Cucurbit[6]uril is an ultrasensitive ¹²⁹Xe NMR contrast agent

Abstract

A lack of molecular contrast agents has slowed the application of ultrasensitive hyperpolarized ¹²⁹Xe NMR methods. Here, we report that commercially available cucurbit[6]uril (CB[6]) undergoes rapid xenon exchange kinetics at 300 K, and is detectable by Hyper-CEST NMR at 1.8 pM in PBS and at 1 μM in human plasma where many molecules, including polyamines, can compete with xenon for CB[6] binding.

Hyperpolarized (HP) ¹²⁹Xe is being investigated for many NMR spectroscopy and imaging applications that require significant enhancements in detection sensitivity. The long-lived ¹²⁹Xe HP state is readily obtained by a process of spin-exchange optical pumping.¹ HP ¹²⁹Xe is non-toxic, can be delivered to living organisms via inhalation or Xe-solution injection,^{2, 3} and has been employed for imaging the lungs and brain of living mammals, including human.^4-6 Xenon is very soluble in organic solvents and accumulates in vivo in lipid environments, while exhibiting low affinity for endogenous proteins and other biomolecules. Cryptophane-A and its derivatives (Scheme 1) are the most studied Xe-binding cages,^{7, 8} and water-soluble versions exhibit association constants in excess of 30,000 M⁻¹ at rt.^9-11 However, multi-step syntheses yield just milligram quantities of water-soluble cryptophane.¹² New xenon-binding contrast agents are needed to expand applications of HP ¹²⁹Xe in chemical sensing, biophysical chemistry, and biomedical imaging.

Scheme 1.

Top: Chemical structures of CB[6] and TAAC. Bottom: Hyper-CEST mechanism involving xenon-binding molecules represented by hexagons.

The unique hollow structures and molecular recognition properties of the cucurbit[n]uril (CB[n]) family have made CB[n] and functionalized CB[n] useful candidates as drug delivery vehicles, components of enzyme assays, and other sensing applications.^{13, 14} Commercially available CB[6] (Scheme 1) possesses hexagonal symmetry with a hydrophobic cavity that is accessible through two carbonyl-fringed portals of ~4-Å diameter.^{15, 16} CB[6] binds xenon with modest affinity but is poorly soluble in pure water. Interestingly, CB[6] becomes water soluble in the presence of monovalent cations (as found in biological fluids), however cation binding at the portals has been proposed to block xenon binding.¹⁷ Here, we consider whether the CB[6] cavity, which is hydrophobic, rigidly open, and of similar dimensions to Xe (diameter ≈ 4.3 Å), can promote rapid Xe exchange interactions, as required for detection by HP ¹²⁹Xe chemical exchange saturation transfer (Hyper-CEST, Scheme 1).

Hyper-CEST NMR has recently enabled the ultrasensitive detection of cryptophanes,^18-25 gas-vesicle proteins,²⁶ and bacterial spores.²⁷ For example, our lab demonstrated 1.4 picomolar detection of a water-soluble tri-acetic acid cryptophane (TAAC, Scheme 1) at 320 K.²⁸ In Hyper-CEST, encapsulated HP ¹²⁹Xe is selectively depolarized by radiofrequency (rf) pulses, and the depolarized ¹²⁹Xe rapidly exchanges with HP ¹²⁹Xe to accumulate in the solvent pool, where loss of signal can be readily monitored. Stevens et al. reported a perfluorocarbon nanoemulsion contrast agent for ¹²⁹Xe NMR, with each droplet encapsulating multiple xenon atoms, depending on droplet size.²⁹ PFOB nanodroplets were recently applied for multiplexed detection using Hyper-CEST NMR in mammalian cells.³⁰ In order to advance many applications we have sought new molecular scaffolds for Hyper-CEST NMR. Here, the rapid, reversible complexation of xenon by CB[6] was investigated in physiologically relevant buffer solution (where CB[6] is soluble to greater than 10 mM), and exploited for Hyper-CEST NMR experiments in human plasma. Through selective saturation and magnetization transfer, the ¹²⁹Xe-CB[6] peak was encoded and amplified in the ¹²⁹Xe-solution peak (Scheme 1).

