Multidimensional Spectroscopy of Nuclear Spin Clusters in Diamond.

Optically active spin defects in solids offer promising platforms to investigate nuclear spin clusters with high sensitivity and atomic-site resolution. To leverage near-surface defects for molecular structure analysis in chemical and biological contexts using nuclear magnetic resonance (NMR), further advances in spectroscopic characterization of nuclear environments are essential. Here, we report Fourier spectroscopy techniques to improve localization and mapping of the test bed ^{13}C nuclear spin environment of individual, shallow nitrogen-vacancy centers at room temperature. We use multidimensional spectroscopy, well-known from classical NMR, in combination with weak measurements of single-nuclear-spin precession. We demonstrate two examples of multidimensional NMR: (i) improved nuclear spin localization by separate encoding of the two hyperfine components along spectral dimensions and (ii) spectral editing of nuclear-spin pairs, including measurement of internuclear coupling constants. Our work adds important tools for the spectroscopic analysis of molecular structures by single-spin probes.

Fragment Screening and Fast Micromolar Detection on a Benchtop NMR Spectrometer Boosted by Photoinduced Hyperpolarization.

Fragment-based drug design is a well-established strategy for rational drug design, with nuclear magnetic resonance (NMR) on high-field spectrometers as the method of reference for screening and hit validation. However, high-field NMR spectrometers are not only expensive, but require specialized maintenance, dedicated space, and depend on liquid helium cooling which became critical over the recurring global helium shortages. We propose an alternative to high-field NMR screening by applying the recently developed approach of fragment screening by photoinduced hyperpolarized NMR on a cryogen-free 80 MHz benchtop NMR spectrometer yielding signal enhancements of up to three orders in magnitude. It is demonstrated that it is possible to discover new hits and kick-off drug design using a benchtop NMR spectrometer at low micromolar concentrations of both protein and ligand. The approach presented performs at higher speed than state-of-the-art high-field NMR approaches while exhibiting a limit of detection in the nanomolar range. Photoinduced hyperpolarization is known to be inexpensive and simple to be implemented, which aligns greatly with the philosophy of benchtop NMR spectrometers. These findings open the way for the use of benchtop NMR in near-physiological conditions for drug design and further life science applications.

High T-cell infiltration in tumor tissue and younger age predict the response to pembrolizumab in recurrent urothelial cancer.

Targeting the programmed cell death-1 signaling pathway has been approved for the anti-cancer therapy in several cancers including urothelial cancer. To determine predictive factors of the responsiveness to pembrolizumab in urothelial cancer patients, a retrospective study that used clinical information and paraffin-embedded samples obtained from patients diagnosed with urothelial cancer between 2015 and 2020 were performed. Seventeen patients who underwent total cystectomy or nephroureterectomy of the primary lesion and were treated with pembrolizumab for chemo-resistant disease were enrolled, and immunohistochemical analysis was performed. A key difference in the characteristics between the non-responder group and the responder group was the age of the patients (74 vs. 63 years, p = 0.0194). Although there was no statistically significant difference, the histological subtype with sarcomatoid and micropapillary components was only seen in the non-responder group, and squamous differentiation and lymph node metastasis were only seen in cases with a complete response. In the results of immunohistochemistry, the density of CD8-positive T-cells and Tregs was significantly increased in the responder group than in the non-responder group. In conclusion, younger age and a high number of tumor-infiltrating lymphocytes were predictive factors of a good response to immune checkpoint inhibitors, although further studies with more enrolled patients are necessary.

Water accessibility in a membrane-inserting peptide comparing Overhauser DNP and pulse EPR methods.

