Mutant lipase-catalyzed kinetic resolution of bulky phenyl alkyl sec-alcohols: A thermodynamic analysis of enantioselectivity

Article abstract of DOI:10.1002/cbic.200900635

The size of the stereoselectivity pocket of Candida antarctica lipase B limits the range of alcohols that can be resolved with this enzyme. These steric constrains have been changed by increasing the size of the pocket by the mutation W104A. The mutated enzyme has good activity and enantioselectivity toward bulky secondary alcohols, such as 1-phenylalkanols, with alkyl chains up to eight carbon atoms. The S enantiomer was preferred in contrast to the wild-type enzyme, which has R selectivity. The magnitude of the enantioselectivity changes in an interesting way with the chain length of the alkyl moiety. It is governed by interplay between entropic and enthalpic contributions and substrates with long alkyl chains are resolved best with E values higher than 100. The enantioselectivity increases with temperature for the small substrates, but decreases for the long ones.

Full text of DOI:10.1002/cbic.200900635

DOI: 10.1002/cbic.200900635
Mutant Lipase-Catalyzed Kinetic Resolution of Bulky
Phenyl Alkyl sec-Alcohols: A Thermodynamic Analysis of
Enantioselectivity
Michaela Vallin, Per-Olof Syrꢀn, and Karl Hult*[a]
Dedicated to Jaap Jongejan on the occasion of his 65th birthday.
The size of the stereoselectivity pocket of Candida antarctica li-
pase B limits the range of alcohols that can be resolved with
this enzyme. These steric constrains have been changed by in-
creasing the size of the pocket by the mutation W104A. The
mutated enzyme has good activity and enantioselectivity to-
ward bulky secondary alcohols, such as 1-phenylalkanols, with
alkyl chains up to eight carbon atoms. The S enantiomer was
preferred in contrast to the wild-type enzyme, which has R se-
lectivity. The magnitude of the enantioselectivity changes in an
interesting way with the chain length of the alkyl moiety. It is
governed by interplay between entropic and enthalpic contri-
butions and substrates with long alkyl chains are resolved best
with E values higher than 100. The enantioselectivity increases
with temperature for the small substrates, but decreases for
the long ones.
Introduction
Enantioenriched secondary alcohols are ubiquitous in the
structures of natural products and pharmaceuticals. Several ap-
proaches to their preparation are available, such as asymmetric
transfer hydrogenation, catalytic hydrogenation[1] and CBS re-
duction[2] (oxazaborolidines first reported by Corey, Bakshi and
Shibata) of ketones and metal-catalyzed alkylation of alde-
hydes.[3] Biocatalysis is now becoming a key component in the
toolbox of the synthetic chemist.[4–5] Biocatalytic methods to
chiral sec-alcohols include lipase-catalyzed transacylation and
hydrolysis reactions of alcohols and esters, and oxidoreduc-
tase-catalyzed hydrogen transfer reactions of racemic alcohols
or prochiral ketones. The scope of the biocatalytic methods
available today is restricted to substrates with only one bulky
substituent at the stereocenter. To the best of our knowledge,
there is no lipase in general use for kinetic resolution (KR) of
bulky chiral sec-alcohols bearing two substituents of similar
size. Nevertheless, a systematic study on lipase-promoted
transacylation (five different wild-type lipases were screened)
concluded that Pseudomonas aeruginosa lipase is active and R
selective toward bulky phenylalkanols.[6] Pseudomonas stutzeri
lipase-catalyzed dynamic kinetic resolution (DKR) of 1,2-diaryl-
ethanols[7] and benzoins[8–9] (1,2-diaryl-2-hydroxyethanone
structures) have been recently reported. In addition, substrates
that have very bulky side groups flanking the carbonyl, such as
phenyl isopropyl ketone, tend to be poor substrates for most
known alcohol dehydrogenases.[10] Hence, explorations of
enzyme substrate promiscuity[11–12] are important within this
field.
