Mutated variant of Candida antarctica lipase B in (S)-selective dynamic kinetic resolution of secondary alcohols

Article abstract of DOI:10.1039/c0ob00748j

An (S)-selective dynamic kinetic resolution of secondary alcohols, employing a mutated variant of Candida antarctica lipase B (CalB) gave products in 84-88% yield and in 90-97% ee.

Full text of DOI:10.1039/c0ob00748j

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Mutated variant of Candida antarctica lipase B in (S)-selective dynamic
kinetic resolution of secondary alcohols†
Karin Engstro¨m,a Michaela Vallin,b Per-Olof Syre´n,b Karl Hultb and Jan-E. Ba¨ckvall*a
Received 20th September 2010, Accepted 29th October 2010
DOI: 10.1039/c0ob00748j
An (S)-selective dynamic kinetic resolution of secondary
alcohols, employing a mutated variant of Candida antarctica
lipase B (CalB) gave products in 84–88% yield and in 90–97%
ee.
in 2005.7,8 This variant of CalB, where Trp104 was changed
into an alanine (CalB W104A), has an enlarged stereospecificity
pocket. As predicted by modelling,5 this variant can accept
larger substrates than wild type CalB. Furthermore, it shows (S)-
selectivity towards 1-phenylethanol and other secondary alcohols.
A recent study of CalB W104A investigated its use in kinetic
resolution of 1-phenylalkanols.9 This study showed that its
stereoselectivity pocket can accommodate 1-phenylalkanols with
an alkyl chain of up to eight carbon atoms and the (S)-enantiomer
was preferred. The enantioselectivity was unsatisfactory for 1-
phenylethanol (E = 3) but acceptable for the longer phenylalkanols.
The highest E values were obtained for substrates with the long
alkyl chains.
Based on these results, we wanted to explore the possibility
to apply the (S)-selective CalB W104A in a dynamic kinetic
resolution (DKR). (S)-selective DKR’s of alcohols are rare and
there are only two examples known in the literature.10,11 These
examples employ the serine protease Subtilisin Carlsberg as the
(S)-selective enzyme. The latter enzyme is quite fragile and can not
be used at elevated temperatures. It was therefore highly desirable
to develop an (S)-selective DKR with a less sensitive enzyme.
We first applied CalB W104A in a kinetic resolution of 1-
phenylpropanol (1a) and 1-phenylheptanol (1b), to find the proper
conditions for the DKR. Isopropenyl acetate was employed as
acyl donor. The kinetic resolution (KR) with CalB W104A had
previously been reported in cyclohexane. This is not a suitable
solvent for DKR as it does not dissolve catalyst 4 used for the
racemization of the alcohol. A more suitable solvent for DKR
is toluene, and therefore this solvent was used for the KR of
1a and 1b (Table 1). CalB W104A had a significantly higher E
value towards 1-phenylpropanol (1a) in toluene at 60 ◦C (E =
82, Table 1, entry 1) compared to that previously reported in
cyclohexane.12 The KR of 1b was run at different temperatures
and the enantioselectivity (E) was determined (Table 1, entries
2–5). The enantioselectivity was higher in toluene (E = 176) than
in cyclohexane13 at 60 ◦C. The enantioselectivity increased with
temperature in toluene. This temperature effect was previously
reported also for 1-phenylethanol with CalB W104A in various
solvents,7 but is otherwise quite unusual, and opposite to the
temperature effect observed for 1b in cyclohexane.9
There is an increasing demand of enantiomerically pure com-
pounds in organic synthesis, and chiral secondary alcohols are
important building blocks in the preparation of natural products,
fine chemicals and pharmaceuticals. Enzymatic resolution with
lipases has become one of the most popular methods for the
preparation of enantiomerically pure alcohols.1 Lipases are widely
used in organic synthesis due to their high enantioselectivity
towards a wide range of substrates and their ability to work
efficiently in organic solvent.2 Lipases require no cofactors; they
acylate alcohols in organic solvent, and are especially enantioselec-
tive towards secondary alcohols. The enantioselectivity of lipases
follows Kazlauskas rule, which predicts (R)-selectivity for most
substrates.3
Candida antarctica lipase B (CalB) is a thermostable lipase
that shows a high natural enantioselectivity towards secondary
alcohols and chiral primary amines and has been used as an
enantioselective biocatalyst in a large number of transformations.4
The high enantioselectivity can be explained based on the different
binding modes for the enantiomers in the active site.5,6 The fast-
reacting enantiomer positions its medium-sized group in the so
called stereoselectivity pocket and its large group towards the
active-site entrance. The size of the stereoselectivity pocket of
CalB is limited by the sidechains of three residues; Thr42, Ser47
and Trp104 and the largest alkyl group that can smoothly fit into
this pocket is ethyl. The slow-reacting enantiomer needs to put
its large group in the stereospecificity pocket to be in the correct
position for catalytic activity. It does not fit however, and this steric
limitation is what makes CalB highly enantioselective towards
secondary alcohols when one of the groups are ethyl or smaller.
A variant of CalB, with a mutation in one of the residues limiting
the size of the stereoselectivity pocket, Trp104 was published