Water accessibility is a key parameter for the understanding of the structure of biomolecules, especially membrane proteins. Several experimental techniques based on the combination of electron paramagnetic resonance (EPR) spectroscopy with site-directed spin labeling are currently available. Among those, we compare relaxation time measurements and electron spin echo envelope modulation (ESEEM) experiments using pulse EPR with Overhauser dynamic nuclear polarization (DNP) at X-band frequency and a magnetic field of 0.33 T. Overhauser DNP transfers the electron spin polarization to nuclear spins via cross-relaxation. The change in the intensity of the (1)H NMR spectrum of H2O at a Larmor frequency of 14 MHz under a continuous-wave microwave irradiation of the nitroxide spin label contains information on the water accessibility of the labeled site. As a model system for a membrane protein, we use the hydrophobic α-helical peptide WALP23 in unilamellar liposomes of DOPC. Water accessibility measurements with all techniques are conducted for eight peptides with different spin label positions and low radical concentrations (10-20 µM). Consistently in all experiments, the water accessibility appears to be very low, even for labels positioned near the end of the helix. The best profile is obtained by Overhauser DNP, which is the only technique that succeeds in discriminating neighboring positions in WALP23. Since the concentration of the spin-labeled peptides varied, we normalized the DNP parameter ϵ, being the relative change of the NMR intensity, by the electron spin concentration, which was determined from a continuous-wave EPR spectrum.

Indium Oxide as a Superior Catalyst for Methanol Synthesis by CO2 Hydrogenation.

Methanol synthesis by CO2 hydrogenation is attractive in view of avoiding the environmental implications associated with the production of the traditional syngas feedstock and mitigating global warming. However, there still is a lack of efficient catalysts for such alternative processes. Herein, we unveil the high activity, 100 % selectivity, and remarkable stability for 1000 h on stream of In2 O3 supported on ZrO2 under industrially relevant conditions. This strongly contrasts to the benchmark Cu-ZnO-Al2 O3 catalyst, which is unselective and experiences rapid deactivation. In-depth characterization of the In2 O3 -based materials points towards a mechanism rooted in the creation and annihilation of oxygen vacancies as active sites, whose amount can be modulated in situ by co-feeding CO and boosted through electronic interactions with the zirconia carrier. These results constitute a promising basis for the design of a prospective technology for sustainable methanol production.

Copper ESEEM and HYSCORE through ultra-wideband chirp EPR spectroscopy.

The main limitation of pulse electron paramagnetic resonance (EPR) spectroscopy is its narrow excitation bandwidth. Ultra-wideband (UWB) excitation with frequency-swept chirp pulses over several hundreds of megahertz overcomes this drawback. This allows to excite electron spin echo envelope modulation (ESEEM) from paramagnetic copper centers in crystals, whereas up to now, only ESEEM of ligand nuclei like protons or nitrogens at lower frequencies could be detected. ESEEM spectra are recorded as two-dimensional correlation experiments, since the full digitization of the electron spin echo provides an additional Fourier transform EPR dimension. Thus, UWB hyperfine-sublevel correlation experiments generate a novel three-dimensional EPR-correlated nuclear modulation spectrum.

How to tickle spins with a fourier transform NMR spectrometer.

In the long bygone days of continuous-wave nuclear magnetic resonance (NMR) spectroscopy, a selected transition within a multiplet of a high-resolution spectrum could be irradiated by a highly selective continuous-wave (CW) radio-frequency (rf) field with a very weak amplitude ω(2)/(2π)≤J. This causes splittings of connected transitions, allowing one to map the connectivities of all transitions within the energy-level diagram of the spin system. Such "tickling" experiments stimulated the invention of two-dimensional spectroscopy, but seem to have been forgotten for nearly 50 years. We show that tickling can readily be achieved in homonuclear systems with Fourier transform spectrometers by applying short pulses in the intervals between the sampling points. Extensions to heteronuclear systems are even more straightforward since they can be carried out using very weak CW rf fields.

Nanoscale quantum sensing with Nitrogen-Vacancy centers in nanodiamonds - A magnetic resonance perspective.

Nanodiamonds containing fluorescent Nitrogen-Vacancy (NV) centers are the smallest single particles, of which a magnetic resonance spectrum can be recorded at room temperature using optically-detected magnetic resonance (ODMR). By recording spectral shift or changes in relaxation rates, various physical and chemical quantities can be measured such as the magnetic field, orientation, temperature, radical concentration, pH or even NMR. This turns NV-nanodiamonds into nanoscale quantum sensors, which can be read out by a sensitive fluorescence microscope equipped with an additional magnetic resonance upgrade. In this review, we introduce the field of ODMR spectroscopy of NV-nanodiamonds and how it can be used to sense different quantities. Thereby we highlight both, the pioneering contributions and the latest results (covered until 2021) with a focus on biological applications.