cially available, stable in organic solvents and tolerates high
temperatures. CalB is active and highly enantioselective in KR
of a wide range of alcohols containing one large and one
small substituent. In hydrolysis/synthesis of esters, the enantio-
preference is predicted by Kazlauskas’ rule: if the large sub-
stituent has the higher priority according to the CIP nomencla-
ture, the R enantiomer reacts preferentially.[18] The crystal struc-
ture of CalB has been known since 1994.[19] CalB belongs to
the family of serine hydrolases with an a/b-hydrolase fold.[20]
The active site hosts a catalytic triad consisting of Ser105,
His224 and Asp187 and the catalytically important oxyanion
hole.[19] In a catalytically active conformation, the alcohol posi-
tions its large substituent toward the entrance of the active
site of CalB, and fits its small substituent into a cavity called
the stereoselectivity pocket (defined by Thr42, Ser47 and
Trp104). The steric constraints on the substrate are set by the
size of this pocket; a drastic drop in both activity and selectivi-
ty is seen when both substituents are larger than an ethyl
group.[17] Due to this constraint the substrates 1c–g are not
substrates for the wild-type enzyme. In CalB, Trp104 forms the
bottom of the steroselctivity pocket. As part of our ongoing
program on the rational design of CalB for new or improved
activities, the CalB W104A mutant was previously identified by
molecular modeling as a biocatalyst of potential use toward
more sterically demanding sec-alcohols than those that fit into
the wild-type enzyme.[21–22] The mutant was prepared and
[a] Dr. M. Vallin, P.-O. Syrꢀn, Prof. K. Hult
Department of Biochemistry, School of Biotechnology
Royal Institute of Technology (KTH), AlbaNova University Center
10691 Stockholm (Sweden)
Candida antarctica lipase B (CalB, EC 3.1.1.3, sometimes re-
ferred to as Pseudozyma antarctica lipase) is one of the most
frequently used enzymes in organic synthesis for the prepara-
tion of optically active alcohols and amines.[13–17] It is commer-
Fax: (+46)8-5537-8468
E-mail: kalle@biotech.kth.se
ChemBioChem 2010, 11, 411 – 416
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
411

K. Hult et al.
esterification reactions were suc-
cessfully performed with 4-hep-
tanol and 5-nonanol.[21] When
evaluated against phenylethanol,
phenylpropanol and 2-hexanol,
which are substrates tolerated
also by the wild type, the
W104A mutation was found to
reverse the enantiopreference of
CalB.[22] The E values were low
(2<E<12). The E value of the
mutant toward phenylethanol
could, however, be increased
from 8 to 44 by the appropriate
choice of solvent in combination
with a higher temperature.
Table 1. Kinetic resolution of sec-alcohols.[a]
Substrate
n
T
[8C]
t
Conversion
[%][b]
eep
E[d]
568C
Trac
[8C]
[min]
[%][c]
288C
1.4 (R)
17 (S)
48 (S)
13 (S)
36 (S)
100 (S)
400 (S)
100 (S)
100 (S)
708C
1a
1b
1c
1d
1e
1 f
1g
1 f[f]
1 f[g]
0
1
2
3
4
5
6
5
5
28
28
28
28
28
28
28
56
70
120
120
120
120
120
120
120
90
17
19
32
22
19
15
6[e]
32
43
17
86
92
83
93
98
99
96
94
2.3 (S)
3.4 (S)
36
ꢀ68
28 (S)
54 (S)
15 (S)
36 (S)
86 (S)
220 (S)
86 (S)
86 (S)
36 (S)
52 (S)
16 (S)
36 (S)
75 (S)
210 (S)
75 (S)
75 (S)
ꢀ223
ꢀ153
ꢀ273
ꢀ559
ꢀ4158
ꢀ559
ꢀ559
90
[a] Conditions: immobilized enzyme (50 mg) and vinyl acetate (0.50 mmol) were added at room temperature
to a solution of alcohol (0.10 mmol) and dodecane (0.10 mmol), which was used as internal standard, in cyclo-
hexane (1 mL) before the mixture was heated to the indicated temperature in a heat shaker. [b] Determined by
GC with use of dodecane as internal standard. [c] Enantiomeric excess of the product measured by chiral GC.
[d] Enantiomeric ratio calculated as an average of eight samples at conversions 0–50% according to Rakels
method from the eep and ees or conversion.[33] The preferred enantiomer is depicted in parenthesis. [e] Deduced
from ees and eep: conv=ees/(ees+eep); [f] 25 mg immobilized enzyme was used; [g] 15 mg immobilized enzyme
was used.
