Preparation of passion fruit-typical 2-alkyl ester enantiomers via lipase-catalyzed kinetic resolution

Article abstract of DOI:10.1021/jf100432s

The preparation of ester enantiomers (acetates, butanoates, hexanoates and octanoates) of the secondary alcohols 2-pentanol, 2-heptanol and 2-nonanol via lipase-catalyzed kinetic resolutions was investigated. Conversion rates and stereochemical courses of esterification and hydrolysis reactions catalyzed by commercially available enzyme preparations were followed for the homologous series of these passion fruit-typical 2-alkyl esters by capillary gas chromatography using heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)- β-cyclodextrin as chiral stationary phase. An efficient method was developed to prepare the ester enantiomers via lipase-catalyzed esterifications: optically pure (R)-2-alkyl esters (ee > 99.9%) were obtained by esterification of the racemic alcohols with enantioselective Candida antarctica lipase B (immobilized) as catalyst. The subsequent esterification of the unreacted alcohols using lipase from Candida cylindracea yielded the optically enriched (S)-esters (ee > 81.4%). The separation of the products via liquid solid chromatography using a mixture of silica gel and aluminum oxide (basic) resulted in high chemical purities and yields (> 40 mol %).

Full text of DOI:10.1021/jf100432s

6328 J. Agric. Food Chem. 2010, 58, 6328–6333
DOI:10.1021/jf100432s
Preparation of Passion Fruit-Typical 2-Alkyl Ester Enantiomers
via Lipase-Catalyzed Kinetic Resolution
HEDWIG
STROHALM, SUSANNE
DOLD, KATHRIN
P
ENDZIALEK, MONIKA
W
EIHER AND
,
K
ARL-HEINZ NGEL*
E
€
€
€
Lehrstuhl fur Allgemeine Lebensmitteltechnologie, Technische Universitat Munchen,
Maximus-von-Imhof-Forum 2, D-85350 Freising-Weihenstephan, Germany
The preparation of ester enantiomers (acetates, butanoates, hexanoates and octanoates) of the
secondary alcohols 2-pentanol, 2-heptanol and 2-nonanol via lipase-catalyzed kinetic resolutions
was investigated. Conversion rates and stereochemical courses of esterification and hydrolysis
reactions catalyzed by commercially available enzyme preparations were followed for the homo-
logous series of these passion fruit-typical 2-alkyl esters by capillary gas chromatography using
heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)-β-cyclodextrin as chiral stationary phase. An
efficient method was developed to prepare the ester enantiomers via lipase-catalyzed esterifica-
tions: optically pure (R)-2-alkyl esters (ee > 99.9%) were obtained by esterification of the racemic
alcohols with enantioselective Candida antarctica lipase B (immobilized) as catalyst. The subse-
quent esterification of the unreacted alcohols using lipase from Candida cylindracea yielded the
optically enriched (S)-esters (ee > 81.4%). The separation of the products via liquid solid chroma-
tography using a mixture of silica gel and aluminum oxide (basic) resulted in high chemical purities
and yields (> 40 mol %).
KEYWORDS: Secondary alcohols; 2-alkyl esters; kinetic resolution; Candida antarctica lipase B;
Candida cylindracea lipase
INTRODUCTION
The use of porcine pancreas lipase as biocatalyst for the
esterification of odd-numbered racemic secondary alcohols with
various short-chain (8, 9) and medium-chain (10) fatty acids
showed preference for the (R)-alcohol, but reaction rate and
enantioselectivity were not useful for preparative scale. Suitable
esterification rates and high enantioselectivity were only achieved
with higher laboratory efforts, such as removal of water by
azeotropic distillation and immobilization of the enzyme (11).
The esterifications of secondary alcohols with Candida antarctica
lipase B, known as highly enantioselective for a broad range of
chiralsubstrates(12), using activated acyldonors, suchas succinic
anhydride or vinyl acetate, exhibited high reaction rates while
leaving the (S)-alcohol nearly unreacted (ee >99%) (13). However,
the direct application of short-chain fatty acids as substrates and
the influence of the chain lengths of homologous secondary alco-
hols acid have not been pursued.
