Protein–ligand binding interactions are characterized by the para-H2 based hyperpolarization technique SABRE and flow-NMR. Binding to the protein is identified by R2 change of a ligand first interacting with the Ir polarization transfer catalyst.
Hyperpolarization through signal amplification by reversible
exchange
(SABRE) provides a facile means to enhance nuclear magnetic resonance
(NMR) signals of small molecules containing an N-heterocycle or other
binding site for a polarization transfer catalyst. A purpose-designed
reporter ligand, which is capable of binding both to a target protein
and to the catalyst, makes the sensitivity enhancement by this technique
compatible with the measurement of a range of biomolecular interactions.
The 1H polarization of the reporter ligand 4-amidinopyridine,
which is targeting trypsin, is used to screen ligands that are not
themselves hyperpolarizable by SABRE. The respective protein–ligand
dissociation constants (K
D) are determined
by an observed change in the R
2 relaxation
rate of the reporter. A calculation of expected signal changes indicates
that the accessible ligand K
D values extend
over several orders of magnitude, while the concentrations of target
proteins and ligands can be reduced considering the sensitivity gains
from hyperpolarization. In general, the design of a single, weakly
binding ligand for a target protein enables the use of SABRE hyperpolarization
for ligand screening or other biophysical studies involving macromolecular
interactions.
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 the ortho-position of the heterocycle, however, result in low polarization using a typical Ir catalyst with a bismesityl 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, the 1 H signal enhancement increased in several substrates with ortho NH 2 substitutions. For example, for a proton in 2,4-diaminopyrimidine, the enhancement factors increased from À 7 � 1 to À 210 � 20 with allylamine or to À 160 � 10 with acetonitrile. CH 3 substituted molecules yielded maximum signal enhancements of À 25 � 7 with acetonitrile addition, which is considerably less than the corresponding NH 2 substituted molecules, despite exhibiting similar steric size. With the more electron-donating NH 2 substitution resulting in greater enhancement, it is concluded that steric hindrance is not the only dominant factor in determining the polarizability of the CH 3 substituted 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 À 70.
A series of iridium catalysts provides NMR sensitivity enhancement using para-hydrogen. The substrate exchange rate can be tuned for optimal polarization by the choice of an aryl and a nucleophilic moiety in the catalyst.
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