We anticipated the CalB
W104A mutant to be of poten-
tial value for also more sterically
demanding chiral sec-alcohols,
which are too bulky to fit in the
wild type. In this paper, a systematic study on CalB W104A-cat-
alyzed KR of phenylalkanols of varying alkyl chain length is
presented. The length of the substrate alkyl substituent was
found to have an interesting influence on the enantioselectivi-
ty of the catalyst. The underlying reason for this variation in
enzyme selectivity has been studied.
alcohols 1c–g that show little or no reactivity with the wild-
type enzyme were indeed successfully resolved with the CalB
W104A catalyst (Table 1). Alcohols 1a–e have been resolved by
P. aeruginosa lipase in a previous study.[6] Further, KR of alcohol
1c has previously been reported by CalB-catalyzed transacyla-
tion[23] and aminolysis,[24] and by lipase QL (from Alcaligenes sp.)
catalyzed transacylation.[25] These transacylation reactions re-
quire long reaction times and are less selective (CalB: 17%
conversion after seven days, E=19; lipase QL: 30% yield after
63 h, E=12) than KR with the W104A mutant presented here.
Transacylation of alcohol 1d has been reported by use of Burk-
holderia sp. lipase (four days, 52% conversion, E=28)[26] and by
an immobilized preparation from Manihot esculenta roots (five
days, 20% conversion, E=4).[27] Alcohol 1 f has been previously
subjected to Burkholderia cepacia lipase-catalyzed hydrolysis[28]
and transacylation,[29] and alcohol 1g to hydrolysis by the
same enzyme.[28,30] Products 2c–g obtained in the present
study were of the S configuration. This is in contrast to the cor-
responding wild-type catalyzed reactions in the literature,
which all are R selective. Hence, the W104A mutant represents
an enantiocomplementary catalyst to the known wild-type li-
pases. In the mutant, the substrates were oriented with the
phenyl group down into the new, rationally designed, stereo-
selectivity pocket. The immobilized mutant enzyme could also
be used at elevated temperatures (Table 1).
Results and Discussion
Kinetic resolution of sec-alcohols
The performance of the immobilized mutant enzyme was stud-
ied in KR of a homologous series of phenyl alkyl alcohols 1
(Scheme 1). Vinyl acetate was used as acyl donor and cyclohex-
Scheme 1. Kinetic resolution of sec-alcohols.
ane was chosen as the solvent. Phenylethanol (1a) reacted
with the mutant enzyme and gave the R enantiomer of acetate
2a at 288C, but the S enantiomer of acetate 2a reacted at 56
and 708C (Table 1). KR of phenylpropanol (1b) afforded the S
enantiomer of acetate 2b at all temperatures investigated. This
is in contrast to the reactivity of the same alcohols in the wild-
type enzyme, which gives the corresponding R enantiomers. In
the mutant lipase, the activity of the slow-reacting enantiomer
has been increased dramatically.[22] The fast-reacting substrates
now orient the phenyl substituents down in the stereoselectiv-
ity pocket, the size of which has been enlarged by the W104A
mutation.[21] The small alkyl substituents are instead directed
toward the active-site entrance. The more sterically demanding
The enantioselectivities observed were all S configured,
except for 1a at 288C, and varied up and down in an interest-
ing way within the homologous series of substrates (Table 1).
Further, the enantioselectivities of the smallest (1a–b) and
largest (1 f–g) alcohols were temperature dependent, whereas
the selectivities of the medium-sized substrates (1c–e) were
(nearly) independent of temperature. Thermodynamic analysis
of these reactions was performed and the results show how
the relative contributions from enthalpy and entropy dictate
the observed E values (see below).
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ChemBioChem 2010, 11, 411 – 416

Resolution of Bulky Secondary Alcohols
To determine the absolute rate of the lipase-catalyzed KRs,
the active-site concentration of CalB W104A had to be known.
Active-site titration[31–32] was performed on the immobilized
enzyme in organic solvent by use of the fluorophore-releasing,
irreversible active-site inhibitor methyl 4-methylumbelliferyl
hexylphosphonate. The active-site concentration of the immo-
bilized enzyme was found to be 76 nmol per gram of carrier,
which corresponds to 0.26% enzyme load. Absolute rates were
calculated at 568C as the number of moles of product per mol
of catalyst per unit of time, and were found to be: 1a=3 sꢀ1,
1b=4 sꢀ1, 1c=9 sꢀ1, 1d=6 sꢀ1, 1e=6 sꢀ1, 1 f=3 sꢀ1 and
1g=3 sꢀ1. The relative specificity constants (kcat/KM) of the
racemates 1a–g followed the same trend as the absolute
rates. Thus, it can be concluded that there were no major dif-
ferences in substrate affinity to the enzyme within the series of
alcohols. The absolute rates and the relative specificity con-
stants obtained showed that CalB W104A is a successful S-se-
lective catalyst for KR of otherwise difficult, bulky alcohols with
two substituents of similar size next to the chiral center.
