Determination of enantiomeric excess of alpha-hydroxy-3-phenoxybenzeneacetonitrile and its n-butyl ester by chiral high-performance liquid chromatography.

Determination of enantiomeric excess of alpha-hydroxy-3-phenoxybenzeneacetonitrile, an important intermediate in the production of several pyrethroid insecticides, is usually done after derivatization and gas chromatographic analysis on a beta-cyclodextrin-based column. In this communication we report a direct determination of enantiomeric excess of alpha-hydroxy-3-phenoxybenzeneacetonitrile and its n-butyl ester by chiral HPLC on Chiralcel OJ (Daicel, Japan) in a single run without derivatization.

Reactivity of trypsin in reverse micelles: pH-effects on the W0 versus enzyme activity profiles.

pH-Dependence of hydrolytic activity of trypsin has been studied in cationic reverse micellar system of cetyltrimethylammonium bromide (CTAB) in (50% v/v) chloroform/isooctane using a positively charged substrate N(alpha)-benzoyl-L-arginine ethyl ester (BAEE). The pH of the medium was varied from 4.0 to 8.5 with addition of 0.025 M citrate-phosphate buffer containing 1 mM CaCl2. Optimum pH for maximum enzyme activity, pH(opt) in reverse micelles is found to be similar to that observed in bulk aqueous solution (8.0-8.5). However, changes in activity of trypsin (k(cat)) as a function of water content W0 (W0 = [H2O]/[CTAB]) in reverse micelles are found to be pH dependent. At low pH (4.0) and low water content (W0 = 5) the enzyme is more active in reverse micelles than in bulk aqueous solution by a factor of 2. This 'superactivity' is lost at higher W0 values and the k(cat) in reverse micelles is found to be similar to that observed in aqueous bulk. At pH 5, the enzyme activity is found to be independent of W0 while at pH 6.0-6.5 the enzyme activity is low at W0 5 and increases with water content to a constant value which is still 50% lower than that in aqueous buffer. Above pH 7, the W0-activity profile becomes distinctly bell shaped with W0 optimum around 10-15. The enzyme activity at optimum W0 is close to that observed in aqueous bulk.

Reverse micellar extraction of antibiotics from aqueous solutions.

Several antibiotics such as erythromycin, oxytetracyclin, benzylpenicillin, and actidione were extracted from aqueous buffers into reverse micellar solution of bis(2-ethylhexyl) sulfosuccinate sodium salt (AOT) in isooctane and recovered with high efficiency under mild conditions. Preliminary experiments with oxytetracycline dissolved in a fermentation broth indicate that the antibiotic can be selectively extracted from the broth and recovered efficiently without serious loss of potency.

Comparative pharmacokinetic evaluation of compressed suppositories of diclofenac sodium in humans.

Diclofenac sodium (CAS 15307-79-6) suppositories were formulated using polyethylene glycol 4000 as the base by dispersing the drug in the molten base, congealing the mass, followed by pulverization, sieving and subsequent compression of the resultant granules. These suppositories were evaluated with respect to their pharmacokinetic behaviour in 12 healthy, male human volunteers. The results were compared with those obtained after oral administration of a commercial enteric coated tablet. Bioequivalence between rectal suppositories and commercial tablets was observed with respect to AUC0-infinity and Cmax. However, tmax differed significantly (p < 0.05) in case of rectal administration (0.625 +/- 0.065 h) compared to oral tablet (1.58 +/- 0.06 h). The relative rectal bioavailability was 107.19 +/- 3.2.

Protein-protein interactions in reverse micelles: trypsin shows superactivity towards a protein substrate alpha-chymotrypsinogen A in reverse micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in isooctane.

Protein-protein interactions in reverse micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT), in isooctane containing varying amounts of Tris buffer are studied by using activation of alpha-chymotrypsinogen A by trypsin (EC 3.4.21.4) to alpha-chymotrypsin (EC 3.4.21.1) as a model reaction. It has been found that protein-protein interactions are strongly dependent on the water content of the medium. At an optimum water content the activation reaction in reverse micelles is faster than reaction in water by a factor of 21.3. At lower water content both reaction rates and the conversion of alpha-chymotrypsinogen A into alpha-chymotrypsin decrease with decrease in water content of the reaction medium.

