Uniformly labeled, synthesis

Dichlorodibenzo-p-dioxin was prepared from isotopic potassium 2,4-dichlorophenate uniformly labeled with Ullman conditions gave a 20.5% yield. Small amounts of dichlorophenoxy chlorophenol were removed from the product by extraction with sodium hydroxide before purification by fractional sublimation and recrystallization from anisole. Chlorination of 2,7-dichlorodibenzo-p-dioxin in chloroform solution containing trace amounts of FeCls and 12 yielded a mixture of tri-, tetra-, and pentachloro substitution products. Purification by digestion in boiling chloroform, fractional sublimation, and recrystallization from anisole was effective in refining this product to 92% 2,3,7,8-tetrachloro isomer, which also contained 7% of the tri- and 1% of the penta-substituted dibenzo-p-dioxin. Mass spectroscopy was used exclusively to monitor the quality of the products during the synthesis. 

TAetection of the highly potent impurity, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), necessitated an environmental assessment of the impact of this contaminate. Information was rapidly needed on movement, persistence, and plant uptake to determine whether low concentrations reaching plants, soils, and water posed any threat to man and his environment. Because of the extreme toxicity of TCDD, utmost precautions were taken to reduce or minimize the risk of exposure to laboratory personnel. Synthesis of uniformly labeled C-TCDD by Muelder and Shadoff (I) greatly facilitated TCDD detection in soil and plant experiments. For unlabeled experiments it seemed wise to use only small quantities of diluted solutions in situations where decontamination was feasible and to rely on the sensitivity afforded by electron capture gas chromatography... 

Uniformly labelled 1 was prepared by the Reichstein-Griissner synthesis (see Scheme 4), starting with unifonnly labelled D-glu-cose.597-599 L-[2,3,4,5,6-14C]Ascorbic acid was prepared 800 from L-[U-14C]xylose by way of L-threo-pentos-2-ulose (9) (see Scheme 2). The L-[U-14C]xylose was prepared from D-[U-,4C]glucitol by using the route shown in Scheme 21. 

The ability of the rat to use lactate, pyruvate, and D-glucose to equal extents in the synthesis of 1-naphthyl D-glucosiduronic acid was demonstrated by Packham and Butler.158 The lactate and pyruvate were labeled with C14 at the carboxyl carbon lactate labeled at Cl and C2 was also used and the D-glucose was uniformly labeled. D-Glucuronate-C14 was utilized for D-glucuronic conjugation on oral administration, but was twenty times as effective after intraperitoneal injection. 

The mechanism of synthesis of hyaluronic acid in mammalian tissues appears to resemble that present in bacteria. The synthesis of hyaluronic acid in tissue culture has been widely studied (GIO, H7), and labeled hyaluronic acid is synthesized in vitro from uniformly labeled D-glucose by explants of human synovial tissue (Yl). An enzyme system has recently been found in mesodermal tissues which catalyzes the synthesis of hyaluronic acid directly from UDP-GNAc and UDP-GA (A5). 

Radioactive acetyl CoA can be generated by direct synthesis from C-acetate or from (3 oxidation of radioactive fatty acids, such as uniformly labeled palmitate. Examination of the reactions of the citric acid cycle reveals that neither of the two carbons that enter citrate horn acetate is removed as carbon dioxide during the first pass through the cycle. Labeled carbon from C-methyl-labeled acetate appears in C-2 and C-3 of oxaloacetate, because succinate is symmetrical, with either methylene carbon in that molecule labeling C-2 or C-3 of oxaloacetate. The conversion of oxaloacetate to phosphoenolpyruvate yields PEP labeled at C-2 or C-3 as well. Formation of glyceraldehyde 3-phosphate and its isomer dihydroxyacetone phosphate gives molecules, both labeled at carbons 2 and... 

Radioactive compounds are obtained in various ways 1. by synthesis in the laboratory 2. biosyntheti-cally, using the specific synthetic capacities of organisms 3. by radiochemical methods, e.g. tritiated compounds by Wilzbach tritiation. The second and third technique often yield unspecific, uniform labeling, but not necessarily an equal degree or labeling of all atoms. For example, in Wilzbach labeling, the C-T bonds are stable, but the O-T and N-T bonds are labile. 