Figure 1. Enthalpy and entropy contributions to enantioselectivity calculated
for T=308C.
minor (1c–d) or no (1e) contribution to the enantiodiscrimina-
tion. The very strong effect of entropy can be illustrated by cal-
culation of the enantioselectivity based on enthalpy only. Such
a calculations affords an enzyme with an R selectivity of
330000. On the other hand if only entropy would set the
enantioselectivity one would have seen an S selectivity of
260000.
The racemic temperature (Trac =DS–RDH/DS–RDS)[44] at which
the enantioselectivity is 1 was 368C for the mutant toward
phenylethanol (1a), that is, within the experimental range
(Table 1). Below this temperature the mutant was R selective,
and above it was S selective. Substrates 1 f–g had racemic
temperatures lower than ꢀ2738C, which is indicative for those
more rare cases in which the enthalpic and entropic contribu-
tions favor the same enantiomer.
Enthalpy and entropy contributions to enantioselectivity
Enantioselectivity is caused by a difference in activation free
energy between the enantiomers that might be separated in
an enthalpic and an entropic term. By determination of the
temperature dependence of the enantiomeric ratio E, the
contributions of differential activation enthalpy, DS–RDH°, and
entropy, DS–RDS°, to enantioselectivity can be determined
[Eq. (1)]:
DSꢀRDH°
1
T
DSꢀRDS°
ð1Þ
ln E ¼ ꢀ
ꢁ
þ
It is known that the R and S enantiomers have different ori-
entations to fulfill the necessary network of hydrogen bonds in
transition state when reacting with CalB.[45] For the phenyl alkyl
sec-alcohols the R alcohol has its alkyl substituent pointing
into the stereoselectivity pocket and the phenyl group point-
ing to the active site entrance (Figure 2A), while the S alcohol
R
R
In 1992 Phillips pointed out that the differential activation en-
tropy can in fact contribute significantly to enantioselectivity.[34]
Since then, Hult[22,35–38] and others[39–43] have shown the impor-
tance of entropy in enzyme catalysis in general and in enantio-
selectivity in particular. In the
present investigation, the tem-
perature dependence of E was
determined for transacylation to
sec-alcohols 1a–g. The range of
the experimental temperatures
and observed E values are pre-
sented in Table 1. Figure 1 shows
the results from the thermody-
namic analysis.
For six of the seven substrates
studied, the entropic contribu-
tion is of similar size or greater
than the enthalpic contribution
to the enantioselectivity. These
experimental findings stress that
Figure 2. The active site of the CalB W104A mutant with the acetate ester of: A) (R)-1-phenylbutanol, and B) (S)-1-
phenylbutanol covalently bound to the catalytic serine in the tetrahedral reaction intermediate. The substrate is
presented with a stick model. The stereoselectivity pocket is represented by residues Ala104, Thr42, Ser47, Leu228
and Leu277 and is shown as a molecular surface at the bottom of the figure. The active site entrance is indicated
entropy is an important factor in
enzyme selectivity. In fact, for
medium-sized alcohols it was
the enthalpic term that gave a by the molecular surface of residues Ile189, Leu278 and Ile285 at the top of the figure.
ChemBioChem 2010, 11, 411 – 416
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413

K. Hult et al.
residual solvent peak in CDCl3 as internal standard. Conversion and
enantiomeric excess of reactions with alcohols 1a–f were deter-
mined by gas chromatography employing a CP Chirasil-Dex CB
column, and of reactions with alcohol 1g by using a J&W CycloSil-
B column. For the determination of relative specificity constants,
relative rates were measured on an achiral GC equipped with a CP-
Sil 5CB column. Optical rotations, [a]D, were measured at the so-
dium D line at 208C. Unless otherwise noted, reagents are com-
mercially available and were used without further purification. Phe-
nylethanol (1a) and phenylpropanol (1b) were purchased, and al-
cohols 1c–g were prepared according to the general procedure
described below. For SDS-PAGE, samples were diluted in SDS load-
ing buffer in 4:1 ratio and denatured at 958C for 10 min. Electro-
phoresis was performed by using NuPAGEꢃ 10% Bis-Tris Gel (Invi-
trogen) in a Protein Gel Chamber (Bio-Rad) at 150 V. SeeBlue Plus2
prestained marker (Invitrogen) was used as a molecular weight
marker and MOPS pH 7.7 as running buffer. Proteins were visual-
ized with SimplyBlue SafeStain (Invitrogen). E. coli cells were grown
on LB agar plates and in liquid LB media supplemented with
Zeocin (25 mgmLꢀ1). P. pastoris cells were grown on YPD agar
plates containing Zeocin (100 mgmLꢀ1) if not stated otherwise.
