Identification, classification, and signal amplification capabilities of high-turnover gas binding hosts in ultra-sensitive NMR

Abstract

Nuclear Magnetic Resonance (NMR) can be a powerful tool for investigating exchange kinetics of host-guest interactions in solution. Beyond conventional direct NMR detection, radiofrequency (RF) saturation transfer can be used to enhance the study of such chemical exchange or to enable signal amplification from a dilute host. However, systems that are both dilute and labile (fast dissociation/re-association) impose specific challenges to direct as well as saturation transfer detection. Here we investigate host-guest systems under previously inaccessible conditions using saturation transfer techniques in combination with hyperpolarized nuclei and quantitative evaluation under different RF exposure. We further use that information to illustrate the consequences for signal amplification capabilities and correct interpretation of observed signal contrast from comparative exchange data of different types of hosts. In particular, we compare binding of xenon (Xe) to cucurbit[6]uril (CB6) with binding to cryptophane-A monoacid (CrA) in water as two different model systems. The Xe complexation with CB6 is extremely difficult to access by conventional NMR due to its low water solubility. We successfully quantified the exchange kinetics of this system and found that the absence of Xe signals related to encapsulated Xe in conventional hyperpolarized ¹²⁹Xe NMR is due to line broadening and not due to low binding. By introducing a measure for the gas turnover during constant association-dissociation, we demonstrate that the signal amplification from a dilute pool of CB6 can turn this host into a very powerful contrast agent for Xe MRI applications (100-fold more efficient than cryptophane). However, labile systems only provide improved signal amplification for suitable saturation conditions and otherwise become disadvantageous. The method is applicable to many hosts where Xe is a suitable spy nucleus to probe for non-covalent interactions and should foster reinvestigation of several systems to delineate true absence of interaction from labile complex formation.

Fig. 1. Probing reversible, labile binding in a molecular cavity with xenon: free xenon atoms (pool A, where blue indicates hyperpolarized Xe and gray indicates depolarized Xe) undergo constant exchange with the binding site/host (i.e., cucurbit[6]uril, CB6; transparent overlay of the ball-stick-model of the molecule with its van der Waals radius representation; molecular modeling in Fig. S1 in the ESI†). The NMR signal of bound Xe shows a remarkably large chemical shift (indicated by orange xenon atoms; pool B). The host geometry with the two opposing portals facilitates fast dissociation of the complex. This causes detrimental line broadening, precluding conventional NMR detection.

Fig. 2. Direct and indirect (Hyper-CEST) ¹²⁹Xe NMR measurements for cucurbit[6]uril (CB6) at a concentration of 3.4 μM dissolved in pure water. (a) ¹²⁹Xe NMR spectrum with 64 averages at T = 295 K. Retrospectively, the Xe–CB6 resonance is expected to appear at ca. δ_B = –95 ppm (red dashed line). The insert shows the CB6 structure as top and side view including the Xe exchange, k_AB,BA. (b) Hyper-CEST z-spectra (dots) for continuous-wave (cw) saturation of B₁/t_sat = {1.1/5 (green), 2.2/10 (orange), 3.3/15 (blue)} μT/s including fitting curves of the full Hyper-CEST (FHC) solution (solid lines); results are listed in Table 1.

Fig. 3. Hyper-CEST effect mapping for equal concentrations of [CB6] = [CrA] = 12.9 μT in water at T = 295 K. (a) shows the proton (¹H)-MRI as a cross-section (tilted black square) of the double bubbling phantom and the region-of-interest (ROI) definition for histogram analysis. (b) Hyper-CEST effect maps of CrA for 5.5 μT (“low RF exposure”) and 33.3 μT (“high RF exposure”) both for 2 s of cw saturation calculated as the difference of the on-resonant image (saturation at – 132 ppm) and the off-resonant image (saturation at + 132 ppm) with respect to the free Xe in solution resonance. The Hyper-CEST effect of 60 % for both RF exposures is significant but unchanged. (c) CB6 Hyper-CEST effect maps for identical RF exposure but the on-resonant image (saturation at – 96 ppm) and the off-resonant image (saturation at + 96 ppm) with respect to the free Xe in solution resonance. Whereas the Hyper-CEST effect for 5.5 μT saturation strength was below 30 %, the stronger saturation resulted in ∼ 100 % Hyper-CEST effect, thus revealing significantly higher gas turnover for the Xe–CB6 complex. The Xe-MR images were acquired with 64² resolution and cubic spline interpolated to 256². The slight blurring in phase encoding direction originated from faster T₂ relaxation with CB6 in the outer compartment.