Phosphate esters isolation

Chemical synthesis of 1-phosphate esters of 2-amino-2-deoxy-n-glucose has been described.37 127 Reaction of 3,4,6-tri-0-acetyl-2-amino-2-deoxy-a-D-glucosyl bromide hydrobromide (XLII) with triethylammonium diphenyl phosphate gave the l-(diphenyl phosphate) ester, isolated as its hydrochloride (XLIII). Cleavage of the phenyl groups and subsequent deacetylation gave 2-amino-2-deoxy-D-glucosyl phosphate as the crystalline, dipolar-ionic, monopotassium salt (XLV). 

Phosphate Esters. The phosphorylation of sucrose using sodium metaphosphate has been reported (78). Lyoptulization of a sodium metaphosphate solution of sucrose at pH 5 for 20 hours followed by storage at 80°C for five days produced a mixture of sucrose monophosphates. These products were isolated by preparative hplc, with a calculated yield of 27% based on all organic phosphate as sucrose monoesters. Small proportions of glucose and fmctose were also formed. 

The flame retardant mechanism for phosphorus compounds varies with the phosphorus compound, the polymer and the combustion conditions (5). For example, some phosphorus compounds decompose to phosphoric acids and polyphosphates. A viscous surface glass forms and shields the polymer from the flame. If the phosphoric acid reacts with the polymer, e.g., to form a phosphate ester with subsequent decomposition, a dense surface char may form. These coatings serve as a physical barrier to heat transfer from the flame to the polymer and to diffusion of gases in other words, fuel (the polymer) is isolated from heat and oxygen. 

Identification of the energy source for muscle contraction and determination of the order in which the phosphate esters were metabolized was helped by the use of inhibitors. These inhibitors blocked different stages in glycolysis and caused preceding substrates to accumulate in quantities which could greatly exceed those normally present. The compounds were then isolated, identified, and used as specific substrates to identify the enzymes involved in their metabolism. Iodoacetic acid (IAc) was one of the most important inhibitors used to analyze glycolysis. 

Biological. Incubation of C-ring labeled endothall (10 mg/mL) by Arthwbactersp., which was isolated from pond water and a hydrosol, in aerobic sediment-water suspensions revealed that after 30 d, 40% evolved as CO2. Glutamic acid was the major transformation product. Minor products were alanine, citric, and aspartic acids and unidentified products, some of which were tentativeiy identified as phosphate esters (Sikka and Saxena, 1973). In pond water treated with endothall (2 and 4 ppm), detectable levels were found after 7 d (Sikka and Rice, 1973). Biodegradation was rapid in an Ontario soil sample. After 1 wk, 70% of endothall added was converted to carbon dioxide (Simsiman et al, 1976). 

Schistosoma japonicum. The carbobenzoxy (CBz) protected template 160 was initially converted to the a, p-dehydrolactone 161 via the phosphate ester, before undergoing cycloaddition to ylide 162, generated in situ by acidic treatment of A(-benzyl-A(-(methoxymethyl)trimethylsilyl amine. The resultant cycloadduct (163) was isolated in 94% yield as a single diastereoisomer. Destructive template removal, by catalytic hydrogenation, released (5)-( )-cucurbitine, after ion-exchange chromatography, as the free amino acid in 90% yield (Scheme 3.46). 

Research to date focused on isolating insecticidal prototype leads from marine origin has resulted in the report of about 40 active compounds.44 In an attempt to summarize these compounds and their activity margins, they have been categorized into seven classes of chemical structures polyhalogenated monoterpenes, polyhalogenated C15-metabolites, diterpenes, peptides and amino acids, phosphate esters, sulfur-containing derivatives, and macrolides. 

Kumar, F. A., Ferchmin, P. A. and Caputto, R. 1965. Isolation and identification of a lactose phosphate ester from cow colostrum. Biochem. Biophys. Res. Commun. 20, 60-62. 

