Directed evolution and axial chirality: Optimization of the enantioselectivity of Pseudomonas aeruginosa lipase towards the kinetic resolution of a racemic allene

Article abstract of DOI:10.1039/b700849j

Directed evolution of Pseudomonas aeruginosa lipase by the use of combinatorial active site saturation test (CAST) criteria provided a highly enantioselective mutant (Leu162Phe) for kinetic resolution of an axially chiral allene, p-nitrophenyl 4-cyclohexy

Full text of DOI:10.1039/b700849j

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Directed evolution and axial chirality: optimization of the
enantioselectivity of Pseudomonas aeruginosa lipase towards the kinetic
resolution of a racemic allene{
a
Jos e´ Daniel Carballeira, Patrik Krumlinde, Marco Bocola, Andreas Vogel, Manfred T. Reetz* and
b
a
a
a
b
Jan-E. B a¨ ckvall*
Received (in Cambridge, UK) 18th January 2007, Accepted 2nd February 2007
First published as an Advance Article on the web 21st February 2007
DOI: 10.1039/b700849j
Directed evolution of Pseudomonas aeruginosa lipase by the use
of combinatorial active site saturation test (CAST) criteria
provided a highly enantioselective mutant (Leu162Phe) for
kinetic resolution of an axially chiral allene, p-nitrophenyl
the enantioselectivity in the kinetic resolution of p-nitrophenyl
2-methyldecanoate. The wild-type enzyme gave a very low
enantioselectivity (E = 1.2 (S)) with the latter substrate.
Although several of the libraries presented some interesting results,
the mutants showing a real improvement were all from the same
library (library D).
4-cyclohexyl-2-methylbuta-2,3-dienoate (E = 111); the high
enantioselectivity of the Leu162Phe mutant was rationalized by
We therefore decided to try the CAST strategy for the kinetic
resolution of an allene structure and to point out that it is possible
to apply directed evolution by means of CASTing to increase the
enantioselectivity of an enzyme versus axial chirality.
p–p stacking.
Biocatalysis has emerged as one of the most important areas of
1,2
enantioselective organic synthesis. The scaling up of biocatalytic
processes is rather uncomplicated since many enzymes are readily
accessible to a low cost, and furthermore, they can often be easily
The p-nitrophenyl ester 1 was chosen as substrate, due to its
similarity with the non-allenic substrates, such as 2 (Fig. 1), that
16a
3
immobilized and reused. Recently, enzymatic resolution has been
previously led to positive results with the designed libraries.
successfully combined with in situ metal-catalyzed racemization of
The wild-type lipase of Pseudomonas aeruginosa gave a
moderate E value (E = 8.5) in the enantioselective hydrolysis of
1 showing preference for the (+)-enantiomer (cf. Table 1, entry 6).
The corresponding kinetic resolution of substrate 2 with the wild-
type enzyme is almost stereorandom (E = 1.2).
the substrate, which leads to highly efficient dynamic kinetic
4–7
resolution (DKR). It is of great interest to broaden the substrate
tolerance of these enzymes as well as to extend their scope to new
