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Micromolar Concentration Affinity Study on a Benchtop NMR Spectrometer with Secondary 13C Labeled Hyperpolarized Ligands
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  • Olivier Cala
    Olivier Cala
    Universite Claude Bernard Lyon 1, CRMN UMR-5082, CNRS, ENS Lyon, Villeurbanne 69100, France
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    Orcidhttps://orcid.org/0000-0003-3579-0973
  • Charlotte Bocquelet
    Charlotte Bocquelet
    Universite Claude Bernard Lyon 1, CRMN UMR-5082, CNRS, ENS Lyon, Villeurbanne 69100, France
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  • Chloé Gioiosa
    Chloé Gioiosa
    Universite Claude Bernard Lyon 1, CRMN UMR-5082, CNRS, ENS Lyon, Villeurbanne 69100, France
    TotalEnergies OneTech, Centre de recherche de Solaize, BP 22, Chemin du Canal, 69360 Solaize, France
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  • Felix Torres
    Felix Torres
    ETH, CH-8093 Zürich, Switzerland
    NexMR GmbH, 8952 Schlieren, Switzerland
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  • Samuel F. Cousin
    Samuel F. Cousin
    Universite Claude Bernard Lyon 1, CRMN UMR-5082, CNRS, ENS Lyon, Villeurbanne 69100, France
    Institut de Chimie Radicalaire (UMR CNRS 7273), Aix-Marseille Université, Service 511, ST JEROME, Avenue Escadrille Normandie Niémen, 13013 Marseille, France
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    Orcidhttps://orcid.org/0000-0002-7021-478X
  • Sylvie Guibert
    Sylvie Guibert
    Universite Claude Bernard Lyon 1, CRMN UMR-5082, CNRS, ENS Lyon, Villeurbanne 69100, France
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  • Morgan Ceillier
    Morgan Ceillier
    Universite Claude Bernard Lyon 1, CRMN UMR-5082, CNRS, ENS Lyon, Villeurbanne 69100, France
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  • Venita Busse
    Venita Busse
    Bruker Switzerland AG, Fällanden 8117, Switzerland
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  • Frank Decker
    Frank Decker
    Bruker Biospin GmbH, Silberstreifen 4, 76287 Rheinstetten, Germany
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  • James G. Kempf
    James G. Kempf
    Bruker Biospin Corp., Billerica, Massachusetts 01821, United States
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  • Stuart J. Elliott
    Stuart J. Elliott
    Universite Claude Bernard Lyon 1, CRMN UMR-5082, CNRS, ENS Lyon, Villeurbanne 69100, France
    Molecular Sciences Research Hub, Imperial College London, London W120BZ, U.K.
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    Orcidhttps://orcid.org/0000-0002-8726-0635
  • Quentin Stern
    Quentin Stern
    Universite Claude Bernard Lyon 1, CRMN UMR-5082, CNRS, ENS Lyon, Villeurbanne 69100, France
    Department of Chemistry, Northwestern University, Evanston, Illinois 60208, United States
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  • Aurélien Bornet
    Aurélien Bornet
    Universite Claude Bernard Lyon 1, CRMN UMR-5082, CNRS, ENS Lyon, Villeurbanne 69100, France
    École Polytechnique Fédérale de Lausanne, Institut des Sciences et Ingénierie Chimiques, 1015 Lausanne, Switzerland
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  • Sami Jannin*
    Sami Jannin
    Universite Claude Bernard Lyon 1, CRMN UMR-5082, CNRS, ENS Lyon, Villeurbanne 69100, France
    *Email: [email protected]
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    Orcidhttps://orcid.org/0000-0002-8877-4929
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ACS Omega

Cite this: ACS Omega 2025, 10, 4, 3332–3337
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https://doi.org/10.1021/acsomega.4c05101
Published January 24, 2025

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Abstract

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Benchtop NMR is becoming an increasingly important tool, sometimes providing a simple and low-cost alternative to high-field NMR. The Achilles heel of NMR and even more critically of benchtop NMR is its limited sensitivity. However, when combined with hyperpolarization techniques, the sensitivity boost can provide excellent sensitivity that can even make benchtop NMR compatible with affinity studies for drug discovery. Hyperpolarization by dissolution dynamic nuclear polarization (dDNP) provides a route to enhancing 13C nuclear magnetic resonance (NMR) sensitivity by more than 5 orders of magnitude for a wide range of small molecules on a benchtop NMR system. We show here how ligands can be secondarily labeled with 13C tags and hyperpolarized with conventional dDNP methods. These hyperpolarized ligands display long nuclear spin–lattice relaxation time constants and can therefore be used to probe interactions with target proteins in conventional dDNP settings. The boost in sensitivity combined with the simplicity of the 13C spectra (one peak per ligand) enables detection on an 80 MHz benchtop NMR spectrometer at micromolar concentrations, which may ultimately provide a way of improving and accelerating the discovery of new drug candidates.

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Introduction

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Identifying intermolecular interactions is at the core of life science, from biological mechanisms in living organisms to the interaction between biomolecules and exogenous molecules, such as drugs. In drug discovery, identifying protein–ligand interactions is a crucial step in the further design of more potent and specific ligands to achieve drug candidates. This identification step is called screening, as several thousands of molecules are tested to identify which interact with a target biomolecule, often a protein. Screening requires a sufficient throughput to test these libraries, and it must be accurate to deliver high-quality primary data sets that will be leveraged by medicinal chemists for drug design. The ideal screening method should be fast and require a low sample production and preparation effort. In the past 20 years, fragment-based drug design (FBDD) has been established as a valid strategy to tackle challenging biomolecular targets. (1) FBDD requires screening methods that can detect the weakly interacting hits that are mostly achieved with fragments. These initial hits consisting of fragments are later assembled through fragment merging, linking, or growing to form higher affinity (typically μM) ligands. (2,3)
Nuclear magnetic resonance (NMR) has emerged over the past decades as a powerful method for screening, providing full protein–ligand characterizations, particularly for weakly interacting systems, (4) in complement to other techniques. (5−9) Due to its high robustness, NMR is the gold standard used in pharmaceutical research to identify and validate hits from compound libraries in drug discovery.
NMR screening experiments can be classified into two families: protein-observed experiments or ligand-observed experiments. (8,10) Protein-observed experiments often require 13C/15N labeling, which is not always feasible for large proteins (mass exceeding 30 kDa with intense relaxation). The classical ligand-observed 1H NMR methods [saturation transfer difference (STD), water-ligand observed through gradient spectroscopy (waterLOGSY), nuclear Overhauser spectroscopy (NOESY), and relaxation-based methods] do not require isotopic labeling of the protein but do require ligand concentrations on the order of hundreds of micromolar (typically for a 20 min experiment). Such NMR screening is systematically performed on high-field NMR spectrometers (ω0/2π > 500 MHz) and remains incompatible with low-field benchtop NMR because of a lack of sensitivity. One efficient approach is to involve hyperpolarization methods, yielding out-of-Boltzmann polarization and enhanced sensitivity.
Dissolution dynamic nuclear polarization (dDNP) (11) is a hyperpolarization method that can boost NMR sensitivity up to 5 orders of magnitude in the context of benchtop NMR. It was successfully used in interaction studies, for example, by Hilty et al. (12) using hyperpolarized fluorine-containing molecules, by Lerche et al. (13) using natural abundance 13C molecules and relaxation-based methods, by Kurzbach et al. (14) by exploring hyperpolarized long-lived state relaxation in deuterated methyl groups, or by Jannin et al. (15) using a waterLOGSY experiment combined with hyperpolarized water to reveal weak binding. Although successful and promising, these approaches still suffer from some critical limitations: (i) 1H and 19F have short nuclear spin–lattice relaxation time constants (T1) on the order of a second, meaning that hyperpolarization is short-lived and that the large enhancement brought by dDNP rapidly vanishes; (ii) 13C nuclear spins have longer values of T1, which make them much more interesting candidates, but the natural abundance of only 1.1% translates into a suboptimal sensitivity, and the very long hyperpolarization time (up to 3 h to hyperpolarize a single sample) translates into a very low throughput; and (iii) all presented methods were limited to weak affinity.
In the past decade, Raftery and co-workers presented the chemical derivatization of amines in biofluids with [1,1′-13C2] acetic anhydride. (16) This easy and robust reaction takes place rapidly using [1,1′-13C2] acetic anhydride in an aqueous solution at ambient temperature on the amine.
Here, we propose to revisit this secondary labeling approach in the context of NMR drug screening by exploiting a chemical derivation of NH2-containing molecules to develop a new 13C-labeled ligand library that is chemically distinct and with different binding properties and couple it with our most recent 1H → 13C cross-polarization (CP)-assisted approaches leading to high (>30%) and fast (10 min) 13C hyperpolarization. Similar to Torres et.al., (17) we restrict for now our chemical space to NH2-containing molecules, thus limiting the possible chemical diversity. However, this approach could be generalized to many more molecules later by competition binding experiments or by 13C enrichment. We used hyperpolarized 13C-based relaxation studies at a few micromolar ligand concentrations against a model protein on an 80 MHz benchtop NMR spectrometer. We show that despite the inherent low sensitivity of the system and its limited resolution, and thanks to the high hyperpolarization brought by dDNP and the simplicity of the resulting 13C spectra (one peak per ligand), our method enables the detection and identification of ligands at a few μM concentration within seconds after 20 min of hyperpolarization.

Results and Discussion

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Figure 1a illustrates the chemical reaction used to tag NH2-containing molecules. When acetic anhydride is added in aqueous solution at room temperature, NH2-containing molecules derivatize rapidly. Here, 6-amino-2-naphthoic acid and glycine were used as test molecules. The N-acetylation occurs on the primary amine group simply by adding an excess of acetic anhydride at room temperature, with a yield greater than 95%. The new products are N-Acetyl [1-13C]-6 amino-2-naphthoic acid and N-Acetyl [1-13C]-glycine, here named Ac-L30 and Ac-L08, respectively. Their 13C assignments are shown in Figure 1b. The 13C NMR spectra of the N-acetylated derivatives show the presence of the isotopically labeled carbonyl sites that appear in the 173–182 ppm region. It can be seen that the individual N-acetyl-molecules are well resolved with a single peak for each molecule with a full-width half-maximum (fwhm) < 1 Hz at 171.75 ppm for Ac-L30 and at 172.55 ppm for Ac-L08. An additional peak is observed at 180 ppm corresponding to [1-13C] acetic acid (resulting from the excess of anhydride acetic acid during synthesis). The derivatization procedure results in a direct 13C NMR sensitivity gain of 91 with respect to a nonlabeled molecule. Any primary amine group containing molecule can be labeled with the presented approach, which results in a new molecule that can be used as a candidate for interaction tests. These compounds containing a 13C center by 13C acetylation of amines evidently become chemically distinct and have different binding properties. These new compounds have been tested with conventional approaches, i.e., STD and 13C T1 relaxation measurements.

