Bimodal Fluorescence/Magnetic Resonance Molecular Probes with Extended Spin Lifetimes.

Bimodal molecular probes combining nuclear magnetic resonance (NMR) and fluorescence have been widely studied in basic science, as well as clinical research. The investigation of spin phenomena holds promise to broaden the scope of available probes allowing deeper insights into physiological processes. Herein, a class of molecules with a bimodal character with respect to fluorescence and nuclear spin singlet states is introduced. Singlet states are NMR silent but can be probed indirectly. Symmetric, perdeuterated molecules, in which the singlet states can be populated by vanishingly small electron-mediated couplings (below 1 Hz) are reported. The lifetimes of these states are an order of magnitude longer than the longitudinal relaxation times and up to four minutes at 7 T. Moreover, these molecules show either aggregation induced emission (AIE) or aggregation caused quenching (ACQ) with respect to their fluorescence. In the latter case, the existence of excited dimers, which are proposed to use in a switchable manner in combination with the quenching of nuclear spin singlet states, is observed.

Spontaneous Enhancement of Magnetic Resonance Signals Using a RASER.

Nuclear magnetic resonance is usually drastically limited by its intrinsically low sensitivity: Only a few spins contribute to the overall signal. To overcome this limitation, hyperpolarization methods were developed that increase signals several times beyond the normal/thermally polarized signals. The ideal case would be a universal approach that can signal enhance the complete sample of interest in solution to increase detection sensitivity. Here, we introduce a combination of para-hydrogen enhanced magnetic resonance with the phenomenon of the RASER: Large signals of para-hydrogen enhanced molecules interact with the magnetic resonance coil in a way that the signal is spontaneously converted into an in-phase signal. These molecules directly interact with other compounds via dipolar couplings and enhance their signal. We demonstrate that this is not only possible for solvent molecules but also for an amino acid.

Gradient selected pure shift EASY-ROESY techniques facilitate the quantitative measurement of 1H,1H-distance restraints in congested spectral regions.

For elucidating molecular structure and dynamics in solution, NMR experiments such as NOESY, ROESY and EXSY have been used excessively over the past decades, to provide interatomic distance restraints or rates for chemical exchange. The extraction of such information, however, is often prohibited by signal overlap in these spectra. To reduce this problem, pure shift methods for improving the spectral resolution have become popular. We report on pure shift EASY-ROESY experiments and their application to extract cross-relaxation rates, proton-proton distances and exchange rates. Homonuclear decoupling (pure shift) is applied in the indirect dimension using the PSYCHE or the perfectBASH technique, to enhance the spectral resolution of severely overcrowded spectral regions. The spectral quality is further improved by using a gradient selected F1-PSYCHE-EASY-ROESY, which produces significantly less t1-noise than the experiment used previously, as also demonstrated by employing the recently introduced SAN (signal-artefact-noise) plots. Applications include the quantification of distance restraints in a peptide organocatalyst and the extraction of a number of distance restraints in cyclosporine A, which were previously not available for analysis, because they were either located in overcrowded spectral regions or hidden under t1-noise. Distances extracted and exchange rates obtained are accurate. Also, the 2D gradient-selected F1-perfectBASH-EASY-ROESY with the additional gradient selection proposed herein, which is superior in terms of sensitivity, can be used to accurately quantify cross-relaxation.

Hyperpolarization of 15N-pyridinium and 15N-aniline derivatives by using parahydrogen: new opportunities to store nuclear spin polarization in aqueous media.

Hyperpolarization techniques hold the promise to improve the sensitivity of magnetic resonance imaging (MRI) contrast agents by over 10 000-fold. Among these techniques, para-hydrogen induced polarization (PHIP) allows for generating contrast agents within seconds. Typical hyperpolarized contrast agents are traceable for 2-3 minutes only, thus prolonging tracking-times holds great importance for the development of new ways to diagnose and monitor diseases. Here, we report on the design of perdeuterated 15N-containing molecules with longitudinal relaxation times (T 1) of several minutes. T 1 is a measure for how long hyperpolarization can be stored. In particular, we introduce two new hyperpolarizable families of compounds that we signal enhanced with para-hydrogen: tert-amine aniline derivatives and a quaternary pyridinium compound with 15N-T 1 of about 8 minutes. Especially the latter compound has great potential for applicability since we achieved 15N-polarization up to 8% and the pyridinium motif is contained in a variety of drug molecules and is also used in drug delivery systems.

Hyperpolarization of Amino Acids in Water Utilizing Parahydrogen on a Rhodium Nanocatalyst.

