Equilibrium-controlled reverse hydrolysis

Reversed Hydrolysis vs. Transglycosylation. Glycosidases have been used for several years for the selective hydrolysis of carbohydrates, such as for the hydrolysis of starch and for the analysis of glycoconjugate carbohydrate structures. These enzymes also can be used for the regioselective and stereospecific synthesis of oligosaccharides in equilibrium-controlled (reversed-hydrolysis. Equation 1) or kinetically controlled (transglycosylation. Equation 2) reactions ... 

Hydrolysis (Sections 20 10 and 20 11) Ester hydrolysis may be catalyzed either by acids or by bases Acid catalyzed hydrolysis is an equilibrium controlled process the reverse of the Fischer esterification Hydrolysis in base IS irreversible and is the method usual ly chosen for preparative purposes... 

Reverse hydrolysis a thermodynamically controlled equilibrium process, in which a free monosaccharide reacts with a nucleophile under exclusion of a water molecule and hence chemically, can be considered a condensation reaction. 

The initial products of organic reactions are formed under conditions of kinetic control - the products are formed in proportions governed by the relative rates of the parallel (forward) reactions leading to their formation. Subsequently, product composition may become thermodynamically controlled (equilibrium controlled), i.e. when products are in proportions governed by the equilibrium constants for their interconversion under the reaction conditions. The reaction conditions for equilibrium control could involve longer reaction times than those for kinetic control, or addition of a catalyst. The mechanism of equilibrium control could simply involve reversal of the initial product-forming reactions (as in Scheme 2.4, see below), or the products could interconvert by another process (e.g. hydrolysis of an alkyl chloride could produce a mixture of an alcohol and an alkene, and the HsO"1" by-product could catalyse their interconversion). 

The two main approaches to glycosidase-catalyzed synthesis of glycosidic linkages involve direct reversal of hydrolysis (equilibrium-controlled synthesis) and trapping of a glycosyl-enzyme intermediate (kinetically-controlled process) [217]. 

For the direct reversal of catalytic hydrolysis of peptides, discussed in this chapter, the term equilibrium-controlled approach should be preferred. Because of the thermodynamic control of both equilibria in Eq. (1) the reversal of proteolysis is often denoted as a thermodynamic approach. In order to increase the product yield of this endergonic process various manipulations are required. In addition to those mentioned above, reverse micellesI1031, anhydrous media containing minimal water concentrations11041, water mimics110S, and reaction conditions promoting product precipitation as discussed in first part of this chapter are often employed. 

There are two basic methods of glycoside synthesis transglycosylation and condensation (reverse hydrolysis) (Figure 24.1) [1,2]. The reactions are also termed the kinetically controlled method and the equilibrium-controlled method, respectively. 

Proteases for peptide synthesis are selected on the basis of their specificity against amino acid residues and include the majority of the commercially available proteases of the four classes mentioned above [58]. Protease-catalyzed bond synthesis can be carried out either as an equilibrium-controlled process which is the direct reversal of the protease-catalyzed hydrolysis or a kinetically controlled process. In the latter case weakly activated carboxy components are employed [61]. 

That said, metabolic interconversion of the amine backbone linkage of the ANPs would require oxidation, and thus would be a redox process rather than condensation/hydrolysis. Water remains a necessity for life and certainly life on Earth exploits condensation in the linkages of the nucleic acid monomers (phosphoester bonds), the amides of polypeptides, and the acetals of polysaccharides, and utilizes the reverse reaction of hydrolysis for metaboUsm. Therefore we have sought to extend the principles learned with the amine backbones to possible backbone chemistries that allow equilibrium control of template-directed polymerization through condensation and hydrolysis reactions. 

In the first control point, citrate synthase catalyzes the condensation of acetyl-CoA with oxaloacetate to produce citrate (AG° = -32.2 kJ mol ). Although the reaction is reversible, the equilibrium lies very much in favor of citrate formation because of the hydrolysis of a bond in the intermediate compound, citroyl-CoA (Fig. 12-4). Citroyl-CoA is bound to citrate synthase, and the hydrolysis of the thioester bond, to produce citrate and coenzyme A, is an exergonic process. Citrate synthase is inhibited by its substrates (acetyl-CoA and oxaloacetate), and its activity is affected by... 

Acid-catalysed ester formation and hydrolysis are the exact reverse of one another the only way we can control the reaction is by altering concentrations of reagents to drive the reaction the way we want it to go. The same principles can be used to convert to convert an ester of one alcohol into an ester of another, a process known as transesterification. It is possible, for example, to force this equilibrium to the right by distilling methanol (which has a lower boiling point than the other components of the reaction) out of the mixture,... 

In the early 1960s, seminal work by Jencks and coworkers demonstrated that formation and hydrolysis of C=N bonds were proceeding via a carbinolamine intermediate, thus leading to a more general mechanism of addition reactions on carbonyl groups [17-19]. The dynamic nature of the reaction of imine formation can be exploited to drive the equilibrium either forward or backwards. Since the reaction involves the loss of a molecule of water, adding or removing water from the reaction mixture proved an efficient way to shift the equilibrium in either direction. The responsive behavior of imines to external stimuli makes the reversible reaction of imine formation perfectly suited for DCC experiments [20], Thermodynamically controlled reactions based on imine chemistry include (1) imine condensation/hydrolysis, (2) transiminations, and (3) imine-metathesis reactions... 

For esters, all of the steps of the hydrolysis reaction are reversible, and the mechanism of ester formation is the reverse of ester hydrolysis. The course of the reaction is controlled by adjusting the reaction conditions, chiefly the choice of solvent and the concentration of water, to drive the equilibrium in the desired direction. For hydrolysis, the reaction is carried out in an excess of water for ester formation, the reaction is carried out with an excess of the alcohol component under anhydrous conditions. Frequently, an experimental set-up is designed to remove water as it is formed in order to favor ester formation. 

Esterification is the reverse of the hydrolysis process. It is carried out by reacting fatty acids with glycerol. In addition to esters, water is also a product of esterification. The reaction is reversible and proceeds to completion only if water is removed from the medium. The equilibrium between the forward reaction (hydrolysis) and the reverse reaction (esterification) is controlled by water content of the reaction mixture. In the presence of excess water, hydrolysis predominates, whereas under water-eliminating conditions, esterification is favored (3, 4). 

It is generally accepted that an enzymatic reaction is virtually reversible, and hence, the equilibrium can be controlled by appropriately selecting the reaction conditions. On the basis of this view, many hydrolases, which are enzymes catalyzing a bond-cleavage reaction by hydrolysis, have been employed as catalysts for the reverse reaction of hydrolysis, leading to polymer production by a bond-forming reaction. 

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