Figure 1

Figure 1. (a) Chemical reaction for the labeling of a ligand containing an -NH2 group as described in the experimental section. (b) Relevant portions of the experimental 13C NMR spectra for the 164 mM tagged ligand of N-Acetyl [1-13C]-6-amino-2-naphthoic acid (N–Ac-L30, red) and N-Acetyl [1-13C]-glycine (Ac-L08, blue) dissolved in a deuterated phosphate buffer and acquired at 20 MHz (1.88 T) and 298 K.

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For the current study, HSA was used as a model protein target. Figure 2 shows the result of a conventional STD interaction test with the HSA protein measured at 600 MHz. We see that Ac-L30 (1H 1D reference spectrum (b) in black and STD spectrum (a) in red) interacts with HSA with a strong STD signal (dashed area), whereas Ac-L08 (1H 1D reference spectrum (d) in black and STD spectrum (c) in blue) has a much lower STD signal translating into an insignificant interaction with HSA.

Figure 2

Figure 2. Relevant portions of the experimental STD and 1H NMR spectra of 2 mM Ac-L08 with 5 μM HSA in 0.5% DMSO-d6, 10% D2O, and 89.5% phosphate buffer pH 8.5 (v:v:v) (a, b) and 2 mM Ac-L30 with 5 μM HSA in 0.5% DMSO-d6, 10% D2O, and 89.5% phosphate buffer pH 8.5 (v:v:v) (c, d) acquired at 600 MHz (14.1 T) and 298 K with 16 and 512 scans, respectively. Assignments are indicated for the two compounds (1′-4′ for Ac-L08 and 1–8 for Ac-L30). * indicates the signal corresponding to DMSO-d6. The dashed boxes represent the area of interest for the STD signal.

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Figure 3 shows the 13C signal decays of hyperpolarized ligands measured at 600 μM concentration after hyperpolarization and injection, with and without the HSA protein at 40 μM concentration. T1 measurements were acquired on an 80 MHz benchtop NMR spectrometer after hyperpolarization with our previously developed dDNP methodology, (18) including low-temperature DNP and 1H → 13C cross-polarization, (19−22) fast dissolution and transfer (23) through a magnetic tunnel, (24) and injection (see Methods section for experimental details). These standardized methods were previously applied in the field of metabolomics (25−27) and will soon be part of a commercial polarizer. Note that using hyperpolarized T1 variation as a probe for interactions entirely lifts the issue of repeatability in the initial polarization. Here, we observe notable changes in 13C T1’s caused by ligand-protein interactions. This is not surprising and confirms that the study is designed appropriately and that our method works straightforwardly. Without protein, we measured 13C T1 values of 11.9 ± 1.0 s for Ac-L30 and 29.3 ± 0.2 s for Ac-L08. When HSA was present in the NMR tube before ligand injection and mixed during injection, we observed a drop in 13C T1 for Ac-L30 down to 6.3 ± 0.8 s, while the 13C T1 of the noninteracting control, Ac-L08, was not significantly affected. The contrast (ξ) in 13C R1 = 1/T1 can be defined as the difference between 13C R1 without (free) and with protein (bound), normalized by 13C R1 with protein:
ξ=R1bound−R1freeR1bound
(1)

Figure 3

Figure 3. Experimental decays for the hyperpolarized 13C NMR signal of 600 μM Ac-L30 and 600 μM Ac-L08 dissolved in deuterated-phosphate buffer pH 8.5 acquired at a 20 MHz 13C frequency (1.88 T) and ∼303 K with and without mixing with a protein solution (40 μM HSA) after 2 s transfer to the NMR spectrometer. (a) 13C T1 determination for Ac-L30 without and with HSA. (b) 13C T1 determination for Ac-L08 without and with HSA. For both ligands, hollow circles represent experimental data (integrals of 1D 13C NMR spectra measured with 5 or 2.5 s intervals and pulse flip angles of 15°), and the solid lines represent fits of the experimental data with a monoexponential decay function: P0·exp(−t/T1) where P0 is a fitting constant.

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ξ was found to be 1.40 ± 0.02% for the noninteracting Ac-L08 and 47 ± 8% for Ac-L30. These results qualitatively agree with thermal equilibrium experiments shown in Supporting Information. Note that, for sensitivity reasons, these thermal equilibrium experiments were performed with excessively high concentrations of ligands (164 mM instead of 600 μM, thus 273 times higher) and with 1 mM HSA. While these control experiments took 8.5 h at 1.88 T with 164 mM ligands, they would have taken 2732more time, equivalent to more than 72 years, with 600 μM ligands and without hyperpolarization at 1.88 T, instead of about 1 h with hyperpolarization. Finally, these results show that our hyperpolarization and dissolution procedure does not influence the sample health and longitudinal relaxation time constants and therefore yields useful interaction information (Ac-L08 interacts significantly less than Ac-L30 with the protein). This demonstrates both efficient labeling of NH2-containing molecules and possible interaction studies based on the 13C T1 decay measurement following hyperpolarization by state-of-the-art CP-assisted dDNP methods. We were able to hyperpolarize these compounds and use their 13C T1 values as a probe of protein–ligand interaction with increased sensitivity at concentrations down to 30 μM with a signal-to-noise ratio (SNR) exceeding 100, even on a benchtop NMR spectrometer operating at 1.88 T. Extra orders of magnitude in sensitivity could evidently be afforded on high field spectrometers if needed.
This method can potentially bring significant benefits in terms of throughput. Furthermore, if we consider the work of previous groups (16,28,29) where up to 20 labeled amino acids were mixed, we speculate that such an approach could be extended to the study of more than 30 mixed ligands simultaneously screened in one shot (as sometimes done for 19F, while for 1H, it is classically only 5–10 ligands (30,31) that are mixed due to 1H NMR signal superposition). Hyperpolarizing such ligand mixtures can typically be repeated every 30 min to an hour depending on the skills of the operator of the dDNP setup. Further automation in the future might bring this number down to 10–20 min, which would translate into two to four thousand ligands potentially screened per day.
The described screening method benefits from the dramatically enhanced 13C NMR signal intensity engendered by implementing hyperpolarization methodologies, in this case, dDNP, leading to reduced protein and ligand concentrations in the μM range. These concentrations may open the way to targeting proteins that are difficult to express or poorly soluble and ligands that have higher affinities and therefore need to be at concentrations close to the protein.
Here, NH2-containing molecules are tagged in a way that applies to hundreds of molecules in our library. Other tags or chemical labeling schemes and the 13C-enrichment of ligands could be envisaged, in particular, for competition experiments with a known ligand. Furthermore, the recent developments in isotope swap will open the way to a further growing number of specifically 13C-labeled small molecules at affordable costs, (32) and other carbon insertion methods are expandable to 13C insertion from economical precursors (33) to provide sufficient diversity in fragment libraries. The small molecule selective labeling approach can also be expanded to other nuclear spins, such as 15N, which also benefits from long spin–lattice relaxation time constants, a comprehensive chemical shift range, and CP-assisted dDNP methodologies developed in our laboratories. (34) Recently, the insertion of 15N atoms into diverse aromatic skeletons has demonstrated the possibility of generating chemically diverse scaffolds containing single 15N atoms. (35−37) Finally, here, we measured T1 as a contrast parameter to detect the binding; however, extending the method to measurements of transverse relaxation might yield an even higher contrast.

Conclusions

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In conclusion, we have shown that NH2-containing ligands can be labeled efficiently and that their interactions with the HSA protein can be measured by either classical 1H NMR approaches (such as STD) or hyperpolarized 13C T1 contrast. We were able to hyperpolarize these compounds and measure their T1 values with sufficient sensitivity at concentrations down to 30 μM in a benchtop NMR spectrometer operating at an 80 MHz frequency for protons, which provided evidence of binding in a single experiment. The method might be extended in the future to the simultaneous study of many ligands mixed and screened in one shot, as 13C NMR spectra are simple (one peak per ligand) and better resolved than conventional 1H spectra. The required sample concentrations are compatible with targeting proteins that are difficult to express or poorly soluble and ligands that have higher affinity and are potentially better drug candidates. This approach might have the potential to become a useful screening method, in particular, in the context of FBDD. In the future, we plan on applying this method to a larger library of ligands and extending it to competitive studies either by using the tag approach or by directly incorporating a 13C label into known ligands.

Methods

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Chemicals

All chemicals were purchased from Sigma-Aldrich.

Labeling

For the derivatization of ligands, stock solutions of 2 commercial molecules (200 mM glycine and 6-amino-2-naphthoic acid in 400 mM deuterated phosphate buffer (pH 8.5) and DMSO, respectively) were prepared in separate tubes. For the derivatization of individual molecules, 250 μL of each compound was reacted 5 times with 1 μL of 1,1′-13C2 acetic anhydride and 10 μL of NaOD 1 M at room temperature, producing 305 μL at 164 mM of each labeled molecule.

DNP Sample Preparation

For N–Ac-glycine, 100 μL of labeled compound was added to 20 μL of D2O + 240 μL of DMSO-d6 + 40 μL of H2O + 1.7 mg of TEMPOL producing 400 μL of 44 mM sample and 25 mM radical in 60:30:10 DMSO-d6:D2O:H2O (v:v:v). For N–Ac-6-amino-2-naphthoic acid, 100 μL of the labeled compound was added to 120 μL of D2O + 140 μL of DMSO-d6 + 40 μL of H2O + 1.7 mg of TEMPOL producing 400 μL of 44 mM sample and 25 mM radical in 60:30:10 DMSO-d6:D2O:H2O (v:v:v). For the mixture of the 2 compounds, 100 μL of each labeled molecule was added to 20 μL of D2O + 140 μL of DMSO-d6 + 40 μL of H2O + 1.7 mg of TEMPOL producing 400 μL of 44 mM of each compound and 25 mM radical in 60:30:10 DMSO-d6:D2O:H2O (v:v:v).

