Decacationic Pillar[5]arene: A New Scaffold for the Development of 129Xe MRI Imaging Agents

A decacationic water-soluble pillar[5]arene possessing a nonsolvated hydrophobic core has been designed and synthesized. This supramolecular host is capable of binding xenon, as evidenced by hyperCEST depletion experiments. Fluorescence-based studies also demonstrate that xenon binds into the cavity of the pillararene with an association constant of 4.6 × 103 M–1. These data indicate that the water-soluble pillararene is a potential scaffold for building contrast agents that can be detected by xenon-129 magnetic resonance imaging.


Aqueous Xenon Solutions for Fluorescence Quenching Experiments.
A 3.32 x 10 -3 M aqueous solution of xenon was prepared according to the method of Dmochowski: 2 Deionized water (25 ml) was added to an acid-washed 50 mL round bottom flask. The flask was capped with a septum, bubbled with nitrogen for 20 minutes, and then evacuated through a 22-gauge needle for 5 minutes. After degassing, the needle was pushed to the bottom of the flask, and xenon was bubbled through the solution for 5 minutes. A 26-gauge needle was used to relieve excess pressure. Both needles were removed, and the solution was set aside. Next, a Chemware Tedlar TM gas sampling bag with a septum valve (6" x 6", Fisher Scientific) was connected by ¼" tubing and a three-way stopcock to both a xenon tank and a needle. The entire system was evacuated through the needle using a separate round bottom flask, its septa, and the house vacuum line. The Tedlar bag was filled with xenon, its nozzle closed, and the three-way stopcock was removed. The needle was placed directly onto the tubing connecting the Tedlar bag, and then pierced through the septum into the headspace of the round bottom flask that contained the xenon solution.
The flask and attached Tedlar bag was placed in a 37 o C incubator for a few hours to equilibrate. This

Fluorescence quenching of 3 with Xenon
A 5.1 x 10 -4 M stock solution of P5A in deionized water was made using a 100 mL volumetric flask. The solution was then added into a 100 mL round bottom flask with a septum. The solution in the round bottom flask was gently bubbled with nitrogen for 5 minutes, and vacuum was applied for 5 minutes to degas the solution. The same dilution and degassing process was used to prepare a 1 x 10 -3 M phosphate buffer solution. A stock solution of 3 was added to a reduced volume cuvette with a septum seal (1.5 mL, 1 cm path length, Starna Cells), and then phosphate buffer was added in order to dilute the 3 concentration to 15 µM at 1.5 mL. The solution in the cuvette was bubbled with nitrogen for 5 minutes and evacuated for 5 minutes in order to ensure degassing. Varying amounts of a 3.32 mM aqueous xenon solution (see above) was added so the xenon concentration varied from 0 to 9.8 x 10 -4 M through the titration at 1.5 mL total volume, keeping the headspace above the solution to a minimum. The cuvette solution was allowed to equilibrate at 25 o C for 15 min in the fluorimeter before emission spectra were obtained (excitation at 292 nm, emission lmax observed at 321 nm).
A saturated xenon measurement was made by directly bubbling xenon into a degassed solution of 3 in the cuvette for 5 minutes. The solution was allowed to equilibrate for at 25 o C in the fluorimeter before spectra were obtained. 2 All measurements were triplicated. S18 Figure S13: Fluorescence quenching phenomena when Xe was added in to a solution of 3 in phosphate buffer. Figure 4 in the manuscript shows the same data following Gaussian curve smoothing using Origin lab software. S19 http://app.supramolecular.org/bindfit/view/ef59fc0f-0786-4811-8258-0ad6bd25392c The fluorescence quenching of P5A is caused by the creation of a host guest complex between P5A and xenon in solution according to reaction (1): (1) 5 ( + ) + ( + ) ⇋ @ 5 The association constant, Ka, for this reaction is defined as,

P5A
Xe Then, they were graphed as a function of xenon solution concentration, and the occupancy curve was developed. The standard error for these data is ~5%.

Dynamic Light Scattering (DLS)
A 100 µM sample of deca-imidazolium functionalized pillar[5]arene was prepared in ultrapure water (pH 6.998). The temperature was set to 25°C and the intensity distributions were analyzed.
Measurements were averaged over three trials without disruption of the sample. DLS data was acquired on a Zetasizer Nano ZS90 system, using software from Malvern Instruments.

Xenon NMR studies
Naturally abundant Xe gas was placed into a 1.0 L Tedlar bag and polarized to 60-80% via the spin exchange optical pumping (SEOP) technique using a Xemed polarizer (Xemed, Durham, NH, USA).
It was then placed into a Tedlar bag which was immediately moved into a pressurized chamber within the bore of a Philips Achieva 3.0 T clinical MRI scanner to preserve its polarization. The pressure inside of the chamber was maintained between 20-45 kPa above atmospheric pressure using a pressuresensitive ventilation device connected to a nitrogen (N2) source, to facilitate the flow of HP 129 Xe gas from the Tedlar bag into the glass-fritted cell containing pillararene solution.
Following polarization of 129 Xe gas, 2.5 mL of solution (10 mM) was transferred into a custom- The data sampling number was 2048, which corresponds to the spectral resolution of 0.44 ppm.
Saturation pre-pulse frequency was automatically adjusted to range from -110 ppm to 30 ppm, where 0 ppm represents dissolved-phase 129 Xe, with a predetermined step before each of the subsequent excitation pulses. The frequency changing step was equal to 2 ppm (70.6 Hz).

Figure S18:
The pulse diagram shown above was used to acquire HyperCEST depletion spectra using saturation pre-pulse train including sinusoidal pulses. Here, fj represents the frequency of saturation pulses during FID acquisition number j. All spoiler gradients along x, y, z axis are also illustrated.
S26 Figure S19: Experimental HyperCEST setup for in vivo studies. Above setup was used to obtain the Xe-spectra and to obtain the final depletion spectra. [Photographs on this page are courtesy of Yurii Shepelytskyi. Copyright 2020. Images are free domain.]