An elegant example of a protease-catalyzed hydrolysis of a carboxylic ester was
demonstrated by the dynamic resolution of the antiinflammatory agent ‘ketorolac’
via hydrolysis of its ethyl ester by an alkali-stable protease derived from Streptomyces griseus (Scheme 2.39) [85]. When the hydrolysis was carried out at pH > 9,
base-catalyzed in-situ racemization of the substrate ester provided more of the
enzymatically hydrolyzed (S)-enantiomer from its (R)-counterpart, thereby raising
the theoretical yield of this racemate resolution to 100%.
Optimization of Selectivity
Stereoselective enzymatic hydrolysis of nonnatural esters often shows imperfect
selectivities with moderate to good Enantiomeric Ratios of about E ¼ 3–20, which
translates into e.e. P values of 50–90%. In order to avoid tedious and materialconsuming processes to enhance the optical purity of the product, e.g., by crystallization or via repeated kinetic resolution, several methods exist to improve the
selectivity of an enzymatic transformation itself [24, 292]. In principle, they can be
applied to all types of enzymes.
Since every catalytic system consists of three main components – (bio)catalyst,
substrate, and medium – there are three possibilities for the tuning of the
selectivity:
• Substrate modification is a straightforward and widely employed strategy.
• Altering the properties of the medium – pH, temperature, cosolvents – within
certain limits is a simple and powerful technique to enhance enzyme
selectivities.
• The ability to choose a different biocatalyst with a superior selectivity for a
given substrate depends on the number of available candidates from the same
CO 2 Me
Ph
HO
CF 3
CO 2 H
Ph
HO
CF 3
CO 2 Me
Ph
HO
CF 3
rac
+
buffer
protease
subtilisin
Aspergillus oryzae protease
25
88
2
46
protease
e.e. [%]
E
25
88
e.e. [%]
Scheme 2.38 Resolution of bulky esters by subtilisin and Aspergillus oryzae protease
base-catalyzed
pH >9
in-situ racemization
e.e. 85%, yield 92%
Ketorolac
protease
Streptomyces griseus
S
R S
N
Ph
O
CO 2 Et
N
Ph
O
CO 2 H
Scheme 2.39 Dynamic resolution with in-situ racemization by protease from Streptomyces
griseus
72
2 Biocatalytic Applications
demonstrated by the dynamic resolution of the antiinflammatory agent ‘ketorolac’
via hydrolysis of its ethyl ester by an alkali-stable protease derived from Streptomyces griseus (Scheme 2.39) [85]. When the hydrolysis was carried out at pH > 9,
base-catalyzed in-situ racemization of the substrate ester provided more of the
enzymatically hydrolyzed (S)-enantiomer from its (R)-counterpart, thereby raising
the theoretical yield of this racemate resolution to 100%.
Optimization of Selectivity
Stereoselective enzymatic hydrolysis of nonnatural esters often shows imperfect
selectivities with moderate to good Enantiomeric Ratios of about E ¼ 3–20, which
translates into e.e. P values of 50–90%. In order to avoid tedious and materialconsuming processes to enhance the optical purity of the product, e.g., by crystallization or via repeated kinetic resolution, several methods exist to improve the
selectivity of an enzymatic transformation itself [24, 292]. In principle, they can be
applied to all types of enzymes.
Since every catalytic system consists of three main components – (bio)catalyst,
substrate, and medium – there are three possibilities for the tuning of the
selectivity:
• Substrate modification is a straightforward and widely employed strategy.
• Altering the properties of the medium – pH, temperature, cosolvents – within
certain limits is a simple and powerful technique to enhance enzyme
selectivities.
• The ability to choose a different biocatalyst with a superior selectivity for a
given substrate depends on the number of available candidates from the same
CO 2 Me
Ph
HO
CF 3
CO 2 H
Ph
HO
CF 3
CO 2 Me
Ph
HO
CF 3
rac
+
buffer
protease
subtilisin
Aspergillus oryzae protease
25
88
2
46
protease
e.e. [%]
E
25
88
e.e. [%]
Scheme 2.38 Resolution of bulky esters by subtilisin and Aspergillus oryzae protease
base-catalyzed
pH >9
in-situ racemization
e.e. 85%, yield 92%
Ketorolac
protease
Streptomyces griseus
S
R S
N
Ph
O
CO 2 Et
N
Ph
O
CO 2 H
Scheme 2.39 Dynamic resolution with in-situ racemization by protease from Streptomyces
griseus
72
2 Biocatalytic Applications