The synthesis of cholesteryl alkyl ethers

Seventeen cholesteryl alkyl ethers were synthesized through alcoholysis of cholesterol p-toluenesulfonate. This method was found superior to the etherification of sodium or potassium cholesterylate with alkyl halides or methanesulfonates, especially for the preparation of long-chain unsaturated alkyl ethers of [7(n)-³H]cholesterol of high specific activity.     

A simple chemical method for the synthesis of catechol estrogens

The preparation of 2-hydroxyestrone, 2-hydroxyestradiol-17, 4-hydroxyestrone and 4-hydroxyestradiol-17β by a simple one-step chemical reaction, treatment with potassium nitrosodisulfonate, is described. The structures of the products were established by nmr, ultraviolet, infrared and mass spectra as well as from their chemical and chromatographic properties.     

A simple method for the synthesis of benzyl 4-O-benzylhexopyranosides

The kinetics of hydrolysis in dilute sulfuric acid of xylo-oligosaccharides ranging between the di- and penta-oligosaccharides has been studied. One of the two terminal bonds and each internal bond of all xylo-oligosaccharides tested were hydrolysed at the same rate. The hydrolytic rate of the other terminal bond was the same as that of xylobiose, which was 1.8 times greater than that of an internal bond. The rates of hydrolysis of xylo-oligosaccharides have been described as functions of the reaction temperature and concentration of sulfuric acid. It has been shown that the yield of xylose in hydrolysis of xylo-oligosaccharides by sulfuric acid may be calculated from the ratio ( 1.8) of the rate for xylobiose to that of an internal bond and the empirical equation that describes the rate-constant for xylobiose.     

A simple and convenient method for the synthesis of pyranoid glycals

A simple, mild, and environmentally benign synthesis procedure of pyranoid glycals is described. In a novel fashion, protected glycopyranosyl bromides undergo the reductive elimination in the presence of zinc in phosphate buffer at room temperature. The pyranoid glycals were obtained in good-to-excellent yields (18 examples).     

A simple and convenient method for acyclonucleoside synthesis.

Introduction of acyclic chain for synthesis of acyclonucleoside derivatives was achieved in a simple and convenient way. Silylated pyrimidine or purine bases were treated with 1,3-dioxolane, trimethyl chlorosilane and metal iodide, such as KI and NaI, all together at room temperature. By this method, 2-thiopyrimidine derivatives were also obtained in good yield, using 2 molecular equivalents of 1,3-dioxolane.     

Radiolabeled cholesteryl ethers: A need to analyze for biological stability before use

Radiolabeled cholesteryl ethers are widely used as non-metabolizable tracers for lipoproteins and lipid emulsions in a variety of in vitro and in vivo experiments. Since cholesteryl ethers do not leave cells after uptake and are not hydrolyzed by mammalian cellular enzymes, these compounds can act as markers for cumulative cell uptakes of labeled particles. We have employed [³H]cholesteryl oleoyl ether to study the uptake and distribution of triglyceride-rich emulsion particles on animal models. However, questionable unexpected results compelled us to analyze the stability of these ethers. We tested the stability of two commercially available radiolabeled cholesteryl ethers - [³H]cholesteryl oleoyl ether and [³H]cholesteryl hexadecyl ether from different suppliers, employing in vitro, in vivo and chemical model systems. Our results show that, among the two cholesteryl ethers tested, one ether was hydrolyzed to free cholesterol in vitro, in vivo and chemically under alkaline hydrolyzing agent. Free cholesterol, unlike cholesteryl ether, can then re-enter the circulation leading to confounding results. The other ether was not hydrolyzed to free cholesterol and remained as a stable ether. Hence, radiolabeled cholesteryl ethers should be analyzed for biological stability before utilizing them for in vitro or in vivo experiments.     

A simple, convenient method for the synthesis of acyclic analogs of guanosine

A simple convenient method for the synthesis of guanosine acyclic analogues with the nucleic base attached to the primary carbon atom is proposed. The method involves treatment of trimethylsilyl derivative of guanine with agents of general formula ROCH2Cl in the absence of Lewis acids. 9-Substituted derivatives of guanine were prepared by this method with 50-65% yields.     