The objectives of this study were (i) to screen commercially
available lipases and esterases regarding their potential to catalyze
enantioselective hydrolysis and synthesis, respectively, of second-
ary alcohols and short-chain fatty acids, (ii) to investigate the
influence of the chain lengths of the acyl and alcohol moieties,
and (iii) to develop a method to prepare the passion fruit-typical
2-alkyl esters as optically pure compounds in a preparative scale.
The odd-numbered secondary alcohols 2-pentanol, 2-heptanol
and 2-nonanol have been described as volatiles in both purple
(Passiflora edulis Sims) and yellow (Passiflora edulis f. flavicarpa)
passion fruits (1). Short-chain fatty acid esters (acetates, butano-
ates, hexanoates and octanoates) of these alkan-2-ols were shown
to be suitable for a differentiation of the two varieties (2). Deter-
minations of the natural enantiomeric ratios showed that nearly
optically pure (R)-enantiomers of the 2-alkyl esters are character-
istic volatiles of the purple variety and not detectable or present
only at trace levels in the yellow fruits (3).
Enzyme-catalyzed kinetic resolutions of racemic substrates are
well established approaches for the preparation of optically pure
compounds (4). Hydrolytic enzymes, in particular lipases, that
catalyze hydrolyses in aqueous medium as well as esterifications
and transesterifications in organic solvents are widely employed.
These biocatalysts accept a broad range of substrates and exhibit
high regio- and enantioselectivity (5). Due to their importance in
pharmaceutical and pesticide industry the lipase-catalyzed pre-
parations of optically pure secondary alcohols are among the
most extensively studied approaches in kinetic resolution (6).
Based on the observed enantioselectivity of lipases toward secon-
dary alcohols, a rule was proposed to predict which enantiomer
reacts faster in lipase-catalyzed reactions (7).
MATERIALS AND METHODS
Enzymes. The following enzyme preparations were purchased
from Sigma-Aldrich (Taufkirchen, Germany) and Fluka (Taufkirchen,
Germany): Candida antarctica lipase B (CAL-B, Fluka 62288; 10.9
*Author to whom correspondence should be addressed. Tel: þ49
(0)8161 71 4250. Fax: þ49 (0)8161 71 4259. E-mail: k.h.engel@
wzw.tum.de.
pubs.acs.org/JAFC
Published on Web 04/23/2010
© 2010 American Chemical Society

Article
J. Agric. Food Chem., Vol. 58, No. 10, 2010 6329
U/mg), Candida antarctica lipase B immobilized on acrylic resin (CAL-B
imm, Sigma-Aldrich L-4777; g10 U/mg), Candida antarctica lipase (CAL,
Fluka 62299; 2.9 U/mg), Candida cylindracea lipase (CCL, Fluka 62316;
7.29 U/mg), Penicillium roqueforti lipase (PRL, Fluka 62308; 0.65 U/mg),
Aspergillus oryzae lipase (AOL, Fluka 62285; 2.5 U/mg), porcine pancreas
lipase (PPL, Sigma-Aldrich L-3126; 30-90 U/mg), esterase from Mucor
miehei (MME, Fluka 46059; 1.1 U/mg) and pig liver esterase immobilized
on Eupergit C (PLE, Fluka 46064; 0.2 U/mg).
Chemicals. 2-Pentanol (>98%), 2-heptanol (>99%), 2-nonanol
(>97%), acetic acid (>99%), butyric acid (>99.5%), hexanoic acid (>98%)
and octanoic acid (>98%) were obtained from Fluka (Taufkirchen,
Germany). (S)-2-Pentanol, (R)-2-heptanol and (S)-2-nonanol were pur-
chased from Aldrich, Steinheim, Germany. (S)-2-Heptanol was obtained
from Fluka, Steinheim, Germany. (R)-2-Pentanol and (R)-2-nonanol were
obtained from Wako Pure Chemical Industries, Ltd., Tokyo, Japan. Silica
gel 60 (0.063-0.200 mm) was purchased from Merck (Darmstadt,
Germany) and aluminum oxide (basic; Brockman activity I) from Fluka
(Taufkirchen, Germany).
