Spectroelectrochemical nuclear magnetic resonance (NMR) experiments are faced with numerous challenges originating from shielding effects and susceptibility gradients in samples, leading to inhomogeneities in the static magnetic fields B0 and the radio frequency (rf) fields B1. Moreover, magnetic feedback caused by eddy currents in conductors can obstruct precise measurements. Previous works have shown that these eddy-current-induced magnetic field distortions can be accurately predicted by finite element method (FEM) simulations. In this work, we present a workflow combining FEM predictions with quantum optimal control (QOC) to tailor custom NMR pulses that exploit specific magnetic field distortions for selective excitation of affected sample regions. The desired selectivity was achieved using pattern pulses optimized for either a particular B1 or Larmor frequency ν0. Experimental validation was performed on a heterogeneous phantom consisting of two cavities filled with two spectroscopically distinguishable liquids, one between copper disks to mimic an electrochemical cell and one between polymer disks as a reference. An over 30-fold suppression of the reference resonance in between polymer compared to the resonance in between copper disks was achieved, demonstrating how QOC-tailored pulses can selectively address FEM-predicted B1 distortions in the vicinity of electrical conductors to achieve spatial selectivity with simultaneous ν0 robustness. It was also demonstrated how QOC-tailored pulses can selectively excite specific ν0 despite B0 distortions, which implies that difficulties with conventional solvent suppression techniques in electrochemical setups can be mitigated using the adjustable robustness of QOC-tailored pulses. The presented approach sets the stage for gradient-free, localized in operando NMR in electrochemistry and material sciences, with the prospect of surface selectivity down to the detection limit of the setup.
The segmented-overlap Fourier filtering and averaging (SOFFA) data acquisition method is described in detail for magnetic resonance spectroscopy. In this work, the four processes that encompass the SOFFA data acquisition method are detailed: (i) oversampling spectral segments, (ii) Fourier block-filtering, (iii) segment-overlap averaging, and (iv) decimation. Three experimental examples are shown: first, conventional continuous-wave (CW) electron paramagnetic resonance (EPR) is compared to SOFFA-CW of a single reduced [ 4 Fe - 4 S ] + ( S = 1 / 2 ) at concentrations of 1 mM, 100 µ M , and 10 µ M , showing an average increase in concentration sensitivity by a factor of 5.6 in a 100 min measurement time. Second, an experimental comparison of CW and SOFFA non-adiabatic rapid-scan (SOFFA-NARS) data with similar filter parameters and field modulation amplitude demonstrates a factor of 10.3 in signal-to-noise (SNR) improvement (32 min measurement time) for a 150 µ M site-directed spin-labeled hemoglobin in 82 % glycerol at 18 ° C . Finally, an SNR-matched experiment of free TEMPO at 10 µ M concentration is presented, where CW was performed at 400 scans (273 min) compared to SOFFA-CW with an overlap factor of 100 (20 min). Also shown is the effect of 1 / f noise on these CW and SOFFA-CW experiments. Ultimately, the SOFFA algorithm achieves sensitivity enhancement by combining massive digital oversampling and out-of-band noise filtration with coherent spatial accumulation of highly overlapped spectral segments. The gains reported here are phenomenological and are grounded in established digital signal processing principles but are validated through experiments rather than closed-form analytical prediction. This fundamental restructuring of the acquisition chain successfully decouples high-frequency filtering from low-frequency averaging while providing a data collection scheme that suppresses 1 / f noise. The SOFFA method can be implemented to perform real-time segmented processing and, combined with more sophisticated averaging methods, will push the state-of-the-art sensitivity in magnetic resonance spectroscopy.
Long-lived states (LLSs) can be excited in geminal protons of aliphatic chains by mono- or poly-chromatic spin-lock-induced crossings (SLICs), i.e., by application of one or more selective radio frequency (RF) fields, to create delocalized population imbalances between states belonging to different symmetry under spin permutations. At low fields (in this work at 1.4 T or 60 MHz for proton NMR), these experiments are challenging due to the proximity of the chemical shifts and the need to consider the full untruncated J -coupling Hamiltonian. Five molecules were studied in this work: ethanolamine, lysine, vitamin B1, metronidazole, and phenoxyethylamine (POEA). For POEA and metronidazole, the LLSs are reported for the first time. Measurements were carried out at low and high magnetic fields (1.4 and 11.7 T or 60 and 500 MHz for protons) using 60 MHz Magritek and 500 MHz Bruker NEO spectrometers. The rates R LLS = 1 / T LLS and R 1 = 1 / T 1 were determined using monochromatic SLIC excitation at both fields. We describe strategies for optimizing SLIC conditions in cases where the signals of neighboring CH2 groups are relatively close to each other.
