Separation of secondary alcohols via enzymatic kinetic resolution using fatty esters as reusable acylating agents

Article abstract of DOI:10.1016/j.tetasy.2010.05.024

A two consecutive step procedure for the resolution-separation of secondary alcohols employing ethyl tetradecanoate in the presence of lipase allowed the enzymatic kinetic resolution of two target molecules, 1-phenylethanol and 6-methylhept-5-en-2-ol. (S)-1-Phenylethanol was isolated in a yield of 47% with an ee of 94% and (R)-1-phenylethanol in a yield of 51% with an ee of 95%. (S)-6-Methylhept-5-en-2-ol was isolated in a yield 47% and an ee of 87% and (R)-6-methylhept-5-en-2-ol in a yield 49% and an ee of 90%.

Full text of DOI:10.1016/j.tetasy.2010.05.024

Tetrahedron: Asymmetry 21 (2010) 952–956
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Separation of secondary alcohols via enzymatic kinetic resolution using
fatty esters as reusable acylating agents
a,c,
Carlos M. Monteiro a, Nuno M. T. Lourenço b, , Carlos A. M. Afonso
*
*
a CQFM, Centro de Química-Física Molecular, IN—Institute of Nanosciences and Nanotechnology, Instituto Superior Técnico, 1049-001 Lisboa, Portugal
b IBB: Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal
c iMed. UL, Faculdade de Farmacia da Universidade de Lisboa, Av. Prof. Gama Pinto, 1640-003 Lisboa, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
A two consecutive step procedure for the resolution-separation of secondary alcohols employing ethyl
tetradecanoate in the presence of lipase allowed the enzymatic kinetic resolution of two target molecules,
1-phenylethanol and 6-methylhept-5-en-2-ol. (S)-1-Phenylethanol was isolated in a yield of 47% with an ee
of 94% and (R)-1-phenylethanol in a yield of 51% with an ee of 95%. (S)-6-Methylhept-5-en-2-ol was isolated
in a yield 47% and an ee of 87% and (R)-6-methylhept-5-en-2-ol in a yield 49% and an ee of 90%.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 19 April 2010
Accepted 14 May 2010
Available online 22 June 2010
1. Introduction
sic,8–10 alkyl carboxylates,11 supercritical fluids,12,13 and ionic liq-
uids,14,15 and all these have become attractive as alternatives to
The design of chemical methods that minimize the usage of
hazardous or toxic substances and production of waste is a major
focus of modern organic synthesis.1 In recent years this has be-
come a major concern for the chemical industries as they are re-
quired to adhere to increasingly stringent guidelines and policies.
This has provided an impetus for the development of new sustain-
able chemical processes.2,3
more traditional approaches.7,16,17 Most recently there has been con-
siderable interest in the use of fatty esters as reaction media,18–22 be-
cause of their availability in large scale from sustainable sources.23
The first reports on lipase-catalyzed reaction on fatty acids were
reported by Sym in 1930 on the esterification of oleic acid in the
presence of pancreatic lipase.24 The physical properties of fatty es-
ters make them a good reaction media for performing enzymatic
esterification or transesterification reactions and they can allow
the continuous removal of volatile compounds under vacuum.25
Moreover, they can themselves act as reagents with the ester func-
tionality used as an acyl donor for lipases.18–20
As part of the drive for sustainability, chemists have continued
to be attracted to the area of biocatalysis, which has many useful
features, particularly for asymmetric synthesis. In recent years,
the biocatalysis approach has become widely utilized for the syn-
thesis of complex enantiomerically pure molecules, and there has
been particular interest from the industrial sector. A number of
important developments have helped to increase the utility of bio-
catalysis including for example: (1) rapid access to new classes of
enzymes through high throughput sequencing; (2) development of
robust screening technologies in order to identify more specific,
