Improvement of mechanical properties of orodispersible hyaluronic acid film by carboxymethyl cellulose addition.

Orodispersible films (ODF) were prepared with mixtures of hyaluronic acid (HA) and carboxymethyl cellulose (CMC), and the effect of CMC addition on the disintegration and mechanical properties of the composite films were examined. Low molecular weight HA (10 kDa) appeared more acceptable for ODF than high molecular weight HA (800 kDa) because of its rapid disintegration in the oral cavity. The composite films appeared similar to pullulan film with excellent transparency and surface smoothness. The disintegration time as well as mechanical properties of the films such as tensile strength and elongation at break were increased by the addition of CMC. Overall, the CMC addition, up to 35%, improved the mechanical properties of low molecular weight HA film within a proper range of disintegration time for ODF.

Structural and Thermodynamic Characteristics of Amyloidogenic Intermediates of ß-2-Microglobulin.

ß-2-microglobulin (ß2m) self-aggregates to form amyloid fibril in renal patients taking long-term dialysis treatment. Despite the extensive structural and mutation studies carried out so far, the molecular details on the factors that dictate amyloidogenic potential of ß2m remain elusive. Here we report molecular dynamics simulations followed by the solvation thermodynamic analyses on the wild-type ß2m and D76N, D59P, and W60C mutants at the native (N) and so-called aggregation-prone intermediate (IT) states, which are distinguished by the native cis- and non-native trans-Pro32 backbone conformations. Three major structural and thermodynamic characteristics of the IT-state relative to the N-state in ß2m protein are detected that contribute to the increased amyloidogenic potential: (i) the disruption of the edge D-strand, (ii) the increased solvent-exposed hydrophobic interface, and (iii) the increased solvation free energy (less affinity toward solvent water). Mutation effects on these three factors are shown to exhibit a good correlation with the experimentally observed distinct amyloidogenic propensity of the D76N (+), D59P (+), and W60C (-) mutants (+/- for enhanced/decreased). Our analyses thus identify the structural and thermodynamic characteristics of the amyloidogenic intermediates, which will serve to uncover molecular mechanisms and driving forces in ß2m amyloid fibril formation.

Stitched α-helical peptides via bis ring-closing metathesis.

Conformationally stabilized α-helical peptides are capable of inhibiting disease-relevant intracellular or extracellular protein-protein interactions in vivo. We have previously reported that the employment of ring-closing metathesis to introduce a single all-hydrocarbon staple along one face of an α-helical peptide greatly increases α-helical content, binding affinity to a target protein, cell penetration through active transport, and resistance to proteolytic degradation. In an effort to improve upon this technology for stabilizing a peptide in a bioactive α-helical conformation, we report the discovery of an efficient and selective bis ring-closing metathesis reaction leading to peptides bearing multiple contiguous staples connected by a central spiro ring junction. Circular dichroism spectroscopy, NMR, and computational analyses have been used to investigate the conformation of these "stitched" peptides, which are shown to exhibit remarkable thermal stabilities. Likewise, trypsin proteolysis assays confirm the achievement of a structural rigidity unmatched by peptides bearing a single staple. Furthermore, fluorescence-activated cell sorting (FACS) and confocal microscopy assays demonstrate that stitched peptides display superior cell penetrating ability compared to their stapled counterparts, suggesting that this technology may be useful not only in the context of enhancing the drug-like properties of α-helical peptides but also in producing potent agents for the intracellular delivery of proteins and oligonucleotides.

Elucidating the Molecular Origin of Hydrolysis Energy of Pyrophosphate in Water.