Re-esterification. Fraction II containing the nonesterified alcohol was
concentrated to a volume of ∼3 mL using a Vigreux column. The sample
was transferred to a 100 mL round-bottom flask and diluted in 40 mL of
˚
n-pentane (dried over molecular sieves 3 A). After addition of an
equimolar amount (∼8 mmol) of organic acid, the reaction was started
with 1.1 g of CCL (∼8000 units). For the monitoring of conversion,
aliquots of 5 μL were taken before addition of the enzyme and after
termination of the reaction, diluted in 500 μL of diethyl ether, dried with
anhydrous sodium sulfate and subjected to GC analysis (system I). After
filtration of the enzyme through a round filter and washing of the enzyme
the solution was dried over anhydrous sodium sulfate and concentrated at
∼35 °C to a final volume of 5 mL using a Vigreux column. The (S)-ester
was obtained by subsequent fractionation and removal of the solvent as
described above.
Calculation of Conversion Rates. Three methods were used to
determine the conversion rate (c):
(i) The amount of produced ester was calculated via determination
of the residual alcohol, using 2-heptanone as internal standard
and taking into account the following FID response factors ex-
perimentally determined for the commercially available 2-alkanols
(GC system II): 2-pentanol 1.07; 2-heptanol 1.0; 2-nonanol 0.66;
2-heptanone 1.07.
Esters of the racemic secondary alcohols as well as esters of the
enantiopure secondary alcohols were synthesized from the corresponding
acyl chlorides using 4-dimethylaminopyridine as catalyst (3). Retention
indices and mass spectrometric data were in accordance with those
previously determined (3).
(ii) The conversion rate starting from a racemic substrate was
calculated on the basis of the enantiomeric excesses of the
substrate (eeS) and the product (eeP) (14):
Solvents were distilled prior to use.
Enzyme Screening. In a screw-capped glass vial, 200 μmol of
2-heptanol and 200 μmol of butanoic acid were dissolved in 1 mL of
˚
n-heptane (dried over molecular sieves 3 A). The reactions were started by
eeS
c ð%Þ ¼
ꢀ 100
addition of 80 units of enzyme and carried out on a rotary shaker for 24 h
at room temperature (20-25 °C). For determination of conversion rate
and enantiomeric ratios of substrate and product, aliquots of 20 μL
were taken from the solution and diluted in 1000 μL of diethyl ether. The
enzyme was removed using a syringe filter. After drying over anhydrous
sodium sulfate, the sample was analyzed by capillary gas chromatography
(system I).
Enzyme-Catalyzed Esterification. In a screw-capped glass vial,
200 μmol of secondary alcohol, 200 μmol of organic acid and the internal
standard (2-heptanone, 22.8 mg) were dissolved in 1 mL of n-heptane
eeS þ eeP
(iii) The conversion rate starting from an optically enriched substrate
was calculated on the basis of the peak areas of the faster
reacting substrate (A) and the corresponding product (P) and
the slower reacting substrate (B) and the corresponding product
(Q) (14):
A þ B
c ð%Þ ¼ 1 -
ꢀ 100
ðA þ PÞ þ ðB þ QÞ
˚
(dried over molecular sieves 3 A). After addition of 8 mg of CAL-B or
Determination of Enantioselectivities. The enantioselectivities (E)
of the reactions were calculated on the basis of the conversion rate (c) and
the enantiomeric excess of the substrate (eeS) or the product (eeP),
respectively (14):
CAL-B imm (80units), the mixture was continuously stirred on a magnetic
stirrer (300 rpm) at room temperature (20-25 °C). For the monitoring of
esterification rates, aliquots of 20 μL were taken after defined intervals and
treated as described above (enzyme screening). The samples were subjected
to GC analysis (system II).
Enzyme-Catalyzed Ester Hydrolysis. In a screw-capped glass vial,
200 μmol of racemic ester was dissolved in 1 mL of potassium phosphate
buffer solution (pH 7.4). After addition of 8 mg of CAL-B imm (80 units),
the mixture was continuously stirred on a magnetic stirrer (300 rpm) at
room temperature (20-25 °C). Aliquots of 20 μL were taken after defined
intervals and extracted with 1000 μL of a mixture of n-pentane and diethyl
ether [1:1, v/v]. The organic layer was dried over anhydrous sodium sulfate
and analyzed by GC (system I).
