Enzyme-assisted preparation of D-tert.-leucine.

Optically pure D-tert.-leucine was obtained by the enzymatic hydrolysis of (+/-)-N-acetyl-tert. leucine chloroethyl ester after about 50% conversion, this being catalyzed by a protease from Bacillus licheniformis (Alcalase), and subsequent acidic saponification of the recovered ester. Among the methyl, ethyl, octyl, chloroethyl and trichloroethyl esters, the chloroethyl ester exhibited the highest rate of hydrolysis.     

A modified method for the preparation of agarose.

A simple procedure for the preparation of agarose suitable for electrophoresis is developed in which anionic polysaccharides are removed by extracting the agar gel-granules with phosphate buffer (0.03 M, pH 6.8) containing urea (4 M), followed by electrophoresis in the same buffer system. Further, alkali treatment in the presence of sodium borohydride, eliminates electroendosmosis, giving essentially a neutral agarose, as judged by the electrophoretic behaviour of basic substances like crystal violet and cytochrome C. The purified agarose with yields 60-65%, has a sulphur content less than 0.1%, and forms rigid, transparent gels.     

Reprecipitation during the preparation of demineralized sections. II. Experimental observations.

Teeth were incompletely demineralized by immersion in unchanged 10% formic acid for 7 days. Reprecipitation deposits of secondary calcium phosphate were present in the dentin and soft tissues of the dental pulp and, if the final pH was 3 or greater, in the remnants of the periodontal ligament. The deposits in the dentin appeared to be intratubular. Deminieralized sections of teeth suspended in supersaturated solutions of brushite contained similar deposits in the soft tissues. It is suggested that reprecipitation of secondary calcium phosphates is a frequent intermediate stage during demineralization with formic acid.     

The enzymic preparation of diphosphoinositides.

A procedure for the preparation of diphosphoinositides is described. Triphosphoinositides isolated from bovine brain are hydrolysed by the triphosphoinositide phosphatase (EC 3.1.3.36) from Crithidia fasciculata in the presence of MgC12 and cetyltrimethyl-ammonium bromide. The diphosphoinositides produced are not degraded further and can be recovered from the reaction mixture in greater than 80% yield. The product is chromatographically pure and has the same structure (1-phosphatidylinositol 4-phosphate) as naturally occurring diphosphoinositides.     

Chemoenzymatic synthesis of spacer-linked oligosaccharides for the preparation of neoglycoproteins.

In the present work, the combination of chemical and enzymatic methods to obtain neoglycoproteins is described. Three bovine serum albumin (BSA)-conjugates, BSA-[GalNAc alpha-], BSA-[Gal(beta 1-3)GalNAc(alpha-], and BSA-[Neu5Ac(alpha 2-3)Gal(beta 1-3)GalNAc(alpha-], were prepared. alpha GalNAc derivatives were galactosylated employing crude beta-galactosidase from bovine testes. The use of oversaturated donor solutions (pNP beta Gal) enhanced the yields up to 60%. This method was verified using divalent structures as acceptors, that rendered di- and tri-galactosylated products. Further treatment of the disaccharides with CMP-Neu5Ac and alpha 2-3 sialyltransferase from pork liver led to formation of trisaccharides. Finally, mono-, di-, and trisaccharides were coupled to BSA employing a thiolic group introduced into the protein for Michael addition to a maleinimide group in the spacer-arm of the saccharide components. The results were monitored by HPLC and MALDI-TOF.     

Cyclic AMP and the preparation of the mouse uterus for implantation.

Experiments are described that demonstrate the ability of dibutyryl cyclic AMP (dcAMP) to induced oestrogen-like changes in the uteri of ovariectomized mice treated with progesterone. A single intraluminal injection of 6-14 micrograms dcAMP in 0-02 ml phosphate-buffered saline promoted cell division in the uterine stroma and sensitized the endometrium so that a decidual reaction could be induced by a subsequent injection of arachis oil into the uterine lumen. Since dcAMP has already been shown to induce implantation of diapausing mouse blastocysts in the absence of oestrogen, it is suggested that cyclic AMP is involved in egg implantation in rodents.     

Improved preparation method for lysogangliosides.

Lyso-GM3 and -GM1 gangliosides were prepared from the corresponding N,N'-dideacylated gangliosides using N-trifluoroacetylation at the sphingosine moiety, followed by N-acetylation and mild saponification. The blocking reaction was performed using a water-ether bilayer system at alkaline medium, in which the N-trifluoroacetylation occurred predominantly at the lipid moiety. Through the procedure, lysoGM3 and lysoGM1 were obtained with higher yields from the corresponding dideacylated gangliosides than through the previous method using 9-fluorenylmethoxycarbonyl chloride as a blocking group or of direct N-acetylation of it on liposomes containing starting ganglioside and other lipid. Chemical structures of the lysogangliosides and the synthetic intermediates were confirmed by the proton nuclear magnetic resonance spectrometry and negative fast atom bombardment-mass spectrometry.