selective and stable enzyme(s); and (3) the availability of a wider
range of expression systems required for the large-scale produc-
tion of biocatalysts in an efficient and cost-effective manner.4–6
Another benefit of using biocatalysts in organic synthesis is that
they can often be carried out under conditions which allow the min-
imization of undesirable organic solvents. The development of new
solventsinthegreenchemistryarenaisimportantbecauseitcanhelp
to improve the overall efficiency of a reaction and enhance the pro-
cess, for example, by allowing catalyst recovery.7 Examples of recent
developments include the use of aqueous biphasic, fluorous bipha-
Several acylating agents have already been described,26 from
whichthemoreused onesare vinylesters. However, eventhose have
theirlimitations, suchastheformationofsideproducts, suchasacet-
aldehyde, that can deactivate the enzyme or react in the reaction
media and form new products.26 In recent years, new acylating
agents have been developed, namely succinic anhydride,27,28 car-
bonates29 and fatty esters.18,19 Additionally, more elaborated sys-
tems have been described as facilitators in the separation process
of free alcohol enantiomer and the other enantiomer as an ester,30,31
such as fluorous solvent,32 ionic liquids,33 ionic liquids-scCO2,
34
membrane35 or PEG.36
Recently we described a new approach to enzymatic kinetic res-
olution for the separation of secondary alcohols in which we used
ionic liquids to assist in the enantiomer separation. This allowed us
to use a sequence of simple extraction methods to achieve an effi-
cient resolution-separation process.33 However, this approach is
not without some limitations, particularly the requirements for a
specific ionic liquid as acylating agent and reaction medium, and
the use of an organic solvent for the extraction step.
* Corresponding authors. Tel.: +351 218419137; fax: +351 218419062
(N.M.T.L.);Tel.: +351 218419785; fax: +351 218464455/7 (C.A.M.A.).
E-mail addresses: nmtl@ist.utl.pt (N.M.T. Lourenço), carlosafonso@ff.utl.pt
(C.A.M. Afonso).
Herein, we report a more general and effective process for the
resolution and isolation of both enantiomers of secondary alcohols
0957-4166/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2010.05.024

C. M. Monteiro et al. / Tetrahedron: Asymmetry 21 (2010) 952–956
953
OUT
OH
OH
O
IN
R
R
OR'
rac
(R)
12
1) Enzymatic
Enzyme
transesterification
(irreversible process by
ethanol removal under
low vacuum)
4) Distillation
Reaction medium:
fatty ester
3) Enzymatic
transesterification
2) Distillation
O
OUT
R'OH
OH
(R)
O
IN
Ethanol
12
R
R
(S)
Scheme 1. One-pot resolution of sec-alcohols, based in the usage of fatty esters as solvent and acylating agent.
derived from resolution, without the use of organic solvents
(Scheme 1). The strategy employs two steps; in the first an enzy-
matic reaction allows the isolation of the (S)-alcohol enantiomer
by distillation. In the second step another enzymatic reaction is
utilized, in order to release the (R)-enantiomer that is also isolated
by distillation.
fatty esters, a sustainable source. These acylating agents were
deemed ideal because of their low melting point and high boiling
point characteristics, which should allow the reactions to be car-
ried out without any additional solvent and also offering the pros-
pect of product isolation via distillation.
In the event, a two-step resolution-separation method was
evaluated using two model substrates, 1-phenylethanol 1 and 6-
methylhept-5-en-2-ol (sulcatol) 2. Using the enzyme Candida ant-
arctica lipase B (CAL-B), a variety of fatty acids were evaluated
including, ethyl tetradecanoate (ethyl myristate) 3 and 2,2,2-tri-
2. Results and discussion
The choice of long chain fatty acid-derived esters as acyl donors
for the resolution of secondary alcohols was based on a number of
factors including physico-chemical properties and its origin from
fluoroethyl tetradecanoate (2,2,2-trifluoroethyl myristate)
(Table 1).
