Fast-pulsing LED-enhanced NMR: A convenient and inexpensive approach to increase NMR sensitivity

Abstract

Low-concentration photochemically induced dynamic nuclear polarization (LC-photo-CIDNP) has recently emerged as a powerful technology for the detection of aromatic amino acids and proteins in solution in the low-micromolar to nanomolar concentration range. LC-photo-CIDNP is typically carried out in the presence of high-power lasers, which are costly and maintenance-heavy. Here, we show that LC-photo-CIDNP can be performed with light-emitting diodes (LEDs), which are inexpensive and much less cumbersome than lasers, laser diodes, flash lamps, or other light sources. When nuclear magnetic resonance (NMR) sample concentration is within the low-micromolar to nanomolar range, as in LC-photo-CIDNP, replacement of lasers with LEDs leads to no losses in sensitivity. We also investigate the effect of optical-fiber thickness and compare excitation rate constants of an Ar ion laser (488 nm) and a 466 nm LED, taking LED emission bandwidths into account. In addition, importantly, we develop a novel pulse sequence (13C RASPRINT) to perform ultrarapid LC-photo-CIDNP data collection. Remarkably, 13C RASPRINT leads to 4-fold savings in data collection time. The latter advance relies on the fact that photo-CID nuclear hyperpolarization does not suffer from the longitudinal-relaxation recovery requirements of conventional NMR. Finally, we combine both the above improvements, resulting in facile and rapid (≈16 s–2.5 min) collection of 1 and 2D NMR data on aromatic amino acids and proteins in solution at nanomolar to low micromolar concentration.

Selective Isotope Labeling and LC-Photo-CIDNP Enable NMR Spectroscopy at Low-Nanomolar Concentration

“…Earlier identification of chemically induced dynamic nuclear polarization (CIDNP) , in organic reactions led to the development of a photochemically enhanced NMR approach known as photo-CIDNP. While originally employed to assess solvent exposure, this technique has more recently been employed to significantly increase NMR sensitivity in solution at a high applied field. − Photo-CIDNP is devoid of most of the drawbacks of other approaches listed in the previous paragraph. This technique requires aromatic moieties and benefits from an O 2 -free environment.…”

“…The use of photosensitizers characterized by a long triplet-state lifetime (e.g., 20 ms for fluorescein , ) gave rise to a branch of photo-CIDNP known as low-concentration photo-CIDNP (LC-photo-CIDNP). Replacement of costly and hard-to-maintain high-power laser with light-emitting diodes (LEDs) (Figure C) with no sensitivity losses led to enhanced ease of access, portability, and safety …”

“…The latter features enable long-term signal averaging and multidimensional NMR of extremely dilute samples. This technology leads to an enhanced signal-to-noise ratio (S/N) of tryptophan (Trp) and tyrosine (Tyr), either as free amino acids or within polypeptides and proteins. ,− Remarkably, high LC-photo-CIDNP enhancements are observed for C α nuclei of aromatic amino acids, likely due to their high hyperfine coupling constants, , enabling the efficient detection of 1 H α – 13 C α pairs. The C α chemical shifts are particularly useful because they are diagnostic of the backbone secondary structure in proteins .…”

“…To take advantage of proton’s higher sensitivity, 13 C-to- 1 H polarization transfer via reverse INEPT is typically employed . In addition, two-dimensional (2D) data collection is carried out to preserve information on 13 C α chemical shifts. , The recently developed 13 C RASPRINT pulse sequence is optimized for ultrafast data collection in an aqueous solvent . This sequence exploits minimal recycle delays, given that most nuclear-spin polarization is generated during the optical irradiation time, thus abrogating the need for lengthy interscan intervals .…”

Universal approach to in situ light-coupled NMR spectroscopy without fibre optics

“…Photo-chemically induced dynamic nuclear polarization (photo-CIDNP), where observed NMR signal intensities of aromatic compounds in the presence of a photosensitiser are modulated upon illumination, has been previously demonstrated using illumination with LASERs, 18,20,21,36 and more recently, with LEDs. 13,31,32 Here, we explored the suitability and effectiveness of NMRtorch for the photo-CIDNP experiments, by recording 19 F NMR spectra of 6-uoroindole (6FI) with photosensitization by avin mononucleotide (FMN) under blue light illumination. With the NMRtorch setup, 6FI exhibited emissive photo-CIDNP, with negative 19 F peaks upon illumination and a maximum 64-fold enhancement achieved with the lighthead containing a single LED with nominal 470 nm peak emission, 3W power consumption (Figure 2A).…”