Comprobación de hipótesis especifica 6

As early as 1937 Bergman and Fraenkel-Conrat161-162 suggested that the reversibility of protease catalysed reactions could be utilised for the formation of peptide bonds. During the past decade such enzymatic catalysis has attracted much attention. This has led to the development of many systems that successfully attain enzymatic activity in predominantly organic media. These include the direct suspension of enzyme particles in the organic phase, covalent modifications to render the enzyme soluble in organic solvents and the use of biphasic media as well as water miscible organic solvents. All these have their advantages and disadvantages, though so far a direct comparison of enzymatic stability and activity has not been possible.

4.5.1 Anhydrous organic solvents.

The use of lipases and proteases in essentially anhydrous solvents has received much attention.163-164' 165 The range of reactions (Scheme 71) includes: general amide synthesis166 using various biocatalysts in hexane and the

production of amides enantioselectively167' 168 and transesterification169 using such media such as hexane, 1,4-dioxane, or diisopropyl ether. N-Acetylations, however, are rarer than O-acetylations and have mostly been used in the field of peptide synthesis.170 N-Acetylation has been accomplished for primary165-171

aromatic172 and secondary amines173 using ethyl acetate as both solvent and transfer reagent.

[372] [373] Scheme 71

Surprisingly, even hydrolysis can be performed in "anhydrous" diisopropyl ether.174 Kanerva and Klibanov175 undertook mechanistic studies using Subtilisin carlsberg as a model as it had been shown to have protease activity in anhydrous organic solvents. They concluded that there were "profound" mechanistic similarities in organic media and aqueous media. This suggested that the transition state in both media is essentially the same.

4.5.2 Biphasic systems

Numerous groups have achieved the synthesis of peptides in two-phase reaction mixtures, 176 viz. an aqueous phase (containing the enzyme) and a water- immiscible organic phase. The equilibrium is shifted due to the preferential solubility of the peptide formed in the organic phase with respect to the starting material. The main limitation of the biphasic system seems to be biocatalyst stability, which often results in the precipitation of the enzyme as a gel. Hailing showed that biocatalyst stability increased as the polarity of the solvent decreased.177

Many groups have investigated the use of water miscible co- solvents178' 179 and the changes they induced in the catalytic activity of a -

chymotrypsin and laccase. The best co-solvents were found to be the polyalcohols ethylene glycol and glycerol (in which a-chymotrypsin inactivation is reversible). The reasons proposed for the changes in catalytic activity of the biocatalysts178' 180' 181 in these systems are summarised below:

• some organic co-solvents (and water) may act as substrates for, and thus competitive inhibitors of enzyme activity;

• the co-solvent may act as a specific inhibitor;

• addition of a co-solvent changes the bulk properties of the solution, thus changing the interactions between, enzyme, substrate and solution;

• the co-solvent may denature the enzyme.

Mozhaev et al produced a quantitative correlation of denaturing ability vs. hydrophobicity.178 This points to the general rule that the best co-solvents are the most hydrophilic ones; The authors add a cautionary note that this relationship can only be applied to organic solvents with the same homological group.

Enzymes have been covalently modified by the attachment of polyethylene glycol residues.182 This rendered the enzyme soluble in benzene both for peptide formation and hydrolysis. This was found to be quite satisfactory, the catalytic activity of the enzymes being only three to four fold lower than in aqueous buffer. Reasonable evidence was found that the enzyme structure is more compact in water restricted environments, limiting conformational mobility. Another interesting discovery was that the minimum amount of water required for enzymatic activity in anhydrous organic media was ca 0.04%, the optimum being 0.09% (by volume).183 Enzymes in 'anhydrous' systems, require a minimum amount of water to maintain their shape and remain active. The conformation of an enzyme is maintained by hydrogen bonds and salt bridges. All organic solvents will allow a certain amount of water to dissolve

in them. If this is not present then the water maintaining the shape of the enzyme w ill dissolve in the solvent and the enzyme will be denatured and thus deactivated.

4.5.3 Emulsions.

The final way to overcome poor solubility of organic reagents in aqueous systems or to maintain favourable equilibrium changes is to use oil-in-water emulsions containing for example, non-ionic surfactants. Hughes et a/,184 in the enzymatic hydrolysis of dithioacetal esters (Scheme 72) using lipase from

Pseudomonas sp., found hydrolysis occurred very slowly.

lipase ^ ^ c o n m«2 ^ - ^ ^ C O N M e 2 COzM» + ^ s ^ COjH 1375] [376] Scheme 72

This was attributed to the presence of a liquid-solid interface produced by the insolubility of the esters, so Triton X-100 was used to aid their solubility. It was found that the addition of water-miscible solvents such as DMF, ethanol and 2-propanol decreased the already sluggish rate of hydrolysis. So, in cases where the optimum organic co-solvent proves elusive the addition of Triton X-100 or

other surfactants (though X-405 and X-705 were in this case less effective) provides another approach.

In summary, the precedent of the known metabolism of polyamines may be put forward as proof that enzymes are capable of differentiating the N-1 and N-8 termini of spermidine. This should allow selective protection of one end of the polyamine, or selective deprotection of a fully protected molecule, in order to begin the construction of one of the argiotoxins.