Diphosphorylation

O Phosphorylation of the tertiary hydroxyl and diphosphorylation of the primary hydroxyl, followed by decarboxylation and simultaneous expulsion of phosphate, gives isopentenyl diphosphate, the precursor of terpenoids,... 

Thiamine can be considered to be the product of the quatemization of 4-methyl-5-(2-hydroxymethyl)thiazole (5) by an active derivative of 4-amino-5-(hydroxymethyl)-2-methyl pyrimidine (4) (Scheme 2). In living cells, pyramine can be activated by conversion into the diphosphate 7, via monophosphate 6, and the substrate of the enzyme responsible for the quatemization is not the thiamine thiazole, but its phosphate 8. The product of the condensation, thiamine phosphate (9), is finally converted into diphosphate 2—the biochemically active derivative—by hydrolysis to free thiamine, followed by diphosphorylation, or more directly, in some cases. Enzymes are known for all of the steps depicted in Scheme 2, and adenosine triphosphate (ATP) is, as usual, the phosphate donor. 

In contrast, synthesis of 3,4-diphosphorylthiophenes requires more elaboration because of low reactivity of 3,4-positions of thiophene and unavailability of 3,4-dihalo or dimetallated thiophenes. Minami et al. synthesized 3,4-diphosphoryl thiophenes 16 as shown in Scheme 24 [46], Bis(phosphoryl)butadiene 17 was synthesized from 2-butyne-l,4-diol. Double addition of sodium sulfide to 17 gave tetrahydrothiophene 18. Oxidation of 18 to the corresponding sulfoxide 19 followed by dehydration gave dihydrothiophene 20. Final oxidation of 20 afforded 3,4-diphosphorylthiophene 16. 3,4-Diphosphorylthiophene derivative 21 was also synthesized by Pd catalyzed phosphorylation of 2,5-disubstituted-3,4-dihalothiophene and converted to diphosphine ligand for Rh catalysts for asymmetric hydrogenation (Scheme 25) [47],... 

Lo 10-3 Ouellet et al. (1952) have reported a kinetics analysis of the enzymatic diphosphorylation of... 

Diethylaminosulfur trifluoride, 1684 Diphosphoryl chloride, 4168 Diselenium dichloride, 4115 Disulfur dibromide, 0282 Disulfur dichloride, 4114 Disulfuryl dichloride, 4103... 

Reetz et al. described the solid-phase enzymatic synthesis of oligonucleotides on Kieselguhr-PDMA-resins via T4 RNA ligase. Goncomitantly, they found that RNase A selectively cleaves the last bound nucleotide at the ribose sugar leaving a 3, 5 - diphosphorylated ohgomer on the resin, but application in synthesis has not yet been undertaken [22]. 

Qureshi N, Takayama K, Kurtz R. (1991) Diphosphoryl lipid A obtained from the nontoxic hpopolysaccharide of Rhodopseudomonas Sphaeroides is an endotoxin antagonist in mice. Infect Immun 59 441 44. 

PF had been proposed as the terminal complex (23) and associated pores were reported on the outer membrane EF (24). Due to their proximity to the site of cellulose ribbon extrusion from the cell surface, these structures were assumed to be responsible for cellulose synthesis. A model was advanced in which cellulose synthase was localized on the outer membrane, which invoked adhesion sites between the outer and plasma membranes as a mechanism to explain the transfer of uridine-diphosphoryl-glucose (UDPG) from the cytoplasm to the cellulose synthases (25,26). However, when the outer and plasma membranes of Acetobacter were isolated separately by density-gradient centrifugation, the cellulose synthase activity was localized only in the plasma membrane fraction (27). Therefore, the linear structures observed on the Acetobacter outer membrane, while they may be associated in some manner with cellulose biosynthesis, are probably not the cellulose synthase terminal complexes. Since no ultrastructural evidence for adhesion sites between the outer and plasma membranes has been presented, a thorough investigation of the mechanism of / (1-4) glucan chain translocation from the cytoplasmic membrane to the outer membrane in Acetobacter xylinvm is now in order. 

Phosphoryl radicals [10, 18, 38-42] tend to add to double bonds. Owing to the exceptionally high constants of hyperfine coupling of the unpaired electron with the phosphorus nucleus, phosphoryl radicals can be utilized as paramagnetic reporters [10]. Phosphoryl radicals have been prepared by photolysis of diphosphoryl mercury compounds (Scheme 6.5). 

