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Abstract Hyperpolarization of N‐heterocycles with signal amplification by reversible exchange (SABRE) induces NMR sensitivity gains for biological molecules. Substitutions with functional groups, in particular in theortho‐position of the heterocycle, however, result in low polarization using a typical Ir catalyst with a bis‐mesityl N‐heterocyclic carbene ligand for SABRE, presumably due to steric hindrance. With the addition of allylamine or acetonitrile as coligands to the precatalyst chloro(1,5‐cyclooctadiene)[4,5‐dimethyl‐1,3‐bis(2,4,6‐trimethylphenyl)imidazol‐2‐ylidene] iridium, the¹H signal enhancement increased in several substrates withorthoNH₂substitutions. For example, for a proton in 2,4‐diaminopyrimidine, the enhancement factors increased from −7±1 to −210±20 with allylamine or to −160±10 with acetonitrile. CH₃substituted molecules yielded maximum signal enhancements of −25±7 with acetonitrile addition, which is considerably less than the corresponding NH₂substituted molecules, despite exhibiting similar steric size. With the more electron‐donating NH₂substitution resulting in greater enhancement, it is concluded that steric hindrance is not the only dominant factor in determining the polarizability of the CH₃substituted compounds. The addition of allylamine increased the signal enhancement for the 290 Da trimethoprim, a molecule with a 2,4‐diaminopyrimidine moiety serving as an antibacterial agent, to −70. 

Nuclear spin hyperpolarization by parahydrogen enables the measurement of biomolecular interactions without the need for a superconducting or permanent magnet. Observed is a fluorine signal of a purpose-designed reporter ligand for a target protein. 

Nuclear spin hyperpolarization through signal amplification by reversible exchange (SABRE), the non-hydrogenative version of para -hydrogen induced polarization, is demonstrated to enhance sensitivity for the detection of biomacromolecular interactions. A target ligand for the enzyme trypsin includes the binding motif for the protein, and at a distant location a heterocyclic nitrogen atom for interacting with a SABRE polarization transfer catalyst. This molecule, 4-amidinopyridine, is hyperpolarized with 50% para -hydrogen to yield enhancement values ranging from −87 and −34 in the ortho and meta positions of the heterocyclic nitrogen, to −230 and −110, for different solution conditions. Ligand binding is identified by flow-NMR, in a two-step process that separately optimizes the polarization transfer in methanol while detecting the interaction in a predominantly aqueous medium. A single scan Carr–Purcell–Meiboom–Gill (CPMG) experiment identifies binding by the change in R 2 relaxation rate. The SABRE hyperpolarization technique provides a cost effective means to enhance NMR of biological systems, for the identification of protein–ligand interactions and other applications. 

A series of bidentate N-heterocyclic carbene (NHC) iridium catalysts, [Ir(κC,N-NHC)H 2 L 2 ]BPh 4 , are proposed for SABRE hyperpolarization. The steric and electronic properties of the NHCs are used to tune substrate affinity and thereby SABRE efficiency. The sterically hindered substrates 2,4-diaminopyrimidine and trimethoprim yielded maximum proton NMR signal enhancements of ∼300-fold and ∼150-fold, respectively.