1D NMR Homodecoupled 1H Spectra with Scalar Coupling Constants from 2D NemoZS‐DIAG Experiments

A two‐dimensional liquid‐state NMR experiment cleanly separating chemical shifts and scalar couplings information is introduced. This DIAG experiment takes advantage of a drastic reduction of the spectral window in the indirect dimension to be quickly recorded and of a new non‐equidistant modulation of the selective pulse to improve the sensitivity of the broadband homodecoupling Zangger–Sterk sequence element by one order of magnitude. A simple automatic analysis results in 1D spectra displaying singlets and lists of the scalar couplings for first‐order multiplets. This facilitates the analysis of 1D spectra by resolving multiplets based on their differences in chemical shifts and coupling structures.     

Accelerated NMR Spectroscopy with Low‐Rank Reconstruction

Accelerated multi‐dimensional NMR spectroscopy is a prerequisite for high‐throughput applications, studying short‐lived molecular systems and monitoring chemical reactions in real time. Non‐uniform sampling is a common approach to reduce the measurement time. Here, a new method for high‐quality spectra reconstruction from non‐uniformly sampled data is introduced, which is based on recent developments in the field of signal processing theory and uses the so far unexploited general property of the NMR signal, its low rank. Using experimental and simulated data, we demonstrate that the low‐rank reconstruction is a viable alternative to the current state‐of‐the‐art technique compressed sensing. In particular, the low‐rank approach is good in preserving of low‐intensity broad peaks, and thus increases the effective sensitivity in the reconstructed spectra.     

Target‐Based Whole‐Cell Screening by 1H NMR Spectroscopy

An NMR‐based approach marries the two traditional screening technologies (phenotypic and target‐based screening) to find compounds inhibiting a specific enzymatic reaction in bacterial cells. Building on a previous study in which it was demonstrated that hydrolytic decomposition of meropenem in living Escherichia coli cells carrying New Delhi metallo‐β‐lactamase subclass 1 (NDM‐1) can be monitored in real time by NMR spectroscopy, we designed a cell‐based NMR screening platform. A strong NDM‐1 inhibitor was identified with cellular IC₅₀ of 0.51 μM , which is over 300‐fold more potent than captopril, a known NDM‐1 inhibitor. This new screening approach has great potential to be applied to targets in other cell types, such as mammalian cells, and to targets that are only stable or functionally competent in the cellular environment.     

Proton‐Detected Solid‐State NMR Spectroscopy of Natural‐Abundance Peptide and Protein Pharmaceuticals

The natural way : A sensitive NMR spectroscopic method is developed to obtain well‐resolved two‐dimensional spectra (¹⁵N–¹H and ¹³C–¹H) for natural‐abundance (that is, without the need for isotopic enrichment) large‐molecule samples, such as biopharmaceuticals. This method gives structural insights on two lyophilized aprotinin samples and three insulin samples in lyophilized, microcrystalline suspension formulation (red; see picture) and fibril (green) forms.

     

1H NMR‐Guided Isolation of Formyl‐Phloroglucinol Meroterpenoids from the Leaves of Eucalyptus robusta

Nine formyl‐phloroglucinolmeroterpenoids (FPMs), namely, eucalrobusones A–I ( 1 – 9 ), were isolated from the leaves of Eucalyptus robusta by tracking the phenolic hydroxyl ¹H NMR peaks. The Snatzke helicity rules for the Cotton effects of twisted benzene rings were applied to elucidate the absolute configurations of the FPMs. These findings, along with NMR spectroscopy, the circular dichroism (CD) exciton chirality method, and CD calculations, allowed complete structures for the FPMs to be assigned. Eucalrobusones A–F ( 1 – 6 ) are novel adducts formed between a formyl‐derived carbon atom on the phloroglucinol ring and monoterpene and sesquiterpene components. Eucalrobusones G–I ( 7 – 9 ) are the first examples of FPMs with cubebane part structures connected by an unusual 1‐oxaspiro[5.5]undecane subunit. Among these isolates, eucalrobusone C ( 3 ) showed significant cytotoxicity against HepG2, MCF‐7, and U2OS cancer cell lines, with IC₅₀ values less than 10 μm . Compound 3 significantly blocks cell proliferation in MCF‐7 cells and induces MCF‐7 cell death through apoptosis.     