aDepartment of Organic Chemistry, Arrhenius Laboratory, Stockholm
University, SE-106 91, Stockholm, Sweden. E-mail: jeb@organ.su.se;
Fax: (+46) 8-154908; Tel: (+46) 8-6747178
bDepartment of Biochemistry, School of Biotechnology, Royal Institute of
Technology (KTH), AlbaNova University Center, SE-106 91 Stockholm,
Sweden
As both the activity and the enantioselectivity were highest at
60 ◦C, this was the temperature that was chosen for the initial
DKR. The first DKR was performed with 1-phenylpropanol (1a)
as substrate. The reaction showed good enantioselectivity and
† Electronic supplementary information (ESI) available: Supplementary
data. See DOI: 10.1039/c0ob00748j
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Table 1 Kinetic resolution of 1-phenylpropanol (1a) and 1-phenyl-
heptanol (1b)a
Formation of ketone has previously been observed dur-
ing racemization by catalyst 4 for bicyclic diols at elevated
temperatures.14 This ketone formation is believed to be a result
of substitution of the intermediate substrate ketone on the metal
center by acetone, a byproduct from the acyl donor. Cross-
over experiments have shown that this kind of ketone–ketone
exchange takes place to some extent if the racemization is left
after completion at elevated temperatures.15
In an attempt to reduce the ketone-formation by reducing the
amounts of by-product acetone present in the solution from the
Entry Substrate T/◦C Time/h eep (%)b ees (%)b Conv.(%)c
E
◦
˚
1d
2
3
4
5
1a
1b
1b
1b
1b
60
60
40
30
r.t.
24
6
24
72
72
96.4
98.4
98.0
97.7
98.0
44.2
36.0
31.5
42.6
14.8
32
33
23
29
13
85 ( 10)
acyl donor, a DKR was run at 60 C in the presence of 4 A
molecular sieves. This reduced the amount of ketone to 7% (Table
2, entry 5). These conditions were then also employed to the DKR
of 1-phenylpentanol (1c), which gave the product 2c in 88% yield
and 90% ee (Table 2, entry 6).
177 ( 25)
134 ( 15)
127 ( 15)
114 ( 15)
a Conditions: 1 (0.5 mmol), isopropenyl acetate (1.5 equiv.), CalB W104A
(20 mg), toluene (1.0 mL) b Determined by chiral GC. c Determined by 1H
NMR. d 1 (0.25 mmol), CalB W104A (5 mg), toluene (0.5 mL).
To conclude, an efficient (S)-selective DKR of 1-phenylalkanols
has been developed. 1-Phenylpropanol was efficiently acylated at
60 ◦C to give the product in 96.5% enantiomeric excess. DKR
of 1-phenylheptanol required lower temperatures (and longer
reaction times) or addition of molecular sieves to suppress ketone
formation. The product was obtained in 97% ee in high yield. This
is the first (S)- selective DKR of a sec-alcohol performed with an
enzyme that can be used at elevated temperatures.
Table 2 (S)-Selective dynamic kinetic resolution of secondary alcoholsa
Acknowledgements
Financial support from the Swedish Research Council, the
Swedish Foundation for Strategic Research and the K&A Wal-
lenberg Foundation is gratefully acknowledged.
Entry Substrate
T/◦C Time/h eep (%)b
Conv. (%)c % 3c
Notes and references
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2 T. Ema, Curr. Org. Chem., 2004, 8, 1009–1025.
3 When the medium-sized group has a lower priority than the large
group, the compound will have (R)-stereochemistry according to the
Cahn–Ingold–Prelog rules: R. J. Kazlauskas, A. N. E. Weissfloch,
A. T. Rappaport and L. A. Cuccia, J. Org. Chem., 1991, 56, 2656–
2665.
4 E. M. Anderson, K. M. Larsson and O. Kirk, Biocatal. Biotransform.,
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1
2
3
1a
1b
1b
1b
1b
1c
60
60
40
30
60
60
36
26
88
6 days
24
96.5
94
95
97
96.5
90
99 (85)d
93
10
38
24
1
7
8
97
4e
5e,f
6e,f
99 (84)d
97(84)d
99 (88)d
24
a Conditions: 1 (0.5 mmol), isopropenyl acetate (1.5 equiv.), Na2CO3 (1
equiv.), Ru-catalyst 4 (0.05 equiv.), tBuOK (0.05 equiv.), CalB W104A (20
mg), toluene (1.0 mL). b Determined by chiral GC. c Determined by 1H
NMR. d Isolated yields in parenthesis. e CalB W104A (30 mg). f 350 mg
˚
molecular sieves (4 A) added.
6 D. Rotticci, F. Haeffner, C. Orrenius, T. Norin and K. Hult, J. Mol.
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after 36 h full conversion was reached (Table 2, entry 1). The
product ester (2a) had an enantiomeric excess of 96.5%, and also
contained 10% of the corresponding ketone 3a.
DKR of 1-phenylheptanol (1b) was also performed at 60 ◦C.
However, this DKR yielded as much as 38% of the corresponding
ketone, 1-phenylheptanone (3b) (Table 2, entry 2). To avoid ketone
formation in the DKR of 1-phenylheptanol (1b), the temperature
was decreased. At 40 ◦C, the amount of ketone was reduced to
24%, and at 30 ◦C, only 1% was formed (Table 2, entries 3–
4) However, longer reaction times were required to reach full
conversion at decreased temperatures. At 30 ◦C, 6 days was
required to reach full conversion (99%). Under these conditions,
(S)-2b was obtained in 97% ee.
11 L. Bore´n, B. Mart´ın-Matute, Y. Xu, A. Co´rdova and J.-E. Ba¨ckvall,
Chem.–Eur. J., 2006, 12, 225–232.
12 The E value was previously reported as 28 at 56 ◦C in cyclohexane9.
13 The E value in cyclohexane at 60 ◦C under the same conditions was
determined to 76.
14 P. Krumlinde, K. Boga´r and J.-E. Ba¨ckvall, Chem. Eur. J., 2010, 16,
4031–4036.
15 B. Mart´ın-Matute, M. Edin, K. Bogar, F. B. Kaynak and J.-E. Ba¨ckvall,
J. Am. Chem. Soc., 2005, 127, 8817–8825.
82 | Org. Biomol. Chem., 2011, 9, 81–82
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