Chloroformates, reaction with polysaccharides

Chitin hydrogels, partially deacetylated, degradation, 182-184 Chloroformates, reaction with polysaccharides, 191,193-195... 

Nitrophenyl chloroformate activation. The reaction of polysaccharides with chloroformates forming reactive carbonate derivatives has often been used to link bioactive compounds, such as affinity ligands and enzymes, onto polysaccharide matrixes. It was reported that upon reaction with ethyl chloroformate cyclic carbonate as well as ethyl-carbonate structures are formed (9-13). Activation with N-succinimi-do chloroformate (14), 2,4,5-trichloroformate (14) and 4-nitrophenyl chloroformate (14, 15) reportedly led to the introduction of pending carbonate moieties. 

Interesting possibilities for the synthesis of new types of polysaccharide derivatives are offered by the reaction of addition to double bonds, which proceeds by a free-radical mechanism. The presence of initiators of free-radical polymerization (benzoyl peroxide, tert-butyl peroxide, dicumyl peroxide, dinitrile of azodiisobutyric acid), also irradiation with ultraviolet light, has effected the addition to 5,6-cellulosene of chloroform, carbon tetrachloride, methylmonochloroacetate, dimethyl-phosphite and other compounds that decompc under the conditions of a reaction with the formation of free radicals (45,46). The reaction proceeds as follows ... 

Methylation- or combined methylation-ethylation reactions were used for structure analysis of polysaccharides. The alkylation of dextran can be applied to the investigation of the branching pattern, i.e. the number and length of side chains (Sect. 2.2) [22,23]. The methylation is carried out in liquid ammonia with sodium iodide and methyl iodide, yielding products that are soluble in chloroform and tetrachloroethane [257]. 

Extraction of lipid A from LPS takes advantage of the acido-labile ketosidic bond between the lipid A and the ketodeoxyoctanoate (Kdo) in the LPS. Acid and heat are sufficient to disrupt the linkage. The lipid A is insoluble in water and therefore can be readily collected by centrifugation. Harsh hydrolysis such as 0.1 M hydrochloric acid at 100°C, and milder hydrolysis treatment with 1 % acetic acid have been used to liberate lipid A moiety from LPS molecules (Fensom and Meadow, 1970 Morrison and Leive, 1975 Oertelt et al., 2001 Osborn, 1963). The harsh hydrolytic conditions could result in partially dephosphorylation and O-deacylation of lipid A (Karibian et al., 1995). This could affect the biological activities of the lipid A, but is useful for the extraction of monophosphoryl lipid A (Qureshi et al., 1982). Milder hydrolysis conditions, such as sodium acetate at pH 4.5, have been proved to be efficient to cleave the lipid A-polysaccharide bond (Rosner et al., 1979). When the hydrolysis is ineffective, 1% SDS can be added to the system (Caroff et al., 1988). The lipid A can be extracted from the hydrolytic reaction mixture using the solvent of chloroform and methanol (2 1, v/v). 

Other approaches for insolubilization have been proposed by Francotte more stable CSPs could be obtained by cross-linking polysaccharide derivatives photochemical I y initiated ] 163] or thermally initiated ] 164] (Tables 9.5 and 9.6) using a radical reaction. The new CSPs exhibited improved separations for many racemates, predominantly through the ability to use chloroform and other co-solvents. Further, compounds that were insoluble in the commonly used /)-heptane-2-propanol eluents could be easily separated into individual enantiomers, and for many enantioseparations run times can be reduced with chloroform, ethyl acetate or THF containing mobile phases. These improved CSPs are about to be commercialized. 

Although cyclic and acyclic carbonates of monosaccharides have been known for some time, little interest had been taken in such derivatives of polysaccharides, probably because the conditions could not be controlled to give any degree of specificity. The formation of cyclic carbonate rings involves treatment of the polysaccharide with ethyl chloro-formate heterogeneous reactions occur, and acyclic carbonate (ethy-oxycarbonyl) groups are also formed. The reaction conditions suitable for maximum cyclic carbonate and minimum acyclic substitution have been investigated in detail for cellulose. Other chloroformates have... 

The procedure employed for hydrolysis and acetylation of polysaccharides is based on the method of Jones and Albersheim [118], Samples (2-5 mg) are hydrolyzed as follows The polysaccharide samples are dissolved in 1 M NH4OH (1 ml) containing 6 mg of NaBELj and kept at room temperature for 1-3 h. The samples are acidified with acetic acid until no bubbles are produced and 1 ml of methanol is added and then evaporated to dryness at 40 °C with N2 gas. Acetic acid and methanol mixture (10%, w/v) are then added to samples and evaporated tmder N2 gas. The samples and standards (which are usually fiiictose, glucose, xylose, arbinose, galoctose, ribose, rhamnose, and fucose) were treated with acetic anhydride in sealed test tubes at 121 °C for 3 h. After completion of the reaction, the samples were extracted with toluene followed by chloroform and washed with water. The chloroform layer was separated, evaporated, and redissolved in methanol prior to GC analysis. 

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