Quadruple‐Resonance Magic‐Angle Spinning NMR Spectroscopy of Deuterated Solid Proteins

¹H‐detected magic‐angle spinning NMR experiments facilitate structural biology of solid proteins, which requires using deuterated proteins. However, often amide protons cannot be back‐exchanged sufficiently, because of a possible lack of solvent exposure. For such systems, using ²H excitation instead of ¹H excitation can be beneficial because of the larger abundance and shorter longitudinal relaxation time, T₁, of deuterium. A new structure determination approach, “quadruple‐resonance NMR spectroscopy”, is presented which relies on an efficient ²H‐excitation and ²H‐¹³C cross‐polarization (CP) step, combined with ¹H detection. We show that by using ²H‐excited experiments better sensitivity is possible on an SH3 sample recrystallized from 30 % H₂O. For a membrane protein, the ABC transporter ArtMP in native lipid bilayers, different sets of signals can be observed from different initial polarization pathways, which can be evaluated further to extract structural properties.     

Crystal‐Structure Determination of Powdered Paramagnetic Lanthanide Complexes by Proton NMR Spectroscopy

Shifts for crystals : Solid‐state NMR spectroscopy can be used for structure determination of microcrystalline paramagnetic solids at natural isotopic abundance. The protocol makes use of paramagnetic effects, measured on suitably recorded ¹H NMR spectra, to define the conformation of a molecule in the lattice and the intermolecular packing in the solid phase. The method is illustrated with a family of lanthanide compounds (see picture).

     

A General Method for Extracting Individual Coupling Constants from Crowded 1H NMR Spectra

Couplings between protons, whether scalar or dipolar, provide a wealth of structural information. Unfortunately, the high number of ¹H‐¹H couplings gives rise to complex multiplets and severe overlap in crowded spectra, greatly complicating their measurement. Many different methods exist for disentangling couplings, but none approaches optimum resolution. Here, we present a general new 2D J‐resolved method, PSYCHEDELIC, in which all homonuclear couplings are suppressed in F₂, and only the couplings to chosen spins appear, as simple doublets, in F₁. This approaches the theoretical limit for resolving ¹H‐¹H couplings, with close to natural linewidths and with only chemical shifts in F₂. With the same high sensitivity and spectral purity as the parent PSYCHE pure shift experiment, PSYCHEDELIC offers a robust method for chemists seeking to exploit couplings for structural, conformational, or stereochemical analyses.     

Solid‐State NMR Spectroscopy Reveals that E. coli Inclusion Bodies of HET‐s(218–289) are Amyloids

Protein deposition frequently occurs as inclusion bodies (IBs) during heterologous protein expression in E. coli. The structure of these E. coli IBs of the prion‐forming domain from the fungal prion HET‐s is the same as that previously determined for fibrils assembled in vitro, and show prion infectivity. These results demonstrate that the IBs of HET‐s(218–289) are amyloids.

     

1H‐Detected Solid‐State NMR Studies of Water‐Inaccessible Proteins In Vitro and In Situ

¹H detection can significantly improve solid‐state NMR spectral sensitivity and thereby allows studying more complex proteins. However, the common prerequisite for ¹H detection is the introduction of exchangeable protons in otherwise deuterated proteins, which has thus far significantly hampered studies of partly water‐inaccessible proteins, such as membrane proteins. Herein, we present an approach that enables high‐resolution ¹H‐detected solid‐state NMR (ssNMR) studies of water‐inaccessible proteins, and that even works in highly complex environments such as cellular surfaces. In particular, the method was applied to study the K⁺ channel KcsA in liposomes and in situ in native bacterial cell membranes. We used our data for a dynamic analysis, and we show that the selectivity filter, which is responsible for ion conduction and highly conserved in K⁺ channels, undergoes pronounced molecular motion. We expect this approach to open new avenues for biomolecular ssNMR.     