4
Table 1
Effect of the acyl donor on the enzymatic resolution of 1-phenylethanol 1 and 6-methylhept-5-en-2-ol (sulcatol) 2
O
OR'
OH
OH
OH
12
CAL B, 35ºC
R
R
R
i) Enzymatic transesterification, 48h; (S)-
enantiomer removal by distillation
ii) Enzymatic transesterification, 2.5 eq
EtOH or CF3CH2OH, 24h; (R)-enantiomer
removal by distillation
(R)
(S)
from i)
rac-1 or 2
from ii)
OH
OH
rac-2
rac-1
Entrya
Step (i)
Step (ii)
(R)-Alcohol yield/eeb (%)
Acylating agent R0
Racemic alcohol
(S)-Alcohol yield/eeb (%)
Primary alcohol
1
2
3
4
5
6
7
8
Et
Et
1
2
1
1
1
1
1
2
48/86
47/87
45/93
47/95
47/94
49/95
48/96
49/94
EtOHe
52/90
49/90
0/-
EtOHe
CH2CF3
CH2CF3
CH2CF3
CH2CF3
CH2CF3
CH2CF3
CF3CH2OH
CF3CH2OHc
CF3CH2OHd
0/-
8/62
31/87
46/98
47/87
EtOHe
f
f
a
All reactions were carried out with 4.1 mmol alcohol, 4.1 mmol of acylating agent and 160 mg of CAL B, 35 °C, 100 mm Hg. To perform the second step, 2.5 equiv of
primary alcohol was added.
b
Enantiomeric excess determined by GLC.
2,2,2-Trifluoroethanol added reduced to 1.1 equiv.
1.1 equiv of 2,2,2-trifluoroethanol added slowly over 6 h.
2.5 equiv of EtOH.
c
d
e
f
Basic hydrolysis, KOH/MeOH.

954
C. M. Monteiro et al. / Tetrahedron: Asymmetry 21 (2010) 952–956
The first enzymatic reaction performed with ethyl tetradecano-
was possible to isolate (S)-2 in a good yield and ee (49%, 94% ee),
and we isolated (R)-2 after basic hydrolysis, in a 47% yield and
87% ee (entry 8).
ate 3 demonstrating that (S)-1 could be obtained in good yield and
ee (48%, 86% ee, entry 1). We observed that the second step also
proceeded well to give (R)-1 in good yield and ee (52%, 90% ee,
entry 1). When we used as an alternative 2,2,2-trifluoroethyl
tetradecanoate 4, (S)-1 was isolated in moderate yield and good
ee (45%, 93% ee, entry 3), but no further reaction was observed
indicating perhaps that enzyme inhibition was occurring due to a
high concentration of 2,2,2-trifluoroethanol.37
In an attempt to improve the efficiency of the protocol we
decreased the concentration of 2,2,2-trifluoroethanol in solution
(entry 4), and modified the addition of the alcohol such that it
was slowly added over 6 h (entry 5). However, in both cases we
did not observe any improvement in the protocol. We also replaced
2,2,2-trifluoroethanol with ethanol in the second transesterifica-
tion step (entry 6). Under these conditions, (R)-1 was isolated in
a 31% yield and 87% ee. However, these results are lower than
might be expected given our assumption that excess 2,2,2-trifluo-
roethanol was compromising the efficiency of the process.
In order to quantify the yield and enantiomeric excess of the
(R)-enantiomer, a basic hydrolysis was performed, providing (R)-
1 in a very good yield and excellent enantiomeric excess (46%,
98% ee, entry 7).
It is known that the particular structure of the acylating agent
is important for the enzymatic resolution of sec-alcohols, by its
influence on the equilibrium of reaction. We assumed that the
structure of the acylating agent would be particularly important
in the present methodology because of its dual role as an acylat-
ing agent and solvent. A study was therefore carried out on the
fatty ester/substrate ratio and as shown in Table 2, increasing
the acylating agent/ substrate ratio from 1 to 10 resulted in a
moderate increase in the enantiomeric excess of (S)-1 and (R)-1
obtained (entry 1 vs entry 2). Identical conditions were used on
the enzymatic resolution of 2, and similar results were obtained
for (S)-2 (entry 3 vs entry 4). On the other hand, we noted that
under these conditions (R)-2 presented a slight decrease in ee
(48% yield, 86% ee, entry 4). These unexpected results could be
attributed to the incomplete distillation of the (S)-enantiomer
during the first step, and consequent erosion of the enantiomeric
excess on the second step.