A simple enzymic method for the synthesis of [32P]phosphoenolpyruvate

A rapid and simple enzymic method is described for the synthesis of [³²P]phosphoenolpyruvate from [³²P]P_i, with a reproducible yield of 74%. The final product was shown to be a good substrate for pyruvate kinase (EC 2.7.1.40).     

A simple method for the synthesis of cholesterol esters in high yield.

A simple and convenient synthesis of cholesterol esters from cholesterol and fatty acid anhydrides is described. The high, reproducible yield of cholesterol ester and the small excess of fatty acid used makes this method attractive for the preparation of cholesterol esters using difficultly obtained fatty acids.     

A method of direct measurement for the enzymatic determination of cholesteryl esters

A direct measurement method for the enzymatic determination of cholesteryl esters (CEs) without measuring total cholesterol (TC) and free cholesterol (FC) is described. In the first step, hydrogen peroxide generated by cholesterol oxidase from FC was decomposed by catalase. In the second step, CE was measured by enzymatic determination using a colorimetric method or a fluorometric method. The measurement sensitivity of the fluorometric method was more than 20 times that of the colorimetric method. Optimal conditions of the assay were determined, and examples of measured CE in human plasma, rat liver, and cultured cells are indicated. The method of directly measuring CE was simple and has exceptional reproducibility compared with the technique of subtracting FC from TC using each measured TC and FC.     

A simple method for the solid phase synthesis of oligodeoxynucleotides containing O4-alkylthymine.

A route to prepare the cyanoethyl-phosphoramidite monomer of O4-alkylthymine and a method for the routine solid-phase synthesis of oligodeoxynucleotides containing O4-alkylthymine are described. This method has been used to make DNA sequences up to 48 bases in length. The amino function of the adenine and guanine in the sequence were protected with the phenoxyacetyl group, and that of cytosine with the isobutyryl group. The phosphodiesters were protected with the cyanoethyl group. This allowed complete deprotection of the oligomer with alkoxide ions (methanol/1,8- diazabicyclo[5.4.0]undec-7-ene (DBU) for the oligomers containing O4-methylthymine, or ethanol/DBU for those containing O4-ethylthymine) thus avoiding the use of ammonia which is known to attack the O4-alkylthymine to form 5-methylcytosine. There was no detectable loss of the alkyl group to form thymine.     

A simple method for following the fate of alanine-containing components in murein synthesis inEscherichia coli

The fate of the alanine-containing components in murein synthesis was followed by incorporation of¹⁴C-l-alanine inE. coli under conditions allowing cell-wall synthesis while preventing protein synthesis. The components were separated by chromatography and detected by autoradiography.Spots containing murein, cell-wall precursors, alanine andd-alanyl-d-alanine were identified. A further component was probably identical to pyruvic acid. Two unidentified spots were found in the region where lipid-intermediates in cell-wall synthesis are usually found. However, the absence of turnover of these two components was at variance with the proposed properties of the lipidintermediates. d-Alanyl-d-alanine and the component which is probably identical to pyruvic acid were excreted into the medium, whereas murein and cell-wall precursors were found in the cellular fraction.The influence of the concentration of alanine, and of the number of cells per ml, on the acid-precipitable activity were studied. The latter increased during, at least, the first two hours and represented mainly lysozyme-degradable material.Significant turnover of murein could be detected neither in the presence nor in the absence of protein synthesis.A time course of the activity of the radioactive components is provided. The influence of a number of antibiotics inhibiting cell-wall synthesis on the acid-precipitable activity and on the activity of the main intermediates in murein synthesis was studied.We thank Mrs. Arna van Schijndel-van Dam and Mr. A. A. G. Verweij for excellent assistance. We thank Dr. P. E. Reynolds (University of Cambridge) for teaching one of us (E J. J. L.) several techniques in the field of bacterial cell walls, and Dr. H. J. W. Wijsman for stimulating discussions.