NMR Experiments

NMR samples for STD experiments consisted of a 2 mM fragment in 0.5:10:89.5 DMSO-d6,:D2O:phosphate buffer pH 8.5 (v:v:v) together with a protein concentration of 5 μm for HSA. Standard 1D, STD, NMR spectra were acquired at 20 °C with an Avance III HD Bruker Biospin 600 MHz NMR spectrometer (14.1 T), equipped with a 5 mm triple-resonance inverse cryoprobe with a z-axis field gradient. NMR experiments were performed at 298 K with excitation sculpting to suppress peaks from water. 1D and STD NMR experiments were performed using 16 and 512 scans, respectively, with 2 s of recycling delay and 2 s of acquisition time. Specific parameters for the STD experiments (saturation frequency and saturation time) were identical for both samples. Selective saturation of the protein NMR spectrum was achieved with the decoupler offset at 0.7 ppm, and nonsaturation control was performed at −50 ppm. The saturation time was set to 2 s for all experiments with 2 s of recycling delay, 2 s of acquisition time, and 512 scans.
44 mM of the sample was mixed with 25 mM TEMPOL in 60:30:10 DMSO-d6:D2O:H2O (v:v:v). For each experiment, 100 μL of the sample was placed in a PEEK sample cup, which was then immersed in the liquid helium bath of the cryostat of our DNP polarizer. The rapid insertion of the sample into the cryostat ensures the formation of a glassy frozen sample. All low-temperature DNP experiments were performed with a prototype DNP polarizer developed by Bruker Biospin operating at variable temperatures of 1.2 ≤ T ≤ 4.2 K in a magnetic field B0 = 7.05 T.

1H and 13C DNP Experiments

DNP was performed by shining frequency-modulated microwave irradiation at 197.648 GHz ± 144 MHz, with a modulation frequency of 500 Hz, onto the sample. Cross-polarization (CP) was used to transfer hyperpolarization from 1H to 13C nuclear spins using a protocol detailed elsewhere. (18,19) Four 6.5 ms CP contacts were performed with a 10 min interval to allow enough time for the protons to repolarize between CP contacts (40 min total time).

Dissolution, Transfer, and Injection Experiments

A solenoid of 1.5 m length was wound around the transfer capillary and fed with a current of 2.5 A, thus generating a 4 mT field along the transfer line. Once maximum polarization was reached, 7 mL of D2O was pressurized at 6 bar and heated up until it reached a pressure of 9 bar (∼180 °C). This hot and pressurized solvent was rapidly transferred to the sample space, achieving sample melting and fast transfer to a homemade prototype NMR injector directly placed in the Bruker Biospin Fourier 80 benchtop NMR spectrometer where 40 μL of 400 mM deuterated-phosphate buffer pH 8.5 was waiting in the NMR tube for experiments without protein. For experiments with protein, 40 μL of 550 μM of HSA in 400 mM deuterated-phosphate buffer pH 8.5 was waiting in the NMR tube such that the final concentration was 40 μM after dissolution (550 μL final volume).

Hyperpolarized Liquid-State NMR Measurements

Hyperpolarized liquid-state NMR spectra were measured by using a Bruker Biospin Fourier 80 benchtop spectrometer (1H nuclear Larmor frequency = 80.222 MHz and 13C nuclear Larmor frequency = 20.172 MHz). After injection, 13C NMR signals were recorded every 5 s for Ac-L30 and 2.5 s for Ac-L08 with a 15° pulse flip angle. Final hyperpolarization enhancements and final concentrations were calculated by cross-calibration with a highly concentrated reference sample. All experimental data were processed in Matlab.

Data Availability

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The experimental data presented in this work can be downloaded from Zenodo 10.5281/zenodo.10995066.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.4c05101.

  • Nuclear spin–lattice relaxation times (T1) measurements (PDF)

Accession Codes

The MATLAB codes used to analyze the data can be downloaded from Zenodo 10.5281/zenodo.10995062.

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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  • Corresponding Author
  • Authors
    • Olivier Cala - Universite Claude Bernard Lyon 1, CRMN UMR-5082, CNRS, ENS Lyon, Villeurbanne 69100, France;  Orcidhttps://orcid.org/0000-0003-3579-0973
    • Charlotte Bocquelet - Universite Claude Bernard Lyon 1, CRMN UMR-5082, CNRS, ENS Lyon, Villeurbanne 69100, France
    • Chloé Gioiosa - Universite Claude Bernard Lyon 1, CRMN UMR-5082, CNRS, ENS Lyon, Villeurbanne 69100, France;  TotalEnergies OneTech, Centre de recherche de Solaize, BP 22, Chemin du Canal, 69360 Solaize, France
    • Felix Torres - ETH, CH-8093 Zürich, Switzerland;  NexMR GmbH, 8952 Schlieren, Switzerland
    • Samuel F. Cousin - Universite Claude Bernard Lyon 1, CRMN UMR-5082, CNRS, ENS Lyon, Villeurbanne 69100, France;  Institut de Chimie Radicalaire (UMR CNRS 7273), Aix-Marseille Université, Service 511, ST JEROME, Avenue Escadrille Normandie Niémen, 13013 Marseille, France;  Orcidhttps://orcid.org/0000-0002-7021-478X
    • Sylvie Guibert - Universite Claude Bernard Lyon 1, CRMN UMR-5082, CNRS, ENS Lyon, Villeurbanne 69100, France
    • Morgan Ceillier - Universite Claude Bernard Lyon 1, CRMN UMR-5082, CNRS, ENS Lyon, Villeurbanne 69100, France
    • Venita Busse - Bruker Switzerland AG, Fällanden 8117, Switzerland
    • Frank Decker - Bruker Biospin GmbH, Silberstreifen 4, 76287 Rheinstetten, Germany
    • James G. Kempf - Bruker Biospin Corp., Billerica, Massachusetts 01821, United States
    • Stuart J. Elliott - Universite Claude Bernard Lyon 1, CRMN UMR-5082, CNRS, ENS Lyon, Villeurbanne 69100, France;  Molecular Sciences Research Hub, Imperial College London, London W120BZ, U.K.;  Orcidhttps://orcid.org/0000-0002-8726-0635
    • Quentin Stern - Universite Claude Bernard Lyon 1, CRMN UMR-5082, CNRS, ENS Lyon, Villeurbanne 69100, France;  Department of Chemistry, Northwestern University, Evanston, Illinois 60208, United States
    • Aurélien Bornet - Universite Claude Bernard Lyon 1, CRMN UMR-5082, CNRS, ENS Lyon, Villeurbanne 69100, France;  École Polytechnique Fédérale de Lausanne, Institut des Sciences et Ingénierie Chimiques, 1015 Lausanne, Switzerland
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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This research was supported by ENS Lyon, the French CNRS, Claude Bernard Lyon 1 University, Bruker Biospin, the European Research Council under the European Union’s Horizon 2020 research and innovation program (ERC Grant Agreements No. 714519/HP4all and Marie Skłodowska-Curie Grant Agreement No. 766402/ZULF). The authors gratefully acknowledge Bruker Biospin for providing the prototype dDNP polarizer, and particularly Dmitry Eshchenko, Roberto Melzi, Marc Rossire, and Marco Sacher for scientific and technical support. The authors graciously acknowledge Bruno Knittel for lending assistance with the operation of the Bruker Biospin Fourier 80 benchtop NMR system. The authors additionally acknowledge Catherine Jose and Christophe Pages for use of the ISA Prototype Service, and Stéphane Martinez of the UCBL mechanical workshop for machining parts of the experimental apparatus.