Bilinear rotations imply differing rotations on a spin I depending on the presence or absence of a bilinear coupling Hamiltonian in connection to a heteronucleus S. As such, spin system selective inversions using BIRD elements, excitations using TANGO, or general (effective) rotations using BANGO and/or BIG-BIRD, as well as multiplicity-edited rotations, are achievable. So far, the well-defined rotations were only imposed on a single spin, e.g., I, while the coupled heteronucleus experienced only an inversion or no rotation at all. Here, we introduce dual bilinear rotations that simultaneously allow spin system selective manipulations on both spins I and S as compared to the coupled spin system IS. Particularly with the advent of multi-receive experiments and/or super-sequences with the necessity of exciting and storing specific spin systems in a flexible way, this may open new possibilities in pulse sequence design. A general derivation of the approach is given, and a quadruple-J-resolved-type experiment for obtaining fully decoupled spectra optimized for different spin systems is introduced for demonstration.
This work presents a theoretical framework for quantitative, scalable modeling of signal amplification by reversible exchange (SABRE) experiments under zero- and ultralow-field (ZULF) conditions. SABRE exploits the singlet spin order of parahydrogen to hyperpolarize nuclear spins of substrates without chemical modification, enhancing NMR signals. In the ZULF SABRE method, polarization transfer occurs in ultralow magnetic fields where Zeeman interactions are comparable to or weaker than scalar couplings, enabling coherent mixing of spin states and revealing interactions often suppressed at high fields. Our approach captures the full quantum dynamics of SABRE, including coherent evolution, chemical exchange, and relaxation, within a Liouville space formalism. We demonstrate that the Hamiltonian, relaxation, and exchange superoperators possess symmetry with respect to the total spin, allowing the dynamics to be rigorously restricted to the zero-quantum coherence subspace. This symmetry-based reduction yields a scalable framework for efficient simulation of multi-spin SABRE systems, allowing the treatment of arbitrary spin ensembles, including those containing 15 N , 13 C , 1 H , and other nuclei. The approach is validated against full Liouville space calculations for small systems and is further applied to a 14-spin SABRE complex, demonstrating its ability to treat spin systems of a complexity well beyond the reach of conventional full Liouville space simulations. The framework thus provides a predictive tool for optimal polarization fields, ZULF NMR spectra, and the design of novel hyperpolarization experiments.
The advantageous characteristics attributed to the 19F nucleus have made it a popular target for nuclear magnetic resonance (NMR) once again in recent years. Aside from solution NMR, an increasing number of studies have been conducted applying solid-state magic-angle spinning (MAS) NMR to fluorine-labelled samples. Here, the high chemical shift anisotropy and strong dipolar couplings can be utilised to get structural insights into proteins and measure long distances. Despite increasing popularity and promising benefits, the sensitivity of biomolecular 19F MAS NMR often suffers from slow longitudinal T1 relaxation and therefore long recycle delays. In this work, we expand paramagnetic doping, an approach commonly used to reduce proton T1 relaxation times, to 19F-labelled biological samples. We study the effect of Gd(DTPA) and Gd(DTPA-BMA) on 19F T1 and T2, and 13C T1 and T2 relaxation in a [5-19F13C]-tryptophan-labelled protein via 19F-detected MAS NMR experiments. The observed paramagnetic relaxation enhancement substantially reduces measurement times of 19F MAS NMR experiments without compromising resolution. Additionally, we report the chemical shift assignments of all four fluorotryptophan signals in the 12×39 kDa-large protein TET2 using a mutagenesis approach.