ln½ð1 - cÞ ꢀ ð1 - eeSÞꢁ
E ¼
ln½ð1 - cÞ ꢀ ð1 þ eeSÞꢁ
ln½ð1 - cÞ ꢀ ð1 þ eePÞꢁ
E ¼
ln½ð1 - cÞ ꢀ ð1 - eePÞꢁ
E was also calculated on the basis of eeS and eeP (15):
Preparation of Optically Pure Esters. Kinetic Resolution. In a 100 mL
round-bottom flask, 15 mmol of secondary alcohol and 15 mmol of
organic acid were diluted in 75 mL of n-pentane (dried over molecular
ln½ð1 - eeSÞ=ð1 þ eeS=eePÞꢁ
E ¼
ln½ð1 þ eeSÞ=ð1 þ eeS=eePÞꢁ
˚
Capillary Gas Chromatography (HRGC-FID). System I. A
Carlo Erba Mega 5160 series gas chromatograph equipped with a flame
ionization detector (230 °C) was used. A chiral column was installed
with 25% heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)-β-cyclo-
dextrin in SE54 (30 mꢀ0.32 mm i.d., 0.25 μm film thickness). Synthesis of
the cyclodextrin derivative and column preparation were carried out in-
house (16). The temperature was programmed from 40 °C (2 min hold)
to 200 at 2 °C/min. Injection was done in the split mode (220 °C;
split ratio 1:15). Carrier gas used was hydrogen at a constant inlet pres-
sure of 110 kPa. Data acquisition was done via Chromcard software
(Thermo Fisher Scientific). The order of elution of the enantiomers was
assigned by injection of optically pure commercially available (2-alkanols)
and chemically synthesized (2-alkyl esters) reference compounds, respec-
tively.
sieves 3 A). The reaction was started by addition of 300 mg of CAL-B imm
(3000 units) and carried out on a rotary shaker for 24 h (2-pentyl- and
2-heptyl esters) and 48 h (2-nonyl esters) at room temperature (20-25 °C).
For the monitoring of conversion, aliquots of 5 μL were taken before
addition of the enzyme and after termination of the reaction, diluted in
500 μL of diethyl ether, dried with anhydrous sodium sulfate and subjected
to GC analysis (system I). After filtration of the enzyme through a round
filter and washing of the enzyme, the solution was dried over anhydrous
sodium sulfate and concentrated at ∼35 °C to a final volume of 5 mL using
a Vigreux column (30 cmꢀ2 cm i.d.).
Liquid-Solid Chromatography (LSC). The sample was placed in a
water-cooled glass column (40ꢀ1.6 cm i.d.) filled with a mixture of silica
gel and aluminum oxide (basic) [1:1; w/w]. Fractions Ia [pentane/dichloro-
methane (2:1; v/v); 300 mL] and Ib [pentane/diethyl ether (9:1; v/v);
150 mL] contained the eluted ester. In fraction II (diethyl ether; 350 mL)
the nonesterified alcohol was eluted. After removal of the solvent using a
Vigreux column and a nitrogen stream the (R)-ester was obtained.
System II. Achiral analyses were performed on a Carlo Erba Mega II
8575 series gas chromatograph equipped with a split/splitless injector
(215 °C, split ratio 1:10) and a flame ionization detector operating at 230 °C.

6330 J. Agric. Food Chem., Vol. 58, No. 10, 2010
Strohalm et al.
The column used was a 60 mꢀ0.25 mm (i.d.) fused silica capillary column
coated with DB-Wax (0.25 μm film thickness; J&W Scientific). The oven
temperature was programmed from 40 °C (5 min hold) at 4 °C/min to
230 °C (25 min hold). Carrier gas used was hydrogen at a constant pressure
of 105 kPa. Data acquisition was done via Chromcard software (Thermo
Fisher Scientific).