positions the phenyl substituent in the stereoselectivity pocket
and the alkyl group toward the active site entrance (Fig-
ure 2B). The consequence is that the R enantiomer has a limit-
ed space to put its alkyl chain, but the S enantiomer has no
such limitation. In the wild-type enzyme the stereoselectivity
pocket is much smaller and it cannot accommodate a phenyl
easily.
The enthalpic component of enantioselectivity can be relat-
ed to direct interactions to the protein in form of electrostatic
and van der Waals interactions as well as spatial restrictions. It
is clearly seen from the results in Figure 1 that the enthalpic
contributions favor the S enantiomer with increasing chain
length. We suggest that the R enantiomer fills up the stereose-
lectivity pocket more and more with a longer alkyl chain. For
reaction with 1e the pentyl and the phenyl moieties seem to
fill the pocket equally good while longer alkyl chains in 1 f–g
are too long and the S enantiomer is favored. With this model
we predict that substrates with even longer alkyl chains will
afford high S selectivity.
The entropic component of enantioselectivity is much less
understood. It should be related to differential solvation or de-
grees of freedom for the enzyme and substrate during cataly-
sis. In Figure 1 it is seen that entropy strongly favors the S
enantiomer for substrates with short alkyl chains, but with
longer chains the effect more or less disappears. It would be
expected that the R enantiomer with its alkyl chain pointing
into the restricted space of the stereoselectivity pocket would
face less and less degrees of freedom with increasing chain
length. That would have an opposite effect on entropy than
seen. Instead we have to take into account the empty space
that a short alkyl chain would leave in the stereoselectivity
pocket. We hypothesize that solvent or water molecules are
trapped in the pocket and lose much of their degrees of free-
dom. This would not happen for the S enantiomer as its
phenyl group fills the pocket by itself. In an earlier investiga-
tion it was shown that one water molecule was trapped in the
stereoselectivity pocket of wild-type CalB and decreased the
enantioselectivity towards 2-pentanol.[46]
Vector construction: The CalB pGAPZaB vector was constructed
as reported elsewhere.[47]
Site-directed mutagenesis: The W104A mutation was introduced
according to the QuikChange site-directed mutagenesis instruction
manual with forward primer 5’-CCC GTG CTT ACC GCT TCC CAG
GGT GGT CTG G-3’ and reverse primer 5’-CC CTG GGA AGC GGT
AAG CAC GGG AAG CTT GTT GTT GC-3’, where the site of mutation
is underlined. The parental plasmid was digested with DpnI and
the amplified DNA fragments were transformed into E. coli XL1-
Blue cells through heat shock. This strain was used for the mainte-
nance and propagation of plasmids. The mutation was confirmed
by sequencing.
Transformation of Pichia pastoris: P. pastoris SMD1168H cells were
made electrocompetent according to the manufacturer’s protocol
(Invitrogen). The vector CalB pGAPZaB W104A was linearized with
restriction enzyme BspHI. The cleaved plasmid DNA (14 mg) was
used to transform the cells according to the Invitrogen protocol in
a Gene Pulser II (Bio-Rad). The transformed cells were plated on
YPDS plates containing Zeocin (100 mgmLꢀ1) and were incubated
at 308C for three days.
Protein production: The W104A mutant was expressed by cultiva-
tion of P. pastoris as described previously,[48] with a few modifica-
tions. BMGY medium (10 mL) was inoculated with a colony of the
transformed P. pastoris and was incubated at 298C and 220 rpm,
overnight. The preculture (0.5 mL) was used to inoculate BMGY
medium (500 mL) in 5 L flasks. The main cultures were incubated
at 228C and 220 rpm. Aqueous glycerol (10 g, 80%) was added
after 72 h of cultivation. After a total time of 96 h (OD600 =60–80)
cell cultures were centrifuged (3800g, 20 min, 48C) and the super-
natant was harvested.
Conclusions
The scope of biocatalysis in organic synthesis is expanded by
the use of CalB W104A in KRs of bulky chiral sec-alcohols. The
products are of S configuration in these reactions. This is in
contrast to the R selectivity of the wild type toward its accept-
ed, less sterically demanding, substrates. Entropy is an ex-
tremely important part of enantioselectivity, which cannot be
neglected when designing new substrate specificities.
