NMR is a noninvasive spectroscopic technique that is ideally suited to probe biomolecular structure and dynamics at atomic resolution. The impact of NMR, however, is curtailed by poor sensitivity, large sample quantities, long acquisition times and multimillion-dollar high-field (>400 MHz) spectrometers. These challenges hinder the study of dilute biomolecules under physiological conditions in liquids. Benchtop NMR (60–90 MHz) spectroscopy offers a cost-effective alternative, yet it comes with severe sensitivity trade-offs. Here, we address this bottleneck by integrating benchtop NMR (80 MHz) with low-concentration photochemically induced dynamic nuclear polarization (LC-photo-CIDNP). LC-photo-CIDNP increases NMR sensitivity by generating transient non-Boltzmann polarization across solvent-exposed aromatic molecules including tryptophan (Trp) or tyrosine (Tyr), either as free species or within proteins. Importantly LC-photo-CIDNP is theoretically predicted to yield stronger hyperpolarization at low field. We highlight two benchtop LC-photo-CIDNP strategies for enhancing 13 C and 1 H signals under physiologically relevant conditions. First, a 1 H-detected- 13 C-photo-CIDNP experiment enabled a 13 C α enhancement of >3000 on a 5 μM deuterated quasi-isolated-spin-pair bearing Trp (QISP Trp) within ∼1 min, extending benchtop-NMR detection limit to 2 μM. We are particularly interested in the 1 H α - 13 C α pair because 1 H α / 13 C α chemical shift deviations from random coils are highly sensitive to protein secondary structure. Second, a 1 H-photo-CIDNP experiment enabled 250 nM detection of epinephrine at natural abundance in aqueous buffer. This marks the lowest sample concentration reported on a benchtop NMR spectrometer in water to date. The above methodologies perform robustly with neurotransmitters, pharmaceuticals, bacterial media, and biofluids (e.g., human serum) affording low-μM detection of these and other biomarkers within minutes. Ongoing work is extending LC-photo-CIDNP to peptides and proteins containing QISP residues. In summary, these advances elevate benchtop NMR from a teaching-lab-oriented instrument into a frontier platform capable of fostering chemical, analytical and biomedical discoveries.
Halder et al. (Sun,) studied this question.
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