Discovery of potent small-molecule inhibitors of lipoprotein(a) formation.

Lipoprotein(a) (Lp(a)), an independent, causal cardiovascular risk factor, is a lipoprotein particle that is formed by the interaction of a low-density lipoprotein (LDL) particle and apolipoprotein(a) (apo(a)). Apo(a) first binds to lysine residues of apolipoprotein B-100 (apoB-100) on LDL through the Kringle IV (K) 7 and 8 domains, before a disulfide bond forms between apo(a) and apoB-100 to create Lp(a) (refs. ).

Activation of the human insulin receptor by non-insulin-related peptides.

The human insulin receptor signalling system plays a critical role in glucose homeostasis. Insulin binding brings about extensive conformational change in the receptor extracellular region that in turn effects trans-activation of the intracellular tyrosine kinase domains and downstream signalling. Of particular therapeutic interest is whether insulin receptor signalling can be replicated by molecules other than insulin.

Free-viewing as experimental system to test the Temporal Correlation Hypothesis: A case of theory-generative experimental practice.

Theory-free characterizations of experimental systems miss normative and conceptual components that sometimes are crucial to understanding their historical development. In the following paper, we show that these components may be part of the intrinsic capacities of experimental systems themselves. We study a case of non-exploratory and theory-oriented research in experimental neuroscience that concerns the construction of free-viewing as an experimental system to test one particular pre-existing hypothesis, the Temporal Correlation Hypothesis (TCH), at a laboratory in Santiago de Chile, during 2002-2008.

Optimization of Hydroxyethylamine Transition State Isosteres as Aspartic Protease Inhibitors by Exploiting Conformational Preferences.

NMR conformational analysis of a hydroxyethylamine peptide isostere developed as an aspartic protease inhibitor shows that it is a flexible architecture. Cyclization to form pyrrolidines, piperidines, or morpholines results in a preorganization of the whole system in solution. The resulting conformation is similar to the conformation of the inhibitor in the active site of BACE-1.

Protein-Observed Fluorine NMR Is a Complementary Ligand Discovery Method to H CPMG Ligand-Observed NMR.

To evaluate its potential as a ligand discovery tool, we compare a newly developed 1D protein-observed fluorine NMR (PrOF NMR) screening method with the well-characterized ligand-observed H CPMG NMR screen. We selected the first bromodomain of Brd4 as a model system to benchmark PrOF NMR because of the high ligandability of Brd4 and the need for small molecule inhibitors of related epigenetic regulatory proteins. We compare the two methods' hit sensitivity, triaging ability, experiment speed, material consumption, and the potential for false positives and negatives.

Conformational flexibility around the Gal-β-(1 → 3)-Glc linkage: Experimental evidence for the existence of the anti-ψ conformation in aqueous solution.

NOE-based analysis of the disaccharide β-Gal-(1 → 3)-β-Glc-OMe (1), especially a diagnostic Gal1-Glc4 NOE detected in a HSQC-NOESY spectrum, reveals the existence of the anti-ψ conformer in aqueous solution in addition to the major syn conformer. This result provides experimental proof of conformational flexibility around the aglyconic bond of β-(1 → 3) disaccharides, in contrast to previous studies that suggested that the flexibility around this linkage was restricted to the syn conformational region.

Two-Site Evaluation of the Repeatability and Precision of an Automated Dual-Column Hydrogen/Deuterium Exchange Mass Spectrometry Platform.

Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) is an information-rich biophysical method for the characterization of protein dynamics. Successful applications of differential HDX-MS include the characterization of protein-ligand binding. A single differential HDX-MS data set (protein ± ligand) is often comprised of more than 40 individual HDX-MS experiments.

Conformational features of secondary N-cyclopropyl amides.

NMR studies in conjunction with ab initio calculations revealed unexpected conformational behavior of N-cyclopropylacetamide (1). This secondary amide displays 16-19% E-rotamer (cis) around the carbonyl-nitrogen bond in apolar solvents, in contrast to other aliphatic secondary acetamides in which significant E-rotamer populations are rare due to steric contacts between the substituents on the amide bond. In addition, 1 adopts an ortho conformation around the N-cPr bond instead of the anti conformation generally preferred by secondary acetamides.

Determination of magnitudes and relative signs of 1H-19F coupling constants through 1D- and 2D-TOCSY experiments.