In addition to the development of various resonators, the concept of a probehead equipped with an additional low-noise amplifier (LNA) is becoming increasingly popular to enhance the sensitivity of electron paramagnetic resonance (EPR) spectrometers. The low-noise-detection amplifier makes it possible to measure pulsed EPR signals with high sensitivity. However, a strong reflected pulse signal can cause saturation and deterioration of the LNA characteristics, which requires protection of the LNA (for example, by using a protection switch in front of the LNA), which, in turn, reduces the signal-to-noise ratio. To overcome these limitations, we propose using an EPR probehead based on a bimodal cavity with strong isolation between the input and output ports in combination with a low-noise amplifier connected to the cavity output. The experiments demonstrate a 4-fold increase in the signal-to-noise ratio (SNR) of a bimodal probehead operating in transmission mode compared to its operation in reflection mode, which was achieved thanks to the additional use of LNA. The performance of the probe was also compared with the Bruker EN 5107D2 probe available in our laboratory, which showed an improvement that can be achieved by increasing the SNR by 2 times due to additional LNA and isolation of the detection channel from the input signal and by 3.3 times due to a larger sample volume in the bimodal probe (∼ 20 µL) at Q-band frequencies compared to the Bruker one (∼ 6 µL). The developed probehead can be used together with commercial Bruker ELEXYS EPR spectrometers without modification of the microwave bridge.
A connection between the symmetry of high-field nuclear magnetic resonance (NMR) spectra, including higher-order spectra, and the properties of the spin system has been established. It is shown that, for a spectrum to be symmetric about the mid-resonance frequency ( ν 0 ), two conditions must be satisfied: (1) the resonance frequencies of the spins must be symmetrically positioned about ν 0 , and (2) there must exist at least one spin ordering with a monotonic increase (or decrease) in resonance frequencies such that the spectrum is invariant under the reflection of the J -coupling matrix about its anti-diagonal (one way to satisfy this condition is for the J -coupling matrix to be explicitly persymmetric). The results were validated by calculating theoretical spectra for three-, four-, five-, and six-spin systems.
Bilinear rotations are essential building blocks in modern NMR spectroscopy. They allow the rotation of an isolated spin without couplings (i.e., bilinear interactions) in one way, while rotating spins with a matched coupling in another way. Different classes of rotations form the different bilinear rotations, with the acronyms BIRD, TANGO, BANGO, and BIG-BIRD. All original elements have in common hard pulses limiting bandwidths and defined rotations for coupled spins that are possible only for a narrow range of coupling constants. We recently introduced the COB-BIRD with a general optimization procedure to obtain robust bilinear rotations that are well compensated for couplings, offsets, and B1 inhomogeneities . Here we show a fundamental principle on how the COB-BIRD can be used to construct all types of bilinear rotations, with the same improved robustness covering a coupling range of 120-250 Hz. In addition, a construction principle for universal rotation pulses is adapted to produce bilinear rotations from INEPT-type transfer elements, allowing the construction of bilinear rotations also for higher coupling ranges from, for example, COB3-INEPT, with coupling compensation in the range of 120-750 Hz. After introducing the two fundamental design principles, example sequences of the four classes of bilinear rotations and different degrees of robustness are derived and characterized in theory and experiment. In addition, a highly useful HMBC/ASAP-HSQC-IPE-COSY supersequence is introduced with a (COB-)BANGO element for Ernst-angle-type excitation. Finally, BIRD-decoupled J -resolved INEPT experiments with extreme compensation for partially aligned samples, with total couplings ranging from 47 Hz up to 434 Hz, are demonstrated.
Abstract. Using cell-free protein synthesis, the protein G B1-domain (GB1) was prepared with uniform high-level substitution of leucine by (2S,4S)-5-fluoroleucine, (2S,4R)-5-fluoroleucine, or 5,5’-difluoroleucine. 19F nuclear magnetic resonance (NMR) spectra showed chemical shift ranges spanning more than 9 ppm. Through-space scalar 19F-19F couplings between CH2F groups arising from transient fluorine-fluorine contacts are readily manifested in [19F,19F]-TOCSY spectra. The 19F chemical shifts correlate with the three-bond 1H–19F couplings (3JHF), confirming the γ-gauche effect as the predominant determinant of the 19F chemical shifts of the CH2F groups. Different 3JHF couplings of different CH2F groups indicate that the rotation of the CH2F groups can be sufficiently restricted in different protein environments to result in the preferential population of a single rotamer. The 3JHF couplings also show that CH2F groups populate the different rotameric states differently in the 5,5’-difluoroleucine residues than in the monofluoroleucine analogues, showing that two CH2F groups in close proximity influence each other’s conformation. Nonetheless, the 19F resonances of the Cδ1H2F and Cδ2H2F groups of difluoroleucine residues can be assigned stereospecifically with good confidence by comparison with the 19F chemical shifts of the enantiomerically pure fluoroleucines. 1H-19F NOEs observed with water indicate hydration with subnanosecond residence times.