References

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    A review. Fragment-based drug discovery (FBDD) is a powerful method to develop potent smallmol. compds. starting from fragments binding weakly to targets. As FBDD exhibits several advantages over high-throughput screening campaigns, it becomes an attractive strategy in target-based drug discovery. Many potent compds./inhibitors of diverse targets have been developed using this approach. Methods used in fragment screening and understanding fragment-binding modes are crit. in FBDD. This review elucidates fragment libraries, methods utilized in fragment identification/confirmation, strategies applied in growing the identified fragments into drug-like lead compds., and applications of FBDD to different targets. As FBDD can be readily carried out through different biophys. and computer-based methods, it will play more important roles in drug discovery.
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    Murray, C. W.; Rees, D. C. The Rise of Fragment-Based Drug Discovery. Nature Chem. 2009, 1 (3), 187– 192,  DOI: 10.1038/nchem.217
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    The rise of fragment-based drug discovery
    Murray, Christopher W.; Rees, David C.
    Nature Chemistry (2009), 1 (3), 187-192CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)
    A review. The search for new drugs is plagued by high attrition rates at all stages in research and development. Chemists have an opportunity to tackle this problem because attrition can be traced back, in part, to the quality of the chem. leads. Fragment-based drug discovery (FBDD) is a new approach, increasingly used in the pharmaceutical industry, for reducing attrition and providing leads for previously intractable biol. targets. FBDD identifies low-mol.-wt. ligands (∼150 Da) that bind to biol. important macromols. The three-dimensional exptl. binding mode of these fragments is detd. using X-ray crystallog. or NMR spectroscopy, and is used to facilitate their optimization into potent mols. with drug-like properties. Compared with high-throughput-screening, the fragment approach requires fewer compds. to be screened, and, despite the lower initial potency of the screening hits, offers more efficient and fruitful optimization campaigns. Here, we review the rise of FBDD, including its application to discovering clin. candidates against targets for which other chem. approaches have struggled.
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    Qin, J.; Gronenborn, A. M. Weak Protein Complexes: Challenging to Study but Essential for Life. FEBS Journal 2014, 281 (8), 1948– 1949,  DOI: 10.1111/febs.12744
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    Fragment-Based Drug Discovery and X-Ray Crystallography; Davies, T. G.; Hyvönen, M., Eds.; Topics in Current Chemistry; Springer Berlin Heidelberg: Berlin, Heidelberg, 2012; Vol. 317.
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    Pellecchia, M.; Sem, D. S.; Wüthrich, K. Nmr in Drug Discovery. Nat. Rev. Drug Discov 2002, 1 (3), 211– 219,  DOI: 10.1038/nrd748
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    NMR in drug discovery
    Pellecchia, Maurizio; Sem, Daniel S.; Wuthrich, Kurt
    Nature Reviews Drug Discovery (2002), 1 (3), 211-219CODEN: NRDDAG ISSN:. (Nature Publishing Group)
    A review. NMR spectroscopy has evolved into an important technique in support of structure-based drug design. Here, we survey the principles that enable NMR to provide information on the nature of mol. interactions and, on this basis, we discuss current NMR-based strategies that can identify weak-binding compds. and aid their development into potent, drug-like inhibitors for use as lead compds. in drug discovery.
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    Fernández, C.; Jahnke, W. New Approaches for NMR Screening in Drug Discovery. Drug Discovery Today: Technologies 2004, 1 (3), 277– 283,  DOI: 10.1016/j.ddtec.2004.10.003
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    Aguirre, C.; Cala, O.; Krimm, I. Overview of Probing Protein-Ligand Interactions Using NMR. Curr. Protoc. Protein Sci. 2015, 81 (1), 17.18.1,  DOI: 10.1002/0471140864.ps1718s81
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    Cala, O.; Krimm, I. Ligand-Orientation Based Fragment Selection in STD NMR Screening. J. Med. Chem. 2015, 58 (21), 8739– 8742,  DOI: 10.1021/acs.jmedchem.5b01114
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    Cala, O.; Guillière, F.; Krimm, I. NMR-Based Analysis of Protein–Ligand Interactions. Anal Bioanal Chem. 2014, 406 (4), 943– 956,  DOI: 10.1007/s00216-013-6931-0
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    NMR-based analysis of protein-ligand interactions
    Cala, Olivier; Guilliere, Florence; Krimm, Isabelle
    Analytical and Bioanalytical Chemistry (2014), 406 (4), 943-956CODEN: ABCNBP; ISSN:1618-2642. (Springer)
    A review. Physiol. processes are mainly controlled by intermol. recognition mechanisms involving protein-protein and protein-ligand (low mol. wt. mols.) interactions. One of the most important tools for probing these interactions is high-field soln. NMR through protein-obsd. and ligand-obsd. expts., where the protein receptor or the org. compds. are selectively detected. NMR binding expts. rely on comparison of NMR parameters of the free and bound states of the mols. Ligand-obsd. methods are not limited by the protein mol. size and therefore have great applicability for analyzing protein-ligand interactions. The use of these NMR techniques has considerably expanded in recent years, both in chem. biol. and in drug discovery. We review here 3 major ligand-obsd. NMR methods that depend on the nuclear Overhauser effect-transferred nuclear Overhauser effect spectroscopy, satn. transfer difference spectroscopy and water-ligand interactions obsd. via gradient spectroscopy expts. - with the aim of reporting recent developments and applications for the characterization of protein-ligand complexes, including affinity measurements and structural detn.
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    Ardenkjaer-Larsen, J. H. Hyperpolarized MR–What’s up Doc?. J. Magn. Reson. 2019, 306, 124– 127,  DOI: 10.1016/j.jmr.2019.07.017
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    Lee, Y.; Zeng, H.; Ruedisser, S.; Gossert, A. D.; Hilty, C. Nuclear Magnetic Resonance of Hyperpolarized Fluorine for Characterization of Protein–Ligand Interactions. J. Am. Chem. Soc. 2012, 134 (42), 17448– 17451,  DOI: 10.1021/ja308437h
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    Nuclear Magnetic Resonance of Hyperpolarized Fluorine for Characterization of Protein-Ligand Interactions
    Lee, Youngbok; Zeng, Haifeng; Ruedisser, Simon; Gossert, Alvar D.; Hilty, Christian
    Journal of the American Chemical Society (2012), 134 (42), 17448-17451CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Fluorine NMR spectroscopy is widely used for detection of protein-ligand interactions in drug discovery because of the simplicity of fluorine spectra combined with a relatively high likelihood for a drug mol. to include at least one fluorine atom. In general, an important limitation of NMR spectroscopy in drug discovery is its sensitivity, which results in the need for unphysiol. high protein concns. and large ligand:protein ratios. An enhancement in the 19F signal of several thousand fold by dynamic nuclear polarization allows for the detection of submicromolar concns. of fluorinated small mols. Techniques for exploiting this gain in signal to detect ligands in the strong-, intermediate-, and weak-binding regimes are presented. Similar to conventional NMR anal., dissocn. consts. are detd. However, the ability to use a low ligand concn. permits the detection of ligands in slow exchange that are not easily amenable to drug screening by traditional NMR methods. The relative speed and addnl. information gained may make the hyperpolarization-based approach an interesting alternative for use in drug discovery.
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    Lerche, M. H.; Meier, S.; Jensen, P. R.; Baumann, H.; Petersen, B. O.; Karlsson, M.; Duus, J. Ø.; Ardenkjær-Larsen, J. H. Study of Molecular Interactions with 13C DNP-NMR. J. Magn. Reson. 2010, 203 (1), 52– 56,  DOI: 10.1016/j.jmr.2009.11.020
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    Study of molecular interactions with 13C DNP-NMR
    Lerche, Mathilde H.; Meier, Sebastian; Jensen, Pernille R.; Baumann, Herbert; Petersen, Bent O.; Karlsson, Magnus; Duus, Jens O.; Ardenkjaer-Larsen, Jan H.
    Journal of Magnetic Resonance (2010), 203 (1), 52-56CODEN: JMARF3; ISSN:1090-7807. (Elsevier B.V.)
    NMR spectroscopy is an established, versatile technique for the detection of mol. interactions, even when these interactions are weak. Signal enhancement by several orders of magnitude through dynamic nuclear polarization alleviates several practical limitations of NMR-based interaction studies. This enhanced non-equil. polarization contributes sensitivity for the detection of mol. interactions in a single NMR transient. We show that direct 13C NMR ligand binding studies at natural isotopic abundance of 13C gets feasible in this way. Resultant screens are easy to interpret and can be performed at 13C concns. below μM. In addn. to such ligand-detected studies of mol. interaction, ligand binding can be assessed and quantified with enzymic assays that employ hyperpolarized substrates at varying enzyme inhibitor concns. The phys. labeling of nuclear spins by hyperpolarization thus provides the opportunity to devise fast novel in vitro expts. with low material requirement and without the need for synthetic modifications of target or ligands.
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    Kress, T.; Walrant, A.; Bodenhausen, G.; Kurzbach, D. Long-Lived States in Hyperpolarized Deuterated Methyl Groups Reveal Weak Binding of Small Molecules to Proteins. J. Phys. Chem. Lett. 2019, 10 (7), 1523– 1529,  DOI: 10.1021/acs.jpclett.9b00149
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    Long-Lived States in Hyperpolarized Deuterated Methyl Groups Reveal Weak Binding of Small Molecules to Proteins
    Kress, Thomas; Walrant, Astrid; Bodenhausen, Geoffrey; Kurzbach, Dennis
    Journal of Physical Chemistry Letters (2019), 10 (7), 1523-1529CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
    The authors introduce a method for the detection of weak interactions of small mols. such as metabolites or medicaments that contain deuterated Me groups with proteins in soln. The technique relies on long-lived imbalances of spin state populations, which are generated by dissoln. dynamic nuclear polarization (D-DNP) and feature lifetimes that depend on the frequency of internal rotation of deuterated Me groups. The authors demonstrate the technique for interactions between deuterated DMSO (DMSO-d6) and bovine serum albumin (BSA) or trypsin, where the Me group rotation is slowed down upon protein binding, which causes a marked redn. in the lifetime of the population imbalances.
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    Stern, Q.; Milani, J.; Vuichoud, B.; Bornet, A.; Gossert, A. D.; Bodenhausen, G.; Jannin, S. Hyperpolarized Water to Study Protein–Ligand Interactions. J. Phys. Chem. Lett. 2015, 6 (9), 1674– 1678,  DOI: 10.1021/acs.jpclett.5b00403
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    Hyperpolarized Water to Study Protein-Ligand Interactions
    Stern, Quentin; Milani, Jonas; Vuichoud, Basile; Bornet, Aurelien; Gossert, Alvar D.; Bodenhausen, Geoffrey; Jannin, Sami
    Journal of Physical Chemistry Letters (2015), 6 (9), 1674-1678CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
    The affinity between a chosen target protein and small mols. is a key aspect of drug discovery. Screening by popular NMR methods such as Water-LOGSY suffers from low sensitivity and from false positives caused by aggregated or denatured proteins. This work demonstrates that the sensitivity of Water-LOGSY can be greatly boosted by injecting hyperpolarized water into solns. of proteins and ligands. Ligand binding can be detected in a few seconds, whereas about 30 min is usually required without hyperpolarization. Hyperpolarized water also enhances proton signals of proteins at concns. below 20μM so that one can verify in a few seconds whether the proteins remain intact or have been denatured.
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    Shanaiah, N.; Desilva, M. A.; Nagana Gowda, G. A.; Raftery, M. A.; Hainline, B. E.; Raftery, D. Class Selection of Amino Acid Metabolites in Body Fluids Using Chemical Derivatization and Their Enhanced 13 C NMR. Proc. Natl. Acad. Sci. U.S.A. 2007, 104 (28), 11540– 11544,  DOI: 10.1073/pnas.0704449104
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    Class selection of amino acid metabolites in body fluids using chemical derivatization and their enhanced 13C NMR
    Shanaiah, Narasimhamurthy; Desilva, M. Aruni; Gowda, G. A. Nagana; Raftery, Ichael A.; Hainline, Bryan E.; Raftery, Daniel
    Proceedings of the National Academy of Sciences of the United States of America (2007), 104 (28), 11540-11544CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The authors report a chem. derivatization method that selects a class of metabolites from a complex mixt. and enhances their detection by 13C NMR. Acetylation of amines directly in aq. medium with 1,1'-13C2 acetic anhydride is a simple method that creates a high sensitivity and quant. label in complex biofluids with minimal sample pretreatment. Detection using either 1 D or 2D 13C NMR expts. produces highly resolved spectra with improved sensitivity. Expts. to identify and compare amino acids and related metabolites in normal human urine and serum samples as well as in urine from patients with the inborn errors of metab. tyrosinemia type II, argininosuccinic aciduria, homocystinuria, and phenylketonuria demonstrate the method. The use of metabolite derivatization and 13C NMR spectroscopy produces data suitable for metabolite profiling anal. of biofluids on a time scale that allows routine use. Extension of this approach to enhance the NMR detection of other classes of metabolites has also been accomplished. The improved detection of low-concn. metabolites shown here creates opportunities to improve the understanding of the biol. processes and develop improved disease detection methodologies.
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    Torres, F.; Bütikofer, M.; Stadler, G. R.; Renn, A.; Kadavath, H.; Bobrovs, R.; Jaudzems, K.; Riek, R. Ultrafast Fragment Screening Using Photo-Hyperpolarized (CIDNP) NMR. J. Am. Chem. Soc. 2023, 145 (22), 12066– 12080,  DOI: 10.1021/jacs.3c01392
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    Ultrafast Fragment Screening Using Photo-Hyperpolarized (CIDNP) NMR
