Diphosphoryl lipid

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. 

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 ...

Takayama K, Qureshi N, Beutler B, Kirkland TN. 1989. Diphosphoryl lipid A from Rhodopseudomonas spaeroides ATCC 17023 blocks induction of cachectin in macrophages by lipopolysaccharide. Infect. Immun. 57 1336-38... 

Mvo-inositol 1,4-diphosphate (38), the racemate of a metabolite of second messenger myo-inositol 1,4,5-triphosphate and its diphosphorothioate analogue (39) have been synthesised from f 1 -1 2.4 S-di-O-isopropylidene-mvo-inositol by way of phosphite intermediates. Similarly, (-mvo-inositol 1,3,4-triphosphate has been obtained from f W2.4.S-tri-O-benzyl-myo-inositol Phosphite chemistry was also employed in the preparation of diphosphorylated and diphosphonylated Lipid A monosaccharide analogues (40). Related Lipid A components are mentioned in Chapter 9. Methyl 3-deoxv-a-D-manno-oct-2-ulopyranosidonic acid 4-phosphate has been synthesised and its chemical stability studied. ... 

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