We also examined the influence of temperature on the overall
reaction and although we noted a modest decrease in the enantio-
meric purity with an increase in temperature from 35 °C to 40 °C
(entry 1 vs entry 5). However, the considerable reduction of reac-
tion time is also noteworthy (entry 1 vs entries 5 and 6).39
We examined the ability to reuse the acylating agent and car-
ried out experiments using recycled acylating agent and enzyme.
These experiments were carried out at 40 °C, using ethyl tetrade-
canoate 3 as acylating agent, 1-phenylethanol as substrate in a
1:1 ratio in the presence of C. antarctica lipase B (CAL B) (Fig. 1).
As shown in Figure 1, the efficiency of the enzyme was re-
tained for at least five cycles (>43% yield, >80% ee). We note that
the loss of 3–7% yield in each step should be noted which is prob-
ably due to incomplete distillation, and product loss during this
process. Since the overall process of five cycles consisted of ten
sequential enzymatic reactions with the same enzyme, fatty ester
and ten distillations of each enantiomer (0.6 g scale), these results
show the simplicity, feasibility and robustness of the overall
process.
We then decided to test the scope of the methodology using an-
other substrate. Hence 6-methylhept-5-en-2-ol (sulcatol) 238 was
evaluated under the previous optimum conditions. Using ethyl
tetradecanoate 3, (S)-2 was isolated in good yields and enantiose-
lectivities: 47%, 87% ee for (S)-2 and 49%, 90% ee for (R)-2 (entry 2).
The use of 2,2,2-trifluoroethyl tetradecanoate 4 confirmed that it
Table 2
Study of the reaction conditions for the resolution/separation of 1-phenylethanol 1
and 6-methylhept-5-en-2-ol (sulcatol) 2
Entry Acylating
Racemic Temp Time (h) (S)-
(R)-
agent/racemic alcohol
alcohol ratio
(°C)
1st/2nd
steps
Alcohol
Alcohol
yield/eec yield/eec
(%)
(%)
1a
2a
3a
4a
5b
6b
1:1
10:1
1:1
10:1
1:1
1
1
2
2
1
1
35
35
35
35
40
40
48/24
48/24
48/24
48/24
24/24
24/12
48/86
47/94
47/87
46/95
45/85
49/90
52/90
51/95
49/90
48/86
45/86
44/80
3. Conclusions
In conclusion a simple, effective and practical methodology for
efficient kinetic resolution and enantiomer separation of secondary
alcohols has been developed. The key features of the approach are
(1) the use of fatty esters as acylating agents and reaction solvent
medium; (2) a second enzymatic reaction to liberate the alcohol
from the ester produced in step 1; and (3) the ability to recycle
1:1
a
The reactions at 35 °C were carried out with 4.1 mmol alcohol, and 160 mg of
CAL B, 100 mm Hg.
The reactions at 40 °C were carried out with 10.35 mmol alcohol and 500 mg of
CAL B. To perform the second step, 2.5 equiv of absolute ethanol was added.
b
c
Enantiomeric excess was determined by GLC.
Figure 1. Reuse process for one-pot resolution of 1: (solid bar) yield, (Ç) ee.

C. M. Monteiro et al. / Tetrahedron: Asymmetry 21 (2010) 952–956
955
both the acylating agent and the enzyme. The simplicity of this
4.3. Preparation of 2,2,2-trifluoroethyl tetradecanoate 4
process offers potential advantages over other kinetic resolution
protocols and we believe that it will find significant utility as a
strategy for the large-scale production of enantiomerically pure
volatile products.
