Signal amplification by reversible exchange (SABRE) employs the non-equilibrium spin order of parahydrogen as a source of strong nuclear magnetic resonance (NMR) signal enhancement, with the objective of increasing NMR sensitivity. In SABRE, a parahydrogen molecule and a substrate form a transient polarization transfer complex. Performed within the high magnetic field of an NMR spectrometer, SABRE enables the hyperpolarization of nuclear spins without additional polarizers. Nevertheless, it requires thorough pulse sequence design. The high-field polarization transfer strategy strongly depends on the type of the spin system formed by the parahydrogen-nascent protons in the SABRE complex: chemically equivalent or non-equivalent. SABRE hyperpolarization in chemically equivalent spin systems has been the subject of considerable attention, even after being relevant only for a limited number of substrates. Efficient hyperpolarization in chemically non-equivalent complexes remained a key challenge, hindering the full potential of high-field SABRE and the ability to polarize a broader range of SABRE substrates. This work reports the multinuclear 1H-15N pulse sequence for efficient 15N hyperpolarization in chemically non-equivalent SABRE complexes. This approach relies on the simultaneous 1H and 15N radiofrequency excitation of the complex-bound nuclei with weak continuous wave magnetic fields. The proposed pulse sequence enabled the hyperpolarization of the 15N nuclei in a mixture of the antimicrobial drugs containing a 5-nitroimidazol moiety at their natural 15N isotopic abundance (0.76% of 15N polarization). Furthermore, it permitted the precise assignment of the SABRE complexes responsible for the polarization transfer.
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