Methyl proton contacts obtained using heteronuclear through-bond transfers in solid-state NMR spectroscopy

A two-dimensional proton-mediated carbon-carbon correlation experiment that relies on through-bond heteronuclear magnetization transfers is demonstrated in the context of solid-state NMR of proteins. This new experiment, dubbed J-CHHC by analogy to the previously developed dipolar CHHC techniques, is shown to provide selective and sensitive correlations in the methyl region of 2D spectra of crystalline organic compounds. The method is then demonstrated on a microcrystalline sample of the dimeric protein Crh (2 x 10.4 kDa). A total of 34 new proton-proton contacts involving side-chain methyl groups were observed in the J-CHHC spectrum, which had not been observed with the conventional experiment. The contacts were then used as additional distance restraints for the 3D structure determination of this microcrystalline protein. Upon addition of these new distance restraints, which are in large part located in the hydrophobic core of the protein, the root-mean-square deviation with respect to the X-ray structure of the backbone atom coordinates of the 10 best conformers of the new ensemble of structures is reduced from 1.8 to 1.1 A.     

Chiral tetranuclear mu3-alkoxo-bridged copper(II) complex with 2 + 4 cubane-like Cu4O4 core framework and ferromagnetic ground state

The sugar-modified Schiff base ligand benzyl 2-deoxy-2-salicylideneamino-alpha-D-glucopyranoside H 2L, prepared by condensation of salicylaldehyde and the monomeric chitosan analogue benzyl 2-deoxy-2-amino-alpha-D-glucopyranoside, reacts with copper(II) acetate to form a self-assembled, alkoxo-bridged tetranuclear homoleptic copper(II) complex [{Cu(L)}4] (4) with Cu4O4 heterocubane core. The chiral complex 4 crystallizes in the space group P2 12 12 1. The tetranuclear complex 4 is composed of two dinuclear {Cu(L)}2 entities linked by the four mu 3-bridging C-3 alkoxide oxygen atoms of the sugar backbone. The preorganization of the dimeric {Cu(L)}2 entities is enforced by strong hydrogen bonds between the phenolate oxygen atom and the C-4 hydroxy group of the two constituting chiral monomeric building blocks. Therefore the Cu4O4 core can be classified as a type I or 2 + 4 cubane. The chirality of the structure is confirmed by circular dichroism (CD) spectra, which reveal a significant dichroism associated with the copper centered transitions at around 600 nm. Temperature dependent magnetic susceptibility measurements indicate ferromagnetic exchange interactions in complex 4. Fitting of the experimental data with a two J model based on the 2 + 4 topology ( H = - J1(S1S3 + S2S4) - J2(S1 + S3)(S2 + S4)) leads to exchange coupling constants of J1 = 64 and J2 = 4 cm(-1). The observed ferromagnetic coupling can be attributed to the very small Cu-O-Cu bridging angles within the Cu2O2 core of the constituting dimeric entities, which are a result of the conformational requirements introduced by the sugar backbone. 4 is not only the first example of an alkoxo-bridged tetranuclear copper(II) complex with Cu4O4 core representing the 2 + 4 cubane class with ferromagnetic ground state but also a rare example for the class of molecules combining a ferromagnetic ground state with optical activity. The ferromagnetic S = 2 ground state of 4 is confirmed by magnetization measurements and ESR spectroscopy.     

Generalized electronic diabatic approach to structural similarity in two‐dimensional potential energy surfaces of various topologies