Abstract. Using cell-free protein synthesis, the protein G B1-domain (GB1) was prepared with uniform high-level substitution of valine by (2S,3S)-4-fluorovaline, (2S,3R)-4-fluorovaline, or 4,4'-difluorovaline. The 19F nuclear magnetic resonance (NMR) signals are distributed over a wide spectral range. The fluorinated samples maintain the relative 1H chemical shifts of the wild-type protein, opening a convenient route to assigning the 19F NMR signals. For the singly fluorinated residues, the 13C chemical shifts of the remaining CH3 group are subject to a γ-effect that depends on the population of different rotameric states of the CH2F group and correlates with 3JFC coupling constants. In addition, the preferentially populated rotamers are reflected by the γ-gauche effect on 19F chemical shifts, which correlates with 3JHF couplings. Some of the side-chain conformations determined by these restraints position the fluorine atom near a backbone carbonyl group, a non-intuitive finding that has previously been observed in the high-resolution crystal structure of a different protein. Through-space scalar 19F–19F couplings due to transient fluorine–fluorine contacts are observed between residues 39 and 54.
Understanding spatially heterogeneous molecular diffusion in semicrystalline polymers is critical for elucidating interfacial dynamics in soft materials. This study employs static-gradient nuclear magnetic resonance (NMR) imaging to capture the depth-resolved translational motion of polymer chains in a polytetrafluoroethylene (PTFE) film. By focusing on spin–spin relaxation behavior in amorphous regions near crystalline lamellae, we identify multiple diffusion regimes consistent with Bloch–Torrey analysis. The results reveal that molecular mobility at the substrate interface of PTFE film, immobilized on a glass substrate using epoxy resin, is significantly constrained, likely due to interfacial pinning, while the air-side surface shows signs of enhanced mobility. Our findings highlight the utility of static-gradient field NMR for probing nanoscale dynamical heterogeneity in semicrystalline systems.
Fast and accurate arbitrary waveform generators (AWGs) for generating shaped pulses in electron paramagnetic resonance (EPR) have been commercially available for over a decade now. However, while the use of chirp pulses as inversion pulses in pulsed electron double resonance (PELDOR) experiments has become common, their application for generating broadband phase-sensitive transverse magnetization is not widely adopted within the community. Here, we give a detailed insight into optimization procedures and instrumental challenges when using chirped pulses for broadband Fourier transform (FT) detection of electron spin echo signals, particularly the two-dimensional frequency-correlated single-frequency technique for refocusing (SIFTER) experiment. To better understand the influence of chirped pulses on the generation of broadband transverse magnetization, we investigated the phase and amplitude of chirped echoes for different time bandwidth products while varying the number of refocusing pulses, particularly under the influence of B1 inhomogeneity. Following our optimization procedures, we were able to perform EPR-correlated 2D-SIFTER measurements using rigid nitroxide spin labels on an RNA duplex. Finally, we also demonstrate the first experiments with two novel SIFTER pulse sequences, which could be of interest for the detection of either shorter or longer distances.
Long-lived states (LLSs) have lifetimes TLLS that exceed longitudinal spin-lattice relaxation times T1. In this study, lifetimes TLLS(19F) have been measured in three different achiral per- and polyfluoroalkyl substances (PFAS) containing two or three consecutive CF2 groups. In a static magnetic field B0=11.7 T, the lifetimes TLLS(19F) exceed the longitudinal relaxation times T1(19F) by about a factor of 2. The lifetimes TLLS(19F) can be strongly affected by binding to macromolecules, a feature that can be exploited for the screening of fluorinated drugs. Both TLLS(19F) and T1(19F) should be longer at lower fields where relaxation due to the chemical shift anisotropy (CSA) of 19F is less effective, which is demonstrated here by running experiments at two fields of 11.7 and 7 T.