The authors experience is that the enzymic phosphorylation and diphosphorylation of nucleoside monophosphates is very efficient the yields are nearly quantitative and the immobilized enzyme system appears reusable for at least three months. 

Figure 1 shows the scheme for the preparation of purified lipid A from endotoxin. S. typhimurium G30/C21 was extracted by the method of Galanos t aK (24) and submitted to one of two different conditions of hydrolysis (a) 0.1 N HC1 [in methanol-water (1 1, v/v)], 100 °C, 45 min, to yield the crude monophosphoryl lipid A (nontoxic), and (b) 0.02 M sodium acetate, pH 4.5, 100 °C for 30 min (two cycles) to yield the crude diphosphoryl lipid A (toxic). The 0.1 N HC1 hydrolysis product was fractionated on a Sephadex LH-20 column (23). Each of these fractions was then separated by preparative thin layer chromatography (TLC) on silica gel H (500 ym), with the solvent system chloroform-methanol-waterconcentrated ammonium hydroxide (50 25 4 2, v/v) as previously described (23) to yield TLC fractions 1-7 and 1-9 respectively. 

Table VII. Chemical Analyses of Mono- and Diphosphoryl Lipid A...

Figure 1. Scheme for preparing and purifying mono- and diphosphoryl lipid A from endotoxin obtained from S. typhimurium G30/ G21. 

The TLC purified diphosphoryl lipid A was hydrolyzed in 0.1 N HCl at 100 °C for 30 min to yield the corresponding monophosphoryl lipid A derivatives as previously described (23). These products were then compared with previously characterized monophosphoryl lipid A fractions by TLC using silica gel H (250 Vim) and the previously mentioned solvent system. The TLC fractions -3, -5, -7 of the acid hydrolyzed diphosphoryl lipid A series corresponded with a similarly numbered series of monophosphoryl lipid A fractions (TLC-3, -5, and -7). There appeared to be some breakdown of the monophosphoryl lipid A products to the lower homologues presumably by the acid catalyzed hydrolysis of some fatty ester linkages. TLC-1 and -9 were not analyzed due to small sample sizes. 

The TLC purified diphosphoryl lipid A fractions (TLC -3, -5, and -7) were analyzed by FAB mass spectrometry in the negative mode as previously described (23) and the results are shown in Table X. TLC-3 gave a molecular ion (M-H) at m/z 1796 TLC-5, m/z 1586 TLC-7, m/z 1360. As expected, these values were 80 amu or (P03H2 H) larger than those for the corresponding monophosphoryl lipid A s of the series as shown in Table VIII. These results established the structural relationship between the mono- and diphosphoryl lipid A s. 

Table X. FAB Mass Spectral Analysis and Molecular Weight Determination of the Series of Purified Diphosphoryl Lipid A Fractions ...

Diphosphinoethane, 0953 Diphosphoryl chloride, 4162 Dipotassium /(-cyclooctatetraene, 2942 Dipotassium diazirine-3,3-dicarboxylate, 1336 Dipotassium hexafluoronickelate(IV), 4357b Dipotassium aci-nitroacetate, 0668 Dipotassium phosphinate, 4425 Dipotassium triimidotellurite, 4489 Dipropargyl ether, see Di(2-propynyl) ether, 2320 f Di-2-propenyl ether, see Diallyl ether, 2425... 

Turnquist RL, Hansen RG(I973) Uridine diphosphoryl glucose pyrophosphorylase. In Boyer PD (ed) The Enzymes Vol VIII. Academic Press, New York, 51... 

Recently we have developed a more general approach to molecules exemplified by III. Thus the Diels-Alder cycloaddition of alkyne II and ct-pyrone, followed by aromatization by loss of carbon dioxide, led to the isolation of III (72%) (5). Alkyne II was obtained in high yields, in two steps from dichloroacetylene and triethylphosphite via Arbuzov-type reactions (5). Since the intermediate chloroalkyne phosphonate I was isolable (90%), phosphorus nucleophiles other than triethylphosphite could be used to give unsymmetrical alkyne diphosphoryl species. We have demonstrated this approach by the reaction of I with PhaPOEt and PhP(OEt)2 (5). 

The phosphorylation of potassiopyrrole by the chlorides of phosphorous acid shows [21] that the process is selective and leads to the products from N-phosphorylation. The reaction of 2,4-dimethyl- and 2,3,5-trimethylpyrroles with chlorophosphites makes it possible to obtain mono- and diphosphorylated alkylpyrroles, depending on the ratio of the phosphorylating agent and alkylpyrrole [22, 23]. 

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