Determination of Optical Rotations. Optical rotations were mea-
sured on a Polartronic-E polarimeter (Schmidt & Haensch; Berlin,
Germany) fitted with a 16.5 mL measuring cell (path length 2 dm) and a
sodium lamp (wavelength 589 nm). Samples were diluted in acetone at
concentrations of 3 g/100 mL. Specific rotations were calculated as [RD] =
(Rm ꢀ 100)/(c ꢀ l), where Rm is the optical rotation measured, c is the
concentration [g/100 mL], and l is the length [dm] of the measuring cell.
enantiomeric excesses determined for the reactions of the acetates
are in accordance with previously reported data (13). However,
with increasing chain lengths of the acyl moieties the conversion
rates decreased, resulting in significantly reduced enantiomeric
excesses of the unreacted ester substrates. Exemplarily, Figure 1
shows the time-course of the conversion of 2-heptyl esters with
immobilized CAL-B as catalyst; for 2-heptyl octanoate the
conversion rate leveled off at approximately 20%. Duplicate
experiments performed with 2-heptyl hexanoate also resulted in
consistently low hydrolysis rates of 27.7% and 25.5%, respec-
tively, after 8 h. Accordingly, this approach is not useful for a
preparative scale. Addition of sodium taurocholate, known as a
useful emulsifying agent in enzyme-catalyzed processes, e.g. with
cholesterol esterases (17), did also not result in increased conver-
sion rates.
Kinetic Resolution via Esterification. As an alternative ap-
proach the enzyme-catalyzed esterification of the secondary
alcohols was investigated. Several commercially available lipases
and esterases were screened using the kinetic resolution ofracemic
2-heptanol via esterification with butanoic acid in heptane as a
test system (Table 2). Most of the employed enzyme preparations
exhibited high enantioselectivity for the (R)-configured alcohol
substrate. Only the lipase from Penicillium roqueforti showed a
preference for the (S)-alcohol; Candida cyclindracea lipase and
pig liver esterase did not react enantioselectively. As outlined in
Table 2, the conversion rates determined after 24 h ranged from
0.1 to 56.6%. The highest yields were obtained with lipase from
Candida cylindracea and with free and immobilized CAL-B
lipase. CAL-B is known as an efficient biocatalyst in organic
synthesis (12) showing high activity and enantioselectivity toward
a wide range of secondary alcohols. All examples reported follow
the empirical rule according to ref 7, which implies that enantio-
selectivity is proportional to the difference in size between the
large (L) and the medium (M) substituents of the substrate.
Experiments with a series of varying M and L groups showed that
CAL-B is highly enantioselective toward secondary alcohols with
an M substituent smaller than n-propyl and an L substituent
larger than n-propyl (18). Short chain secondary alcohols, such as
the screened substrate 2-heptanol, fulfill the described molecular
requirements.
RESULTS AND DISCUSSION
Kinetic Resolution via Hydrolysis. Based on the enantioselec-
tivity reported for the hydrolysis of racemic 2-pentyl acetate by
lipase B from Candida antarctica (CAL-B) (13), the potential of
this biocatalyst to prepare optically pure short-chain fatty acid
esters of 2-alkanols via hydrolysis-based kinetic resolutions
was investigated as a first approach. As shown in Table 1,
the hydrolyses of the 2-alkyl esters proceeded with high enantio-
selectivity for the (R)-esters. The conversion rates and the
Table 1. Enantioselectivity of CALB imm toward Esters of Secondary
Alcohols (after 8 h Reaction Time)a
conversionb (%)
eeesterc (%)
eealcohold (%)
Ee
2-pentyl acetate
48.6
42.6
36.8
29.8
48.3
39.3
27.7
21.4
49.0
37.2
27.6
19.0
94.6
74.3
58.3
42.4
93.4
64.7
38.3
27.2
96.2
59.4
38.1
23.5
>99.9
>99.9
>99.9
>99.9
>99.9
>99.9
>99.9
>99.9
f
>100
>100
>100
>100
>100
>100
>100
>100
g
2-pentyl butanoate
2-pentyl hexanoate
2-pentyl octanoate
2-heptyl acetate
2-heptyl butanoate
2-heptyl hexanoate
2-heptyl octanoate
2-nonyl acetate
2-nonyl butanoate
2-nonyl hexanoate
2-nonyl octanoate
f
g
f
g
f
g
a E, enantioselectivity; ee, enantiomeric excess. b Calculated according to ref14.
c Enantiomeric excess of the remaining (S)-enantiomer. d Enantiomeric excess of
the formed (R)-enantiomer. e Calculated on the basis of eeS according to ref 14.
f Enantiomers of 2-nonanol not separated; conversion calculated on the assumption
that only (R)-2-nonanol is formed. g Enantioselectivity not calculated because of the
missing separation of the enantiomers of 2-nonanol.
