Pluronic l81

Hyperpolarized (hp) ¹²⁹Xe was generated using the spin-exchange optical pumping (SEOP) method with a home-built ¹²⁹Xe polarizer based on the IGI.Xe.2000 commercial model by GE. A Shark 65 W tunable ultra-narrow band diode laser (OptiGrate) set to 795 nm was used for optical pumping of Rb vapor. A gas mixture of 88% helium, 10% nitrogen, and 2% natural abundance xenon (Linde Group, NJ) was used as the hyperpolarizer input. ¹²⁹Xe hyperpolarization level was roughly 10–15%. For each data point in the hyper-CEST z-spectra, hp ¹²⁹Xe was bubbled into a 10-mm NMR tube containing 2.5 mL of sample through capillaries for 20 s, followed by a 3-s delay to allow bubbles to collapse. A Dsnob saturation pulse with 690 Hz bandwidth was used. Pulse length t_pulse = 3.80 ms, field strength B_1,max = 77 μT, number of pulses n_pulse = 400, saturation time T_sat = 1.52 s. NMR experiments were performed using a Bruker BioDRX 500 MHz NMR spectrometer and a 10-mm PABBO probe at 300 K. A 90° hard pulse of this probe has a pulse length of 22 μs. For all experiments, the cryptophane and/or protein concentration was 5 μM, in pH 7.2 PBS, with 0.1% DMSO and 0.1% (v/v) Pluronic L81 (Aldrich) to mitigate foaming. Measurements were averaged over 3 trials.

Hp ¹²⁹Xe was cryogenically separated, accumulated, thawed, and collected in controlled atmosphere valve 10 mm NMR tubes (New Era) containing the sample and 0.1% (v/v) Pluronic L81 (Aldrich). Prior to data collection, tubes were vigorously shaken to mix sample solutions with Xe. An EBURP-1 selective pulse centered at 63 ppm with an excitation bandwidth of 680 Hz, pulse length τ_pulse = 2.2 ms, and B_1,max = 288 μT was used, and the signal was averaged over 64 scans, unless otherwise noted. Fourier-transformed spectra were processed with zero-filling and Lorentzian line broadening of 40 Hz. Chemical shifts were referenced to aqueous ¹²⁹Xe in its respective solvent.