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2. Synthesis of glycosides of simple aliphatic and aromatic alcohols and diols The hydrolysis mediated by glycosidases is a multi-step process in which the glycosides
are hydrolyzed via a general acid catalysis forming a covalent glycopyranosyl-enzyme
intermediate. In the kinetically controlled synthesis of glycosides the reactive
glycosylated enzyme generated by the hydrolysis of a carbohydrate donor, can be
intercepted by the glycosyl acceptor or by water (transglycosylation). During the
synthesis of glycosides in the equilibrium controlled approach, a glucose molecule is
attached to the acceptor in an inversion of catabolic route of the enzyme (reverse
hydrolysis) (Scheme 1). Different preparations of the thermophilic archaeon Sulfolobus solfataricus, such as crude homogenate Ref.14 immobilized microrganism Ref.13, homogeneous native and recombinant enzymes Ref.15 were used as catalysts for the kinetically controlled glycosylation of various aglycones
Fig.1B,
The donors used in these reactions include phenyl gluco- galacto- and fuco- pyranosides, and/or disaccharides such as lactose and cellobiose. The study of transglycosylation with other thermophilic enzymes is at beginning and only few examples of model molecules were tested for the synthesis of glycosides
(Table 1). General wide substrate specificity of ß-glycosidase from S. solfataricus was demonstrated by using as acceptors primary (R-OH; C1-C8) and secondary (R1R2CH-OH; R1= Me, R2= C1-C5; R1= Et, R2= C2-C4) alcohols of different chain length Ref.14. The yield of reaction depends upon chain length and OH position but a marked increase can be obtained increasing the molar excess of alcohol. Reaction mixtures in which the alcohol and/or organic cosolvent concentrations are up to 95-97% can be used. Other alcohols of different structures can be accepted by the enzyme Ref.13-15. Tertiary alcohols are not substrates Ref.14. Interestingly, immobilized cells have been used to synthesize n-hexyl-ß-glucoside recycling the biocatalyst Ref.15. Whole cells of the thermophilic biocatalyst S. solfataricus can be also used avoiding immobilization procedures, in fact lyophilized cells have been employed for the high yield (ca 80%) synthesis of HEMA-ß-D-galactoside (2-ß-D-galactopyranosyloxyethyl methacrylate). This anomerically pure galactosylated monomer represents the starting point for further studies in the field of modified polymers for biomedical application Ref.6,16. The use of lyophilized cells of the microorganism has three main advantages, i) improved stability of the cell-entrapped enzyme with respect to a solubilized form, ii) minimized cost for biocatalyst production which has not to be purified from the source, and iii) its possible reuse. The use of mesophilic enzyme (Aspergillus oryzae) did not give good yield of reaction probably due to the denaturation of the biocatalyst in the reaction condition Ref.16. |
Fig. 1A,
Fig.1C. 