On the other hand, the lipase preparation from Candida
cylindracea (CCL; in recent times classified as Candida rugosa)
showed a high conversion rate with only a slight preference for the
(R)-alcohol. Contradictory results concerning the enantioselectivity
Figure 1. Conversion rates (%) determined for the hydrolysis of 2-heptyl esters catalyzed by CALB imm.

Article
J. Agric. Food Chem., Vol. 58, No. 10, 2010 6331
of CCL have been reported in the literature. It has been shown to
be a highly stereospecific enzyme (13, 19), but also an unspecific
biocatalyst (20, 21). In contrary to CAL-B, the predictive rule
according to (7) does not apply to the kinetic resolution of acyclic
alcohols by CCL. Furthermore, it is well-known that the en-
antiopreference of CCL is highly dependent on the polarity of the
reaction medium (22). Because of very low conversion rates the
other screened lipases and the esterases from Mucor miehei and
porcine liver were not considered suitable for an application on
preparative scale.
Figure 2 shows the time-course of the esterification of
2-heptanol with a series of homologous short-chain organic
acids (C2-C8) in heptane using immobilized CAL-B as biocatalyst.
For all synthesized 2-heptyl esters maximum esterification rates
(46-50%) were achieved after a short reaction time of 2-4 h.
Differences in the synthesis rates of the esters could only be seen in
the initial stages of the reaction. Duplicate experiments performed
exemplarily for 2-heptyl butanoate resulted in consistently high
conversion rates of 49.8% and 49.9% after 24 h.
immobilization is well-known in biocatalysis (23). For CAL-B
remarkable differences in enantioselectivity were found depending
Table 3. Enantioselectivity of CALB and CALB imm toward Secondary
Alcohols Using Short-Chain Acids as Acyl Donors (after 24 h Reaction Time)a
conversionb (%) eeesterc (%) eealcohold (%) Ee
CALB
2-pentyl acetate
52.1
53.2
52.0
49.5
46.1
43.9
45.2
47.6
42.3
47.1
46.9
47.2
46.5
49.8
46.0
49.5
46.8
47.5
46.2
46.2
83.6
81.4
85.3
93.0
99.2
99.7
99.1
99.1
99.4
99.8
99.3
99.5
99.8
99.1
99.9
99.8
99.8
99.1
99.8
99.8
85.3
93.1
87.5
86.4
82.0
73.6
73.7
82.8
86.5
88.4
87.4
89.8
83.6
95.5
83.4
95.3
f
30
33
2-pentyl butanoate
2-pentyl hexanoate
2-pentyl octanoate
36
77
CALB imm 2-pentyl acetate
2-pentyl butanoate
2-pentyl hexanoate
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
g
2-pentyl octanoate
2-heptyl acetate
CALB
2-heptyl butanoate
2-heptyl hexanoate
2-heptyl octanoate
CALB imm 2-heptyl acetate
2-heptyl butanoate
2-heptyl hexanoate
Conversion rates and enantioselectivities determined for the
synthesis of the homologous 2-alkyl esters after 24 h are listed in
Table 3. For the 2-pentyl esters significant differences between the
free and the immobilized enzyme were observed. The use of
free CAL-B led to higher conversion rates (>50%) but decrea-
sed enantioselectivity toward the secondary alcohol (E < 100).
The phenomenon of changing the selectivity of enzymes by
2-heptyl octanoate
2-nonyl acetate
CALB
2-nonyl butanoate
2-nonyl hexanoate
2-nonyl octanoate
f
g
f
g
f
g
a E, enantioselectivity; ee, enantiomeric excess. b Calculated via determination of
the residual alcohol using 2-heptanone as internal standard (see Materials and
Methods: Calculation of Conversion Rates). c Enantiomeric excess of the (R)-
enantiomer. d Enantiomeric excess of the (S)-enantiomer. e 2-Pentyl- and 2-heptyl
esters: calculated according to ref15. 2-Nonyl esters: calculated on the basis of eeP
according to ref 14. f Enantiomers of 2-nonanol not separated. g Enantioselectivity
not calculated because of the missing separation of the enantiomers of 2-nonanol.
