Cellobiosyl fluoride

Among other in vitro enzymatic polymerizations that have been studied are the oxidative polymerizations of 2,6-disubstituted phenols to poly(p-phenylene oxide)s (Sec. 2-14b) catalyzed by horseradish peroxidase [Higashimura et al., 2000b] and the polymerization of P-cellobiosyl fluoride to cellulose catalyzed by cellulase [Kobayashi, 1999 Kobayashi et al., 2001],... 

Konstantinidis, A. K., Marsden, I., and Sinnott, M. L., Hydrolyses of alpha- and beta-cellobiosyl fluorides by cellobiohydrolases of Trichoderma reesei. Biochem J1993, 291 (Pt 3), 883-8. 

Enzymatic synthesis of cellulose has been achieved by cel-lulase. For example, incubation of fS-cellobiosyl fluoride with a cellulase from Trichoderma viride can produce cellulose in 54% yield with DP around 22 after 12h. In addition, change of the reaction conditions (substrate concentration or organic solvent concentration) enabled the selective synthesis of the water-soluble celloohgosaccharides (118). 

The endo-glycosynthase obtained from the E197A mutant of the retaining cellulose Cel7B of Humicola insolens could be employed to transfer a-cellobiosyl fluoride (87) to methyl /3-cellobioside (88) to afford the cellotetraoside 89 in virtually quantitative yield [124]... 

Cellulase was found to be effective in the synthesis of artificial cellulose in a single-step reaction by polycondensation of /J-D-cellobiosyl fluoride (Scheme 13).123 The polymerization is a repetition of the transglycosylation reaction, which became predominant over the hydrolysis reaction when the enzymatic polycondensation was carried out in a mixed solvent of acetonitrile/acetate buffer (5 1, pH 5). This synthesis is therefore kinetically controlled as well as equilibrium controlled. The fi configuration of the Cl fluorine atom is necessary to form a reactive intermediate leading to a / (1—4) product via a double displacement mechanism .124 Thus, this method provided the first successful in vitro synthesis of cellulose, the most abundant biomacromolecules on the earth, the synthesis of which had been unsolved for one-half a century.123... 

Thiooligosaccharides can be inhibitors of cellulases and are useful for elucidation of the molecular mechanism of the cellulolytic actions. Hemithiocello-oligosaccharides from tetraose to tetradecaose were synthesized by a cellulase mixture using 4-thio-/ -cellobiosyl fluoride as an activated donor in a buffer/ acetonitrile solvent system.139... 

The in vitro synthesis of cellulose via a nonbiosynthetic path has been achieved for the first time by condensation of [3-D-cellobiosyl fluoride as substrate for cellulase (systematic name 4-[3-D-glucan 4-glucanohydrolase EC 3.2.1.4) in a mixed solvent of acetonitrile/acetate buffer by Kobayashi et al. (see Figure 9.13) [218, 246-249],... 

Kobayashi and coworkers proposed a mechanism for the enzymatic polymerization of 3-D-cellobiosyl fluoride with cellulase following the general accepted mechanism for glycosidase-catalyzed synthesis of glycosides. 

Cellulase hydrolyzes the (1—>4)-/3-glycoside bonds of cellulose, and this enzymatic hydrolysis is essentially reversible. Kobayashi etal succeeded in cellulose synthesis using the reversible reaction of cellulase, where a particular substrate, /3-cellobiosyl fluoride, was used in an aqueous buffer containing acetonitrile (Figure 22). 

Figure 22 Enzymatic synthesis of cellulose from /3-cellobiosyl fluoride in acetonitrile/aqueous buffer mixture.

Oligomerization of the sulfur-linked cellobiosyl fluoride 28 by use of a cellulase gave a set of products with even numbers of glucose residues of general structure... 

Amphiphilic cyclodextrins 56 have been synthesized in high yields in one step from the corresponding bromides 55. In a similar preparation of peracetylated heptakis [6-S-(2,3-dihydroxypropyl)-6-thio]-P-cyclodextrin (58), the heptakis iodide 57 was used as precursor. Oxidation with MCPBA furnished heptakis sulfone 59. Hemithiocyclodextrins (cyclodextrins in which half of the inter-glycosidic oxygen atoms are replaced by sulfur atoms) and hemithiocellodextrins have been obtained in yields of ca. 10 % by exposure of 4-thio-a-maltosyl fluoride to cyclodextringlycosyltransferases and 4-thio-P-cellobiosyl fluoride to cellu-lases, respectively. 

Cellulase-catalyzed polycondensation of 4-thio-)8-cellobiosyl fluoride produced hemithiocellodextrins having 4-thiocellobiosyl repeating units linked by /3(1 4) oxygen linkages (37). A water-soluble oligomer with DP up to 20 was obtained in an aqueous acetonitrile. 

Kobayashi and co-workers [218] reported that cellulose I and cellulose II can be selectively synthesised in vitro using the enzymatic polymerisation of the P-cellobiosyl fluoride monomer, and this selectivity could be controlled by changing the enzyme purity and polymerisation conditions. 

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