The preceding experiments prove that there is an intermediate on the reaction pathway in each case, the measured rate constants for the formation and decay of the intermediate are at least as high as the value of kcat for the hydrolysis of the ester in the steady state. They do not, however, prove what the intermediate is. The evidence for covalent modification of Ser-195 of the enzyme stems from the early experiments on the irreversible inhibition of the enzyme by organo-phosphates such as diisopropyl fluorophosphate the inhibited protein was subjected to partial hydrolysis, and the peptide containing the phosphate ester was isolated and shown to be esterified on Ser-195.1516 The ultimate characterization of acylenzymes has come from x-ray diffraction studies of nonspecific acylenzymes at low pH, where they are stable (e.g., indolylacryloyl-chymotrypsin),17 and of specific acylenzymes at subzero temperatures and at low pH.18 When stable solutions of acylenzymes are restored to conditions under which they are unstable, they are found to react at the required rate. These experiments thus prove that the acylenzyme does occur on the reaction pathway. They do not rule out, however, the possibility that there are further intermediates. For example, they do not rule out an initial acylation on His-57 followed by rapid intramolecular transfer. Evidence concerning this and any other hypothetical intermediates must come from additional kinetic experiments and examination of the crystal structure of the enzyme. 

More recently, isotopic labeling experiments have assumed a major role in establishing the detailed mechanism of enzymic action. It was shown that alkaline phosphatase possesses transferase activity whereby a phos-phoryl residue is transferred directly from a phosphate ester to an acceptor alcohol (18). Later it was found that the enzyme could be specifically labeled at a serine residue with 32P-Pi (19) and that 32P-phosphoserine could also be isolated after incubation with 32P-glucose 6-phosphate (20), providing strong evidence that a phosphoryl enzyme is an intermediate in the hydrolysis of phosphomonoesters. The metal-ion status of alkaline phosphatase is now reasonably well resolved (21-23). Like E. coli phosphatase it is a zinc metalloenzyme with 2-3 g-atom of Zn2+ per mole of enzyme. The metal is essential for catalytic activity and possibly also for maintenance of native enzyme structure. 

The incubation digest (7.0 ml) contained 1 ml of 0.022 M phenyl phosphate 2.5 ml of 0.1 M acetate buffer, pH 5.0 0.5 ml of test enzyme solution and 3.0 ml of solutions of acceptors giving a final concentration as shown in the third column. Incubation time, 30 min. Digests were inactivated by 3.0 ml of 10% trichloroacetic acid solution and were analyzed for phenol and inorganic phosphate. In the case of the standard acceptor, 1,4-butanediol, the expected transfer product, 1,4-butanediol phosphate, was isolated in a yield of 35% from a large-scale experiment. The hydrolysis of this phosphate ester by prostatic acid phosphatase liberated approximately equimolar amounts of 1,4-butanediol and inorganic phosphate. 

The enzyme was purified from Candida utilis in 1965 by Rosen et al. (8Q). Dried yeast was allowed to autolyze in phosphate buffer at pH 7.5 for 48 hr, and the enzyme was isolated in crystalline form from these autolysates by a procedure which included heating to 55° at pH 5.0, fractionation with ammonium sulfate, and purification on phospho-cellulose columns from which the enzyme was specifically eluted with malonate buffer containing 2.0 mM FDP. Crystallization was carried out by addition of ammonium sulfate in the presence of mM magnesium chloride. The Candida enzyme was more active than the mammalian FDPases at room temperature and pH 9.5 the crystalline protein catalyzed the hydrolysis of 83 /nnoles of FDP per minute per milligram of protein. The enzyme was completely inactive with other phosphate esters, including sedoheptulose diphosphate, ribulose diphosphate, and fructose 1- or fructose 6-phosphates. Nor was the activity of the enzyme inhibited by any of these compounds. Optimum activity was observed at concentrations of FDP between 0.05 and 0.5 mM higher concentrations of FDP (5 mM) were inhibitory. 