types of substrates. In this respect directed evolution is a powerful
tool, and substantial progress has recently been made in this
Due to the limitations of the evaluation of the enantioselectivity
by gas chromatography (y1 h/sample), we were forced to
dramatically reduce the size of the library to be screened. From
8,9
area.
Allenes are important compounds that have attracted consider-
able interest during the past decade. They have recently been found
1
6
a previous screening of 15000 variants, a total of 600 mutants
had been selected that showed different behaviour compared to the
wild-type in terms of enantioselectivity towards the kinetic
resolution of a structurally related ester 2 or in terms of substrate
10–12
to participate in a number of spectacular reactions.
Allenes
possess an axial chirality and we recently reported on a palladium-
1
3
catalyzed racemization of allenes. The latter racemization if
combined with an enzymatic resolution may lead to an efficient
DKR process. However, because allenes are rare in nature only
limited examples of enzymatic resolution of axially chiral allenes
1
6
scope towards other p-nitrophenyl esters. These 600 mutants
were screened towards enantioselective hydrolysis of 1.
From the screening, mutants with a higher enantioselectivity
compared to the wild-type in kinetic resolution of 1 were found as
well as mutants that showed the reverse enantiopreference
14,15
are known in the literature.
We now report on a mutant from
directed evolution of Pseudomonas aeruginosa lipase (PAL) that
gives very high enantioselectivity (E . 100) in the kinetic resolution
of an axially chiral allene.
(selectivity for the (2)-enantiomer) (Table 1). As for 2, the best
results for the allenic substrate 1 were obtained with library D
variants (mutants of residues Leu159 and Leu162). The best
mutant obtained harbors a single mutation Leu162Phe (L162F)
that enhances the enantioselectivity of the enzyme from E = 8.5 for
the wild-type to E = 111 (showing the same enantiopreference,
giving the (+)-isomer) (Table 1, entry 1). This mutant was superior
to all lipases tested for the kinetic resolution of 1 (ESI{) and the E
is by far the highest known for axially chiral allenes. Due to the
During the development of combinatorial active site saturation
test (CAST) method using Pseudomonas aeruginosa lipase (PAL)
five different saturation mutagenesis libraries (A–E) were selected
1
6
according to the CAST criteria (see ESI{). These libraries were
tested towards a collection of bulky p-nitrophenyl esters with the
objective to expand the substrate scope, and in parallel to enhance
a
Max-Planck-Institut f u¨ r Kohlenforschung, Kaiser-Wilhelm-Platz 1,
D-45470, M u¨ lheim/Ruhr, Germany.
E-mail: reetz@mpi-muelheim.mpg.de
Department of Organic Chemistry, Arrhenius Laboratory, Stockholm
b
University, SE-106 91, Stockholm, Sweden. E-mail: jeb@organ.su.se
{ Electronic supplementary information (ESI) available: Experimental
procedures and analysis of the products. See DOI: 10.1039/b700849j
Fig. 1
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Chem. Commun., 2007, 1913–1915 | 1913