Sicca syndrome during ipilimumab and nivolumab therapy for metastatic renal cell carcinoma.

Introduction: Dry mouth is the main symptom of sicca syndrome, which rarely occurs as an immune-related adverse event. Here we report a case of sicca syndrome caused by immune checkpoint inhibitor treatment. Case presentation: A 70-year-old man was diagnosed with left renal cell carcinoma after radical left nephrectomy. Nine years later, computed tomography revealed a metastatic nodule in the upper left lung lobe. Subsequently, ipilimumab and nivolumab were administered for recurrent disease. After 13 weeks of treatment, xerostomia and dysgeusia were noted. Salivary gland biopsy revealed lymphocyte and plasma cell infiltration in the salivary glands. Sicca syndrome was diagnosed and pilocarpine hydrochloride was prescribed without corticosteroids, with continuation of immune checkpoint inhibitor therapy. The symptoms alleviated after 36 weeks of treatment, with shrinkage of the metastatic lesions. Conclusion: We experienced sicca syndrome caused by immune checkpoint inhibitors. Sicca syndrome improved without steroids and the immunotherapy could be continued.

Distance measurements between 5 nanometer diamonds - single particle magnetic resonance or optical super-resolution imaging?

5 nanometer sized detonation nanodiamonds (DNDs) are studied as potential single-particle labels for distance measurements in biomolecules. Nitrogen-vacancy (NV) defects in the crystal lattice can be addressed through their fluorescence and optically-detected magnetic resonance (ODMR) of a single particle can be recorded. To achieve single-particle distance measurements, we propose two complementary approaches based on spin-spin coupling or optical super-resolution imaging. As a first approach, we try to measure the mutual magnetic dipole-dipole coupling between two NV centers in close DNDs using a pulse ODMR sequence (DEER). The electron spin coherence time, a key parameter to reach long distance DEER measurements, was prolonged using dynamical decoupling reaching T 2,DD ≈ 20 µs, extending the Hahn echo decay time T 2 by one order of magnitude. Nevertheless, an inter-particle NV-NV dipole coupling could not be measured. As a second approach, we successfully localize the NV centers in DNDs using STORM super-resolution imaging, achieving a localization precision of down to 15 nm, enabling optical nanometer-scale single-particle distance measurements.

Ultrahigh-resolution magnetic resonance in inhomogeneous magnetic fields: two-dimensional long-lived-coherence correlation spectroscopy.

A half-century quest for improving resolution in Nuclear Magnetic Resonance (NMR) and Magnetic Resonance Imaging (MRI) has enabled the study of molecular structures, biological interactions, and fine details of anatomy. This progress largely relied on the advent of sophisticated superconducting magnets that can provide stable and homogeneous fields with temporal and spatial variations below ΔB(0)/B(0)<0.01 ppm. In many cases however, inherent properties of the objects under investigation, pulsating arteries, breathing lungs, tissue-air interfaces, surgical implants, etc., lead to fluctuations and losses of local homogeneity. A new method dubbed "long-lived-coherence correlation spectroscopy" (LLC-COSY) opens the way to overcome both inhomogeneous and homogeneous broadening, which arise from local variations in static fields and fluctuating dipole-dipole interactions, respectively. LLC-COSY makes it possible to obtain ultrahigh resolution two-dimensional spectra, with linewidths on the order of Δν=0.1 to 1 Hz, even in very inhomogeneous fields (ΔB(0)/B(0)>10 ppm or 5000 Hz at 9.7 T), and can improve resolution by a factor up to 9 when the homogeneous linewidths are determined by dipole-dipole interactions. The resulting LLC-COSY spectra display chemical shift differences and scalar couplings in two orthogonal dimensions, like in "J spectroscopy." LLC-COSY does not require any sophisticated gradient switching or frequency-modulated pulses. Applications to in-cell NMR and to magnetic resonance spectroscopy (MRS) of selected volume elements in MRI appear promising, particularly when susceptibility variations tend to preclude high resolution.

