Xenon-Cryptophane And Xenon-Protein Interactions Enabling Ultrasensitive Xenon-129 Nmr

Proton-based magnetic resonance imaging (1H MRI) is a widely used technique for the detection and diagnosis of disease states. However, 1H MRI is limited in imaging low-abundance biomarkers due to the low sensitivity inherent in the technique. To overcome this issue, hyperpolarized (hp) 129Xe NMR and MRI have been investigated as complements to 1H-based techniques. Xenon possesses a high sensitivity to its molecular environment due its large and polarizable electron cloud and offers 104-108-fold signal enhancement over thermal polarization in its hp state. These features allow hp 129Xe to be used in biosensing applications, where the chemical shift or the intensity of the 129Xe NMR signal is modulated when a Xe-binding host interacts with its target. The best-studied of these Xe hosts are the organic cage molecules known as cryptophanes. We recently discovered that cryptophanes are not well-solvated in solution, as previously assumed, but instead form water-soluble aggregates of tens-to-hundreds of nanometers in size. The insights gleaned from dynamic light scattering, fluorescence quenching, and 129Xe NMR analyses of these aggregates suggest that a reevaluation of cryptophane characterization and sensing strategies is necessary. These strategies were further studied using a novel adamantyl-functionalized cryptophane-A (AFCA) with record-high affinity for Xe. Hp 129Xe NMR only revealed signal corresponding to AFCA-encapsulated 129Xe at low AFCA concentrations at which the compound was monomeric, and not at higher concentrations at which small aggregates were observed. Additionally, no change in the 129Xe NMR chemical shift, nor the AFCA aggregation state, was observed upon β-CD binding to the adamantyl moiety, suggesting that changes in the aggregation state are necessary to elicit a 129Xe NMR chemical shift change. AFCA was also investigated as a model system for “turn-on” Xe sensing, where signal is only observed in the presence of analyte or environmental change. These studies helped to identify important limitations in cryptophane-based Xe sensing. In addition to cryptophane-based contrast agents, genetically encoded (GE) MRI reporters have been attracting significant interest. Such reporters have the advantage of being expressed directly in the tissue of interest, as well as being able to be modified to bind specific analytes via directed evolution. We have recently identified two proteins, maltose binding protein and ribose binding protein, as “smart” GE contrast agents capable of detecting and quantifying their respective ligands in solution via 129Xe NMR techniques. These results lead us to conclude that these and similar periplasmic binding proteins can constitute a novel class of GE reporters for 129Xe MRI

Xenon-Cryptophane and Xenon-Protein Interactions Enabling Ultrasensitive Xenon-129 NMR

Proton-based magnetic resonance imaging (1H MRI) is a widely used technique for the detection and diagnosis of disease states. However, 1H MRI is limited in imaging low-abundance biomarkers due to the low sensitivity inherent in the technique. To overcome this issue, hyperpolarized (hp) 129Xe NMR and MRI have been investigated as complements to 1H-based techniques. Xenon possesses a high sensitivity to its molecular environment due its large and polarizable electron cloud and offers 104-108-fold signal enhancement over thermal polarization in its hp state. These features allow hp 129Xe to be used in biosensing applications, where the chemical shift or the intensity of the 129Xe NMR signal is modulated when a Xe-binding host interacts with its target. The best-studied of these Xe hosts are the organic cage molecules known as cryptophanes. We recently discovered that cryptophanes are not well-solvated in solution, as previously assumed, but instead form water-soluble aggregates of tens-to-hundreds of nanometers in size. The insights gleaned from dynamic light scattering, fluorescence quenching, and 129Xe NMR analyses of these aggregates suggest that a reevaluation of cryptophane characterization and sensing strategies is necessary. These strategies were further studied using a novel adamantyl-functionalized cryptophane-A (AFCA) with record-high affinity for Xe. Hp 129Xe NMR only revealed signal corresponding to AFCA-encapsulated 129Xe at low AFCA concentrations at which the compound was monomeric, and not at higher concentrations at which small aggregates were observed. Additionally, no change in the 129Xe NMR chemical shift, nor the AFCA aggregation state, was observed upon β-CD binding to the adamantyl moiety, suggesting that changes in the aggregation state are necessary to elicit a 129Xe NMR chemical shift change. AFCA was also investigated as a model system for “turn-on” Xe sensing, where signal is only observed in the presence of analyte or environmental change. These studies helped to identify important limitations in cryptophane-based Xe sensing. In addition to cryptophane-based contrast agents, genetically encoded (GE) MRI reporters have been attracting significant interest. Such reporters have the advantage of being expressed directly in the tissue of interest, as well as being able to be modified to bind specific analytes via directed evolution. We have recently identified two proteins, maltose binding protein and ribose binding protein, as “smart” GE contrast agents capable of detecting and quantifying their respective ligands in solution via 129Xe NMR techniques. These results lead us to conclude that these and similar periplasmic binding proteins can constitute a novel class of GE reporters for 129Xe MRI

Cryptophane Nanoscale Assemblies Expand ¹²⁹Xe NMR Biosensing

Cryptophane-based biosensors are promising agents for the ultrasensitive detection of biomedically relevant targets via ¹²⁹Xe 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 ¹²⁹Xe 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 ¹²⁹Xe 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