used more widely. Despite numerous efforts directed towards the cloning and
overexpression of microbial esterases, the number of synthetically useful
enzymes – possessing a relaxed substrate specificity by retaining high
enantioselectivity – are limited: many novel esterases showed disappointing selectivities [193, 194].
Fortunately, as mentioned in the foregoing chapter, a large number of proteases
can also selectively hydrolyze carboxylic esters and this effectively compensates
for the limited number of esterases [195]. The most frequently used members of this
group are α-chymotrypsin [196], subtilisin [197] and, to a somewhat lesser extent,
trypsin, pepsin [198], papain [199], penicillin acylase [200, 201] and a protease
from Aspergillus oryzae. The latter enzyme seems to be particularly useful for the
selective hydrolysis of bulky esters. As a rule of thumb, when acting on nonnatural
carboxylic esters, most proteases seem to retain a preference for the hydrolysis of
that enantiomer which mimics the configuration of an L-amino acid more
closely [202].
Since many of the studies on the ester-hydrolysis catalyzed by α-chymotrypsin
and subtilisin have been performed together with PLE in the same investigation,
representative examples are not singled out in a separate section but are incorporated into the following chapter.
The structural features of more than 90% of the substrates which have been
transformed by esterases and proteases can be reduced to the general formulas
given in Scheme 2.20. The following general rules can be applied to the construction of substrates for esterases and proteases:
• For both esters of the general type I and II, the center of chirality (marked by an
asterisk [
* ]) should be located as close as possible to the site of the reaction (that
is, the carbonyl group of the ester) to ensure an optimal chiral recognition. Thus,
α-substituted carboxylates and esters of secondary alcohols are usually more
selectively hydrolyzed than their β-substituted counterparts and esters of chiral
primary alcohols, respectively.
• Both substituents R
1 and R
2 can be alkyl or aryl groups, but they should differ in
size and polarity to aid the chiral recognition process of the enzyme. They may
also be joined together to form cyclic structures.
• Polar or charged functional groups located at R
1 and R
2 , such as –OH, –COOH,
–CONH 2 , or –NH 2 , which are heavily hydrated in an aqueous environment
should be absent, since esterases (and in particular lipases) do not accept highly
polar hydrophilic substrates. If such moieties are required, they should be
masked with an appropriate lipophilic protective group.
• The alcohol moieties R
3 of type-I esters should be as short as possible, preferably
methyl or ethyl. If neccessary, the reaction rate of ester hydrolysis may be
enhanced by attaching electron-withdrawing groups to the alcohol moiety to
give methoxymethyl or 2-haloethyl esters, respectively. In contrast, carboxylates
bearing long-chain alcohols are usually hydrolyzed at reduced reaction rates
with esterases and proteases.
2.1 Hydrolytic Reactions
59
overexpression of microbial esterases, the number of synthetically useful
enzymes – possessing a relaxed substrate specificity by retaining high
enantioselectivity – are limited: many novel esterases showed disappointing selectivities [193, 194].
Fortunately, as mentioned in the foregoing chapter, a large number of proteases
can also selectively hydrolyze carboxylic esters and this effectively compensates
for the limited number of esterases [195]. The most frequently used members of this
group are α-chymotrypsin [196], subtilisin [197] and, to a somewhat lesser extent,
trypsin, pepsin [198], papain [199], penicillin acylase [200, 201] and a protease
from Aspergillus oryzae. The latter enzyme seems to be particularly useful for the
selective hydrolysis of bulky esters. As a rule of thumb, when acting on nonnatural
carboxylic esters, most proteases seem to retain a preference for the hydrolysis of
that enantiomer which mimics the configuration of an L-amino acid more
closely [202].
Since many of the studies on the ester-hydrolysis catalyzed by α-chymotrypsin
and subtilisin have been performed together with PLE in the same investigation,
representative examples are not singled out in a separate section but are incorporated into the following chapter.
The structural features of more than 90% of the substrates which have been
transformed by esterases and proteases can be reduced to the general formulas
given in Scheme 2.20. The following general rules can be applied to the construction of substrates for esterases and proteases:
• For both esters of the general type I and II, the center of chirality (marked by an
asterisk [
* ]) should be located as close as possible to the site of the reaction (that
is, the carbonyl group of the ester) to ensure an optimal chiral recognition. Thus,
α-substituted carboxylates and esters of secondary alcohols are usually more
selectively hydrolyzed than their β-substituted counterparts and esters of chiral
primary alcohols, respectively.
• Both substituents R
1 and R
2 can be alkyl or aryl groups, but they should differ in
size and polarity to aid the chiral recognition process of the enzyme. They may
also be joined together to form cyclic structures.
• Polar or charged functional groups located at R
1 and R
2 , such as –OH, –COOH,
–CONH 2 , or –NH 2 , which are heavily hydrated in an aqueous environment
should be absent, since esterases (and in particular lipases) do not accept highly
polar hydrophilic substrates. If such moieties are required, they should be
masked with an appropriate lipophilic protective group.
• The alcohol moieties R
3 of type-I esters should be as short as possible, preferably
methyl or ethyl. If neccessary, the reaction rate of ester hydrolysis may be
enhanced by attaching electron-withdrawing groups to the alcohol moiety to
give methoxymethyl or 2-haloethyl esters, respectively. In contrast, carboxylates
bearing long-chain alcohols are usually hydrolyzed at reduced reaction rates
with esterases and proteases.
2.1 Hydrolytic Reactions
59