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Cryptophane host molecules provide ultrasensitive contrast agents for 129 Xe NMR/MRI. To investigate key features of cryptophane-Xe sensing behavior, we designed a novel water-soluble cryptophane with a pendant hydrophobic adamantyl moiety, which has good affinity for a model receptor, beta-cyclodextrin (β-CD). Adamantyl-functionalized cryptophane-A (AFCA) was synthesized and characterized for Xe affinity, 129 Xe NMR signal, and aggregation state at varying AFCA and β-CD concentrations. The Xe-AFCA association constant was determined by fluorescence quenching, K A = 114,000 ± 5000 M -1 at 293 K, which is the highest reported affinity for a cryptophane host in phosphate-buffered saline (pH 7.2). No hyperpolarized (hp) 129 Xe NMR peak corresponding to AFCA-bound Xe was directly observed at high (100 μM) AFCA concentration, where small cryptophane aggregates were observed, and was only detected at low (15 μM) AFCA concentration, where the sensor remained fully monomeric in solution. Additionally, we observed no change in the chemical shift of AFCA-encapsulated 129 Xe after β-CD binding to the adamantyl moiety and a concomitant lack of change in the size distribution of the complex, suggesting that a change in the aggregation state is necessary to elicit a 129 Xe NMR chemical shift in cryptophane-based sensing. These results aid in further elucidating the recently discovered aggregation phenomenon, highlight limitations of cryptophane-based Xe sensing, and offer insights into the design of monomeric, high-affinity Xe sensors.

Dysregulation of cellular ribose uptake can be indicative of metabolic abnormalities or tumorigenesis. However, analytical methods are currently limited for quantifying ribose concentration in complex biological samples. Here, we utilize the highly specific recognition of ribose by ribose-binding protein (RBP) to develop a single-protein ribose sensor detectable via a sensitive NMR technique known as hyperpolarized 129 Xe chemical exchange saturation transfer (hyper-CEST). We demonstrate that RBP, with a tunable ribose-binding site and further engineered to bind xenon, enables the quantitation of ribose over a wide concentration range (nM to mM). Ribose binding induces the RBP "closed" conformation, which slows Xe exchange to a rate detectable by hyper-CEST. Such detection is remarkably specific for ribose, with the minimal background signal from endogenous sugars of similar size and structure, for example, glucose or ribose-6-phosphate. Ribose concentration was measured for mammalian cell lysate and serum, which led to estimates of low-mM ribose in a HeLa cell line. This highlights the potential for using genetically encoded periplasmic binding proteins such as RBP to measure metabolites in different biological fluids, tissues, and physiologic states.

We investigated Xe binding in a previously reported paramagnetic metal-organic tetrahedral capsule, [Co 4 L 6 ] 4- , where L 2- = 4,4'-bis[(2-pyridinylmethylene)amino][1,1'-biphenyl]-2,2'-disulfonate. The Xe-inclusion complex, [XeCo 4 L 6 ] 4- , was confirmed by 1 H NMR spectroscopy to be the dominant species in aqueous solution saturated with Xe gas. The measured Xe dissociation rate in [XeCo 4 L 6 ] 4- , k off = 4.45(5) × 10 2 s -1 , was at least 40 times greater than that in the analogous [XeFe 4 L 6 ] 4- complex, highlighting the capability of metal-ligand interactions to tune the capsule size and guest permeability. The rapid exchange of 129 Xe nuclei in [XeCo 4 L 6 ] 4- produced significant hyperpolarized 129 Xe chemical exchange saturation transfer (hyper-CEST) NMR signal at 298 K, detected at a concentration of [XeCo 4 L 6 ] 4- as low as 100 pM, with presaturation at -89 ppm, which was referenced to solvated 129 Xe in H 2 O. The saturation offset was highly temperature-dependent with a slope of -0.41(3) ppm/K, which is attributed to hyperfine interactions between the encapsulated 129 Xe nucleus and electron spins on the four Co II centers. As such, [XeCo 4 L 6 ] 4- represents the first example of a paramagnetic hyper-CEST (paraHYPERCEST) sensor. Remarkably, the hyper-CEST 129 Xe NMR resonance for [XeCo 4 L 6 ] 4- (δ = -89 ppm) was shifted 105 ppm upfield from the diamagnetic analogue [XeFe 4 L 6 ] 4- (δ = +16 ppm). The Xe inclusion complex was further characterized in the crystal structure of (C(NH 2 ) 3 ) 4 [Xe 0.7 Co 4 L 6 ]·75 H 2 O ( 1 ). Hydrogen bonding between capsule-linker sulfonate groups and exogenous guanidinium cations, (C(NH 2 ) 3 ) + , stabilized capsule-capsule interactions in the solid state and also assisted in trapping a Xe atom (∼42 Å 3 ) in the large (135 Å 3 ) cavity of 1 . Magnetic susceptibility measurements confirmed the presence of four noninteracting, magnetically anisotropic high-spin Co II centers in 1 . Furthermore, [Co 4 L 6 ] 4- was found to be stable toward aggregation and oxidation, and the CEST performance of [XeCo 4 L 6 ] 4- was unaffected by biological macromolecules in H 2 O. These results recommend metal-organic capsules for fundamental investigations of Xe host-guest chemistry as well as applications with highly sensitive 129 Xe-based sensors.