A novel methodology based on 1D- and 2D-TOCSY experiments is described for a quick and accurate measurement of proton-fluorine coupling constants in fluorinated organic compounds. The magnitude of the (1)H-(19)F coupling was measured from the displacement between the relayed peaks associated with the α or β spin state of the fluorine, and its relative sign was derived from the sense of the displacement.

Conformational selection in glycomimetics: human galectin-1 only recognizes syn-Ψ-type conformations of β-1,3-linked lactose and its C-glycosyl derivative.

The human lectin galectin-1 (hGal-1) translates sugar signals, that is, β-galactosides, into effects on the level of cells, for example, growth regulation, and has become a model for studying binding of biopharmaceutically relevant derivatives. Bound-state conformations of Galβ-C-(1→3)-Glcβ-OMe (1) and its βGal-(1→3)-βGlc-OMe disaccharide parent compound were studied by using NMR spectroscopy (transferred (TR)-NOESY data), assisted by docking experiments and molecular dynamics (MD) simulations. The molecular recognition process involves a conformational selection event.

Long-range proton-carbon coupling constants: NMR methods and applications.

A general review of novel NMR methods to measure heteronuclear long-range proton-carbon coupling constants ((n)JCH; n>1) in small molecules is made. NMR experiments are classified in terms of NMR pulse scheme and cross-peak nature. A discussion about simplicity, general applicability and accuracy for each particular NMR experiment is presented and exemplified.

CLIP-HSQMBC: easy measurement of small proton-carbon coupling constants in organic molecules.

A user-friendly 2D NMR approach denoted as CLIP-HSQMBC is proposed for the very easy, direct and accurate measurement of long-range proton-carbon coupling constants in organic molecules and natural products. The J value can be extracted directly from the analysis of resolved in-phase (1)H multiplets that show an additional splitting arising from the proton-carbon coupling. In cases of unresolved peaks, a simple fitting analysis using the internal satellite lines as a reference is performed.

A definitive NMR solution for a simple and accurate measurement of the magnitude and the sign of small heteronuclear coupling constants on protonated and non-protonated carbon atoms.

Simply successful: a proton-selective HSQMBC-TOCSY experiment can be used to measure small proton-carbon ((n)J(CH); n>1) coupling constants on both protonated and non-protonated carbon atoms. The method combines in a single pulse scheme all the benefits of the widely used HSQMBC and HSQC-TOCSY experiments. The magnitude and the sign of (n)J(CH) can be determined simply with excellent accuracy.

Accurate measurement of small heteronuclear coupling constants from pure-phase α/β HSQMBC cross-peaks.

A simple proton-selective α/β-HSQMBC experiment is proposed for the accurate measurement of long-range proton-carbon coupling constants (nJCH) in small molecules without need for an individualized and time-consuming post-processing fitting procedure. The method acquires two pure-phase In-phase (IP) and Anti-phase (AP) multiplets completely free of any phase distortion due to the absence of JHH evolution. Accurate nJCH values can be directly measured analyzing the relative displacement of the resulting IPAP cross-peaks.

Measurement of long-range proton-carbon coupling constants from pure in-phase 1D multiplets.

The SELective INverse detection of carbon-proton CORrelation pulse sequence that yields a 1D spectrum of a proton directly bonded to a selected carbon resonance has been converted into a proton and carbon double-selective variant that provides a (1)H spectrum of a selected proton that is long-range coupled to a specific carbon resonance. The resulting 1D proton multiplet exhibits a pure absorptive in-phase lineshape for precise measurement of specific long-range proton-carbon coupling constants in small organic molecules at natural abundance.

Selective 1D HCH experiment: a fast NMR tool that connect protons belonging to different spin systems.

A selective 1D version of the HCH experiment (selHCH) is proposed for the efficient and fast correlation between protons belonging to different spin systems. The experiment consists of two consecutive, doubly selective heteronuclear J(CH) transfer steps that can individually be optimized. As any conventional proton-selective 1D experiment, the successful application of a frequency-selective 180° pulse on a well-isolated proton is the only practical requirement.

IPAP-HSQMBC: measurement of long-range heteronuclear coupling constants from spin-state selective multiplets.

A new NMR approach is proposed for the measurement of long-range heteronuclear coupling constants ((n)J(XH), n>1) in natural abundance molecules. Two complementary in-phase (IP) and anti-phase (AP) data are separately recorded from a modified HSQMBC experiment and then added/subtracted to provide spin-state-selective α/β-HSQMBC spectra. The magnitude of (n)J(XH) can be directly determined by simple analysis of the relative displacement between α- and β-cross-peaks.

