N-α,O-Dibenzyl-L-serine methyl ester hydrochloride

Surfactants with the cationic functionality based on an amino acid structure have been used to prepare novel biocompatible devices for the controlled encapsulation and release of DNA. We report here the formation of DNA gel particles mixing DNA (either single- (ssDNA) or double-stranded (dsDNA)) with two different single-chain amino acid-based surfactants: arginine-N-lauroyl amide dihydrochloride (ALA) and N(alpha)-lauroyl-arginine-methyl ester hydrochloride (LAM). The degree of DNA entrapment, the swelling/deswelling behavior, and the DNA release kinetics have been studied as a function of both the number of charges in the polar head of the amino acid-based surfactant and the secondary structure of the nucleic acid. Analysis of the data indicates a stronger interaction of ALA with DNA, compared with LAM, mainly attributed to the double charge carried by the former surfactant compared to the singly charged headgroup of the latter species. The stronger interaction with amphiphiles for ssDNA compared with dsDNA suggests the important role of hydrophobic interactions in DNA. Data on the microstructure of the complexes obtained from small-angle X-ray scattering (SAXS) of the particles strongly suggests a hexagonal packing. It was found that, the shorter the lattice parameter, the stronger the surfactant-DNA interaction and the slower the DNA release kinetics. Complexation and neutralization of DNA on the DNA gel particles was confirmed by agarose gel electrophoresis measurements.

The supramolecular interactions of Mg(ii) and N(alpha)-4-tosyl-l-arginine methyl ester hydrochloride (TAME) with ATP have been investigated using (1)H and (31)P NMR spectra. Furthermore, the hydrolysis of ATP catalyzed by Mg(ii) and TAME has been studied at 60 degrees C and pH 7 using (31)P NMR spectra. In the Mg(ii)-ATP-TAME ternary system, the binding interaction of Mg(2+) with ATP involves not only N1 and N7 in the adenine ring but also beta- and gamma-phosphate of ATP. The binding forces are mainly electrostatic interaction and cation (Mg(2+))-pi interaction. The guanidinium group and the aromatic ring of TAME interacts with ATP by beta and gamma phosphate and the adenine ring of ATP. The binding forces are mainly electrostatic interactions and pi-pi stacking. A significant difference between the binary and the ternary system indicates that TAME is essential to the stablization of the intermediate. Kinetic studies show that the hydrolysis rate constant of ATP is 2.16 x 10(-2) h(-1) at pH 7 in the Mg(ii)-TAME-ATP ternary system. The Mg(ii) ion and TAME can accelerate the ATP hydrolysis process. A possible mechanism has been proposed that the hydrolysis occurs through an addition-elimination, in which the phosphoramidate intermediate was observed at 3.21 ppm in the (31)P NMR of the ternary system. These results provide further information concerning the effect of the key amino acid residue and metal ions as cofactors of ATPase on ATP synthesis/hydrolysis at the molecular level.

Aspergillus awamori was cultivated in a modified Breccia medium, and the extracellular fraction was obtained, which presented 260 ± 15 µg of protein/mg and specific protease activity of 3.87 ± 0.52 mM.min-1.mg of protein-1 using Nα-p-tosyl-L-arginine methyl ester hydrochloride (L-TAME) as substrate. This fraction showed major proteins about 104 and 44 kDa and maximal protease activity at pH 5.5, 6.5, and 9.0, suggesting that A. awamori secretes acidic, neutral, and alkaline proteases with expressive thermal stability, however, aspartic protease was the most important activity. When yeast extract was supplemented to a modified Breccia medium, A. awamori protein secretion and protease activity were maximal and the affinity chromatography on pepstatin-agarose was employed to isolate the aspartic protease activity, which was called ASPA, with approximately 75 kDa. ASPA maximal activity was obtained at pH 4.5 and 6.5, and 50 °C. Pepstatin inhibited about 80% of ASPA activity, with IC50 and Ki values of 0.154 and 0.072 μM, respectively. ASPA cleaved protein and peptides substrates with the highest activity against gelatin (95 U/mg) and good peptidase activity with KM 0.0589 mM and Vmax 1.909 mM.min-1.mg protein-1, using L-TAME as substrate. A. awamori extracellular fraction is a source of proteases with important activity, and the supplementation of modified Breccia medium increased the aspartic protease production. This enzyme presented different biochemical characteristics from the previously reported A. awamori aspartic proteases. Therefore, ASPA is an excellent candidate for biotechnological application due to its important activity and thermostability.