A QM/MM study on the reaction pathway leading to 2‐Aceto‐2‐hydroxybutyrate in the catalytic cycle of AHAS

The reaction between the intermediate 2‐hydroxyethyl‐thiamin diphosphate (HEThDP^?) and 2‐ketobutyrate, in the third step of the catalytic cycle of acetodydroxy acid synthase, is addressed from a theoretical point of view by means of hybrid quantum/molecular mechanical calculations. The QM region includes one molecule of 2‐ketobutyrate, the HEThDP^? intermediate, and the residues Arg 380 y Glu 139; whereas the MM region includes the rest of the protein. The study includes potential energy surface scans to identify and characterize critical points on it, transition state search and activation barrier calculations. The results show that the reaction occurs via a two‐step mechanism corresponding to the carboligation and proton transfer in the first stage; and the product release in the second step. © 2014 Wiley Periodicals, Inc.     

Synthesis and thermal crosslinking of carbosiloxane and oligo(oxyethylene) polymers

Acyclic diene metathesis (ADMET) polymerization has been used in the synthesis of telechelic materials using alkoxy‐functionalized carbosiloxane or oligo(oxyethylene)‐based polymers, varying from internal to terminal cured materials or the combination of them. Previous investigations demonstrated that introduction of chain‐end crosslinking improves the stress–strain behavior of such materials. A series of saturated and unsaturated carbosiloxane and oligo(oxyethylene)‐based polymers were synthesized by ADMET polymerization using silacyclobutane as chain‐end, thermally induced crosslinker. The carbosiloxane derivatives presented pure amorphous behavior, whereas the oligo(oxyethylene) polymers were semicrystalline. The thermal curing process was monitored by differential scanning calorimetry via the exotherm between 160 and 210 °C. Mechanical properties on thermoset polymers were measured, where cured polymers showed moduli from 0.6 to 9.3 MPa, tensile strengths from 0.3 to 1.0 MPa, and elongations from 12 to 76%. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

Supramolecular structure of 5-methyl-5-phenyl hydantoin and hydrogen-bonding patterns in 5,5′-substituted hydantoins

The crystal structure of heterocyclic compound 5-methyl-5-phenyl hydantoin has been determined from X-ray single crystal structural characterization. This material crystallizes in the orthorhombic system and noncentrosymmetric space group P21 (N°4). The crystal packing is governed by N–H···O hydrogen bond-type intermolecular interactions, forming chains and edge-fused 12-membered rings with graph-set C(4) C(5) C22(8) R33(12) in a similar hydrogen-bonding pattern of another chiral 5,5′-substituted hydantoins.     

Unravelling the Time Scale of Conformational Plasticity and Allostery in Glycan Recognition by Human Galectin-1

The interaction of human galectin-1 with a variety of oligosaccharides, from di-(N-acetyllactosamine) to tetra-saccharides (blood B type-II antigen) has been scrutinized by using a combined approach of different NMR experiments, molecular dynamics (MD) simulations, and isothermal titration calorimetry. Ligand- and receptor-based NMR experiments assisted by computational methods allowed proposing three-dimensional structures for the different complexes, which explained the lack of enthalpy gain when increasing the chemical complexity of the glycan. Interestingly, and independently of the glycan ligand, the entropy term does not oppose the binding event, a rather unusual feature for protein-sugar interactions. CLEANEX-PM and relaxation dispersion experiments revealed that sugar binding affected residues far from the binding site and described significant changes in the dynamics of the protein. In particular, motions in the microsecond-millisecond timescale in residues at the protein dimer interface were identified in the presence of high affinity ligands. The dynamic process was further explored by extensive MD simulations, which provided additional support for the existence of allostery in glycan recognition by human galectin-1.     

