Determination of internuclear distances in uniformly labeled molecules by rotational-resonance solid-state NMR

Rotational-resonance magic-angle spinning NMR experiments are frequently used to measure dipolar couplings and to determine internuclear distances. So far most measurements were performed on samples containing isolated spin pairs. Thus, extensive structure elucidation, for example in biomolecules, requires the preparation of a whole set of doubly labeled samples. Here, we describe the analysis of the rotational-resonance polarization-exchange curves obtained from a single, uniformly labeled sample. It is shown experimentally that, at a magnetic field of 14.09 T, the rotational-resonance conditions in uniformly (13)C-labeled threonine are sufficiently narrow to permit the measurement of five distances between the four carbon spins with an accuracy of better than 10%. The polarization-exchange curves are analyzed using a modified two-spin model consisting of the two active spins. The modified model includes an additional offset in the final polarization, which comes from the coupling to the additional, passive, spins. The validity of this approach is experimentally verified for uniformly (13)C-labeled threonine. The broader applicability of such a model is demonstrated by numerical simulations which quantify the errors as a function of the most relevant parameters in the spin system.     

Stereochemistry of Polynuclear Cadmium(II)thioglycolates: Crystal Structure of [ICd8(SCH2CH2OH)12]3+ · 3I− · H2O

Crystals of the title compound are triclinic, a = 27.87 Å, b = 10.77 Å, c = 12.94 Å, α 73.1°, β 116.1°, γ 120.0°, space group P1 . The structure consists of octanuclear ions: Eight Cd(II) ions are found at the corners of a distorted cube, the center of the cube is occupied by an iodide, the twelve thioglycolate sulfur atoms bridge the twelve edges of the cube thereby forming a distorted icosahedron. Cadmium ions are either five or seven coordinate. The phase problem for this structure was solved using a combination of very high and very low E-values.     

NMR spectra of a microcrystalline protein at 30 kHz MAS

Proteins are not always available in amounts desirable for solid-state magic-angle spinning (MAS) nuclear-magnetic resonance (NMR) spectroscopy. To maximize the signal-to-noise ratio achievable with small samples, the filling factor must be optimized by using small-diameter MAS rotors. These rotors have the added benefit of allowing higher radio frequency field amplitudes during polarization transfer steps and during decoupling periods as well as allowing higher spinning frequencies. We demonstrate the advantages of relatively fast MAS (30 kHz using a 2.5 mm rotor) compared to MAS at 12 kHz for the 10.4 kDa model protein Crh with 93 residues and show that the signal-to-noise ratio in two-dimensional correlation spectra can be significantly improved by taking advantage of optimized pulse sequences available with rapid MAS.     

Two-dimensional Blue Native/sodium dodecyl sulfate gel electrophoresis for analysis of multimeric proteins in platelets

Two-dimensional (2-D) Blue Native/SDS gel electrophoresis combines a first-dimensional separation of monomeric and multimeric proteins in their native state with a second denaturing dimension. These high-resolution 2-D gels aim at identifying multiprotein complexes with respect to their subunit composition. We applied this method for the first time to analyze two human platelet subproteomes: the cytosolic and the microsomal membrane protein fraction. Solubilization of platelet membrane proteins was achieved with the nondenaturing detergent n-dodecyl-beta-D-maltoside. To validate native solubilization conditions, we demonstrated the correct assembly of the Na,K-ATPase, a functional multimeric transmembrane protein, when expressed in Xenopus oocytes. We identified 63 platelet proteins after in-gel tryptic digestion of 58 selected protein spots and liquid chromatography-coupled tandem mass spectrometry. Nine proteins were detected for the first time in platelets by a proteomic approach. We also show that this technology efficiently resolves several known membrane and cytosolic multiprotein complexes. Blue Native/SDS gel electrophoresis is thus a valuable procedure to analyze specific platelet subproteomes, like the membrane(-bound) protein fraction, by mass spectrometry and immunoblotting and could be relevant for the study of protein-protein interactions generated following platelet activation.     