    Torres, Felix; Butikofer, Matthias; Stadler, Gabriela R.; Renn, Alois; Kadavath, Harindranath; Bobrovs, Raitis; Jaudzems, Kristaps; Riek, Roland
    Journal of the American Chemical Society (2023), 145 (22), 12066-12080CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    While NMR (NMR) is regarded as a ref. in fragment-based drug design, its implementation in a high-throughput manner is limited by its lack of sensitivity resulting in long acquisition times and high micromolar sample concns. Several hyperpolarization approaches could, in principle, improve the sensitivity of NMR also in drug research. However, photochem. induced dynamic nuclear polarization (photo-CIDNP) is the only method that is directly applicable in aq. soln. and agile for scalable implementation using off-the-shelf hardware. With the use of photo-CIDNP, this work demonstrates the detection of weak binders in the millimolar affinity range using low micromolar concns. down to 5 μM of ligand and 2 μM of target, thereby exploiting the photo-CIDNP-induced polarization twice: (i) increasing the signal-to-noise by one to two orders in magnitude and (ii) polarization-only of the free non-bound mol. allowing identification of binding by polarization quenching, yielding another factor of hundred in time when compared with std. techniques. The interaction detection was performed with single-scan NMR expts. of a duration of 2 to 5 s. Taking advantage of the readiness of photo-CIDNP setup implementation, an automated flow-through platform was designed to screen samples at a screening rate of 1500 samples per day. Furthermore, a 212 compds. photo-CIDNP fragment library is presented, opening an avenue toward a comprehensive fragment-based screening method.
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    Elliott, S. J.; Stern, Q.; Ceillier, M.; El Daraï, T.; Cousin, S. F.; Cala, O.; Jannin, S. Practical Dissolution Dynamic Nuclear Polarization. Prog. Nucl. Magn. Reson. Spectrosc. 2021, 126–127, 59– 100,  DOI: 10.1016/j.pnmrs.2021.04.002
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    Practical dissolution dynamic nuclear polarization
    Elliott, Stuart J.; Stern, Quentin; Ceillier, Morgan; El Darai, Theo; Cousin, Samuel F.; Cala, Olivier; Jannin, Sami
    Progress in Nuclear Magnetic Resonance Spectroscopy (2021), 126-127 (), 59-100CODEN: PNMRAT; ISSN:0079-6565. (Elsevier B.V.)
    This review article intends to provide insightful advice for dissoln.-dynamic nuclear polarization in the form of a practical handbook. The goal is to aid research groups to effectively perform such expts. in their own labs. Previous review articles on this subject have covered a large no. of useful topics including instrumentation, experimentation, theory, etc. The topics to be addressed here will include tips for sample prepn. and for checking sample health; a checklist to correctly diagnose system faults and perform general maintenance; the necessary mech. requirements regarding sample dissoln.; and aids for accurate, fast and reliable polarization quantification. Herein, the challenges and limitations of each stage of a typical dissoln.-dynamic nuclear polarization expt. are presented, with the focus being on how to quickly and simply overcome some of the limitations often encountered in the lab.
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    Elliott, S. J.; Ceillier, M.; Cala, O.; Stern, Q.; Cousin, S. F.; Jannin, S. Simple and Cost-Effective Cross-Polarization Experiments under Dissolution-Dynamic Nuclear Polarization Conditions with a 3D-Printed 1H-13C Background-Free Radiofrequency Coil. J. Magn. Reson. Open 2022, 10–11, 100033  DOI: 10.1016/j.jmro.2022.100033
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    Bornet, A.; Pinon, A.; Jhajharia, A.; Baudin, M.; Ji, X.; Emsley, L.; Bodenhausen, G.; Ardenkjaer-Larsen, J. H.; Jannin, S. Microwave-Gated Dynamic Nuclear Polarization. Phys. Chem. Chem. Phys. 2016, 18 (44), 30530– 30535,  DOI: 10.1039/C6CP05587G
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    Microwave-gated dynamic nuclear polarization
    Bornet, Aurelien; Pinon, Arthur; Jhajharia, Aditya; Baudin, Mathieu; Ji, Xiao; Emsley, Lyndon; Bodenhausen, Geoffrey; Ardenkjaer-Larsen, Jan Henrik; Jannin, Sami
    Physical Chemistry Chemical Physics (2016), 18 (44), 30530-30535CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)
    Dissoln. dynamic nuclear polarization (D-DNP) has become a method of choice to enhance signals in NMR (NMR). Recently, we have proposed to combine cross-polarization (CP) with D-DNP to provide high polarization P(13C) in short build-up times. In this paper, we show that switching microwave irradn. off for a few hundreds of milliseconds prior to CP can significantly boost the efficiency. By implementing microwave gating, 13C polarizations on sodium [1-13C]acetate as high as 64% could be achieved with a polarization build-up time const. as short as 160 s. A polarization of P(13C) = 78% could even be reached for [13C]urea.
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    Bornet, A.; Milani, J.; Vuichoud, B.; Perez Linde, A. J.; Bodenhausen, G.; Jannin, S. Microwave Frequency Modulation to Enhance Dissolution Dynamic Nuclear Polarization. Chem. Phys. Lett. 2014, 602, 63– 67,  DOI: 10.1016/j.cplett.2014.04.013
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    Microwave frequency modulation to enhance Dissolution Dynamic Nuclear Polarization
    Bornet, Aurelien; Milani, Jonas; Vuichoud, Basile; Perez Linde, Angel J.; Bodenhausen, Geoffrey; Jannin, Sami
    Chemical Physics Letters (2014), 602 (), 63-67CODEN: CHPLBC; ISSN:0009-2614. (Elsevier B.V.)
    Hyperpolarization by Dissoln. Dynamic Nuclear Polarization is usually achieved by monochromatic microwave irradn. of the ESR spectrum of free radicals embedded in glasses at 1.2 K and 3.35 T. Hovav et al. (2014) have recently shown that by using frequency-modulated (rather than monochromatic) microwave irradn. one can improve DNP at 3.35 T in the temp. range 10-50 K. We show in this Letter that this is also true under Dissoln.-DNP conditions at 1.2 K and 6.7 T. We demonstrate the many virtues of using frequency-modulated microwave irradn.: higher polarizations, faster build-up rates, lower radical concns., less paramagnetic broadening, more efficient cross-polarization, and less crit. frequency adjustments.
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    Jannin, S.; Bornet, A.; Melzi, R.; Bodenhausen, G. High Field Dynamic Nuclear Polarization at 6.7T: Carbon-13 Polarization above 70% within 20min. Chem. Phys. Lett. 2012, 549, 99– 102,  DOI: 10.1016/j.cplett.2012.08.017
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    High field dynamic nuclear polarization at 6.7 T. Carbon-13 polarization above 70% within 20 min
    Jannin, Sami; Bornet, Aurelien; Melzi, Roberto; Bodenhausen, Geoffrey
    Chemical Physics Letters (2012), 549 (), 99-102CODEN: CHPLBC; ISSN:0009-2614. (Elsevier B.V.)
    In most applications of dissoln.-DNP, the polarization of nuclei with low gyromagnetic ratios such as 13C is enhanced directly by irradiating the ESR transitions of radicals with narrow ESR lines such as Trityl at low temps. T = 1.2 K in polarizing fields B0 ≤ 5 T. In a field B0 = 6.7 T at T = 1.2 K, DNP with TEMPO leads to a rapid build-up of proton polarization P(1H) = 91% with τDNP(1H) = 150 s. CP at low temps. yields a polarization P(1H → 13C) in excess of 70% within 20 min. After rapid dissoln. to room temp., this is 122 000 times larger than the Boltzmann polarization at 300 K and 6.7 T.
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    Ceillier, M.; Cala, O.; El Daraï, T.; Cousin, S. F.; Stern, Q.; Guibert, S.; Elliott, S. J.; Bornet, A.; Vuichoud, B.; Milani, J.; Pages, C.; Eshchenko, D.; Kempf, J. G.; Jose, C.; Lambert, S. A.; Jannin, S. An Automated System for Fast Transfer and Injection of Hyperpolarized Solutions. J. Magn. Reson. Open 2021, 8–9, 100017  DOI: 10.1016/j.jmro.2021.100017
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    Milani, J.; Vuichoud, B.; Bornet, A.; Miéville, P.; Mottier, R.; Jannin, S.; Bodenhausen, G. A Magnetic Tunnel to Shelter Hyperpolarized Fluids. Rev. Sci. Instrum. 2015, 86 (2), 024101  DOI: 10.1063/1.4908196
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    A magnetic tunnel to shelter hyperpolarized fluids
    Milani, Jonas; Vuichoud, Basile; Bornet, Aurelien; Mieville, Pascal; Mottier, Roger; Jannin, Sami; Bodenhausen, Geoffrey
    Review of Scientific Instruments (2015), 86 (2), 024101/1-024101/8CODEN: RSINAK; ISSN:0034-6748. (American Institute of Physics)
    To shield solns. carrying hyperpolarized nuclear magnetization from rapid relaxation during transfer through low fields, the transfer duct can be threaded through an array of permanent magnets. The advantages are illustrated for solns. contg. hyperpolarized 1H and 13C nuclei in a variety of mols. (c) 2015 American Institute of Physics.
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    Dey, A.; Charrier, B.; Martineau, E.; Deborde, C.; Gandriau, E.; Moing, A.; Jacob, D.; Eshchenko, D.; Schnell, M.; Melzi, R.; Kurzbach, D.; Ceillier, M.; Chappuis, Q.; Cousin, S. F.; Kempf, J. G.; Jannin, S.; Dumez, J.-N.; Giraudeau, P. Hyperpolarized NMR Metabolomics at Natural 13 C Abundance. Anal. Chem. 2020, 92 (22), 14867– 14871,  DOI: 10.1021/acs.analchem.0c03510
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    Hyperpolarized NMR Metabolomics at Natural 13C Abundance
    Dey, Arnab; Charrier, Benoit; Martineau, Estelle; Deborde, Catherine; Gandriau, Elodie; Moing, Annick; Jacob, Daniel; Eshchenko, Dmitry; Schnell, Marc; Melzi, Roberto; Kurzbach, Dennis; Ceillier, Morgan; Chappuis, Quentin; Cousin, Samuel F.; Kempf, James G.; Jannin, Sami; Dumez, Jean-Nicolas; Giraudeau, Patrick
    Analytical Chemistry (Washington, DC, United States) (2020), 92 (22), 14867-14871CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Metabolomics plays a pivotal role in systems biol., and NMR is a central tool with high precision and exceptional resoln. of chem. information. Most NMR metabolomic studies are based on 1H 1D spectroscopy, severely limited by peak overlap. 13C NMR benefits from a larger signal dispersion but is barely used in metabolomics due to ca. 6000-fold lower sensitivity. We introduce a new approach, based on hyperpolarized 13C NMR at natural abundance, that circumvents this limitation. A new untargeted NMR-based metabolomic workflow based on dissoln. dynamic nuclear polarization (d-DNP) for the first time enabled hyperpolarized natural abundance 13C metabolomics. Statistical anal. of resulting hyperpolarized 13C data distinguishes two groups of plant (tomato) exts. and highlights biomarkers, in full agreement with previous results on the same biol. model. We also optimize parameters of the semiautomated d-DNP system suitable for high-throughput studies.
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    Bornet, A.; Maucourt, M.; Deborde, C.; Jacob, D.; Milani, J.; Vuichoud, B.; Ji, X.; Dumez, J.-N.; Moing, A.; Bodenhausen, G.; Jannin, S.; Giraudeau, P. Highly Repeatable Dissolution Dynamic Nuclear Polarization for Heteronuclear NMR Metabolomics. Anal. Chem. 2016, 88 (12), 6179– 6183,  DOI: 10.1021/acs.analchem.6b01094
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    Highly Repeatable Dissolution Dynamic Nuclear Polarization for Heteronuclear NMR Metabolomics
    Bornet, Aurelien; Maucourt, Mickael; Deborde, Catherine; Jacob, Daniel; Milani, Jonas; Vuichoud, Basile; Ji, Xiao; Dumez, Jean-Nicolas; Moing, Annick; Bodenhausen, Geoffrey; Jannin, Sami; Giraudeau, Patrick
    Analytical Chemistry (Washington, DC, United States) (2016), 88 (12), 6179-6183CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    At natural 13C abundance, metabolomics based on heteronuclear NMR is limited by sensitivity. We have recently demonstrated how hyperpolarization by dissoln. dynamic nuclear polarization (D-DNP) assisted by cross-polarization (CP) provides a reliable way of enhancing the sensitivity of heteronuclear NMR in dil. mixts. of metabolites. In this Tech. Note, we evaluate the precision of this exptl. approach, a crit. point for applications to metabolomics. The higher the repeatability, the greater the likelihood that one can detect small biol. relevant differences between samples. The av. repeatability of our state-of-the-art D-DNP NMR equipment for samples of metabolomic relevance (20 mg dry wt. tomato exts.) is 3.6% for signals above the limit of quantification (LOQ) and 6.4% when all the signals above the limit of detection (LOD) are taken into account. This first report on the repeatability of D-DNP highlights the compatibility of the technique with the requirements of metabolomics and confirms its potential as an anal. tool for such applications.
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    Dumez, J.-N.; Milani, J.; Vuichoud, B.; Bornet, A.; Lalande-Martin, J.; Tea, I.; Yon, M.; Maucourt, M.; Deborde, C.; Moing, A.; Frydman, L.; Bodenhausen, G.; Jannin, S.; Giraudeau, P. Hyperpolarized NMR of Plant and Cancer Cell Extracts at Natural Abundance. Analyst 2015, 140 (17), 5860– 5863,  DOI: 10.1039/C5AN01203A
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    Hyperpolarized NMR of plant and cancer cell extracts at natural abundance
    Dumez, Jean-Nicolas; Milani, Jonas; Vuichoud, Basile; Bornet, Aurelien; Lalande-Martin, Julie; Tea, Illa; Yon, Maxime; Maucourt, Mickael; Deborde, Catherine; Moing, Annick; Frydman, Lucio; Bodenhausen, Geoffrey; Jannin, Sami; Giraudeau, Patrick
    Analyst (Cambridge, United Kingdom) (2015), 140 (17), 5860-5863CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)
    Natural abundance 13C NMR spectra of biol. exts. are recorded in a single scan provided that the samples are hyperpolarized by dissoln. dynamic nuclear polarization combined with cross polarization. Heteronuclear 2D correlation spectra of hyperpolarized breast cancer cell exts. can also be obtained in a single scan. Hyperpolarized NMR of exts. opens many perspectives for metabolomics.
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    Katsikis, S.; Marin-Montesinos, I.; Ludwig, C.; Günther, U. L. Detecting Acetylated Aminoacids in Blood Serum Using Hyperpolarized 13C-1Η-2D-NMR. J. Magn. Reson. 2019, 305, 175– 179,  DOI: 10.1016/j.jmr.2019.07.003
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    Wilson, D. M.; Hurd, R. E.; Keshari, K.; Van Criekinge, M.; Chen, A. P.; Nelson, S. J.; Vigneron, D. B.; Kurhanewicz, J. Generation of Hyperpolarized Substrates by Secondary Labeling with [1,1- 13 C] Acetic Anhydride. Proc. Natl. Acad. Sci. U.S.A. 2009, 106 (14), 5503– 5507,  DOI: 10.1073/pnas.0810190106
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  • Abstract