The HP ¹²⁹Xe NMR spectrum obtained with 5 mM CB[6] using a direct detection method showed that the ¹²⁹Xe-CB[6] peak in pH 7.2 PBS (1.058 mM potassium phosphate monobasic, 154 mM sodium chloride, and 5.6 mM sodium phosphate dibasic) was 72 ppm upfield-shifted from the ¹²⁹Xe-water peak (Figure 1). Due to rapid exchange of xenon with CB[6], the line shape of both ¹²⁹Xe NMR peaks appeared broad. Nonetheless, the “bound” ¹²⁹Xe peak was well-separated from the “free” peak, allowing it to be selectively irradiated with rf pulses without perturbing free HP ¹²⁹Xe in solution. Thermodynamic and kinetic parameters associated with the complexation of xenon by CB[6] at 300 K in PBS solution were determined by 2D HP ¹²⁹Xe NMR exchange spectroscopy (Figure S1). 2D-EXSY spectra were recorded with 2048 data points in t2 domain and 16 data points in t1 domain, using States-TPPI method in the t1 dimension. To evaluate the exchange rate constant, equations were used as described previously (Supporting Information).³¹ The extracted rate constants for association and dissociation, k_on and k_off, were 4.1*10⁵ M⁻¹s⁻¹ and 840 s⁻¹, respectively. This result is similar to k_off values determined by Kim et al. for a more water-soluble CB[6] derivative: k_off = 2300 s⁻¹ in water, k_off = 310 s⁻¹ in 0.4 M Na⁺ solution.¹⁷ We determined the association constant (K_A = k_on/k_off) for xenon and CB[6] in PBS at pH 7.2 to be 490 M⁻¹ at 300 K, in accord with previous measurements for this Xe-host interaction,^{17, 32} taking into account the intermediate buffer salt concentration. The k_off value determined by EXSY was similar to the measured exchange rate from line-width analysis for the corresponding ¹²⁹Xe NMR spectrum (k_exch = 1470 s⁻¹, Figure S2). Xe affinity determined for CB[6] in PBS at 300 K was ~40-fold lower than measured previously for TAAC.¹¹ However, the ¹²⁹Xe-CB[6] exchange rate was ~17-fold higher than previously measured for ¹²⁹Xe-TAAC (k_exch = 86 s⁻¹) at 300 K,²⁸ and should afford efficient magnetization transfer, as required for ultrasensitive detection in the Hyper-CEST scheme.

Figure 1.

HP ¹²⁹Xe NMR spectrum with 5 mM CB[6] dissolved in pH 7.2 PBS at 300 K. A 30 degree pulse was used and signal averaged over 8 scans. Fourier-transformed spectra were processed with zero-filling and Lorentzian line-broadening of 20 Hz. Peak width (FWHM) was 463 Hz for ¹²⁹Xe-aq peak, and 570 Hz for ¹²⁹Xe-CB[6] peak.

To test CB[6] for Hyper-CEST NMR spectroscopy, multiple selective Dsnob-shaped saturation pulses were scanned over the chemical shift range of 85-210 ppm in 5-ppm steps. Two saturation responses were observed (Figure 2), centered at 193 ppm (¹²⁹Xe-aq) and 122 ppm (¹²⁹Xe-CB[6]). Similar to the direct detection spectrum with 5 mM CB[6] (Figure 1), both peaks in the Hyper-CEST z-spectrum with 0.8 μM CB[6] appeared broad, which allowed for a broad saturation frequency window.

Figure 2.

