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

Abstract

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.

Spin Hyperpolarization in Modern Magnetic Resonance

“…472 Further, the combination of LED irradiation and selective isotope labeling, in the presence of small amounts of ascorbic acid (2 μM), recently enabled the detection of 20 nM (2.8 ng) Trp in solution. 484 In this study, LC-photo-CIDNP attenuation effects where minimized upon employing Trp-α- 13 C-β,β,β,2,4,5,6,7-d 7 , a Trp isotopologue bearing a 13 C α -1 H α pair surrounded predominantly by non-NMR-active and low-γ nuclei. 484 Given that LC-photo-CIDNP is tailored to very low concentrations of the molecule of interest (low-or submicromolar), dyes with a long triplet-state lifetime and oxygen depletion strategies are an absolute requirement.…”

“…Therefore, the potential advantage of switching magnetic field during a photo-CIDNP experiment needs to be evaluated on a case-by-case basis. , An example of the expected dependence of photo-CIDNP polarization on an applied magnetic field is provided in Figure . The simulations in this figure were carried out according to known procedures, , and employed published values of relevant parameters. − …”

Illuminating the Human Metabolome: Selective Detection of Multiple Metabolites in Unaltered Biofluids via Hyperpolarisation-Enhanced NMR Spectroscopy

“…Significant experimental and theoretical work into increasing the sensitivity of biological photo-CIDNP NMR measurements has been carried out by the group of Cavagnero in the last fifteen years. It has very recently been demonstrated that a combination of some of these setup improvements can lower the photo-CIDNP detection limit down to the low nM concentration range [14,15]. Many of these improvements can be implemented into the photo-CIDNP analysis of metabolomically relevant biofluids.…”

“…less invasive, experimental conditions and involves the instantaneous (low-power) LEDor laser light-induced generation of transient radical pairs in the presence of a small amount of a photosensitiser yielding spin-polarised molecular species [11][12][13]. In addition, photo-CIDNP is a very sensitive homo-and heteronuclear hyperpolarisation method that allows the NMR detection of analytes present in a low nanomolar concentration range provided that certain experimental requirements are met [14,15]. Traditionally, the method has been mainly employed to gauge bio-macromolecular solvent exposure in the steady state as well as in real-time owing to its ability to selectively highlight the three aromatic amino acid side-chains of tyrosine (Tyr), tryptophan (Trp), and histidine (His) as well as the aliphatic amino acid methionine (Met).…”

“…Thus, up to the present, biological photo-CIDNP experiments have been conducted almost exclusively for the analysis of isolated amino acids and proteins in buffered aqueous solution. To the best of our knowledge, only one precedent of the application of photo-CIDNP NMR spectroscopy in the context of (complex) biological media has been reported using specific means, which seem difficult to be implementable into metabolomic workflows: the detection of a singly 13 C-labelled Trp isotopologue in a diluted bacterial cell extract applying a low concentration (LC) photo-CIDNP approach involving multi-step pretreatment procedures of the sample solution prior to acquisition of the hyperpolarisation spectrum [14,15].…”