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

• 15N-labeled metronidazole antibiotic was hyperpolarized via pulsed SABRE-SHEATH method; • 15N polarization of up to 18% was achieved for 20 mM drug in 80 seconds; • Feasibility of spin-relayed polarization transfer between three 15N sites was demonstrated in pulsed SABRE-SHEATH; • 15N relaxation dynamics is reported in a wide range of magnetic fields. 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 [ 15 N 3 ]metronidazole, an FDA-approved antibiotic (in non-enriched and non-hyperpolarized form) and potential hypoxia sensing molecule. Specifically, we demonstrate that 15 N polarization of 18.5% can be obtained in 80 s of parahydrogen bubbling parahydrogen through a solution containing 20 mM [ 15 N 3 ]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 15 N-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 15 N catalyst-binding site, all three 15 N sites of [ 15 N 3 ]metronidazole attained the hyperpolarized state. This spin-relayed polarization transfer becomes possible due to the 15 N 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.