Using cell-free protein synthesis, the protein G B1 domain (GB1) was prepared with uniform high-level substitution of valine by (2 S ,3 S )-4-fluorovaline, (2 S ,3 R )-4-fluorovaline or 4,4'-difluorovaline. The 19 F nuclear magnetic resonance (NMR) signals are distributed over a wide spectral range. The fluorinated samples maintain the relative 1 H chemical shifts of the wild-type protein, opening a convenient route to assign the 19 F -NMR signals. For the singly fluorinated residues, the 13 C chemical shifts of the remaining CH 3 group are subject to a γ effect that depends on the population of different rotameric states of the CH 2 F group and correlates with 3 J FC coupling constants. In addition, the preferentially populated rotamers are reflected by the γ -gauche effect on 19 F chemical shifts, which correlates with 3 J HF couplings. Some of the side-chain conformations determined by these restraints position the fluorine atom near a backbone carbonyl group, a non-intuitive finding that has previously been observed in the high-resolution crystal structure of a different protein. Through-space scalar 19 F - 19 F couplings due to transient fluorine-fluorine contacts are observed between residues 39 and 54.
Conference travel contributes to the climate footprint of academic research. Here, we provide a quantitative estimate of the carbon emissions associated with conference attendance by analyzing travel data from participants of 10 international conferences in the field of magnetic resonance, namely EUROMAR, ENC and ICMRBS. We find that attending a EUROMAR conference produces, on average, more than 1 t CO2 eq.. For the analyzed conferences outside Europe, the corresponding value is about 2–3 times higher, on average, with intercontinental trips amounting to up to 5 t. We compare these conference-related emissions to other activities associated with research and show that conference travel is a substantial portion of the total climate footprint of a researcher in magnetic resonance. We explore several strategies to reduce these emissions, including the impact of selecting conference venues more strategically and the possibility of decentralized conferences. Through a detailed comparison of train versus air travel – accounting for both direct and infrastructure-related emissions – we demonstrate that train travel offers considerable carbon savings. These data may provide a basis for strategic choices of future conferences in the field and for individuals deciding on their conference attendance.
Pulse-dipolar electron paramagnetic resonance (PD-EPR) has emerged as an effective tool in structural biology, enabling distance measurements between spin labels attached to biomolecules. The sensitivity and accessible distance range of these measurements are governed by the phase memory time ( T m ) of the spin labels. Understanding the decoherence mechanisms affecting T m is crucial for optimizing sample preparation and spin-label design. This study investigates the phase relaxation behavior of two Gd(III) spin-label complexes, Gd-PyMTA and Gd-TPMTA, with various degrees of deuteration. These two complexes have significantly different zero-field-splitting (ZFS) parameters. Hahn echo decay and dynamical decoupling (DD) measurements were performed at W-band (95 GHz) in deuterated solvents (D2O / glycerol-d8), both for the free complexes and when conjugated to proteins. The impact of temperature, concentration, and field position within the EPR spectrum on T m was examined. Results indicate that protons within 5 Å of the Gd(III) ion do not contribute to nuclear spin diffusion (NSD), and protein deuteration offers minimal enhancement in T m . The dominant phase relaxation mechanisms identified at low concentrations were direct spin-lattice relaxation ( T 1 ) and transient ZFS (tZFS) fluctuations. Dynamical decoupling (DD) measurements, using the Carr-Purcell sequence with ∼ 140 refocusing pulses, resolved the presence of two populations: one with a long phase relaxation time, T m , s , and the other with a short one, T m , f . The dominating mechanism for the slowly relaxing population is direct- T 1 . T m , s showed no concentration dependence and was longer by a factor of about 2 than T m for both complexes. We tentatively assign the increase in T m , s to full suppression of the residual indirect- T 1 -induced spectral diffusion and NSD mechanisms. For the fast-relaxing population, T m , f is shorter for Gd-TPMTA; therefore, we assign it to populations for which the tZFS mechanism dominates. Because of the relatively short T 1 and the contribution of the tZFS mechanism, protein deuteration does not significantly affect T m .