The six principal B6 vitamers are widely distributed in foods (102,103). They include pyridoxine (PN), pyridoxal (PL), pyridoxamine (PM), and their 5 -phosphate esters, pyridoxine phosphate (PNP), pyridoxal phosphate (PLP), and pyridoxamine phosphate (PMP) (Fig. 5). The predominate B6 vitamer in animal-based foods is PLP, whereas plant products generally contain PN and PM or their phosphorylated forms. Conjugated vitamers in the form of PN-glycosides have also been isolated from plant-based foods. Pyridoxal is readily converted to PM during cooking and food processing. Total vitamin B6 is the sum of the six principal vitamers inclusion of the conjugated forms depends on the extraction procedure. 

Manson and Lampen243 reported that they obtained the phosphorolysis and arsenolysis of hypoxanthine desoxyriboside by enzyme preparations from calf-thymus gland and rat liver. An acid-stable phosphate ester was isolated as a product of phosphorolysis. Results to be outlined suggested that this ester was 2-desoxy-D-ribose 5-phosphate and evidence was obtained for its formation by a mutase type reaction from 2-desoxy-D-ribose 1-phosphate. This evidence was extended and reinforced when Manson and Lampen244 obtained indications for the formation of desoxy-D-ribose 1-phosphate during the phosphorolysis of thymidine. Consequently the conversions outlined may be depicted as shown. 

Analyses of the lipid A components show a general similarity of composition between the lipid A material isolated from each of the seven immunotypes. All contain a B-D-(l->6)-linked 2-amino-2-deoxy-D-glucose disaccharide (17,18), substituted at both amino groups and at most of the hydroxyI—groups by 3-hydroxy fatty acyl chains, and phosphate ester groups are present at 0-1 and 0-4. ... 

The term nucleoside was originally proposed by Levene and Jacobs in 1909 for the carbohydrate derivatives of purines (and, later, of pyrimidines) isolated from the alkaline hydrolyzates of yeast nucleic acid. The phosphate esters of nucleosides are the nucleotides, which, in polymerized forms, constitute the nucleic acids of all cells.2 The sugar moieties of nucleosides derived from the nucleic acids have been shown, thus far, to be either D-ribose or 2-deoxy-D-eri/fAro-pentose ( 2-deoxy-D-ribose ). The ribo-nucleosides are constituents of ribonucleic acids, which occur mainly in the cell cytoplasm whereas 2-deoxyribo -nucleosides are components of deoxypentonucleic acids, which are localized in the cell nucleus.3 The nucleic acids are not limited (in occurrence) to cellular components. They have also been found to be important constituents of plant and animal viruses. 

While these results support a phosphate ester bridge between the glycerol and inositol, it does not prove where the bond is located on the inositol molecule. Inasmuch as the inositol phosphate isolated by alkaline cleavage from the parent phosphatidylinositol or the glycerophosphoinositol was optically active, the phosphate must be attached at the 1 or 4 position. Substitution at the 2 position of inositol would not yield an optically active product. Subsequently as will be discussed, observations from several laboratories have shown that the phosphate bond is between the sn- 3 position of the glycerol and the C-l hydroxyl on myo-inositol. 

While all the above molecular phosphates were prepared starting from phosphonic acids and phosphate esters, there are a few examples of molecular phosphates synthesized from phosphoric acid in aqueous medium. Although under hydrothermal conditions the reactions of phosphoric acid with metal ions generally result in extended open framework structures, it has been possible to isolate molecular zero-dimensional metal phosphates.4142... 

When such strains as E. coli 83-24, which are blocked after shikimic acid, were grown on minimal medium plus aromatic supplement, they accumulated 400-800 mg. of shikimic acid per liter, together with variable amounts of shikimate 5-phosphate. Since no mutants that are blocked between shikimic acid and its phosphorylated form were found, it was considered that the phosphate ester is not on the main path of biosynthesis. As will be pointed out later, enzymic studies showed that shikimate 5-phosphate is actually an intermediate between shikimate and the aromatic compounds. It would appear, therefore, that the block in such strains as E. coli 83-24 is probably immediately after shikimate 5-phosphate. With filtrates from this organism, methods were developed for the isolation of pure shikimate and for its stepwise degradation. ... 

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