Immobilized enzymes in reverse micelles: studies with gel-entrapped trypsin and alpha-chymotrypsin in AOT reverse micelles.

Trypsin and alpha-chymotrypsin were immobilized by gelentrapment in polyacrylamide cross-linked with N,N(1)-methylenebisacrylamide. The immobilized enzymes are catalytically efficient in suspensions of reverse micelles formed in isooctane by bis(2-ethylhexyl) sodium sulfosuccinate (AOT) and water. Both entrapped enzymes are stable in reverse micellar suspension at room temperature and pH 8.2 for 3 days and lose 30-40% activity after 1 week. The enzymes obey Michaelis-Menten kinetics in the investigated concentration range with K(m) values higher than those in solution. Activity of the enzymes is independent of the water content of the micellar solution. No shift in pH optimum was observed for immobilized trypsin activity toward Nalpha-benzoyl-L-arginine ethyl ester. The utility of the procedure, which combines the advantage of enzyme immobilization and enzymology in reverse micelles, is illustrated by an example of peptide synthesis. In particular, peptide synthesis (e. g., Z--Ala--Phe--Leu--NH(2)) using water-insoluble substrate has been performed with gelentrapped alpha-chymotrypsin in reverse micellar suspension with the advantage of efficient enzyme recycling.

Structure and activity of trypsin in reverse micelles.

The kinetic properties of trypsin have been studied in reverse micelles formed by two surfactant systems, namely bis(2-ethylhexyl) sodium sulfosuccinate (AOT) in isooctane, and hexadecyltrimethyl ammonium bromide (CTAB) in chloroform/isooctane (1:1, by vol.). Three substrates have been used, namely N alpha-benzoyl-L-Arg ethyl ester, N alpha-benzoyl-L-Phe-L-Val-L-Arg p-nitroanilide (BzPheValArg-NH-Np) in AOT and N alpha-benzyloxycarbonyl-L-Lys p-nitrophenyl ester (ZLysO-Np) in CTAB. One of the main aims of the work was to compare the behaviour of trypsin in reverse micelles with that of alpha-chymotrypsin, for which an enhancement of kcat had been observed with respect to aqueous solutions. The pH profile is not significantly altered in reverse micelles with respect to water, however the kinetic parameters (kcat and Km) differ widely from one another, and are markedly affected by the micellar conditions, in particular by the water content wo (wo = [H2O]/[AOT]). Whereas in the case of BzPheValArg-NH-Np kcat is much smaller than in water, in the case of ZLysO-Np at pH 3.2 (but not at pH 6.0) a slight enhancement with respect to water is observed. On the basis of rapid kinetic spectrophotometry (stopped-flow) and solvent isotope effect studies, this enhancement is ascribed to a change in the rate-limiting step (acylation rather than hydrolysis). As in the case of alpha-chymotrypsin, the maximal activity is found for all substrates at rather small wo values (below 12), which is taken to suggest that the enzyme works better when is surrounded by only a few layers of tightly bound water. Spectroscopic studies [ultraviolet absorption, circular dichroism (CD) and fluorescence] have been carried out as a function of wo. Whereas the absorption properties are practically unchanged, the CD spectrum in AOT micelles has a lower intensity than in water, which is interpreted as a partial unfolding. The intensity is partly restored when Ca2+ ions are added, indicating that the micellar environment may cause a partial denaturation by depleting it of calcium ions. Fluorescence data show that the emission properties of the protein in reverse micelles match those in aqueous solution at around wo = 13 approx., whereas lambda max shifts towards the red by increasing wo, indicating an exposure of the tryptophan residues and probably an unfolding of the whole protein, at wo values above 15. Finally the reaction between trypsin and its specific macromolecular Kunitz inhibitor from soybeans is studied.(ABSTRACT TRUNCATED AT 400 WORDS)