Figure 8. Synthesis of labeled branched cyclomaltohexaose by reaction of pullulanase or isoamylase with cyclomaltohexaose and (A) miformly labeled maltose, (B) reducing-end labeled maltose, and (C) nonlabeled maltose and uniformly labeled cyclomaltohexaose. Symbols are the same as in Figure 2.

Glutamate, on the other hand, does not appear to be a direct precursor in histidine synthesis. In experiments with yeast grown on a medium containing inert L-glutamate and radioactive uniformly labeled glucose, the histidine was found to be highly radioactive. This appears to demon-... 

The emyme-catal sed synthesis of mono-, oligo- and poly-saccharides has been covered in a major review with 310 references, and a review has appeared on the synthesis of saccharides uniformly labelled with starting from D-[U- C]glucose. ... 

Glucose-C (uniformly labeled) Mice Fatty acids (also palmitic acid-C ) of liver, intestine, carcass Liver primary site of synthesis (239) ... 

One of the advantages of H-phosphonate chemistry is the ease with which phosphate analogs are prepared (Fig. 3). Phosphorothioates are readily prepared by oxidation with Sg in pyridine/CS2. This is a very clean reaction leading to virtually 100% substitution by sulfur on phosphate. This reaction has been extended to the synthesis of oligo-phosphorothioates (7,12,13), as well as uniformly labeled phosphorothioates (29,30). The synthesis of phosphorothioates has been discussed in greater detail in Chapter 8. 

Nick translation is one of the DNA-labeling techniques conventionally used to prepare hybridization probes. Nick translation utilizes combined activities of DNase I which introduces nicks (or single-strand breaks) and the 5 -(exo)nuclease and polymerase activities of E. coli DNA Pol I. While the 5 -nuclease activity of Pol I removes the nucleotides from the 5 -phosphoryl terminus, the polymerase activity carries out the sequential addition of nucleotides to the 3 -hydroxyl terminus, thus translocating the nick. When a highly radioactive nucleotide, e.g., [o - P]dATP, is included during the reaction, nick translation results in the uniform labeling of duplex molecules with a specific activity >10 cpm//zg DNA (1). Nick translation produces labeled DNA probes from both strands. Pol Ik, which lacks 5 -nuclease activity, cannot perform the nick translation, but it can carry out a strand displacement synthesis. 

Figure 9.11 The formation of two examples of the tetraponerines from ants. Glutamic acid, ornithine and y-aminobutyric acid uniformly labelled with 0, sodium acetate labelled in C-1 and C-2 with Q and [l,4- C]putrescine were all used to show the synthesis is as shown...

Frank and deMoss (1959) incubated a mixture of washed cells of P. aeruginosa, salts, and alanine- C for 24 hours, and then isolated and purified the pyocyanine produced. With uniformly-labeled L-alanine C as the substrate, the ratio of the specific activity of the pyocyanine synthesized to the specific activity of the alanine added was close to the value expected if the carbon atoms of the L-alanine were the sole source of carbon for the synthesis of pyocyanine. In another experiment, C-labeled cells were produced by growing P. aeruginosa on a medium containing uniformly-labeled L-alanine- C as the sole source of carbon. Such cells, when incubated with unlabeled alanine and salts, produced pyocyanine which contained little radioactivity. One can conclude from the above results that the pyocyanine synthesized was derived from alanine and not from preformed intermediates in the cells. The authors showed also that when DL-alanine-1- C and DL-alanine-2- C were tested as precursors in the system described, the former compound contributed relatively less radioactive carbon for the biosynthesis of pyocyanine than did the latter. Interpretation of these results is difficult because the distribution of radioactivity in the pyocyanine produced was not known, and because results obtained with DL-alanine may not be directly comparable with those obtained with L-alanine. 

The utilization of pyruvate or glycerol for the synthesis of pyocyanine was investigated in experiments in which uniformly-labeled L-alanine- C, salts, and washed cells were incubated with or without the addition of unlabeled p5n uvate or glycerol (Frank and deMoss, 1959)- After the media had been incubated for 20 or 24 hours, pyocyanine was isolated and its specific activity determined. It had been shown that with only L-alanine as a substrate that essentially 100% of the pyocyanine carbon atoms were derived from L-alanine (see preceding para-... 

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