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    Figure 1

    Figure 1. (a) Chemical reaction for the labeling of a ligand containing an -NH2 group as described in the experimental section. (b) Relevant portions of the experimental 13C NMR spectra for the 164 mM tagged ligand of N-Acetyl [1-13C]-6-amino-2-naphthoic acid (N–Ac-L30, red) and N-Acetyl [1-13C]-glycine (Ac-L08, blue) dissolved in a deuterated phosphate buffer and acquired at 20 MHz (1.88 T) and 298 K.

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    Figure 2

    Figure 2. Relevant portions of the experimental STD and 1H NMR spectra of 2 mM Ac-L08 with 5 μM HSA in 0.5% DMSO-d6, 10% D2O, and 89.5% phosphate buffer pH 8.5 (v:v:v) (a, b) and 2 mM Ac-L30 with 5 μM HSA in 0.5% DMSO-d6, 10% D2O, and 89.5% phosphate buffer pH 8.5 (v:v:v) (c, d) acquired at 600 MHz (14.1 T) and 298 K with 16 and 512 scans, respectively. Assignments are indicated for the two compounds (1′-4′ for Ac-L08 and 1–8 for Ac-L30). * indicates the signal corresponding to DMSO-d6. The dashed boxes represent the area of interest for the STD signal.

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    Figure 3

    Figure 3. Experimental decays for the hyperpolarized 13C NMR signal of 600 μM Ac-L30 and 600 μM Ac-L08 dissolved in deuterated-phosphate buffer pH 8.5 acquired at a 20 MHz 13C frequency (1.88 T) and ∼303 K with and without mixing with a protein solution (40 μM HSA) after 2 s transfer to the NMR spectrometer. (a) 13C T1 determination for Ac-L30 without and with HSA. (b) 13C T1 determination for Ac-L08 without and with HSA. For both ligands, hollow circles represent experimental data (integrals of 1D 13C NMR spectra measured with 5 or 2.5 s intervals and pulse flip angles of 15°), and the solid lines represent fits of the experimental data with a monoexponential decay function: P0·exp(−t/T1) where P0 is a fitting constant.