A double-necked round-bottomed flask equipped with a mag-
netic stirrer bar was filled with tetradecanoic acid (5.0 g;
0.219 mol) and heated to 40 °C with a few drops of DMF. Thionyl
chloride (13.8 g; 0.117 mol) was added slowly and the reaction
mixture was heated to 70 °C and stirred for 1 h to produce tetradec-
anoyl chloride. Volatile compounds were distilled at 120 °C under
vacuum (1 mm Hg). Triethylamine (27 mL, 0.195 mol) was added
to 2,2,2-trifluoroethanol (11,7 g, 0.117 mol) in CH2Cl2 (125 ml) in
another round-bottomed flask. After stirring for 30 min, tetradeca-
noyl chloride was added and the reaction mixture was stirred at
room temperature for 3 h. After this time, the reaction was
quenched with aqueous HCl 5% (250 mL) and the mixture was ex-
tracted with CH2Cl2 (3 ꢀ 100 mL). The organic phase was washed
with an aqueous solution of saturated sodium bicarbonate
(200 mL) and dried with anhydrous magnesium sulfate and filtered.
The solvents were evaporated under reduced pressure in a rotava-
por and the product was isolated by distillation of the volatile
compounds under vacuum (120 °C, 1 mm Hg) yielding the 2,2,2-tri-
fluoroethyl tetradecanoate 4 as a colourless oil (29.99 g, 89%). 1H
NMR d 4.52–4.43 (2H, q, J = 8.6 Hz), 2.45–2.40 (2H, t, J = 7.5 Hz),
1.69–1.64 (2H, t, J = 6.9 Hz), 1.27 (20H, m), 0.92–0.87 (3H, t,
J = 6.6 Hz); 13C NMR d 172.3 (CO), 125.0–121.3 (d, J = 278 Hz, CF3),
61.0–59.5 (q, J = 36.2 Hz, CH2O), 33.79 (CH2(CO)), 32.07 (CH2),
29.81 (CH2), 29.78 (CH2), 29.71 (CH2), 29.55 (CH2), 29.02 (CH2),
29.31 (CH2), 29.11 (CH2), 24.84 (CH2), 22.83 (CH2(CH3)), 14.25
(CH3).
4. Experimental
4.1. General experimental details
4.1.1. Reagents
The reactions were performed in the presence of C. antarctica
lipase B (Novozym 435Ò with 1–2% water w/w and 7000 PLU/g)
produced from Novozymes Co. (Denmark). The fatty ester used
was prepared from tetradecanoic acid (myristic acid) (from Fluka)
or commercially available from SAFC (Ref. W24,450-3-K). The
absolute ethanol and 2,2,2-trifluoroethanol were obtain from
Riedel-de Haën and Sigma–Aldrich, respectively. The secondary
alcohols 1-phenylethanol and 6-methylhept-5-en-2-ol (sulcatol)
were purchased from Sigma-Aldrich and distilled before use.
4.1.2. Analysis
The enantiomeric excesses (ee) were determined by GC analysis,
performed on Trace Focus Unicam, FID detection, using capillary col-
umn Astec chiraldex™ G-TA (30 m ꢀ 0.25 mm ꢀ 0.12
lm) (Ref.
73033AST);injector:250 °C; detector:250 °C; split ratio = 6, column
flow (H2): 60 kPas (1.2 mL/min); 1-phenylethanol: oven: 100 °C for
15 min and ramp 8 °C/min to 155 °C), tR (S) = 12.78 min; tR
(R) = 13.45 min; tR ethyl tetradecanoate = 40.12 min; 6-methylh-
ept-5-en-2-ol (sulcatol): oven: 75 °C for 15 min and ramp 8 °C/min
to 155 °C); tR (S) = 12.43 min; tR (R) = 12.94 min; tR ethyl tetradecan-
oate = 43.72 min; mass spectrometry analysis were performed by
the mass spectrometry service of Santiago de Compostela Univer-
sity, Spain. NMR spectra were recorded at room temperature in a
Bruker AMX 300, CDCl3 as solvent and (CH3)4Si (1H) as internal stan-
dard. Infrared (IR) spectra were recorded with a Jasco FT/IR-430
model as thinly dispersed films on NaCl disks. For the procedures
under vacuum was used a diaphragm pump (1–760 mm Hg).
