Polyhydroxyl compounds, enzyme

Protein drugs have been formulated with excipients intended to stabilize the protein in the milieu of the pharmaceutical product. It has long been known that a variety of low molecular weight compounds have the effect of preserving the activity of proteins and enzymes in solution. These include simple salts, buffer salts and polyhydroxylated compounds such as glycerol, mannitol, sucrose and polyethylene glycols. Certain biocompatible polymers have also been applied for this purpose such as polysaccharides and synthetic polymers such as polyvinyl pyrrolidone and even nonionic surfactants. 

The addition of polyhydroxyl compounds to enzyme solutions have been shown to increase the stabilities of enzymes, (13,16,19,20). This is thought to be due to the interaction of the polyhydroxyl compound, (e.g. sucrose, polyethylene glycols, sugar alcohols, etc), with water in the system. This effectively reduces the protein - water interactions as the polyhydroxy compounds become preferentially hydrated and thus die hydrophobic interactions of the protein structure are effectively strengthened. This leads to an increased resistance to thermal denaturadon of the protein structure, and in the case of enzymes, an increase in the stability of the enzyme, shown by retention of enzymic activity at temperatures at which unmodified aqueous enzyme solutions are deactivated. 

This effect of polyhydroxyl compounds may not be quite as simple as it has been described, as the structure of the polyhydroxyl compound may play some part in effective stabilization of enzymes in wet systems. Thus Fujita et al, (20) reported that inositol was more effective than sorbitol in stabilizing lysozyme in aqueous solutions. Both compounds contain six hydroxyl groups, but inositol is cyclic in structure whereas sorbitol is linear, Fig 10. The interaction of polyhydroxyl compounds with water promotes a change in the molecular structure of water. Inositol was reported to have a larger structure-making effect than sorbitol, which accounted for the greater stabilization effect of this compound. 

Figure 11. Alcohol oxidase. Interaction of polyelectrolytes/polyhydroxyls. The diagrams above represent the postulated interaction of alcohol oxidase with (a) DEAE—Dextran and (b) the same interaction in the presence of polyhydroxyl compounds. The structure of the enzyme was taken from Woodward 1990 (2).

However, in the case of acetone (results shown in Table 9.4) the enzyme shows a preference towards intermediate or high chain length fatty acids, which is in accordance with other reports for the enzymatic acylation of polyhydroxylated compounds catalyzed by immobilized CALB in conventional organic solvents... 

The different enzyme behavior observed in the case of ionic liquids can be attributed to the lower solubility of long chain acyl donors in these media, compared to the less polar organic solvents used for the enzymatic modification of natural polyhydroxylated compounds. Due to the low solubUily of long chain acyl substrates in ionic liquids, a two-phase system was formed, which is expected to decrease the availability of substrates to the enzyme and therefore the biocata-lytic acylation of phenohc compounds [5j. 

Cycloaddition reaction of nitrone (—)-(394) with dimethyl maleate D14 has been used for the synthesis of two new polyhydroxyl pyrrolizidines (687) and (688) (Schemes 2.293, 2.294). These compounds are analogs of alkaloids ros-marinecine and crotanecine, which were assayed for their inhibitory activities toward 22 commercially available glycosidase enzymes. One of them ((-)- a-epi-crotanecine) (—)-(688) is a potent and selective inhibitor of a-mannosidases (310). The reaction of (—)-(394) with dimethyl maleate gave a 9.6 6 1 mixture of cycloadducts (—)-(680), (+ )-(680), and (—)-(681), which arise from anti-exo,... 

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