Utilizing a water-soluble cryptophane with fast xenon exchange rates for picomolar sensitivity NMR measurements

Abstract

Hyperpolarized (129)Xe chemical exchange saturation transfer ((129)Xe Hyper-CEST) NMR is a powerful technique for the ultrasensitive, indirect detection of Xe host molecules (e.g., cryptophane-A). Irradiation at the appropriate Xe-cryptophane resonant radio frequency results in relaxation of the bound hyperpolarized (129)Xe and rapid accumulation of depolarized (129)Xe in bulk solution. The cryptophane effectively "catalyzes" this process by providing a unique molecular environment for spin depolarization to occur, while allowing xenon exchange with the bulk solution during the hyperpolarized lifetime (T(1) ≈ 1 min). Following this scheme, a triacetic acid cryptophane-A derivative (TAAC) was indirectly detected at 1.4 picomolar concentration at 320 K in aqueous solution, which is the record for a single-unit xenon host. To investigate this sensitivity enhancement, the xenon binding kinetics of TAAC in water was studied by NMR exchange lifetime measurement. At 297 K, k(on) ≈ 1.5 × 10(6) M(-1) s(-1) and k(off) = 45 s(-1), which represent the fastest Xe association and dissociation rates measured for a high-affinity, water-soluble xenon host molecule near rt. NMR line width measurements provided similar exchange rates at rt, which we assign to solvent-Xe exchange in TAAC. At 320 K, k(off) was estimated to be 1.1 × 10(3) s(-1). In Hyper-CEST NMR experiments, the rate of (129)Xe depolarization achieved by 14 pM TAAC in the presence of radio frequency (RF) pulses was calculated to be 0.17 μM·s(-1). On a per cryptophane basis, this equates to 1.2 × 10(4)(129)Xe atoms s(-1) (or 4.6 × 10(4) Xe atoms s(-1), all Xe isotopes), which is more than an order of magnitude faster than k(off), the directly measurable Xe-TAAC exchange rate. This compels us to consider multiple Xe exchange processes for cryptophane-mediated bulk (129)Xe depolarization, which provide at least 10(7)-fold sensitivity enhancements over directly detected hyperpolarized (129)Xe NMR signals.

Figure 1

Triacetic acid cryptophane-A derivative (TAAC).

Figure 2

HP ¹²⁹Xe NMR spectrum of 140 µM TAAC dissolved in ultrafiltered water at 320 K, showing: Xe(aq) peak at 194.3 ppm and Xe@TAAC peak at 65.8 ppm (selectively excited). Lorentzian deconvolved spectrum is shown in red.

Figure 3

Hyper-CEST pulse sequence diagram, with parameters provided for in-house 10 mm PABBO probe. Different values were chosen considering TAAC concentration, sample temperature, HP Xe flow rate. From a recent saturation transfer optimization, saturation pulse is DSnob-shaped to enhance power efficiency. Field strength of the shaped pulse was calculated to be 29 µT (cw equivalent).

Figure 4

¹²⁹Xe Hyper-CEST profiles of 14 pM and 1.4 pM TAAC at 320 K plotted as the Xe(aq) peak intensities vs. saturation time. Differences between on- and off-resonance saturation are compared. Exponential fits of data are shown as red and dark curves. Depolarization lifetimes were fitted to be 10 ± 1 s (on) and 38 ± 2 s (off) for 14 pM sample (averaged over two trials); 53 ± 4 s (on) and 65 ± 4 s (off) for 1.4 pM sample (representative of 3 trials), respectively. In each experiment, a series of 2.6 ms DSnob pulses with 20 µs delay was applied. In the Hyper-CEST pulse sequence, the following parameters were used: sp6 = 2.6 ms, d12 = 20 µs, d1 = 1 s, L6 = 8 000 (max, 14 pM) or 10 000 (max, 1.4 pM).

Figure 5

Enthalpogram of 3.31 mM aqueous xenon solution titrated into 131 µM TAAC solution (phosphate buffer, 20 mM, pH 7.5) at 310 K.

Figure 6

Xe-TAAC exchange lifetime measurement by selective EXSY at 297 K. The horizontal axis denotes the mixing time between two consecutive excitations. The vertical axis indicates the ratio of the integrated intensities of Xe@TAAC signals, exchange recovered vs. equilibrium. Data were exponentially fitted to give an exchange lifetime, τ_exch = 22 ± 3 ms. This exchange lifetime is in good agreement with linewidth measurements made at 300 K (Table 1).

Scheme 1

Saturation transfer processes in ¹²⁹Xe Hyper-CEST NMR with cryptophane.