The Stereochemistry of 1,2,3‐Triols Revealed by 1H NMR Spectroscopy: Principles and Applications

The conformational compositions of the tris(α‐methoxy‐α‐phenylacetic acid) ester derivatives of 1,2,3‐prim,sec,sec‐triols are presented. These conformations have been determined by theoretical and experimental data (i.e., energy‐ and chemical‐shift calculations, circular dichroism (CD) experiments, coupling‐constant analysis, enantioselective deuteration experiments, and low‐temperature NMR spectroscopic studies). A detailed analysis of the anisotropic effects due to the most significant conformers in the ¹H NMR spectra supported the correlation between the ¹H NMR spectra (Δδ^RS value of H(3′) and |Δ(Δδ^RS)| parameters) and the absolute configuration of the substrate. The study also allows the identification of the pro‐R and pro‐S methylene protons from their vicinal coupling constants and relative chemical shifts.     

Probing Long‐Range Anisotropic Interactions: a General and Sign‐Sensitive Strategy to Measure 1H–1H 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 ¹H–¹³C RDCs alone often fall short. Long‐range ¹H–¹H 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 ¹H–¹H couplings. Here, we present a generally applicable strategy that allows the measurement of a large number of ¹H–¹H RDCs, including their signs, which is based on a combination of an improved PSYCHEDELIC method and a new selective constant‐time β‐COSY experiment. The potential of ¹H–¹H RDCs to better determine molecular alignment and to discriminate between enantiomers and diastereomers is demonstrated.     

Solution NMR Structure of a Ligand/Hybrid-2-G-Quadruplex Complex Reveals Rearrangements that Affect Ligand Binding

Telomeric G-quadruplexes have recently emerged as drug targets in cancer research. Herein, we present the first NMR structure of a telomeric DNA G-quadruplex that adopts the biologically relevant hybrid-2 conformation in a ligand-bound state. We solved the complex with a metalorganic gold(III) ligand that stabilizes G-quadruplexes. Analysis of the free and bound structures reveals structural changes in the capping region of the G-quadruplex. The ligand is sandwiched between one terminal G-tetrad and a flanking nucleotide. This complex structure involves a major structural rearrangement compared to the free G-quadruplex structure as observed for other G-quadruplexes in different conformations, invalidating simple docking approaches to ligand–G-quadruplex structure determination.     

Enhancing NMR Sensitivity of Natural‐Abundance Low‐γ Nuclei by Ultrafast Magic‐Angle‐Spinning Solid‐State NMR Spectroscopy

Although magic‐angle‐spinning (MAS) solid‐state NMR spectroscopy has been able to provide piercing atomic‐level insights into the structure and dynamics of various solids, the poor sensitivity has limited its widespread application, especially when the sample amount is limited. Herein, we demonstrate the feasibility of acquiring high S/N ratio natural‐abundance ¹³C NMR spectrum of a small amount of sample (≈2.0 mg) by using multiple‐contact cross polarization (MCP) under ultrafast MAS. As shown by our data from pharmaceutical compounds, the signal enhancement achieved depends on the number of CP contacts employed within a single scan, which depends on the T_1ρ of protons. The use of MCP for fast 2D ¹H/¹³C heteronuclear correlation experiments is also demonstrated. The significant signal enhancement can be greatly beneficial for the atomic‐resolution characterization of many types of crystalline solids including polymorphic drugs and nanomaterials.     

NMR analysis of 6‐(2′,3′‐dihydro‐1′H‐inden‐1′‐yl)‐1H‐indene

¹H, ¹³C and two‐dimensional NMR analyses were applied to determine the NMR parameters of 6‐(2′,3′‐dihydro‐1′H‐inden‐1′‐yl)‐1H‐indene. The measurements were accomplished with 0.5 mg of the substance, this quantity being sufficient to determine the chemical shifts of all the H and C atoms, and also the appropriate coupling constants and to give the complete NMR resonance assignments of the molecule. The predicted patterns of the four different H atoms of the methylene groups of the indane structural element coincided completely with the complex patterns in the NMR spectra. Copyright © 2002 John Wiley & Sons, Ltd.