Polychromatic decoupling of a manifold of homonuclear scalar interactions in solution-state NMR.

Window-acquired tetrachromatic irradiation allows one to decouple simultaneously four amide protons in cyclosporine A (wavy arrows; see figure) leading to simplified multiplets of the alpha protons. By inserting a manifold of polychromatic pulses in each dwell time, several subsystems can be decoupled simultaneously.

Subcapsular renal hematoma after ureterorenoscopy.

Introduction: Subcapsular renal hematoma after ureterorenoscopy using a holmium yttrium-aluminum-garnet laser is a rare complication. We experienced a case of subcapsular hematoma after ureterorenoscopy. Case presentation: The patient was a 56-year-old man with a history of hypertension and coronary vasospastic angina, and he was taking antiplatelet drugs. He had the middle and lower calyx stones measured 36 mm in diameter of the right kidney. We performed ureterorenoscopy, which was completed about 2 h without intraoperative complications. We could not remove the stone completely. After the surgery, the patient developed a fever and complained of right back pain. Computed tomography showed several residual stones formed a stone street, obstructing the stent and resulting in grade 3 hydronephrosis. Furthermore, the right subcapsular renal hematoma infection had detected. Percutaneous hematoma drainage and percutaneous nephrostomy were performed. Conclusion: Subcapsular renal hematoma after ureterorenoscopy is an uncommon complication but should be kept in mind.

A simple and soft chemical deaggregation method producing single-digit detonation nanodiamonds.

Detonation nanodiamonds (DNDs) are a class of very small and spherical diamond nanocrystals. They are used in polymer reinforcement materials or as drug delivery systems in the field of nanomedicine. Synthesized by detonation, only the final deaggregation step down to the single-digit nanometer size (<10 nm) unfolds their full potential. Existing deaggregation methods mainly rely on mechanical forces, such as high-power sonication or bead milling. These techniques entail drawbacks such as contamination of the sample and the need for a specialized apparatus. In this paper, we report a purely chemical deaggregation method by simply combining oxidation in air followed by a boiling acid treatment, to produce highly stable single-digit DNDs in a suspension. The resulting DNDs are surface functionalized with carboxyl groups, the final boiling acid treatment removes primary metal contaminants such as magnesium, iron or copper and the nanoparticles remain dispersed over a wide pH range. Our method can be easily carried out in a standard chemistry laboratory with commonly available laboratory apparatus. This is a key step for many DND-based applications, ranging from materials science to biological or medical applications.

Control of cross relaxation of multiple-quantum coherences induced by fast chemical exchange under heteronuclear double-resonance irradiation.

A fully analytical description of the control of the cross-correlated cross relaxation of multiple-quantum coherences in the presence of local dynamics under heteronuclear double-resonance radio-frequency (RF) irradiation is presented. The contribution of chemical exchange to relaxation can be partly or fully quenched by RF fields. We assume a correlated two-site chemical exchange model with arbitrary populations, and show that in the limit of fast exchange the dependence of the effective multiple-quantum cross-relaxation rate on the applied RF amplitude can be described by a compact analytical expression. Numerical simulations and preliminary experiments support our theoretical results. The relaxation dispersion as a function of RF amplitude can provide accurate information on the kinetics of correlated processes.

Extending timescales and narrowing linewidths in NMR.

Among the different fields of research in nuclear magnetic resonance (NMR) which are currently investigated in the Laboratory of Biomolecular Magnetic Resonance (LRMB), two subjects that are closely related to each other are presented in this article. On the one hand, we show how to populate long-lived states (LLS) that have long lifetimes T(LLS) which allow one to go beyond the usual limits imposed by the longitudinal relaxation time T1. This makes it possible to extend NMR experiments to longer time-scales. As an application, we demonstrate the extension of the timescale of diffusion measurements by NMR spectroscopy. On the other hand, we review our work on long-lived coherences (LLC), a particular type of coherence between two spin states that oscillates with the frequency of the scalar coupling constant J(IS) and decays with a time constant T(LLC). Again, this time constant T(LLC) can be much longer than the transverse relaxation time T2. By extending the coherence lifetimes, we can narrow the linewidths to an unprecedented extent. J-couplings and residual dipolar couplings (RDCs) in weakly-oriented phases can be measured with the highest precision.