High resolution magic angle spinning NMR applied to the analysis of organic compounds bound to solid supports.

In situ structural characterization of organic compounds attached to solid supports can be achieved by high-resolution magic angle spinning NMR (HRMAS NMR), a technique that provides solution-like spectra for resin-bound molecules. This review outlines the principles of the technique, the influence of the solid support on data quality, and NMR experiments that are useful for obtaining valuable information. The review describes, with multiple examples mainly from the last 7 years, how HRMAS NMR has been applied to monitor solid-phase reactions, elucidate reaction products and quantify compound loading on a solid support.

Improved NMR methods for the direct 13C-satellite-selective excitation in overlapped 1H-NMR spectra.

Improved pulsed-field gradient echo methods are presented and discussed for the direct selective excitation of the (13)C-satellite lines in overcrowded (1)H NMR spectra of small molecules. Sensitivity enhancements in (13)C spin-state selection can be achieved by combining multiple-proton-frequency excitation and Hadamard phase encoding. Several satellite-selective (SATSEL) NMR experiments are proposed and exemplified by measuring the sign and the magnitude of small, long-range proton-carbon coupling constants for (1)H resonances showing several levels of signal overlapping.

Resin-bound chiral derivatizing agents for assignment of configuration by NMR spectroscopy.

A general methodology for assigning the configuration of chiral mono- and polyfunctional compounds by NMR is presented. The approach is based on the use of polystyrene-bound chiral derivatizing agents (CDA-resins) specifically designed to achieve the high-yield formation of the covalent linkages (amide or ester bonds) between the substrate and the chiral auxiliary within the NMR tube, without the need for other manipulations, on a microscale level and in a short time. The deuterated NMR solvents (CDCl3, CD3CN, CS2/CD2Cl2) are also the reaction solvents and separations, purifications or workups of any kind are not necessary prior to recording the spectra.

Long-range 1H-1H NMR correlation: extending connectivities to remote bonds via an intermediate heterospin.

An out-and-stay 2D proton-proton NMR correlation experiment is proposed to detect long-range proton-proton connectivities up to six and seven bonds away. The magnetization flow pathway is based on a consecutive, dual-step J(CH)-transfer mechanism and it allows one to trace out (1)H-(1)H connectivities between protons belonging to different spin systems. This novel experimental scheme will be particularly useful in cases when carbon resonances overlap, providing connectivity information that could not be obtained in a HMBC experiment.

Assignment of absolute configuration on the basis of the conformational effects induced by chiral derivatizing agents: the 2-arylpyrrolidine case.

A novel approach for determining the absolute configuration of a chiral compound is proposed. The methodology is based on the distinct conformational effects imposed on a chiral substrate by each enantiomer of a chiral derivatizing agent. As a proof of concept, it is shown that the absolute configuration of 2-arylpyrrolidines can easily be determined by inspection of the multiplicity of the NMR signal of the methine proton of the pyrrolidine ring in the corresponding Mosher's amides.

Solid-phase synthesis of novel trimers containing a phenylstatine core and analysis by high-resolution magic angle spinning.

Here we describe a multistep solid-phase synthetic approach for the addition of amino acid residues to both the C- and N-termini of a phenylstatine core, yielding a library aimed at the development of structure-activity relationships in the S2 and S2' regions of the aspartyl proteases. Optimization of the synthetic strategy was performed on the basis of the in situ analysis of the compounds bound to the solid support through high-resolution magic angle spinning NMR Spectroscopy (HR-MAS NMR).

Conformational behaviour of glycomimetics: NMR and molecular modelling studies of the C-glycoside analogue of the disaccharide methyl beta-D-galactopyranosyl-(1-->3)-beta-D-glucopyranoside.

The conformational behaviour of the C-glycoside beta-C-Gal-(1-->3)-beta-Glc-OMe (1) has been studied using a combination of molecular mechanics and NMR spectroscopy (proton-proton coupling constants and nuclear Overhauser effects). It is shown that the C-disaccharide populates two distinctive conformational families in solution, the normal syn-psi conformation, which is the predominating conformation of parent O-glycoside 2, and the anti-psi conformation, which has not been detected for the O-disaccharide.