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      Elliott, S. J.; Stern, Q.; Ceillier, M.; El Daraï, T.; Cousin, S. F.; Cala, O.; Jannin, S. Practical Dissolution Dynamic Nuclear Polarization. Prog. Nucl. Magn. Reson. Spectrosc. 2021, 126–127, 59– 100,  DOI: 10.1016/j.pnmrs.2021.04.002
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      Elliott, Stuart J.; Stern, Quentin; Ceillier, Morgan; El Darai, Theo; Cousin, Samuel F.; Cala, Olivier; Jannin, Sami
      Progress in Nuclear Magnetic Resonance Spectroscopy (2021), 126-127 (), 59-100CODEN: PNMRAT; ISSN:0079-6565. (Elsevier B.V.)
      This review article intends to provide insightful advice for dissoln.-dynamic nuclear polarization in the form of a practical handbook. The goal is to aid research groups to effectively perform such expts. in their own labs. Previous review articles on this subject have covered a large no. of useful topics including instrumentation, experimentation, theory, etc. The topics to be addressed here will include tips for sample prepn. and for checking sample health; a checklist to correctly diagnose system faults and perform general maintenance; the necessary mech. requirements regarding sample dissoln.; and aids for accurate, fast and reliable polarization quantification. Herein, the challenges and limitations of each stage of a typical dissoln.-dynamic nuclear polarization expt. are presented, with the focus being on how to quickly and simply overcome some of the limitations often encountered in the lab.
    19. 19
      Elliott, S. J.; Ceillier, M.; Cala, O.; Stern, Q.; Cousin, S. F.; Jannin, S. Simple and Cost-Effective Cross-Polarization Experiments under Dissolution-Dynamic Nuclear Polarization Conditions with a 3D-Printed 1H-13C Background-Free Radiofrequency Coil. J. Magn. Reson. Open 2022, 10–11, 100033  DOI: 10.1016/j.jmro.2022.100033
      There is no corresponding record for this reference.
    20. 20
      Bornet, A.; Pinon, A.; Jhajharia, A.; Baudin, M.; Ji, X.; Emsley, L.; Bodenhausen, G.; Ardenkjaer-Larsen, J. H.; Jannin, S. Microwave-Gated Dynamic Nuclear Polarization. Phys. Chem. Chem. Phys. 2016, 18 (44), 30530– 30535,  DOI: 10.1039/C6CP05587G
      20
      Microwave-gated dynamic nuclear polarization
      Bornet, Aurelien; Pinon, Arthur; Jhajharia, Aditya; Baudin, Mathieu; Ji, Xiao; Emsley, Lyndon; Bodenhausen, Geoffrey; Ardenkjaer-Larsen, Jan Henrik; Jannin, Sami
      Physical Chemistry Chemical Physics (2016), 18 (44), 30530-30535CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)
      Dissoln. dynamic nuclear polarization (D-DNP) has become a method of choice to enhance signals in NMR (NMR). Recently, we have proposed to combine cross-polarization (CP) with D-DNP to provide high polarization P(13C) in short build-up times. In this paper, we show that switching microwave irradn. off for a few hundreds of milliseconds prior to CP can significantly boost the efficiency. By implementing microwave gating, 13C polarizations on sodium [1-13C]acetate as high as 64% could be achieved with a polarization build-up time const. as short as 160 s. A polarization of P(13C) = 78% could even be reached for [13C]urea.
    21. 21
      Bornet, A.; Milani, J.; Vuichoud, B.; Perez Linde, A. J.; Bodenhausen, G.; Jannin, S. Microwave Frequency Modulation to Enhance Dissolution Dynamic Nuclear Polarization. Chem. Phys. Lett. 2014, 602, 63– 67,  DOI: 10.1016/j.cplett.2014.04.013
      21
      Microwave frequency modulation to enhance Dissolution Dynamic Nuclear Polarization
      Bornet, Aurelien; Milani, Jonas; Vuichoud, Basile; Perez Linde, Angel J.; Bodenhausen, Geoffrey; Jannin, Sami
      Chemical Physics Letters (2014), 602 (), 63-67CODEN: CHPLBC; ISSN:0009-2614. (Elsevier B.V.)
      Hyperpolarization by Dissoln. Dynamic Nuclear Polarization is usually achieved by monochromatic microwave irradn. of the ESR spectrum of free radicals embedded in glasses at 1.2 K and 3.35 T. Hovav et al. (2014) have recently shown that by using frequency-modulated (rather than monochromatic) microwave irradn. one can improve DNP at 3.35 T in the temp. range 10-50 K. We show in this Letter that this is also true under Dissoln.-DNP conditions at 1.2 K and 6.7 T. We demonstrate the many virtues of using frequency-modulated microwave irradn.: higher polarizations, faster build-up rates, lower radical concns., less paramagnetic broadening, more efficient cross-polarization, and less crit. frequency adjustments.
    22. 22
      Jannin, S.; Bornet, A.; Melzi, R.; Bodenhausen, G. High Field Dynamic Nuclear Polarization at 6.7T: Carbon-13 Polarization above 70% within 20min. Chem. Phys. Lett. 2012, 549, 99– 102,  DOI: 10.1016/j.cplett.2012.08.017
      22
      High field dynamic nuclear polarization at 6.7 T. Carbon-13 polarization above 70% within 20 min
      Jannin, Sami; Bornet, Aurelien; Melzi, Roberto; Bodenhausen, Geoffrey
      Chemical Physics Letters (2012), 549 (), 99-102CODEN: CHPLBC; ISSN:0009-2614. (Elsevier B.V.)
      In most applications of dissoln.-DNP, the polarization of nuclei with low gyromagnetic ratios such as 13C is enhanced directly by irradiating the ESR transitions of radicals with narrow ESR lines such as Trityl at low temps. T = 1.2 K in polarizing fields B0 ≤ 5 T. In a field B0 = 6.7 T at T = 1.2 K, DNP with TEMPO leads to a rapid build-up of proton polarization P(1H) = 91% with τDNP(1H) = 150 s. CP at low temps. yields a polarization P(1H → 13C) in excess of 70% within 20 min. After rapid dissoln. to room temp., this is 122 000 times larger than the Boltzmann polarization at 300 K and 6.7 T.
    23. 23
      Ceillier, M.; Cala, O.; El Daraï, T.; Cousin, S. F.; Stern, Q.; Guibert, S.; Elliott, S. J.; Bornet, A.; Vuichoud, B.; Milani, J.; Pages, C.; Eshchenko, D.; Kempf, J. G.; Jose, C.; Lambert, S. A.; Jannin, S. An Automated System for Fast Transfer and Injection of Hyperpolarized Solutions. J. Magn. Reson. Open 2021, 8–9, 100017  DOI: 10.1016/j.jmro.2021.100017
      There is no corresponding record for this reference.
    24. 24
      Milani, J.; Vuichoud, B.; Bornet, A.; Miéville, P.; Mottier, R.; Jannin, S.; Bodenhausen, G. A Magnetic Tunnel to Shelter Hyperpolarized Fluids. Rev. Sci. Instrum. 2015, 86 (2), 024101  DOI: 10.1063/1.4908196
      24
      A magnetic tunnel to shelter hyperpolarized fluids
      Milani, Jonas; Vuichoud, Basile; Bornet, Aurelien; Mieville, Pascal; Mottier, Roger; Jannin, Sami; Bodenhausen, Geoffrey
      Review of Scientific Instruments (2015), 86 (2), 024101/1-024101/8CODEN: RSINAK; ISSN:0034-6748. (American Institute of Physics)
      To shield solns. carrying hyperpolarized nuclear magnetization from rapid relaxation during transfer through low fields, the transfer duct can be threaded through an array of permanent magnets. The advantages are illustrated for solns. contg. hyperpolarized 1H and 13C nuclei in a variety of mols. (c) 2015 American Institute of Physics.
    25. 25
      Dey, A.; Charrier, B.; Martineau, E.; Deborde, C.; Gandriau, E.; Moing, A.; Jacob, D.; Eshchenko, D.; Schnell, M.; Melzi, R.; Kurzbach, D.; Ceillier, M.; Chappuis, Q.; Cousin, S. F.; Kempf, J. G.; Jannin, S.; Dumez, J.-N.; Giraudeau, P. Hyperpolarized NMR Metabolomics at Natural 13 C Abundance. Anal. Chem. 2020, 92 (22), 14867– 14871,  DOI: 10.1021/acs.analchem.0c03510
      25
      Hyperpolarized NMR Metabolomics at Natural 13C Abundance
      Dey, Arnab; Charrier, Benoit; Martineau, Estelle; Deborde, Catherine; Gandriau, Elodie; Moing, Annick; Jacob, Daniel; Eshchenko, Dmitry; Schnell, Marc; Melzi, Roberto; Kurzbach, Dennis; Ceillier, Morgan; Chappuis, Quentin; Cousin, Samuel F.; Kempf, James G.; Jannin, Sami; Dumez, Jean-Nicolas; Giraudeau, Patrick
      Analytical Chemistry (Washington, DC, United States) (2020), 92 (22), 14867-14871CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Metabolomics plays a pivotal role in systems biol., and NMR is a central tool with high precision and exceptional resoln. of chem. information. Most NMR metabolomic studies are based on 1H 1D spectroscopy, severely limited by peak overlap. 13C NMR benefits from a larger signal dispersion but is barely used in metabolomics due to ca. 6000-fold lower sensitivity. We introduce a new approach, based on hyperpolarized 13C NMR at natural abundance, that circumvents this limitation. A new untargeted NMR-based metabolomic workflow based on dissoln. dynamic nuclear polarization (d-DNP) for the first time enabled hyperpolarized natural abundance 13C metabolomics. Statistical anal. of resulting hyperpolarized 13C data distinguishes two groups of plant (tomato) exts. and highlights biomarkers, in full agreement with previous results on the same biol. model. We also optimize parameters of the semiautomated d-DNP system suitable for high-throughput studies.
    26. 26
      Bornet, A.; Maucourt, M.; Deborde, C.; Jacob, D.; Milani, J.; Vuichoud, B.; Ji, X.; Dumez, J.-N.; Moing, A.; Bodenhausen, G.; Jannin, S.; Giraudeau, P. Highly Repeatable Dissolution Dynamic Nuclear Polarization for Heteronuclear NMR Metabolomics. Anal. Chem. 2016, 88 (12), 6179– 6183,  DOI: 10.1021/acs.analchem.6b01094
      26
      Highly Repeatable Dissolution Dynamic Nuclear Polarization for Heteronuclear NMR Metabolomics
      Bornet, Aurelien; Maucourt, Mickael; Deborde, Catherine; Jacob, Daniel; Milani, Jonas; Vuichoud, Basile; Ji, Xiao; Dumez, Jean-Nicolas; Moing, Annick; Bodenhausen, Geoffrey; Jannin, Sami; Giraudeau, Patrick
      Analytical Chemistry (Washington, DC, United States) (2016), 88 (12), 6179-6183CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      At natural 13C abundance, metabolomics based on heteronuclear NMR is limited by sensitivity. We have recently demonstrated how hyperpolarization by dissoln. dynamic nuclear polarization (D-DNP) assisted by cross-polarization (CP) provides a reliable way of enhancing the sensitivity of heteronuclear NMR in dil. mixts. of metabolites. In this Tech. Note, we evaluate the precision of this exptl. approach, a crit. point for applications to metabolomics. The higher the repeatability, the greater the likelihood that one can detect small biol. relevant differences between samples. The av. repeatability of our state-of-the-art D-DNP NMR equipment for samples of metabolomic relevance (20 mg dry wt. tomato exts.) is 3.6% for signals above the limit of quantification (LOQ) and 6.4% when all the signals above the limit of detection (LOD) are taken into account. This first report on the repeatability of D-DNP highlights the compatibility of the technique with the requirements of metabolomics and confirms its potential as an anal. tool for such applications.