NMR offers many possibilities in chemical analysis, structural investigations, and medical diagnostics. Although it is broadly used, one of NMR spectroscopies main drawbacks is low sensitivity. Hyperpolarization techniques enhance NMR signals by more than four orders of magnitude allowing the design of new contrast agents. Parahydrogen induced polarization that utilizes the para-hydrogen's singlet state to create enhanced signals is of particular interest since it allows to produce molecular imaging agents within seconds. Herein, we present a strategy for signal enhancement of the carbonyl 13 C in amino acids by using parahydrogen, as demonstrated for glycine and alanine. Importantly, the hyperpolarization step is carried out in water and chemically unmodified canonical amino acids are obtained. Our approach thus offers a high degree of biocompatibility, which is crucial for further application. The rapid sample hyperpolarization (within seconds) may enable the continuous production of biologically useful probes, such as metabolic contrast agents or probes for structural biology.

perfectBASH: Band-selective homonuclear decoupling in peptides and peptidomimetics.

Band selective techniques offer the highest sensitivity of all pure shift approaches and thus are the best choice for decoupling well-separated 1 H-frequency regions, such as the amide- or the α-proton region of α-peptides. They are inept to fully decouple the amide- and the α-proton region simultaneously, though. Herein, we present a new homonuclear decoupling technique, which extends the capabilities of band selective decoupling using the perfect echo principle. This modification allows a complete backbone decoupling (amide- and α-protons) in peptides and opens band selective homonuclear decoupling to substances with two mutually coupled protons in the spectral range of interest.

A pure shift experiment with increased sensitivity and superior performance for strongly coupled systems.

Motivated by the persisting need for enhanced resolution in solution state NMR spectra, pure shift techniques such as Zangger-Sterk decoupling have recently attracted widespread interest. These techniques for homonuclear decoupling offer enhanced resolution in one- and multidimensional proton detected experiments by simplifying multiplet structures. In this work, a modification to the popular Zangger-Sterk technique PEPSIE (Perfect Echo Pure Shift Improved Experiment) is presented, which decouples pairs of spins even if they share the same volume element. This in turn can drastically improve the sensitivity, as compared to classical Zangger-Sterk decoupling, as larger volume elements can be used to collect the detected signal. Most interestingly, even in the presence of moderate strong coupling, the PEPSIE experiment produces clean and widely artifact free spectra. In order to better understand this - to us initially - surprising behaviour we performed analyses using numerical simulations and derived an (approximate) analytical solution from density matrix formalism. We show that this experiment is particularly suitable to study samples with strong signal clustering, a situation which can render classic Zangger-Sterk decoupling inefficient.

Uncovering Key Structural Features of an Enantioselective Peptide-Catalyzed Acylation Utilizing Advanced NMR Techniques.

We report on a detailed NMR spectroscopic study of the catalyst-substrate interaction of a highly enantioselective oligopeptide catalyst that is used for the kinetic resolution of trans-cycloalkane-1,2-diols via monoacylation. The extraordinary selectivity has been rationalized by molecular dynamics as well as density functional theory (DFT) computations. Herein we describe the conformational analysis of the organocatalyst studied by a combination of nuclear Overhauser effect (NOE) and residual dipolar coupling (RDC)-based methods that resulted in an ensemble of four final conformers. To corroborate the proposed mechanism, we also investigated the catalyst in mixtures with both trans-cyclohexane-1,2-diol enantiomers separately, using advanced NMR methods such as T1 relaxation time and diffusion-ordered spectroscopy (DOSY) measurements to probe molecular aggregation. We determined intramolecular distance changes within the catalyst after diol addition from quantitative NOE data. Finally, we developed a pure shift EASY ROESY experiment using PSYCHE homodecoupling to directly observe intermolecular NOE contacts between the trans-1,2-diol and the cyclohexyl moiety of the catalyst hidden by spectral overlap in conventional spectra. All experimental NMR data support the results proposed by earlier computations including the proposed key role of dispersion interaction.

Distinction of trans-cis photoisomers with comparable optical properties in multiple-state photochromic systems - examining a molecule with three azobenzenes via in situ irradiation NMR spectroscopy.

Photochromic compounds like azobenzenes are widely used for the production of stimuli responsive materials. To analyse cascaded azobenzene switching inside a benzene-tricarboxamide (BTA) with three azobenzene moieties in a site-specific fashion, we used in situ irradiation NMR spectroscopy. Four photoisomers can be distinguished by their chemical shifts. Analysis of 1H, 13C and 15N shifts reveals that the configuration of one sidechain has an influence on the chemical shifts of both the other sidechains. Interconversion kinetics upon irradiation with ultraviolet (UV) light as well as molar fractions in photostationary states (PSS) were examined. Analysis of thermal fading of the different photoisomers into the ground state shows that thermal relaxation rates of all three azobenzene moieties behave as if they were independent of each other.

