 1 from Nicotiana tabacum is investigated in detail by NMR using xenon as a spy. The analysis of the (129)Xe chemical shifts and self-relaxation times gives evidence of protein-xenon interaction. Thermodynamics of the binding is characterized through the study of aliphatic (1)H and (13)C chemical shift variation as a function of xenon pressure. The binding constant is evaluated to 75.5 +/- 1.0 M(-1) at 293 K. The location of xenon inside the cavity is deduced from SPINOE experiments. The noble gas appears to occupy four sites, and xenon self-relaxation experiments indicate that it quickly jumps between different sites. The chemical shifts of amide protons and nitrogens also depend on the xenon concentration, either specifically or nonspecifically for atoms at the external surface of the protein. Yet, contrary to aliphatic atoms, they do not correspond to short-range interactions as confirmed by magnetization transfer experiments between laser-polarized xenon and protons in H(2)O. These (15)N chemical shift variations, used in combination with (15)N transverse self-relaxation rates to determine the lower limit of the binding rate, consequently reveal subtle changes in the structure of the protein upon binding.     

Understanding a Host–Guest Model System through 129Xe NMR Spectroscopic Experiments and Theoretical Studies

Gaining an understanding of the nature of host–guest interactions in supramolecular complexes involving heavy atoms is a difficult task. Described herein is a robust simulation method applied to complexes between xenon and members of a cryptophane family. The calculated chemical shift of xenon caged in a H₂O₂ probe, as modeled by quantum chemistry with complementary‐orbital, topological, and energy‐decomposition analyses, is in excellent agreement with that observed in hyperpolarized ¹²⁹Xe NMR spectra. This approach can be extended to other van der Waals complexes involving heavy atoms.     

NMR-spectroscopic mapping of an engineered cavity in the I14A mutant of HPr from Staphylococcus carnosus using xenon

The interaction between the histidine-containing phosphocarrier protein HPr and xenon atoms in solution is studied in the present paper. Wild-type HPr as well as the exchange mutant I14A have been studied. Specific binding of xenon into an engineered cavity created via the exchange of amino acid residue I14 by alanine could be shown using 1H-15N heteronuclear single-quantum coherence (HSQC) spectroscopy. Xenon binding results in pronounced changes of the 1H and 15N chemical shifts of amide groups close to the cavity. In addition to this observation which allows the NMR-spectroscopic mapping of such cavities, we have shown that the entire molecule is slightly rearranged as a result of xenon binding. In contrast, wild-type HPr only exhibits minor chemical shift changes due to the nonspecific interactions with the xenon atoms in solution.     

An Ultra-Microporous Metal–Organic Framework with Exceptional Xe Capacity

Molecular confinement plays a significant effect on trapped gas and solvent molecules. A fundamental understanding of gas adsorption within the porous confinement provides information necessary to design a material with improved selectivity. In this regard, metal–organic framework (MOF) adsorbents are ideal candidate materials to study confinement effects for weakly interacting gas molecules, such as noble gases. Among the noble gases, xenon (Xe) has practical applications in the medical, automotive and aerospace industries. In this Communication, we report an ultra-microporous nickel-isonicotinate MOF with exceptional Xe uptake and selectivity compared to all benchmark MOF and porous organic cage materials. The selectivity arises because of the near perfect fit of the atomic Xe inside the porous confinement. Notably, at low partial pressure, the Ni–MOF interacts very strongly with Xe compared to the closely related Krypton gas (Kr) and more polarizable CO₂. Further ¹²⁹Xe NMR suggests a broad isotropic chemical shift due to the reduced motion as a result of confinement.     

Novel method for the measurement of xenon gas solubility using 129Xe NMR spectroscopy

A novel method is presented for determining xenon partitioning between a gas phase and a liquid phase. An experimental setup which permits the simultaneous measurement of the 129Xe chemical shift in both the gas and the liquid phases, that is, under the same experimental conditions, has been designed. Xenon solubility is obtained via 129Xe chemical shift measurements in the gas phase. The method was validated against xenon solubility data from the literature; in general, the agreement is found to be within 3%. The solubility of xenon in three solvents for which data have not been previously reported (acetone, acetonitrile, and 1,1,2,2-tetrachloroethane) was determined using this novel method. 129Xe chemical shifts for dissolved xenon are also reported; it is found that xenon-xenon interactions may play a significant role in the liquid phase even at low equilibrium xenon pressures.     

Fluorescence spectra of matrix isolated silver atoms

Silver atoms isolated in noble gas matrices show fluorescence spectra with large Stokes shifts, when optically excited via the 5P ← 5S transition which is three-fold split in the matrix by spin—orbit and crystal field interaction. The shifts are explained by the formation of an excimeric bond between the excited metal atom and the noble gas cage. For Ag in Kr it is demonstrated, that most of the Stokes shift arises from the ground state repulsion.     