    27. 27
      Dumez, J.-N.; Milani, J.; Vuichoud, B.; Bornet, A.; Lalande-Martin, J.; Tea, I.; Yon, M.; Maucourt, M.; Deborde, C.; Moing, A.; Frydman, L.; Bodenhausen, G.; Jannin, S.; Giraudeau, P. Hyperpolarized NMR of Plant and Cancer Cell Extracts at Natural Abundance. Analyst 2015, 140 (17), 5860– 5863,  DOI: 10.1039/C5AN01203A
      27
      Hyperpolarized NMR of plant and cancer cell extracts at natural abundance
      Dumez, Jean-Nicolas; Milani, Jonas; Vuichoud, Basile; Bornet, Aurelien; Lalande-Martin, Julie; Tea, Illa; Yon, Maxime; Maucourt, Mickael; Deborde, Catherine; Moing, Annick; Frydman, Lucio; Bodenhausen, Geoffrey; Jannin, Sami; Giraudeau, Patrick
      Analyst (Cambridge, United Kingdom) (2015), 140 (17), 5860-5863CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)
      Natural abundance 13C NMR spectra of biol. exts. are recorded in a single scan provided that the samples are hyperpolarized by dissoln. dynamic nuclear polarization combined with cross polarization. Heteronuclear 2D correlation spectra of hyperpolarized breast cancer cell exts. can also be obtained in a single scan. Hyperpolarized NMR of exts. opens many perspectives for metabolomics.
    28. 28
      Katsikis, S.; Marin-Montesinos, I.; Ludwig, C.; Günther, U. L. Detecting Acetylated Aminoacids in Blood Serum Using Hyperpolarized 13C-1Η-2D-NMR. J. Magn. Reson. 2019, 305, 175– 179,  DOI: 10.1016/j.jmr.2019.07.003
      There is no corresponding record for this reference.
    29. 29
      Wilson, D. M.; Hurd, R. E.; Keshari, K.; Van Criekinge, M.; Chen, A. P.; Nelson, S. J.; Vigneron, D. B.; Kurhanewicz, J. Generation of Hyperpolarized Substrates by Secondary Labeling with [1,1- 13 C] Acetic Anhydride. Proc. Natl. Acad. Sci. U.S.A. 2009, 106 (14), 5503– 5507,  DOI: 10.1073/pnas.0810190106
      There is no corresponding record for this reference.
    30. 30
      Campos-Olivas, R. NMR Screening and Hit Validation in Fragment Based Drug Discovery. CTMC 2011, 11 (1), 43– 67,  DOI: 10.2174/156802611793611887
      There is no corresponding record for this reference.
    31. 31
      Dalvit, C.; Flocco, M.; Knapp, S.; Mostardini, M.; Perego, R.; Stockman, B. J.; Veronesi, M.; Varasi, M. High-Throughput NMR-Based Screening with Competition Binding Experiments. J. Am. Chem. Soc. 2002, 124 (26), 7702– 7709,  DOI: 10.1021/ja020174b
      31
      High-throughput NMR-based screening with competition binding experiments
      Dalvit, Claudio; Flocco, Maria; Knapp, Stefan; Mostardini, Marina; Perego, Rita; Stockman, Brian J.; Veronesi, Marina; Varasi, Mario
      Journal of the American Chemical Society (2002), 124 (26), 7702-7709CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The Achilles heel of ligand-based NMR screening methods is their failure to detect high-affinity ligands and mols. that bind covalently to the receptor. We have developed a novel approach for performing high-throughput screening with NMR spectroscopy that overcomes this limitation. The method also permits detection of potential high-affinity mols. that are only marginally sol., thus significantly enlarging the diversity of compds. amenable to NMR screening. The techniques developed utilize transverse and/or selective longitudinal relaxation parameters in combination with competition binding expts. Math. expressions are derived for proper setup of the NMR expts. and for extg. an approx. value of the binding const. for the identified ligand from a single-point measurement. With this approach it is possible to screen thousands of compds. in a short period of time against protein or DNA and RNA fragments. The methodol. can also be applied for screening plant and fungi exts.
    32. 32
      Ditzler, R. A. J.; Zhukhovitskiy, A. V. Sigmatropic Rearrangements of Polymer Backbones: Vinyl Polymers from Polyesters in One Step. J. Am. Chem. Soc. 2021, 143 (48), 20326– 20331,  DOI: 10.1021/jacs.1c09657
      32
      Sigmatropic Rearrangements of Polymer Backbones: Vinyl Polymers from Polyesters in One Step
      Ditzler, Rachael A. J.; Zhukhovitskiy, Aleksandr V.
      Journal of the American Chemical Society (2021), 143 (48), 20326-20331CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Polymer modification is a fundamental scientific challenge, as a means of both upcycling plastics and extg. a stimulus response from them. To date, the overwhelming majority of polymer modifications has focused on the polymer periphery. Herein, we demonstrate nearly quant., scission-free modification of polymer backbones, namely, a metamorphosis of polyesters into vinyl polymers resembling commodity materials via the Ireland-Claisen sigmatropic rearrangement. The glass transition temp. (Tg) and thermal stability of the polyesters undergo dramatic changes post-transformation. Beyond polymer modification, our work advances the application of retrosynthetic anal. in polymer synthesis; the nontraditional prodn. of vinyl polymers from lactones opens the door to a slew of previously inaccessible materials.
    33. 33
      Dherange, B. D.; Kelly, P. Q.; Liles, J. P.; Sigman, M. S.; Levin, M. D. Carbon Atom Insertion into Pyrroles and Indoles Promoted by Chlorodiazirines. J. Am. Chem. Soc. 2021, 143 (30), 11337– 11344,  DOI: 10.1021/jacs.1c06287
      33
      Carbon Atom Insertion into Pyrroles and Indoles Promoted by Chlorodiazirines
      Dherange, Balu D.; Kelly, Patrick Q.; Liles, Jordan P.; Sigman, Matthew S.; Levin, Mark D.
      Journal of the American Chemical Society (2021), 143 (30), 11337-11344CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Herein, we report a reaction that selectively generates 3-arylpyridine and quinoline motifs by inserting aryl carbynyl cation equiv. into pyrrole and indole cores, resp. By employing α-chlorodiazirines as thermal precursors to the corresponding chlorocarbenes, the traditional haloform-based protocol central to the parent Ciamician-Dennstedt rearrangement can be modified to directly afford 3-(hetero)arylpyridines and quinolines. Chlorodiazirines are conveniently prepd. in a single step by oxidn. of com. available amidinium salts. Selectivity as a function of pyrrole substitution pattern was examd., and a predictive model based on steric effects is put forward, with DFT calcns. supporting a selectivity-detg. cyclopropanation step. Computations surprisingly indicate that the stereochem. of cyclopropanation is of little consequence to the subsequent electrocyclic ring opening that forges the pyridine core, due to a compensatory homoarom. stabilization that counterbalances orbital-controlled torquoselectivity effects. The utility of this skeletal transform is further demonstrated through the prepn. of quinolinophanes and the skeletal editing of pharmaceutically relevant pyrroles.
    34. 34
      Milani, J.; Vuichoud, B.; Bornet, A.; Melzi, R.; Jannin, S.; Bodenhausen, G. Hyperpolarization of Nitrogen-15 Nuclei by Cross Polarization and Dissolution Dynamic Nuclear Polarization. Rev. Sci. Instrum. 2017, 88 (1), 015109  DOI: 10.1063/1.4973777
      There is no corresponding record for this reference.
    35. 35
      Zhang, C.; Xu, L.; Huang, Q.; Wang, Y.; Tang, H. Detecting Submicromolar Analytes in Mixtures with a 5 min Acquisition on 600 MHz NMR Spectrometers. J. Am. Chem. Soc. 2023, 145 (47), 25513– 25517,  DOI: 10.1021/jacs.3c07861
      There is no corresponding record for this reference.
    36. 36
      Reisenbauer, J. C.; Green, O.; Franchino, A.; Finkelstein, P.; Morandi, B. Late-Stage Diversification of Indole Skeletons through Nitrogen Atom Insertion. Science 2022, 377 (6610), 1104– 1109,  DOI: 10.1126/science.add1383
      36
      Late-stage diversification of indole skeletons through nitrogen atom insertion
      Reisenbauer, Julia C.; Green, Ori; Franchino, Allegra; Finkelstein, Patrick; Morandi, Bill
      Science (Washington, DC, United States) (2022), 377 (6610), 1104-1109CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
      Herein, the skeletal editing of indoles through nitrogen atom insertion, accessing the corresponding quinazoline or quinoxaline bioisosteres by trapping of an electrophilic nitrene species generated from ammonium carbamate and hypervalent iodine was reported. This reactivity relied on the strategic use of a silyl group as a labile protecting group that could facilitate subsequent product release. The utility of this highly functional group-compatible methodol. in the context of late-stage skeletal editing of several com. drugs was demonstrated.
    37. 37
      Woo, J.; Stein, C.; Christian, A. H.; Levin, M. D. Carbon-to-Nitrogen Single-Atom Transmutation of Azaarenes. Nature 2023, 623 (7985), 77– 82,  DOI: 10.1038/s41586-023-06613-4
      37
      Carbon-to-nitrogen single-atom transmutation of azaarenes
      Woo, Jisoo; Stein, Colin; Christian, Alec H.; Levin, Mark D.
      Nature (London, United Kingdom) (2023), 623 (7985), 77-82CODEN: NATUAS; ISSN:1476-4687. (Nature Portfolio)
      When searching for the ideal mol. to fill a particular functional role (for example, a medicine), the difference between success and failure can often come down to a single atom1. Replacing an arom. carbon atom with a nitrogen atom would be enabling in the discovery of potential medicines2, but only indirect means exist to make such C-to-N transmutations, typically by parallel synthesis3. Here, authors report a transformation that enables the direct conversion of a heteroarom. carbon atom into a nitrogen atom, turning quinolines into quinazolines. Oxidative restructuring of the parent azaarene gives a ring-opened intermediate bearing electrophilic sites primed for ring reclosure and expulsion of a carbon-based leaving group. Such a 'sticky end' approach subverts existing atom insertion-deletion approaches and as a result avoids skeleton-rotation and substituent-perturbation pitfalls common in stepwise skeletal editing. Authors show a broad scope of quinolines and related azaarenes, all of which can be converted into the corresponding quinazolines by replacement of the C3 carbon with a nitrogen atom. Mechanistic expts. support the crit. role of the activated intermediate and indicate a more general strategy for the development of C-to-N transmutation reactions.

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    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.4c05101.

    • Nuclear spin–lattice relaxation times (T1) measurements (PDF)

    Accession Codes

    The MATLAB codes used to analyze the data can be downloaded from Zenodo 10.5281/zenodo.10995062.


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