Click and count: specific detection of acid ceramidase activity in live cells

The use of intact cells in medical research offers a number of advantages over employing cell-free systems. In diagnostics, cells isolated from liquid biopsies can be directly used, speeding up the time of analysis and diminishing the risk of protein degradation by sample manipulation. In drug discovery, studies in live cells take into account aspects neglected in cell-free systems, such as uptake, metabolization, and subcellular concentration by compartmentalization of potential drug candidates. Therefore, probes for studies in cellulo are of paramount importance. Acid ceramidase (AC) is a lysosomal enzyme that hydrolyses ceramides into sphingoid bases and fatty acids. The essential role of this enzyme in the outburst and progress of several diseases, some of them still incurable, is well sustained. Despite the great clinical relevance of AC as a biomarker and therapeutic target, the specific monitoring of AC activity in live cells has remained elusive due to the concomitant existence of neutral and alkaline ceramidases. In this work, we report that 1-deoxydihydroceramides are exclusively hydrolysed by AC. Using N-octanoyl-18-azidodeoxysphinganine as a probe and a BODIPY-substituted bicyclononyne, we show the click-reliant predominant staining of lysosomes, with extra-lysosomal labeling also occurring in some cells. Importantly, using pharmacological and genetic tools together with high resolution mass spectrometry, we demonstrate that both lysosomal and extra-lysosomal staining are AC-dependent. These findings are translated into the specific flow cytometry monitoring of AC activity in intact cells, which fills an important gap in the field of diseases linked to altered AC activity.

The use of intact cells in medical research offers a number of advantages over employing cell-free systems.     

Novel Copolymers of Styrene. 15. Phenoxy Ring-Substituted Methyl 2-Cyano-3-Phenyl-2-Propenoates

Electrophilic trisubstituted ethylenes, phenoxy ring-substituted methyl 2-cyano-3-phenyl-2-propenoates, RPhCH=C(CN)CO₂CH₃, where R is 4-(4-BrC₆H₅O), 2-(4-ClC₆H₅O), 3-(4-ClC₆H₅O), 4-(3-ClC₆H₅O), 4-(4-ClC₆H₅O), 4-(4-FC₆H₅O), 2-(3-CH₃OC₆H₅O), 2-(4-CH₃OC₆H₅O), 3-(4-CH₃OC₆H₅O), 4-(4-CH₃OC₆H₅O), 3-(4-CH₃C₆H₅O) were prepared and copolymerized with styrene. The monomers were synthesized by the piperidine catalyzed Knoevenagel condensation of phenoxy ring-substituted benzaldehydes and methyl cyanoacetate, and characterized by CHN analysis, IR, ¹H and ¹³C-NMR. All the ethylenes were copolymerized with styrene (M₁) in solution with radical initiation (ABCN) at 70°C. The compositions of the copolymers were calculated from nitrogen analysis and the structures were analyzed by IR, ¹H and ¹³C-NMR. The order of relative reactivity (1/r₁) for the monomers is 4-(4-CH₃OC₆H₅O) (6.07) > 3-(4-ClC₆H₅O) (3.38) > 3-(4-CH₃OC₆H₅O) (2.78) > 4-(3-ClC₆H₅O) (2.77) > 2-(4-ClC₆H₅O) (2.29) > 3-(4-CH₃C₆H₅O) (1.98) > 4-(4-FC₆H₅O) (1.92) > 4-(4-ClC₆H₅O) (1.89) > 2-(3-CH₃OC₆H₅O) (1.39) > 2-(4-CH₃OC₆H₅O) (0.90) > 4-(4-BrC₆H₅O) (0.77). Relatively high T_g of the copolymers in comparison with that of polystyrene indicates a decrease in chain mobility of the copolymer due to the high dipolar character of the trisubstituted ethylene monomer unit. Decomposition of the copolymers in nitrogen occurred in two steps, first in the 200-500°C range with residue (2.5-8.0% wt), which then decomposed in the 500-800°C range.     

Stability constants of metal complexes of macrocyclic ligands with pendant donor groups

Abstract

In our studies of the stability constants of metal complexes, we have investigated a number of macrocyclic ligands with pendant donor groups. The ligands are characterized by the fact that they have nitrogen donors in the macrocyclic ring and oxygen or sulfur donors in the pendant arms. These ligands represent seven different macrocycles, and by varying the pendant donor groups, ten different ligands are indicated. The affinities of these ligands for fifteen metal ions will be described. The Fe(III) complex of triazanonane with o-hydroxypyridyl or o-hydroxybenzyl pendant donor groups are the most stable ferric complexes ever reported. The In(III) complex of triazacyclononane with pendant mercaptoethyl donor groups, is exceptionally stable. Also, the Ca(II) complex of DOTA probably has the highest stability of any calcium(II) complex. These, and other comparisons will be made on the basis of the thermodynamic stability constant data for the ligands described.