Efficient ¹⁵N hyperpolarization of [¹⁵N₃]metronidazole antibiotic via spin-relayed pulsed SABRE-SHEATH

Signal Amplification by Reversible Exchange in SHield Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH) is an NMR hyperpolarization technique that relies of the simultaneous exchange of parahydrogen and a to-be-hyperpolarized molecule on the metal center of a polarization-transfer catalyst in a microtesla magnetic field. Until recently, this method has been understood to perform hyperpolarization by establishing level anti-crossings between the nuclear spins of the parahydrogen derived hydrides (acting as a source of hyperpolarization) and those of the substrate. Recently, the application of highly non-intuitive pulse sequences (comprising pulses of microtesla DC fields) was predicted to hyperpolarize nuclear spins more efficiently than the canonical (static-field) SABRE-SHEATH approach. Here we show that by employing a basic "on-off" pulse sequence of rectangular microtesla pulses, it is possible to improve the hyperpolarization efficiency for SABRE-SHEATH of [¹⁵N₃]metronidazole, an FDA-approved antibiotic (in non-enriched and non-hyperpolarized form) and potential hypoxia sensing molecule. Specifically, we demonstrate that ¹⁵N polarization of 18.5 % can be obtained in 80 s of parahydrogen bubbling parahydrogen through a solution containing 20 mM [¹⁵N₃]metronidazole. In practice, (1.32 ± 0.14)-fold improvements in $P_{15N}$ was obtained with the pulsed method described here compared to static field technique variant. These results show that pulsed SABRE-SHEATH was successfully applied to ¹⁵N-labeled biologically relevant molecule. Moreover, we also demonstrate that although the pulsed SABRE-SHEATH sequence was designed for polarization transfer from parahydrogen derived hydrides to the metronidazole's ¹⁵N catalyst-binding site, all three ¹⁵N sites of [¹⁵N₃]metronidazole attained the hyperpolarized state. This spin-relayed polarization transfer becomes possible due to the ¹⁵N relay network established by their spin-spin J-couplings. The feasibility of the spin-relayed polarization transfer is demonstrated here for the first time for pulsed SABRE-SHEATH (as opposed to the static-field SABRE-SHEATH reported previously) and it paves the way to broad applicability of the technique.