Biocompatible SABRE Hyperpolarization of 1‐ 13 CKetoleucine for Cellular Metabolic Flux Sensing

ABSTRACT Ketoleucine (α‐ketoisocaproate) is a novel hyperpolarized substrate for noninvasive metabolic imaging, enabling rapid, high‐sensitivity detection of branched‐chain amino acid flux, a pathway that is aberrant in many diseases including cancer and Alzheimer's disease. Utilizing NMR Signal Amplification by Reversible Exchange (SABRE) with an 80:20 acetone:water solvent system, we achieved >11% polarization of 1‐ 13 Cketoleucine (corresponding to signal enhancement over 230,000‐fold at 0.55 T) at 70 mM within 2 min, using parahydrogen as a cheap and fast source of hyperpolarization. A two‐stage liquid–liquid extraction and gas stripping protocol removes excess excipients, yielding a biocompatible aqueous solution of 1‐ 13 Cketoleucine (79 ± 10 mM). When mixed with Saccharomyces cerevisiae (Baker's yeast), hyperpolarized 1‐ 13 Cketoleucine produced strong, long‐lasting signals, where downstream metabolites are observed for >200 s, allowing first‐order kinetic modeling of CO 2 and bicarbonate. These cell experiments demonstrate both the biocompatibility and signal strength of SABRE–hyperpolarized KL, establishing KL as a versatile hyperpolarized agent and opening avenues for real‐time investigation of metabolic dysregulation in cancer, neurodegenerative disorders, and beyond.