Lipase immobilization: The supernatant was concentrated 100
times in 70 mL Centricon Plus-70 filter units (Millipore) with a
10 kDa cutoff. The concentrated enzyme solution was mixed with
Accurel MP1000<1500 mm (Membrana GmbH, Obernburg, Germa-
ny) and was stirred end-over-end at 48C for three days. The
enzyme solution was exchanged for MOPS buffer and the beads
were washed by end-over-end rotation at 48C, overnight. The mix-
ture was filtered and the immobilized enzyme was dried in
vacuum, overnight, before it was equilibrated to a water activity of
0.1 against a saturated aqueous solution of lithium chloride.
Experimental Section
General: Cyclohexane and vinyl acetate were dried over 4 ꢂ mo-
lecular sieves. Analytical TLC was performed with use of aluminum
plates precoated with silica gel 60 F254 and compounds were de-
tected with UV light and phosphomolybdic acid (5% in EtOH).
Flash column chromatography was carried out on 60 ꢂ (40–63 mm)
1
silica gel. H NMR spectra were recorded at 500 MHz by using the
414
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ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2010, 11, 411 – 416

Resolution of Bulky Secondary Alcohols
Active-site titration:[31–32] The amount of active lipase immobilized
on resin was determined by active-site titration with the inhibitor
methyl 4-methylumbelliferyl hexylphosphonate as described earli-
er.[21]
Determination of relative specificity constants: Alcohols 1a, 1b,
1c and 1e and dodecane (0.025 mmol, 200 mL, 125 mm of each in
cyclohexane) were added to a mixture of CalB W104A (15 mg) in
cyclohexane (1 mL). Vinyl acetate (0.125 mmol, 12 mL) was added
and the reaction was heated to 568C in a heat shaker. Samples
were withdrawn, filtered through cotton and diluted with EtOAc
before they were analyzed by achiral GC. The same experiment
was performed by using alcohols 1a, 1d, 1 f and 1g.
General procedure for preparation of sec-alcohols: NaBH4
(1.01 g, 26.8 mmol) was added to a stirred solution of 1-heptano-
phenone (5.10 g, 26.8 mmol) in methanol (30 mL) at room temper-
ature. The exotherm was controlled by an ice bath. The suspension
was stirred at room temperature for 2.5 h, before the reaction mix-
ture was poured into water (20 mL). The aqueous phase was ex-
tracted with Et2O (3ꢄ30 mL). The combined organic phases were
washed with brine (20 mL), dried (MgSO4) and concentrated. Flash
column chromatography (pentane/Et2O, 20:1!3:1) gave alcohol
Acknowledgements
Financial support from the Swedish Research Council (VR) is
gratefully acknowledged. We thank Marianne Wittrup Larsen for
providing us with the CalB pGAPZa B vector and Novozymes A/S
for the generous gift of Candida antarctica lipase B antibodies.
1
1 f (4.90 g, 95%) as a colorless oil. The H NMR spectra of alcohols
1c,[49] 1d,[50] 1e,[51] 1 f,[52] and 1g[53] were all in agreement with
those reported in the literature.
General procedure for preparation of racemic acetates: Triethyl-
amine (75 mL, 0.54 mmol), acetic anhydride (28 mL, 0.30 mmol) and
DMAP (cat.) were added subsequently to a stirred solution of alco-
hol 1 f (52 mg, 0.27 mmol) in CH2Cl2 (2 mL) at room temperature.
The reaction was stirred at room temperature for 2 h. CH2Cl2
(10 mL) was added and the mixture was washed with 1m aq. HCl
(10 mL). The aqueous phase was extracted with CH2Cl2 (3ꢄ10 mL).
The combined organic extracts were washed with saturated aq.
NaHCO3 (20 mL), water (20 mL) and brine (15 mL). Drying (MgSO4)
and concentration gave a colorless oil of 1-phenyl-1-heptyl acetate
(62 mg, 98%).
Keywords: enantioselectivity · enzyme catalysis · lipases ·
thermodynamics
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General procedure for kinetic resolution: Immobilized enzyme
(15 mg including beads) and vinyl acetate (0.50 mmol, 46 mL) were
added at room temperature to
a solution of alcohol 1b
(0.10 mmol, 14 mg) and dodecane (0.10 mmol, 23 mL) in cyclohex-
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Received: October 16, 2009
Published online on January 4, 2010
416
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