The molecular origin of the energy produced by the ATP hydrolysis has been one of the long-standing fundamental issues. A classical view is that the negative hydrolysis free energy of ATP originates from intramolecular effects connected with the backbone P-O bond, so called "high-energy bond". On the other hand, it has also been recognized that solvation effects are essential in determining the hydrolysis free energy. Here, using the 3D-RISM-SCF (three-dimensional reference interaction site model self-consistent field) theory that integrates the ab initio quantum chemistry method and the statistical mechanical theory of liquids, we investigate the molecular origin of hydrolysis free energy of pyrophosphate, an ATP analogue, in water. We demonstrate that our theory quantitatively reproduces the experimental results without the use of empirical parameters. We clarify the crucial role of water in converting the hydrolysis free energy in the gas phase determined solely by intramolecular effects, which ranges from endothermic, thermoneutral, to highly exothermic depending on the charged state of pyrophosphate, into moderately exothermic in the aqueous phase irrespective of the charged state as observed in experimental data. We elucidate that this is brought about by different natures of solute-water interactions depending on the charged state of solute species: the hydration free energy of low-charged state is mainly subjected to short-range hydrogen-bonds, while that of high-charged state is dominated by long-range electrostatic interactions. We thus provide unambiguous evidence on the critical role of water in determining the ATP hydrolysis free energy.

Stereoconversion of amino acids and peptides in uryl-pendant binol schiff bases.

(S)-2-Hydroxy-2'-(3-phenyluryl-benzyl)-1,1'-binaphthyl-3-carboxaldehyde (1) forms Schiff bases with a wide range of nonderivatized amino acids, including unnatural ones. Multiple hydrogen bonds, including resonance-assisted ones, fix the whole orientation of the imine and provoke structural rigidity around the imine C==N bond. Due to the structural difference and the increase in acidity of the alpha proton of the amino acid, the imine formed with an L-amino acid (1-l-aa) is converted into the imine of the D-amino acid (1-D-aa), with a D/L ratio of more than 10 for most amino acids at equilibrium. N-terminal amino acids in dipeptides are also predominantly epimerized to the D form upon imine formation with 1. Density functional theory calculations show that 1-D-Ala is more stable than 1-L-Ala by 1.64 kcal mol(-1), a value that is in qualitative agreement with the experimental result. Deuterium exchange of the alpha proton of alanine in the imine form was studied by (1)H NMR spectroscopy and the results support a stepwise mechanism in the L-into-D conversion rather than a concerted one; that is, deprotonation and protonation take place in a sequential manner. The deprotonation rate of L-Ala is approximately 16 times faster than that of D-Ala. The protonation step, however, appears to favor L-amino acid production, which prevents a much higher predominance of the D form in the imine. Receptor 1 and the predominantly D-form amino acid can be recovered from the imine by simple extraction under acidic conditions. Hence, 1 is a useful auxiliary to produce D-amino acids of industrial interest by the conversion of naturally occurring L-amino acids or relatively easily obtainable racemic amino acids.

Cu2+-induced intermolecular static excimer formation of pyrenealkylamine.

Synthesis of monopyrenylalkylamine derivative 1 and its fluorescence behavior for Cu2+ in H2O/CH3CN (1:1, v/v) were investigated. Upon Cu2+ binding, 1, bearing a sulfonamide group, exhibited a marked excimer emission at 455 nm along with a weak monomer emission at 375 nm. The excimer emission, driven by formation of an intermolecular pyrenyl static excimer upon Cu2+ binding to the sulfonamide group, is rationalized by experimental and theoretical DFT calculation results.

Structural determination of monoacylglycerols extracted from marine sponge by fast atom bombardment tandem mass spectrometry.

Five new monoacylglycerols (MAGs) were isolated from the marine sponge Stelletta sp. by reversed-phase high-performance liquid chromatography and analyzed by positive ion fast atom bombardment mass spectrometry (FAB-MS). FAB mass spectra of these compounds produced abundant sodium-adducted molecules [M+Na]+ from a mixture of 3-nitrobenzyl alcohol and sodium iodide. The structural elucidation of these sponge MAGs was carried out by FAB tandem mass spectrometry (MS/MS). To find diagnostic ions for the characterization of the MAGs, authentic MAGs were initially analyzed by collision-induced dissociation (CID) MS/MS. The CID MS/MS of [M+Na]+ precursor ions resulted in the formation of numerous characteristic product ions via a series of dissociative processes. The product ions formed by charge-remote fragmentation (CRF) provided important information for the characterization of acyl chains substituted at the glycerol backbone, and product ions at m/z 84, 97, 113 and 139 were diagnostic for the sodiated glycerol backbone. On the basis of these fragmentation patterns, the structures of five MAGs extracted from marine sponge were elucidated. In addition, high-resolution mass measurement was performed to obtain the elemental compositions of the MAGs.