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Table 1 Kinetic resolution of the p-nitrophenyl allene ester 1 using
different mutants from Pseudomonas aeruginosa lipase
Table 2 Energies of the presumed tetrahedral intermediates for the
hydrolysis of 1 using the wild-type lipase of Pseudomonas aeruginosa
lipase and the mutants Leu162Gly and Leu162Phe
Library/
source
Conv.
(%)
a
ee
21
Entry Variant
E
Energy/kcal mol
R-tetrahedral
intermediate
S-tetrahedral
intermediate
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
a
Leu162Phe
Leu162Val
Leu162Ile
Leu162Ala
Leu162Thr
P. aeruginosa lipase
Leu162Asn
D
D
D
D
D
WT
D
D
A
96 (+) 44
91 (+) 40
90 (+) 39
82 (+) 29
77 (+) 23
74 (+) 25
75 (+) 20
68 (+) 17
59 (+) 23
56 (+) 31
55 (+) 17
42 (+) 28
20 (2) 18
16 (2) 24
15 (2) 22
26 (2) 15
111
39
33
13
10
8.5
8.4
6
4.2
4.5
3.9
2.8
1.6
1.5
1.4
1.8
Variant
D (R 2 S)
WT
Leu162Gly
Leu162Phe
218.1
223.2
220.7
216.1
228.8
217.4
+2.0
25.4
+3.3
Leu159Trp/Leu162Thr
Met16Ala, Leu17Phe
Val232Ile
mutant 15B10 (V232I, M16L, A34T, P86L, D113G, S237T,
T150A, S147N, V94A, T87S, L208H) were previously found to
give the best (R)- and (S)-selectivity, respectively in the kinetic
0
1
2
3
4
5
6
E
b
15B10
Leu231Ile/Val232Cys
Leu162Gly
E
18
D
D
D
resolution of 2 after several rounds of directed evolution. Also,
for the kinetic resolution of substrate 1 these two mutants show
opposite enantioselectivity, albeit with low E values (Table 1,
entries 11 and 16).
Leu162Gly/Leu159Tyr
Leu162Gly/Leu159Val
1H8
c
Enantiomeric excess of the acid (4-cyclohexyl-2-methylbuta-2,3-
Interestingly, mutant Leu162Phe shows a much larger enhance-
ment of the enantioselectivity in the kinetic resolution of 1
dienoic acid). The first eluting enantiomer of the acid has a positive
rotation (+). 15B10 represents the following mutations: V232I,
b
M16L, A34T, P86L, D113G, S237T, T150A, S147N, V94A, T87S,
L208H. 1H8 represents the following mutations: D20N, S53P,
S155M, L162G, T180I, T234S
(EL162F .100, Ewild-type = 8.5) compared to the kinetic resolution
c
of 2 (EL162F = 5, Ewild-type = 1.2) and this observation may be
explained by the p–p stacking effect between the phenyl side chain
of phenylalanine 162 and the p-electrons of the allene bond (Fig. 2).
This interaction may affect the orientation of the methyl group in
the 2-position of 1, favoring the recognition of one of the
enantiomers. In the case of 2, this type of interaction is not
possible. The p–p stacking effect is very important for the
high degree of enantioselectivity displayed by the best hit in the
initial library D of mutants, it was not necessary to engage in an
evolutionary process by performing iterative CASTing.
The dramatic increase of the E value from 8.5 to an E value of
over 100 on changing leucine to phenylalanine at position 162
1
9
stabilization of the protein structure as previously described and
may also be responsible for the major changes in the interaction
with the substrate and in the conformation of the substrate
(
Leu162Phe), as well as the considerable effects found with other
amino acid exchanges (Leu162Val, E = 39 and Leu162Ile, E = 33)
Table 1, entries 2 and 3), is especially remarkable as position
(
16b,20
binding site.
Leu162 was previously found to be critical for the enantioselec-
tivity in the kinetic resolution of 2. In the latter case the single
mutation responsible for the most dramatic effect on the
enantioselectivity was found by changing leucine for glycine at
this position (Leu162Gly), which improved the E value from 1.2
The energies of the presumed (R) and (S) tetrahedral
intermediates in the hydrolysis of the p-nitrophenyl allene ester 1
catalyzed by the wild type enzyme and variants Leu162Gly and
Leu162Phe were calculated with MOLOC using the same protocol
21
21
as previously published. In that study it was shown that the
tetrahedral intermediate is representative for the rate-limiting
catalytic step in ester hydrolysis. Thus, we can relate the difference
in stabilization energy between the two enantiomers of the
modeled tetrahedral intermediates directly with the observed
enantioselectivity. The results confirmed the inversion in enantio-
selectivity in the case of Leu162Gly and the enhanced enantio-
selectivity with Leu162Phe (Table 2).
(
S-selective) of the wild-type to an E value of 32 (S-selective) in the
17
hydrolysis of 2. Also for the kinetic resolution of 1, variant
Leu162Gly results in a significant enantioselectivity change,
inverting the enantioselectivity to give the (2)-isomer with E =
1
.6 (Table 1, entry 13).
In general, a given mutant provides similar consequences in
terms of enantioselectivity towards substrates 1 and 2. Thus,
mutant 1H8 (D20N, S53P, S155M, L162G, T180I, T234S) and
In conclusion, directed evolution by means of CASTing and
iterative–CASTing has proven to be a fast and reliable way to
2
2
increase the enantioselectivity of an enzyme. In this work a
highly enantioselective mutant for kinetic resolution of an axially
chiral allene was found by screening a reduced library of
600 mutants obtained by CASTing.
We thank the Swedish Foundation for Strategic Research, the
European Community (Network of Excellence IDECAT), the
Fonds der Chemischen Industrie and the German-Israeli Project
Cooperation (DIP) for financial support.
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1
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