Optimising in-cell NMR acquisition for nucleic acids.

Understanding the structure and function of nucleic acids in their native environment is crucial to structural biology and one focus of in-cell NMR spectroscopy. Many challenges hamper in-cell NMR in human cell lines, e.g. sample decay through cell death and RNA degradation. The resulting low signal intensities and broad line widths limit the use of more complex NMR experiments, reducing the possible structural and dynamic information that can be extracted. Here, we optimize the detection of imino proton signals, indicators of base-pairing and therefore secondary structure, of a double-stranded DNA oligonucleotide in HeLa cells, using selective excitation. We demonstrate the reproducible quantification of in-cell selective longitudinal relaxation times (selT1), which are reduced compared to the in vitro environment, as a result of interactions with the complex cellular environment. By measuring the intracellular selT1, we optimize the existing proton pulse sequences, and shorten measurement time whilst enhancing the signal gained per unit of time. This exemplifies an advantage of selective excitation over conventional methods like jump-return water suppression for in-cell NMR. Furthermore, important experimental controls are discussed, including intracellular quantification, supernatant control measurements, as well as the processing of lowly concentrated in-cell NMR samples. We expect that robust and fast in-cell NMR experiments of nucleic acids will facilitate the study of structure and dynamics and reveal their functional correlation.

Pd-Catalyzed Asymmetric Allylic Alkylation of Cyclobutenes: From Double Inversion to Double Retention.

The Pd-catalyzed allylic alkylation of 3,4-disubstituted, racemic cyclobutene electrophiles exhibits a highly unusual stereoselectivity that allows for controlling diastereo- and enantioselectivity only by the choice of ligand and independent of the configuration of the substrate. In order to shed light on the origin of stereoinduction, we performed a systematic mechanistic investigation, including preparation of various putative Pd-allyl intermediates, 1H/31P NMR reaction monitoring, 2H-labeling studies, ESI-HRMS and 31P NMR analysis of reaction mixtures, and DFT structural computations. The mechanism disclosed exhibits several steps with stereospecificities deviating from the commonly accepted "double inversion rule": oxidative addition was found to follow a stereoconvergent course, giving anti-configured η1-Pd-cyclobutene species as detectable on-cycle intermediates irrespective of the configuration of starting material, while the subsequent nucleophilic attack features a stereodivergent behavior. In stark contrast to their highly reactive anti-analogues, syn-Pd-cyclobutene complexes that can be formed as side products are rendered entirely unreactive by strong internal Pd-O chelation, preventing the formation of undesired product diastereomers.

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.

Probing Long-Range Anisotropic Interactions: a General and Sign-Sensitive Strategy to Measure 1 H-1 H Residual Dipolar Couplings as a Key Advance for Organic Structure Determination.

Residual dipolar couplings (RDCs) are amongst the most powerful NMR parameters for organic structure elucidation. In order to maximize their effectiveness in increasingly complex cases such as flexible compounds, a maximum of RDCs between nuclei sampling a large distribution of orientations is needed, including sign information. For this, the easily accessible one-bond 1 H-13 C RDCs alone often fall short. Long-range 1 H-1 H RDCs are both abundant and typically sample highly complementary orientations, but accessing them in a sign-sensitive way has been severely obstructed due to the overflow of 1 H-1 H couplings. Here, we present a generally applicable strategy that allows the measurement of a large number of 1 H-1 H RDCs, including their signs, which is based on a combination of an improved PSYCHEDELIC method and a new selective constant-time ß-COSY experiment. The potential of 1 H-1 H RDCs to better determine molecular alignment and to discriminate between enantiomers and diastereomers is demonstrated.

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.