Hyper-CEST frequency-scan profile of 0.8 μM CB[6] in pH 7.2 PBS at 300 K. When saturation rf pulse was positioned at 121 ppm (-72 ppm from ¹²⁹Xe-aq peak), encapsulated ¹²⁹Xe was depolarized and exchange caused rapid decrease in ¹²⁹Xe-aq signal. The black squares show the experimental data, and the lines show the exponential Lorentzian fits.³³

Ultrasensitive indirect detection of CB[6] was achieved by applying shaped rf saturation pulses at the chemical shift of ¹²⁹Xe in CB[6], and measuring the residual aqueous ¹²⁹Xe signal after spin transfer as on-resonance CEST response (Figure 3 and Figure S3). The observed depolarization response in Hyper-CEST experiments arose from both self-relaxation of HP ¹²⁹Xe and CB[6]-mediated saturation transfer. The depolarization rates were obtained by fitting both on-resonance and off-resonance decay curves to first-order exponential kinetics. Remarkably, 1.8 pM CB[6] was readily detected in PBS at 300 K (Figure 3). Average of three trials gave τ_on = 24.6 ± 1.2 s and τ_off = 58.5 ± 3.7 s. The high S/N at picomolar concentration is comparable to our previous Hyper-CEST measurements with TAAC, which required elevated temperature (320 K) to achieve similar 10³ s⁻¹ exchange kinetics.²⁸ As postulated previously for TAAC,²⁸ CB[6]-mediated exchange is likely enhanced by peripheral Xe atoms undergoing rapid magnetization transfer with the “bound” Xe atom at the primary site. Indeed, the open, tubular structure of CB[6] may promote rapid ¹²⁹Xe(primary)-¹²⁹Xe(periphery) interactions at both portals. Importantly, xenon is very soluble (4.2 mM atm⁻¹) in water at 300 K,³⁴ and working near rt is convenient for many biochemical and cellular assays.

Figure 3.

Representative Hyper-CEST profile of 1.8 pM CB[6] in pH 7.2 PBS at 300 K. Saturation frequencies of Dsnob-shaped pulses were positioned at 122.3 ppm (193.5 – 71.2 ppm) and 264.7 ppm (193.5 + 71.2 ppm), for on- and off-resonance. Pulse length, τ_pulse = 1.05 ms; field strength, B_1,max = 279 μT.

Having established CB[6] as an ultrasensitive ¹²⁹Xe NMR contrast agent in physiologic buffer solution, we investigated the feasibility of using this agent in biological fluids. We first performed Hyper-CEST NMR experiments with 1 μM CB[6] in blood plasma (purchased from Sigma), and observed a peak at the characteristic ¹²⁹Xe-CB[6] chemical shift, 122 ppm (Figure 4). As expected, the aqueous Xe peak was broader, based on the faster exchange of HP ¹²⁹Xe in plasma. The many components of blood plasma that can interact with CB[6] also contributed to the HP ¹²⁹Xe-CB[6] peak being less intense than observed in PBS. Polyamines, for example, are naturally occurring organic molecules found in all living organisms and are known to have high affinity for CB[6] relative to other small molecules.³⁵ Polyamines are present at millimolar concentrations inside living cells, with ~10 percent being free polyamines, and at micromolar concentrations in biological fluids.^{36, 37} Putrescine is believed to be the most abundant polyamine in most biological fluids, and is strongly associated with cancer and chemotherapy.^{38, 39} We confirmed by isothermal titration calorimetry (ITC) that putrescine has high affinity for CB[6] in PBS (K_A = 3.6*10⁶ M⁻¹ at 300 K, Figure S4). To investigate the effect of putrescine on CB[6]-mediated Hyper-CEST signal in this biological fluid, we added 10 μM putrescine to the 1 μM CB[6]-plasma solution. The ¹²⁹Xe-CB[6] Hyper-CEST signal at 122 ppm remained visible but was reduced as a result of less free CB[6] in the sample (Figure 4). These experiments suggest that it is feasible to use CB[6] as a sensitive in vivo ¹²⁹Xe contrast agent in environments where competing polyamines exceed CB[6] concentration by less than 10-fold.

Figure 4.

Hyper-CEST spectra shown for 1 μM CB[6] in PBS (black), in blood plasma (blue), and in blood plasma with 10 μM putrescine (red); all data collected at 300 K.