Additive manufacturing has enabled rapid prototyping of components with minimum investment in specific fabrication infrastructure. These tools allow for a fast iteration from design to functional prototypes within days or even hours. Such prototyping technologies exist in many fields, including three-dimensional mechanical components and printed electric circuit boards (PCBs) for electrical connectivity, to mention two. In the case of nuclear magnetic resonance (NMR) spectroscopy, one needs the combination of both fields; we need to fabricate three-dimensional electrically conductive tracks as coils that are wrapped around a sample container. Fabricating such structures is difficult (e.g., six-axis micro-milling) or simply not possible with conventional methods. In this paper, we modified an additive manufacturing method that is based on the extrusion of conductive ink to fast-prototype solenoidal coil designs for NMR. These NMR coils need to be as close to the sample as possible and, by their shape, have specific inductive values. The performance of the designs was first investigated using electromagnetic field simulations and circuit simulations. The coil found to have optimal parameters for NMR was fabricated by extrusion printing, and its performance was tested in a 1.05 T imaging magnet. The objective is to demonstrate reproducible rapid prototyping of complicated designs with high precision that, as a side effect, hardly produces material waste during production.
Flavoproteins are a versatile class of proteins involved in numerous biological processes, including redox reactions, electron transfer, and signal transduction, often relying on their ability to stabilize different oxidation states of their flavin cofactor. A critical feature of flavin cofactors is their capacity to achieve, within particular protein environments, a semiquinone state that plays a pivotal role in mediating single-electron transfer events and that is key to understanding flavoprotein reactivity. Hyperfine interactions between the unpaired electron and magnetic nuclei in the isoalloxazine ring provide valuable insights into the semiquinone state and its mechanistic roles. This study investigates the hyperfine interactions of isotopically labeled flavodoxin (Fld) with 13 C and 15 N in specific positions of the flavin mononucleotide (FMN) ring using advanced electron paramagnetic resonance (EPR) techniques. The combination of continuous-wave (CW) EPR at the X-band and ELDOR-detected NMR and HYSCORE at the Q-band revealed a strong and anisotropic hyperfine interaction with the nucleus of 13 C at 4a and yielded principal tensor values of 40, - 13.5 , and - 9 MHz, the first of which is associated with the axis perpendicular to the flavin plane. On the other hand, as predicted, the hyperfine interaction with the 13 C nucleus in position 2 was minimal. Additionally, HYSCORE experiments on 15 N -FMN-labeled Fld provided precise axial hyperfine parameters, i.e., (74, 5.6, 5.6) MHz for 15 N (5) and (38, 3.2, 3.2) MHz for 15 N (10). These were used to refine quadrupole tensor values for 14 N nuclei through isotope-dependent scaling. These results showcase the potential of combining CW EPR, ELDOR-detected NMR, and HYSCORE with isotopic labeling to probe electronic and nuclear interactions in flavoproteins. The new data complete and refine the existing experimental map for the electronic structure of the flavin cofactor and expose systematic divergences between the calculated and experimental values of hyperfine couplings of the atoms that contribute most to the semi-occupied orbital (SOMO). This could indicate a slight but significant shift in the unpaired electron density from position 4a towards the central nitrogens of the pyrazine ring as compared with the calculations. These results highlight the importance of integrating computational and experimental approaches to refine our understanding of flavin cofactor reactivity.
The implementation of parallel nuclear magnetic resonance detection aims to enhance measurement throughput in support of high-throughput-screening applications, including, for example, drug discovery. In support of modern pulse sequences and solvent suppression methods, each detection site must have independent pulsed field gradient capabilities. Hereby, a challenge is introduced in which the local gradients applied in parallel detectors introduce field spillover into adjacent channels, leading to spin dephasing and, hence, to signal suppression. This study proposes a compensation scheme employing optimized pulses to achieve coherence locking during gradient pulse periods. The design of coherence-locking pulses utilizes optimal control to address gradient-induced field inhomogeneity. These pulses are applied in a pulsed-gradient spin echo (PGSE) experiment and a parallel heteronuclear single quantum coherence (HSQC) experiment, demonstrating their effectiveness in protecting the desired coherences from gradient field spillover. This compensation scheme presents a valuable solution for magnetic resonance probes equipped with parallel and independently switchable gradient coils.