Longitudinal (1)H relaxation optimization in TROSY NMR spectroscopy

A general method to enhance the sensitivity of the multidimensional NMR experiments performed at high-polarizing magnetic field via the significant reduction of the longitudinal proton relaxation times is described. The method is based on the use of two vast pools of "thermal bath" 1H spins residing on hydrogens covalently attached to carbon and oxygen atoms in 13C,15N labeled and fully protonated or fractionally deuterated proteins to uniformly enhance longitudinal relaxation of the 1HN spins and concomitantly the sensitivity of multipulse NMR experiments. The proposed longitudinal relaxation optimization is implemented in the 2D [15N,1H]-LTROSY, 2D [15N,1H]-LHSQC and 3D LTROSY-HNCA experiments yielding the factor 2-2.5 increase of the maximal signal-to-noise ratio per unit time at 600 MHz. At 900 MHz, the predicted decrease of the 1HN longitudinal relaxation times can be as large as one order of magnitude, making the proposed method an important tool for protein NMR at high magnetic fields.     

The chemistry of cyclic enaminoketones. III. Synthesis of bi- and tricyclic enaminoketones from enamine esters and nitriles

The cyclization of enamines derived from β-aminoesters and β-aminonitriles into bi- and tricyclic enaminoketones ( 6,9,13 and 24 ) has been investigated. The enamines derived from aminonitriles cyclize smoothly with magnesium perchlorate in benzene or toluene, whereas the enamines derived from aminoesters cyclize spontaneously during their formation. The scope and limitation of this process is discussed.     

Ag+ labeling: a convenient new tool for the characterization of hydrogen-bonded supramolecular assemblies by MALDI-TOF mass spectrometry

Herein we describe our results on the characterization of a wide variety of different hydrogen-bonded assemblies by means of a novel matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) technique with Ag+ labeling. The labeling technique with Ag+ ions is extremely mild and provides a nondestructive way to generate charged assemblies that can be detected by mass spectrometry. Up to now more than 25 different single (1(3).2(3)), double (3(3).2(6)), and tetrarosettes (4(3).2(12)) have been successfully characterized by the use of this method. The success of the method entirely depends on the presence of a suitable binding site for the Ag+ ion. A variety of functionalities has been identified that provide strong binding sites for Ag+, either acting in a cooperative way (pi-arene and pi-alkene donor functionalities) or individually (cyano and crown ether functionalities). The method works well for assemblies with molecular weights between 2,000 and 8,000 Da, and most likely far beyond this limit.     

C2-Symmetric 4,4′,5,5′-Tetrahydrobi(oxazoles) and 4,4′,5,5′-Tetrahydro-2,2′-methylenebis[oxazoles] as Chiral Ligands for Enantioselective Catalysis Preliminary Communication

The synthesis of a series of enantiomerically pure, C₂-symmetric 4,4′,5,5′-tetrahydro-2,2′-methylenebis[oxazoles] and 4,4′,5,5′-tetrahydro-2,2′-bi(oxazoles) is reported. Copper complexes with anionic tetrahydromethylenebis[oxazole] ligands are efficient catalysts for the enantioselective cyclopropane formation from olefins and diazo compounds (up to 96% ee in the reaction of styrene with menthyl diazoacetate). Tetrahydrobi(oxazole)iridium(I) complexes were found to catalyze transfer hydrogenations of aryl alkyl ketones with i-PrOH (up to 91% ee). Tetrahydrobi(oxazole)palladium complexes can be used as enantioselective catalysts for allylic nucleophilic substitution (up to 77% ee in the reaction of PhCH?CHCH(OAc)Ph with NaHC(COOMe)₂).     

Fulven-Dimere: Synthese,Strukturbeweis and thermisches Verhalten

Fulvene-Dieters: Synthesis, Structure Elucidation and Thermal Behaviour In contrast to earlier assumptions, thermal reaction of pure fulvene ( 1a ), 6-methylfulvene ( lb ) and 6, 6-dimethylfuivene ( 1c ) at 227deg; gives oligomeric mixtures consisting mainly of the endo-[4 + 2]-eycloaddition products 2a , 2b and 2c . Thermal reactivity of the fulvenes decreases strongly in the series 1a > 1b > 1c . While the dimers 2b and 2c equilibrate very easily in solution above room temperature with 1b and 1c , respectively, 2a equilibrates with the isomer 5a (? 1, 6-Dimethyliden3a α, 3bβ, 6a α, 6bβ-tetrahydro-1-H), 6 H-bi (cyclopentadienylen). This surprising rearrangement envolves a formal 1,3-shift of the 1, 2-dihydrofulvene-unit of 2a (s. Scheme 4).