Table 2. Screening of Enzymes for Kinetic Resolution of Racemic 2-Heptanol
via Esterification with Butanoic Acid in Heptane (after 24 h Reaction Time)a
conversionb eeester
eealcohol
(%)
enzymes
(%)
(%)
Ec
lipases
Candida antarctica lipase B
Candida antarctica lipase B (imm)
Candida antarctica lipase
Candida cylindracea lipase
Penicillium roqueforti lipase
Aspergillus oryzae lipase
porcine pancreas lipase
47.0
49.1
6.0
>99.8 (R) 88.4 (S) >100
>99.1 (R) 95.5 (S) >100
36.3 (R) 2.3 (S)
6.6 (R) 8.6 (S)
57.1 (S) 0.6 (R)
2.2
56.6
1.0
1.2
3.7
0.1
1.7
>99.9 (R) 0.1 (S) >100
85.4 (R) 1.5 (S) >12.9
esterases
Mucor miehei esterase
pig liver esterase
4.7
24.1
>99.9 (R) 4.9 (S) >100
2.7 (S) 2.7(R)
1.2
Figure 3. Kinetic resolution of racemic 2-heptanol via esterification with
hexanoic acid and CALB imm (after 24 h reaction time; for GC conditions
see Materials and Methods).
a E, enantioselectivity; ee, enantiomeric excess. b Calculated according to ref14.
c Calculated according to ref 15.
Figure 2. Conversion rates (%) determined for the synthesis of 2-heptyl esters catalyzed by CALB imm.

6332 J. Agric. Food Chem., Vol. 58, No. 10, 2010
Strohalm et al.
Table 4. Yields, Chemical Purities, Enantiomeric Excesses (ee), and Optical Rotations [RD] of the Prepared (R)- and (S)-Esters
(R)-ester
(S)-ester
(R,S)-alcohol,
initial wt [g]
yield
[g/mol %]
purity
(GC) [%]
opt. purity
(ee) [%]
RD
[deg]
yield
[g/mol %]
purity
(GC) [%]
opt. purity
(ee) [%]
RD
[deg]
RD calcd
[deg]
product
2-pentyl acetate
1.32
1.32
1.32
1.32
1.74
1.74
1.74
1.74
2.16
2.16
2.16
2.16
0.81/41.5
1.02/43.2
1.21/43.4
1.40/43.6
1.04/43.8
1.27/45.6
1.47/45.8
1.57/43.2
1.16/41.4
1.29/40.0
1.46/40.2
1.78/43.8
99.7
99.7
99.3
99.0
99.5
99.6
99.7
99.2
99.8
99.7
99.7
99.1
>99.9
>99.9
>99.9
>99.9
>99.9
>99.9
>99.9
>99.9
>99.9
>99.9
>99.9
>99.9
-16.01
-16.61
-15.01
-13.43
-6.32
-9.40
-9.45
-8.88
-4.51
-7.27
-8.19
-7.89
0.78/40.0
0.96/40.4
1.13/40.3
1.34/41.7
0.95/40.1
1.23/43.9
1.37/42.6
1.46/40.1
1.14/40.9
1.30/40.7
1.46/40.1
1.83/45.1
99.6
99.5
99.6
99.0
99.0
99.3
99.7
99.2
99.8
99.9
99.8
99.4
81.4
83.9
83.1
83.7
82.6
82.7
86.9
82.3
82.9
88.2
84.3
84.0
þ13.08
þ14.02
þ12.49
þ11.22
þ5.29
þ7.81
þ8.37
þ7.44
þ3.67
þ6.51
þ6.77
þ6.61
þ13.03
þ13.94
þ12.47
þ11.24
þ5.22
þ7.77
þ8.22
þ7.31
þ3.74
þ6.41
þ6.90
þ6.63
2-pentyl butanoate
2-pentyl hexanoate
2-pentyl octanoate
2-heptyl acetate
2-heptyl butanoate
2-heptyl hexanoate
2-heptyl octanoate
2-nonyl acetate
2-nonyl butanoate
2-nonyl hexanoate
2-nonyl octanoate
on the immobilization on different supports (24). The enhance-
ment of enantioselectivity of the immobilized enzyme may be
explained by the increased stability of the enzyme conformation
because of interactions with the polymer (macroporous acrylic
resin). Slight shifts in the conformation of the binding pockets of
the enzyme provide more space for a docking of the (S)-enantio-
mer, leading to a decrease in enantioselectivity E (18). Because of
the smaller difference in the size of substituents compared to
2-heptanol, this phenomenon was only determined for 2-pentanol.