We investigate the magnetic resonance properties and exchange kinetics of guest molecules in a series of hetero-bimetallic capsules, [Co n Fe 4- n L 6 ] 4- ( n = 1-3), where L 2- = 4,4'-bis[(2-pyridinylmethylene)amino]-[1,1'-biphenyl]-2,2'-disulfonate. H bond networks between capsule sulfonates and guanidinium cations promote the crystallization of [Co n Fe 4- n L 6 ] 4- . The following four isostructural crystals are reported: two guest-free forms, (C(NH 2 ) 3 ) 4 [Co 1.8 Fe 2.2 L 6 ]·69H 2 O ( 1 ) and (C(NH 2 ) 3 ) 4 [Co 2.7 Fe 1.3 L 6 ]·73H 2 O ( 2 ), and two Xe- and CFCl 3 -encapsulated forms, (C(NH 2 ) 3 ) 4 [(Xe) 0.8 Co 1.8 Fe 2.2 L 6 ]·69H 2 O ( 3 ) and (C(NH 2 ) 3 ) 4 [(CFCl 3 )Co 2.0 Fe 2.0 L 6 ]·73H 2 O ( 4 ), respectively. Structural analyses reveal that Xe induces negligible structural changes in 3 , while the angles between neighboring phenyl groups expand by ca. 3° to accommodate the much larger guest, CFCl 3 , in 4 . These guest-encapsulated [Co n Fe 4- n L 6 ] 4- molecules reveal 129 Xe and 19 F chemical shift changes of ca. -22 and -10 ppm at 298 K, respectively, per substitution of low-spin Fe II by high-spin Co II . Likewise, the temperature dependence of the 129 Xe and 19 F NMR resonances increases by 0.1 and 0.06 ppm/K, respectively, with each additional paramagnetic Co II center. The optimal temperature for hyperpolarized (hp) 129 Xe chemical exchange saturation transfer (hyper-CEST) with [Co n Fe 4- n L 6 ] 4- capsules was found to be inversely proportional to the number of Co II centers, n , which is consistent with the Xe chemical exchange accelerating as the portals expand. The systematic study was facilitated by the tunability of the [M 4 L 6 ] 4- capsules, further highlighting these metal-organic systems for developing responsive sensors with highly shifted 129 Xe resonances.

Cryptophane-based biosensors are promising agents for the ultrasensitive detection of biomedically relevant targets via 129 Xe NMR. Dynamic light scattering revealed that cryptophanes form water-soluble aggregates tens to hundreds of nanometers in size. Acridine orange fluorescence quenching assays allowed quantitation of the aggregation state, with critical concentrations ranging from 200 nM to 600 nM, depending on the cryptophane species in solution. The addition of excess carbonic anhydrase (CA) protein target to a benzenesulfonamide-functionalized cryptophane biosensor (C8B) led to C8B disaggregation and produced the expected 1:1 C8B-CA complex. C8B showed higher affinity at 298 K for the cytoplasmic isozyme CAII than the extracellular CAXII isozyme, which is a biomarker of cancer. Using hyper-CEST NMR, we explored the role of stoichiometry in detecting these two isozymes. Under CA-saturating conditions, we observed that isozyme CAII produces a larger 129 Xe NMR chemical shift change (δ = 5.9 ppm, relative to free biosensor) than CAXII (δ = 2.7 ppm), which indicates the strong potential for isozyme-specific detection. However, stoichiometry-dependent chemical shift data indicated that biosensor disaggregation contributes to the observed 129 Xe NMR chemical shift change that is normally assigned to biosensor-target binding. Finally, we determined that monomeric cryptophane solutions improve hyper-CEST saturation contrast, which enables ultrasensitive detection of biosensor-protein complexes. These insights into cryptophane-solution behavior support further development of xenon biosensors, but will require reinterpretation of the data previously obtained for many water-soluble cryptophanes.