Cryptophane Nanoscale Assemblies Expand ¹²⁹Xe NMR Biosensing

Abstract

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

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

“…However, since recent work on cryptophane derivatives including solubilizing groups reported significant aggregation effects, we performed DLS measurements for compounds 2 and 3 (these showed significant differences in their MT profiles). The determined particle sizes for 2 and 3 were found to be practically identical (see Figure S3) and significantly smaller than that reported for CrA derivatives . It can thus be hypothesized that the difference in MT performance is not primarily due to different aggregation but presumably due to modulation of the exchange rate by the different counterions.…”

“…Due to the excellent water solubility of compounds 2 – 4 , we expect aggregation to be less pronounced than in the CrA derivative case for example. However, since recent work on cryptophane derivatives including solubilizing groups reported significant aggregation effects, we performed DLS measurements for compounds 2 and 3 (these showed significant differences in their MT profiles). The determined particle sizes for 2 and 3 were found to be practically identical (see Figure S3) and significantly smaller than that reported for CrA derivatives .…”

“…132 ppm upfield from free Xe. [32] The reversible binding of Xe to CrA-ma also causes an MT effect around the signal of free Xe with a profile which is similar to that of 3 but reflects significantly slower exchange than for 2 (see Figure S5). A series of MRI scans was taken with different saturation offsets (covering AE 2 kHz in 30 steps symmetrically around the resonance of free Xe for the MT effect and À12.5 kHz … À16.5 kHz in 30 steps around the optimum CEST offset).…”

“…The determined particle sizes for 2 and 3 were found to be practically identical (see Figure S3) and significantly smaller than that reported for CrA derivatives. [30] It can thus be hypothesized that the difference in MT performance is not primarily due to different aggregation but presumably due to modulation of the exchange rate by the different counterions. In addition, note that the CrA derivative which was reported to form higher aggregates compared with those found for 2 and 3 gave a defined CEST peak and no pronounced MT effect.…”

Molecular Sensing with Host Systems for Hyperpolarized 129Xe

“…To make the biosensor more water soluble, a solubilizing linker such as 3-azidopropionic acid was treated with crude monopeptide CryA via a second CuAAC to yield Xe biosensors with two propionic acids and respective peptides in yields approximately between 40–70%. This approach was used to synthesize enantiopure trifunctionalized cryptophanes for various applications [ 206 ], including pH-dependent cell labeling of cancer cells [ 179 ], targeting integrins expressions in cells [ 127 ], sensing human carbonic anhydrase (CA) I and II enzymes [ 177 , 207 , 208 , 209 ] and monitoring Ca(II)-triggered peptide binding to calmodulin [ 178 ], respectively.…”