Using heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)-
β-cyclodextrin as chiral stationary phase, the enantiomers of
the substrates and products were separated by means of capillary
gas chromatography (except for 2-nonanol) and the stereochem-
ical course of the reactions could be examined. As example, the
kinetic resolution of 2-heptanol via esterification with hexanoic
acid and immobilized CAL-B as biocatalyst is shown in Figure 3.
After 24 h of reaction time the (R)-ester is produced selectively
(ee>99.9%), resulting in an optical enrichment of the alcohol
(91.7% (S), 8.3%(R)).
The production of optically pure (S)-esters via application of
“anti-Kazlauskas catalysts” was not pursued, because this type
of biocatalyst is rather uncommon or only available by great
production efforts (25,26). Recently, wheat germ lipase has been
reported as “anti-Kazlauskas catalysts” in nonaqueous biocata-
lysis, but low conversion rates and low enantioselectivities were
determined for aliphatic secondary alcohols as substrates (27).
Preparation of Esters. Optically pure (R)-esters and optically
enriched (S)-esters of secondary alcohols were prepared via
lipase-catalyzed kinetic resolution using a two-step procedure.
The (R)-ester was obtained via esterification of the racemic
secondary alcohol (15 mmol) with an equimolar amount of acid
using CAL-B imm (300 mg) as biocatalyst. Compared to the
microscale experiments, the solvent heptane was replaced by the
more volatile pentane in order to facilitate its subsequent
removal. After gentle concentration of the reaction mixture using
a Vigreux column, the ester was separated from the unreacted
alcohol by fractionation. In order to remove the unreacted acid
substrate, a mixture of silica gel and aluminum oxide, known as
efficient adsorbent for short-chain and aromatic acids (28), was
used. After total removal of the solvent, the (R)-esters were
obtained as optically pure compounds (ee >99.9%) with high
chemical purities; an overview of the data is presented in Table 4.
Only minor losses (yields g40 mol %) were encountered, demon-
strating the effectiveness of the liquid solid chromatography
approach employed for purification. The availability of the
substances at high chemical and optical purities allowed for the
first time the determinatin of the optical rotations [R]D of the
2-alkyl esters.
Neither the hydrolysis of racemic esters (Table 1) nor the
application of an “anti-Kazlauskas catalyst” (27) seemed suit-
able for the preparation of optically pure (S)-esters. Therefore,
the optically enriched (S)-alcohols remaining as unreacted sub-
strates after the esterification with CAL-B imm were employed as
substrates for further esterification. As shown in Table 2, the
lipase preparation from Candida cylindracea (CCL) had exhibited
an acceptable conversion rate in the enzyme screening, while
accepting both enantiomers of the alcohol (E = 1.2). After
addition of an equimolar amount of acid the remaining alcohol
was esterified using this biocatalyst. The (S)-esters were obtained
by subsequent fractionation and removal of the solvent. The
yields, the chemical purities and the enantiomeric excesses (ee)
are listed in Table 4. The optical purities are lower than those
achieved for the (R)-esters, because they are determined by the
alcohol substrates esterified by a nonspecific lipase. In accordance
with the levorotation determined for the (R)-esters, the (S)-esters
were dextrorotatory. The experimentally determined specific
rotations [R]D were in good agreement with the values calculated
on the basis of the optical purities determined by GC analysis.
In conclusion, the developed method allows the preparation of
optically pure (R)- and optically enriched (S)-esters of secondary
alcohols with moderate experimental efforts. The kinetic resolu-
tion-based approach enables the use of the rather inexpensive
racemic alcohols as starting compounds. Due to their chemical
and optical purities, the substances obtained may serve as
materials for subsequent sensory analyses of these esters. Deter-
minations of thresholds via GC/O and sensory evaluations in
water are subjects of ongoing investigations.
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Received for review February 2, 2010. Revised manuscript received April
8, 2010. Accepted April 10, 2010.