Exploring surfaces and cavities in lipoxygenase and other proteins by hyperpolarized xenon-129 NMR

This paper presents an exploratory study of the binding interactions of xenon with the surface of several different proteins in the solution and solid states using both conventional and hyperpolarized (129)Xe NMR. The generation of hyperpolarized (129)Xe by spin exchange optical pumping affords an enhancement by 3-4 orders of magnitude of its NMR signal. As a result, it is possible to observe Xe directly bound to the surface of micromolar quantities of lyophilized protein. The highly sensitive nature of the (129)Xe line shape and chemical shift are used as indicators for the conditions most likely to yield maximal dipolar contact between (129)Xe nuclei and nuclear spins situated on the protein. This is an intermediate step toward achieving the ultimate goal of NMR enhancement of the binding-site nuclei by polarization transfer from hyperpolarized (129)Xe. The hyperpolarized (129)Xe spectra resulting from exposure of four different proteins in the lyophilized, powdered form have been examined for evidence of binding. Each of the proteins, namely, metmyoglobin, methemoglobin, hen egg white lysozyme, and soybean lipoxygenase, yielded a distinctly different NMR line shape. With the exception of lysozyme, the proteins all possess a paramagnetic iron center which can be expected to rapidly relax the (129)Xe and produce a net shift in its resonance position if the noble gas atom occupies specific binding sites near the iron. At temperatures from 223 to 183 K, NMR signals were observed in the 0-40 ppm chemical shift range, relative to Xe in the gas phase. The signals broadened and shifted downfield as the temperature was reduced, indicating that Xe is exchanging between the gas phase and internal or external binding sites of the proteins. Additionally, conventional (129)Xe NMR studies of metmyoglobin and lipoxygenase in the solution state are presented. The temperature dependence of the chemical shift and line shape indicate exchange of Xe between adsorption sites on lipoxygenase and Xe in the solvent on the slow to intermediate exchange time scale. The NMR results are compared with N(2), Xe, and CH(4) gas adsorption isotherms. It is found that lipoxygenase is unique among the proteins studied in possessing a relatively high affinity for gas molecules, and in addition, demonstrating the most clearly resolved adsorbed (129)Xe NMR peak in the lyophilized state.     

Xenon porometry at room temperature

Xenon porometry is a method in which porous material is immersed in a medium and the properties of the material are studied by means of 129Xe nuclear magnetic resonance (NMR) of xenon gas dissolved in the medium. For instance, the chemical shift of a particular signal (referred to as signal D) arising from xenon inside small pockets formed in the pores during the freezing of the confined medium is highly sensitive to the pore size. In the present study, we show that when naphthalene is used as the medium the pore size distribution of the material can be determined by measuring a single one-dimensional spectrum near room temperature and converting the chemical shift scale of signal D to the pore radius scale by using an experimentally determined correlation. A model has been developed that explains the curious behavior of the chemical shift of signal D as a function of pore radius. The other signals of the spectra measured at different temperatures have also been identified, and the influence of xenon pressure on the spectra has been studied. For comparison, 129Xe NMR spectra of pure xenon gas adsorbed to porous materials have been measured and analyzed.     

Application of quadrupolar 131Xe-NMR relaxation to the study of macromolecular systems

¹³¹Xe-NMR longitudinal relaxation rates have been measured by the inversion recovery method for xenon in presence of lecithin vesicles or a small protein charybdotoxin. The transverse relaxation rates in the same spectra have been obtained by spectral deconvolution. The results show that xenon in lecithin vesicles is in a rapid exchange between free and bound sites and that averaging of the electric-field gradient at the Xe nucleus is a two-step process. From these results, estimates have been obtained for the parameters entering the ¹³¹Xe quadrupolar interaction. © 1996 John Wiley & Sons, Inc.     

Structure of a 129Xe-cryptophane biosensor complexed with human carbonic anhydrase II

Cryptophanes represent an exciting class of xenon-encapsulating molecules that can be exploited as probes for nuclear magnetic resonance imaging. The 1.70 A resolution crystal structure of a cryptophane-derivatized benezenesulfonamide complexed with human carbonic anhydrase II shows how an encapsulated xenon atom can be directed to a specific biological target. The crystal structure confirms binding measurements indicating that the cryptophane cage does not strongly interact with the surface of the protein, which may enhance the sensitivity of 129Xe NMR spectroscopic measurements in solution.     

Water soluble cryptophanes showing unprecedented affinity for xenon: candidates as NMR-based biosensors

Cryptophanes bearing OCH(2)COOH groups in place of the methoxy groups represent a new class of xenon-carrier molecules soluble in water at biological pH. By using (1)H and (129)Xe NMR (thermally- and laser-polarized dissolved gas), the structural and dynamical behaviors of these host molecules as well as their interaction with xenon are studied. They are shown to exist in aqueous solution under different conformations in very slow exchange. A saddle form present for one of these conformations could explain the (1)H NMR spectra. Whereas the cryptophanes in such a conformation are unable to complex xenon, unprecedented high binding constants are found for cryptophanes in the other canonical crown-crown conformation. These host molecules could therefore be valuable candidates for biosensing using (129)Xe MRI.