5 - Max-Planck-Institut für Kohlenforschung

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Synthetic Organic Chemistry - M. T. Reetz
• study different types of enzymes such as lipases, epoxide hydrolases and
oxidases
Another goal of paramount importance is to reveal the crucial reasons for improved
enantioselectivity of optimized mutant enzymes, i.e., to learn from directed evolution.
Results: In 1997 we reported the first example of an evolutionary process in the
creation of an enantioselective enzyme, specifically in the lipase (Pseudomonas
aeruginosa)-catalyzed kinetic resolution of the chiral ester 1. Whereas the wild-type
enzyme shows a selectivity factor of only E = 1.1 in favor of the (S)-acid 2, four rounds
of mutagenesis using error-prone PCR provided a mutant lipase displaying improved
enantioselectivity (E = 11.3). The screening was performed using a crude UV/VIS test
on a 96-well microtiter plate which allowed about 400-500 mutants to be assayed per
day.
R CO 2R'
CH 3
H 2O
lipase
R CO 2H
CH 3
+
R CO 2H
CH 3
rac-1 (S)-2 (R)-2
R = C 8H 17; R' = p-NO 2-C 6H 4
In 2001 enantioselectivity was increased to E > 51 by the application of epPCR at
higher mutation rate, saturation mutagenesis and DNA shuffling. The best mutant has
six amino acid substitutions, five of these mutations occurring at remote sites and one
closer to the active site. A major goal of the remaining project was to uncover the
reasons for these surprising observations, which was achieved in a collaborative effort
with the Theory Department (W. Thiel). The MM/QM study shows that the (S)- and
(R)-substrates are not bound enantioselectively in the Michaelis-Menten complex!
Rather, a larger binding pocket has been created which accommodates both
enantiomers, but the (S)-substrate reacts faster due to a relay mechanism originating
from remote positions. The transition state of the reaction of the (S)-ester is stabilized
by an additional H-bond, which is not possible with the (R)-substrate (see report by W.
Thiel).
Recently the best lipase mutant was also tested successfully with alkyl esters of 1, but
upon varying the structure of the acid part, problems arose. Sterically demanding
substrates such as iboprufen ester or achiral substrates such as benzoic ester are not
accepted. The problem of substrate acceptance (not just enantioselectivity) is of