NMR spectroscopy is a powerful tool
to investigate molecular structure
and dynamics. The poor sensitivity of this technique, however, limits
its ability to tackle questions requiring dilute samples. Low-concentration
photochemically induced dynamic nuclear polarization (LC-photo-CIDNP)
is an optically enhanced NMR technology capable of addressing the
above challenge by increasing the detection limit of aromatic amino
acids in solution up to 1000-fold, either in isolation or within proteins.
Here, we show that the absence of NMR-active nuclei close to a magnetically
active site of interest (e.g., the structurally diagnostic 1Hα–13Cα pair
of amino acids) is expected to significantly increase LC-photo-CIDNP
hyperpolarization. Then, we exploit the spin-diluted tryptophan isotopolog
Trp-α-13C-β,β,2,4,5,6,7-d7 and take advantage of the above prediction to experimentally achieve
a ca 4-fold enhancement in NMR sensitivity over regular LC-photo-CIDNP.
This advance enables the rapid (within seconds) detection of 20 nM
concentrations or the molecule of interest, corresponding to a remarkable
3 ng detection limit. Finally, the above Trp isotopolog is amenable
to incorporation within proteins and is readily detectable at a 1
μM concentration in complex cell-like media, including Escherichia coli cell-free extracts.