Room-temperature hyperpolarization of polycrystalline samples with optically polarized triplet electrons: pentacene or nitrogen-vacancy center in diamond?

We demonstrate room-temperature 13C hyperpolarization by dynamic nuclear polarization (DNP) using optically polarized triplet electron spins in two polycrystalline systems: pentacene-doped [carboxyl-13C] benzoic acid and microdiamonds containing nitrogen-vacancy (NV-) centers. For both samples, the integrated solid effect (ISE) is used to polarize the 13C spin system in magnetic fields of 350-400 mT. In the benzoic acid sample, the 13C spin polarization is enhanced by up to 0.12 % through direct electron-to-13C polarization transfer without performing dynamic 1H polarization followed by 1H-13C cross-polarization. In addition, the ISE has been successfully applied to polarize naturally abundant 13C spins in a microdiamond sample to 0.01 %. To characterize the buildup of the 13C polarization, we discuss the efficiencies of direct polarization transfer between the electron and 13C spins as well as that of 13C-13C spin diffusion, examining various parameters which are beneficial or detrimental for successful bulk dynamic 13C polarization.

Transverse relaxation of scalar-coupled protons.

In a preliminary communication (B. Baishya, T. F. Segawa, G. Bodenhausen, J. Am. Chem. Soc. 2009, 131, 17538-17539), we recently demonstrated that it is possible to obtain clean echo decays of protons in biomolecules despite the presence of homonuclear scalar couplings. These unmodulated decays allow one to determine apparent transverse relaxation rates R(2) (app) of individual protons. Herein, we report the observation of R(2) (app) for three methyl protons, four amide H(N) protons, and all 11 backbone H(α) protons in cyclosporin A. If the proton resonances overlap, their R(2) (app) rates can be measured by transferring their magnetization to neighboring (13)C nuclei, which are less prone to overlap. The R(2) (app) rates of protons attached to (13)C are faster than those attached to (12)C because of (13)C-(1)H dipolar interactions. The differences of these rates allow the determination of local correlation functions. Backbone H(N) and H(α) protons that have fast decay rates R(2) (app) also feature fast longitudinal relaxation rates R(1) and intense NOESY cross peaks that are typical of crowded environments. Variations of R(2) (app) rates of backbone H(α) protons in similar amino acids reflect differences in local environments.

Apparent transverse relaxation rates in systems with coupled carbon-13 spins.

In systems with homonuclear scalar couplings, the envelopes of spin echoes obtained with simple refocusing pulses or trains of such pulses are normally modulated so that it is difficult to extract transverse relaxation rates. It has been shown recently that echo modulations can be quenched by cumulative pulse errors that arise after applying a large number of refocusing pulses with moderate rf amplitudes. The resulting unmodulated decays allow one to extract apparent transverse relaxation rates. Early work on systems comprising only two nitrogen-15 nuclei or two carbon-13 spins has recently been extended to systems with coupled protons. This work focuses on systems with three coupled carbon-13 spins, which in turn are coupled to several neighbouring protons. Unmodulated echo trains can be obtained by optimizing the pulse interval, the carrier frequency and the rf amplitude of the refocusing pulses.

How to Identify, Attribute, and Quantify Triplet Defects in Ensembles of Small Nanoparticles.

Nanodiamonds containing negatively charged triplet (having an electron spin S = 1) nitrogen-vacancy (NV-) centers are an extraordinary room-temperature quantum system, whose electron spins may be polarized and read out optically even in a single nanocrystal. In this Viewpoint we promote a simple but reliable method to identify, attribute, and quantify these triplet defects in a polycrystalline sample using electron paramagnetic resonance (EPR) spectroscopy. The characterization relies on a specific "forbidden" transition ("ΔMS = 2"), which appears at about half the central magnetic field and shows a remarkably small anisotropy. In particular, we emphasize that this method is by far not limited to NV- centers in diamond but could become an important characterization tool for novel triplet defects in various types of nanoparticles.