To quantify how polyamines affect CB[6] Hyper-CEST efficiency, we carried out a set of experiments with putrescine in PBS, which has similar salt concentration to plasma but affords longer T₁ (~60 sec) of HP ¹²⁹Xe. Putrescine concentrations of 1 μM to 50 μM were investigated, as this is the relevant range in biological fluids.³⁷ For each putrescine sample, 1 μM CB[6] was added and incubated for 20 min at 300 K. Then, the same Hyper-CEST NMR method was used as shown in Figure 3, with slightly adjusted saturation pulse (see Supporting Information for details). Saturation transfer efficiency (ST),²⁷ which is proportional to MR image contrast, and free CB[6] concentration were calculated for each putrescine sample (Table 1, see Supporting Information for details). With increasing putrescine in solution, the amount of CB[6] available for Xe exchange decreased, and a correspondingly smaller ST contrast value was observed. This experiment further demonstrated that only small excess of CB[6] (e.g., 5 nM CB[6] in PBS) is needed to generate useful Hyper-CEST contrast at intermediate field strength (B_1,max = 92 μT).

Table 1.

Saturation transfer (ST) efficiency for 1 μM CB[6] samples in PBS with varying putrescine concentration.

Putrescine (μM)	Calculated free CB[6] concentration (μM)	ST efficiency
0	1.0	0.68 ± 0.09
1	0.41	0.67 ± 0.10
2	0.19	0.34 ± 0.06
5	0.064	0.25 ± 0.03
10	0.030	0.16 ± 0.03
20	0.014	0.10 ± 0.01
50	0.0056	0.08 ± 0.01

A corollary from this experiment is that CB[6] enables fast and sensitive detection of putrescine in solution, without need for polyamine derivatization, by correlating the difference between on- and off-resonance HP ¹²⁹Xe decay rates to putrescine concentration. (See Figures S5 and S6 for more details.) To date, efforts with Hyper-CEST have focused on targeting proteins,²¹ lipids,¹⁹ or metal ions²⁴ by attaching different recognition moieties to cryptophane. Here, through competing guest encapsulation and “turn off” sensing, CB[6] affords new capabilities in small-molecule detection.

Conclusions

We demonstrated that commercially available cucurbit[6]uril can serve as a Hyper-CEST ¹²⁹Xe NMR contrast agent, both in physiologic buffer solution and a model biological fluid (human plasma). 2D-EXSY experiments confirmed that xenon k_exch with CB[6] is rapid but does not approach the fast exchange limit on the ¹²⁹Xe NMR time scale, which allowed the use of broadband irradiation to achieve efficient saturation of the ¹²⁹Xe-CB[6] complex without affecting free HP ¹²⁹Xe in solution. Efficient saturation transfer enabled low picomolar detection of CB[6] at 300 K, which was equivalent to the previous single-site Hyper-CEST detection record achieved in our laboratory using water-soluble cryptophane TAAC at 320 K.²⁸ Our data suggest that for many applications in aqueous buffer solution near rt, CB[6] should provide superior Hyper-CEST signal to water-soluble cryptophanes. A variety of cucurbituril derivatives⁴⁰ and acyclic variants^{41, 42} have been reported that highlight opportunities for cucurbituril functionalization, as will likely be required to target specific biomolecules in solution.

CB[6] is very soluble in biological fluids and may also prove useful as a MRI/MRS contrast agent for in vivo applications. This will depend on the circulation time and localization of CB[6] in vivo, among other factors. One potential limitation of using CB[6] as a ¹²⁹Xe MR contrast agent is the competition for available xenon binding sites from endogenous small molecules. Importantly, saturation transfer efficiency was found to be strongly correlated with free CB[6] concentration, which is useful for establishing conditions that are amenable to the Hyper-CEST approach, even when the nature of the competing species is not perfectly known. For example, we showed that Hyper-CEST contrast can be achieved for CB[6] in plasma, which contains many competing species including high-affinity polyamines. Finally, we determined that it is possible to exploit the promiscuity of CB[6] to estimate the concentration of a known small molecule (e.g., putrescine) that competes with xenon for the binding cavity. The ready availability and versatile host-guest chemistry of CB[6] opens many in vitro as well as in vivo applications, employing direct detection of ^{HP 129}Xe or Hyper-CEST NMR. Following our work with cryptophanes,^{25, 43-45} we aim to develop cucurbituril xenon biosensors that take advantage of the special Hyper-CEST capabilities of this contrast agent.