SABRE Acethydrazide as a Co-Substrate Agent for Quantitative Analysis of Submicromolar N-Heterocyclic Compounds Using Parahydrogen Hyperpolarization

Abstract

Signal amplification by reversible exchange (SABRE) hyperpolarization is a well-known method for enhancing nuclear magnetic resonance (NMR) signals by transferring para-hydrogen-derived polarization to the analyte of interest. However, due to inherent sensitivity limitations, detecting analyte signals at micromolar (μM) or lower concentrations remains challenging. This study proposes acethydrazide (ACH) as a novel co-substrate, which significantly improves SABRE hyperpolarization efficiency and enables the detection of aromatic N-heterocycles at submicromolar concentrations. ACH enhances signal amplification by stabilizing iridium catalysts through the formation of a pincer complex, as confirmed by non-hydrogenative parahydrogen-induced polarization (nhPHIP) experiments and density functional theory (DFT) calculations. Notably, at the optimal concentration ratio of iridium catalyst:analytes:ACH (1:1:6.5), detection of aromatic N-heterocycles at concentrations below 1.0 μM can be achieved within a single scan using a 400 MHz NMR spectrometer. The enhancement factor and signal-to-noise ratio of the nicotinamide H2-free proton are 422-fold and 2.52 au, respectively, at 0.75 μM using 3 bar of 60% parahydrogen. The ability to detect analytes at a micromolar concentration using SABRE with this new co-substrate expands its potential for biological and industrial applications.