Active site properties in some proteins can be affected by conformational fluctuations of neighbor residues, even when the latter are not involved directly in the binding process. A local environment thus appears to alter the relevant potential energy surface and its reaction paths. Here, some aspects of this phenomenon are simulated within a generalized electronic diabatic (GED) scheme to study the geometry and structural similarity for a class of two‐dimensional (2D) energy surfaces. The electronic quantum state is a linear superposition of diabatic basis functions, each of which is taken to represent a single (pure) electronic state for the isolated material system. Here, we describe a model reaction of isomerization by shifts in amplitudes for three diabatic species (reactant, product, and an open‐shell transition state) coupled in an external field. The “effective” 2D energy surface in the field is characterized in terms of critical points, and the amplitudes along the main reaction paths. A new feature is the introduction of a phase diagram where all possible potential‐energy‐surface topologies (consistent with three‐state systems in two linear coordinates) are matched with actual model parameters. By varying the coupling strengths between diabatic states, we classify regions of this phase diagram in terms of electronic and structural similarities; some regions comprise models whose reaction paths have geometries that belong to the catchment region of the reactant, yet are electronically akin to the diabatic transition state or product. © 2007 Wiley Periodicals, Inc. Int J Quantum Chem, 2008     

Modification of Branched Poly(ethylene imine) with d-Fructose for Selective Delivery of siRNA into Human Breast Cancer Cells

Branched poly(ethylene imine) (bPEI) is frequently used in RNA interference (RNAi) experiments as a cationic polymer for the delivery of small interfering RNA (siRNA) because of its ability to form stable polyplexes that facilitate siRNA uptake. However, the use of bPEI in gene delivery is limited by its cytotoxicity and a need for target specificity. In this work, bPEI is modified with d- fructose to improve biocompatibility and target breast cancer cells through the overexpressed GLUT5 transporter. Fructose-substituted bPEI (Fru−bPEI) is accessible in three steps starting from commercially available protected fructopyranosides and bPEI. Several polymers with varying molecular weights, degrees of substitution, and linker positions on d- fructose (C1 and C3) are synthesized and characterized with NMR spectroscopy, size exclusion chromatography, and elemental analysis. In vitro biological screenings show significantly reduced cytotoxicity of 10 kDa bPEI after fructose functionalization, specific uptake of siRNA polyplexes, and targeted knockdown of green fluorescent protein (GFP) in triple-negative breast cancer cells (MDA-MB-231) compared to noncancer cells (HEK293T).     

Monitoring ssDNA Binding to the DnaB Helicase from Helicobacter pylori by Solid‐State NMR Spectroscopy

DnaB helicases are bacterial, ATP‐driven enzymes that unwind double‐stranded DNA during DNA replication. Herein, we study the sequential binding of the “non‐hydrolysable” ATP analogue AMP‐PNP and of single‐stranded (ss) DNA to the dodecameric DnaB helicase from Helicobacter pylori using solid‐state NMR. Phosphorus cross‐polarization experiments monitor the binding of AMP‐PNP and DNA to the helicase. ¹³C chemical‐shift perturbations (CSPs) are used to detect conformational changes in the protein upon binding. The helicase switches upon AMP‐PNP addition into a conformation apt for ssDNA binding, and AMP‐PNP is hydrolyzed and released upon binding of ssDNA. Our study sheds light on the conformational changes which are triggered by the interaction with AMP‐PNP and are needed for ssDNA binding of H. pylori DnaB in vitro. They also demonstrate the level of detail solid‐state NMR can provide for the characterization of protein–DNA interactions and the interplay with ATP or its analogues.     

Ascospiroketals A and B, unprecedented cycloethers from the marine-derived fungus Ascochyta salicorniae

Chemical investigation of the marine-derived fungus Ascochyta salicorniae led to the isolation of two novel natural products, ascospiroketals A (1) and B (2). From a biosynthetic standpoint, the compounds possess new ring systems. [structure: see text].     

Blank problems in trace analysis of diethylhexyl and dibutyl phthalate: investigation of the sources, tips and tricks

The analysis of phthalates, particularly that of di(2-ethylhexyl)phthalate (DEHP) and dibutyl phthalate (DBP), is notorious for blank problems. Methods and tools are listed to identify the sources and reduce the system contamination to below 1 pg DEHP and DBP or below 1 ng mL⁻¹ of sample solution. Once direct contact with phthalate-containing plastic articles is ruled out, the air is the major source, primarily via absorption to the surfaces of laboratory glass ware. A main improvement was achieved by cleaning solvents with aluminium oxide permanently left in the reservoirs. The data enables to estimate the contamination to be expected and to design methods keeping blanks below a critical threshold.