Extraction of distance restraints from pure shift NOE experiments.

NMR techniques incorporating pure shift methods to improve signal resolution have recently attracted much attention, owing to their potential use in studies of increasingly complex molecular systems. Extraction of frequencies from these simplified spectra enables easier structure determination, but only a few of the methods presented provide structural parameters derived from signal integral measurements. In particular, for quantification of the nuclear Overhauser effect (NOE) it is highly desirable to utilize pure shift techniques where signal overlap normally prevents accurate signal integration, to enable measurement of a larger number of interatomic distances. However, robust methods for the measurement of interatomic distances using the recently developed pure shift techniques have not been reported to date. In this work we discuss some of the factors determining the accuracy of measurements of signal integrals in interferogram-based Zangger-Sterk (ZS) pure shift NMR experiments. The ZS broadband homodecoupling technique is used in different experiments designed for quantitative NOE determination from pure shift spectra. It is shown that the techniques studied can be used for quantitative extraction of NOE-derived distance restraints, as exemplified for the test case of strychnine.

CLIP-COSY: A Clean In-Phase Experiment for the Rapid Acquisition of COSY-type Correlations.

The COSY experiment is an essential homonuclear 2D NMR experiment for the assignment of resonances. Its multiplet line shape, however, is often overly complicated, potentially leads to signal intensity losses, and is responsible for long minimum overall acquisition times. Herein, we present CLIP-COSY, a COSY-type experiment yielding clean in-phase peaks. It can be recorded within a few minutes and benefits from enhanced signal intensities for most cross-peaks. In combination with non-uniform sampling, the experiment times can be further reduced, and the in-phase multiplets enable the application of modern homonuclear decoupling techniques in both dimensions. As antiphase cancelations are avoided, CLIP-COSY can also be applied to macromolecules and other samples with broadened lines.

Accurate determination of one-bond heteronuclear coupling constants with "pure shift" broadband proton-decoupled CLIP/CLAP-HSQC experiments.

We report broadband proton-decoupled CLIP/CLAP-HSQC experiments for the accurate determination of one-bond heteronuclear couplings and, by extension, for the reliable measurement of small residual dipolar coupling constants. The combination of an isotope-selective BIRD((d)) filter module with a non-selective (1)H inversion pulse is employed to refocus proton-proton coupling evolution prior to the acquisition of brief chunks of free induction decay that are subsequently assembled to reconstruct the fully-decoupled signal evolution. As a result, the cross-peaks obtained are split only by the heteronuclear one-bond coupling along the F2 dimension, allowing coupling constants to be extracted by measuring simple frequency differences between singlet maxima. The proton decoupling scheme presented has also been utilized in standard HSQC experiments, resulting in a fully-decoupled pure shift correlation map with significantly improved resolution.

The influence of electronic modifications on rotational barriers of bis-NHC-complexes as observed by dynamic NMR spectroscopy.

There has been much debate about the σ-donor and π-acceptor properties of N-heterocyclic carbenes (NHCs). While a lot of synthetic modifications have been performed with the goal of optimizing properties of the catalyst to tune reactivity in various transformations (e.g. metathesis), direct methods to characterize σ-donor and π-acceptor properties are still few. We believe that dynamic NMR spectroscopy can improve understanding of this aspect. Thus, we investigated the intramolecular dynamics of metathesis precatalysts bearing two NHCs. We chose four systems with one identical NHC ligand (N,N'-Bis(2,4,6-trimethylphenyl)-imidazolinylidene (SIMes) in all four cases) and NHC(ewg) ligands bearing four different electron-withdrawing groups (ewg). Both rotational barriers of the respective Ru-NHC-bonds change significantly when the electron density of one of the NHCs (NHC(ewg)) is modified. Although it is certainly not possible to fully dissect σ-donor and π-acceptor portions of the bonding situations in the respective Ru-NHC-bond via dynamic NMR spectroscopy, our studies nevertheless show that the analysis of the rotation around the Ru-SIMes-bond can be used as a spectroscopic parameter complementary to cyclic voltammetry. Surprisingly, we observed that the rotation around the Ru-NHC(ewg)-bond shows the same trend as the initiation rate of a ring-closing metathesis of the four investigated bis-NHC-complexes.