4.4. Preparation of 1-phenylethyl tetradecanoate 5
A double-necked round-bottomed flask equipped with a mag-
netic stirrer bar was filled with tetradecanoic acid (5.0 g;
0.022 mol) and heated to 40 °C with a few drops of DMF. Thionyl
chloride (2.8 g; 0.023 mol) was added slowly. The reaction mixture
was heated to 80 °C and stirred for 1 h to produce tetradecanoate
chloride. Volatile compounds were distilled at 120 °C under vac-
uum (1 mm Hg). In another round-bottomed flask, triethylamine
(5.4 mL, 0.039 mol) was added to 1-phenylethanol (2.86 g,
0.023 mol) in CH2Cl2 (10 ml). After 30 min stirring, tetradecanoyl
chloride obtained previously was added to the reaction mixture,
which was stirred at room temperature for 3 h. After this time,
the reaction was quenched with aqueous HCl 5% (20 mL) and the
mixture was extracted with CH2Cl2 (3 ꢀ 10 mL). The organic phase
was washed with an aqueous solution of saturated sodium bicar-
bonate (200 mL) and dried with anhydrous magnesium sulfate
and filtered. The solvents were evaporated under reduced pressure
in a rotavapor and the product was isolated by distillation of the
volatile compounds under vacuum (120 °C, 1 mm Hg). 1-Phenyl-
ethyl tetradecanoate was obtained as colourless oil (6.33 g, 87%).
1H NMR d 7.37–7.30 (5H, m), 5.97–5.90 (1H, q, J = 6.7 Hz), 2.38–
2.33 (2H, t, J = 7.5 Hz), 1.68–1.64 (2H, t, J = 6.9 Hz), 1.58–1.56 (3H,
d, J = 6.6 Hz), 1.30 (20H, m), 0.95–0.90 (3H, t, J = 6.6 Hz); 13C NMR
d 173.1 (CO), 142.0 (Ar), 128.5 (Ar), 127.9 (Ar), 126.1 (Ar) 72.08
(CHO), 34.71 (CH2(CO)), 32.04 (CH2), 29.78 (CH2), 29.76 (CH2),
29.70 (CH2), 29.57 (CH2), 29.47 (CH2), 29.37 (CH2), 29.20 (CH2),
25.08 (CH2), 22.80 (CH2), 22.35 (CH2(CH3)), 14.21 (CH3).
4.2. Preparation of ethyl tetradecanoate (ethyl myristate) 3
A single-necked round-bottomed flask equipped with a mag-
netically stir bar was filled with tetradecanoic acid (100 g;
0.438 mol), ethanol 96% (V/V) (573.5 mL; 9.75 mol) and sulfuric
acid (12.0 mL, 0.225 mol). The reaction mixture was stirred at
80 °C (temperature measured in the silicon bath) for 16 h. After
this time, the mixture was cooled at room temperature and the
ethanol was removed under vacuum in a rotavapor. The mixture
was transferred to a separating funnel (1 L) and washed with water
(3 ꢀ 300 mL) and a solution of sodium bicarbonate (50% (v/v),
300 mL). The organic phase was dissolved in hexane (300 mL),
washed with water (300 mL), dried with anhydrous magnesium
sulfate and filtered. The solvents were evaporated under reduced
pressure in a rotavapor and the product was isolated by distillation
under vacuum (120 °C, 1 mm Hg) in order to obtain ethyl tetrade-
canoate as a colourless oil (95.38 g, 86%). 1H NMR d 4.14–4.07 (2H,
q, J = 7.1 Hz), 2.29–2.42 (2H, t, J = 7.5 Hz), 1.62–1.58 (2H, t, J =
7.2 Hz), 1.26–1.21 (23H, m), 0.88–0.84 (3H, t, J = 6.6 Hz); 13C
NMR d 173.9 (CO), 60.21 (CH2O), 34.47 (CH2(CO)), 32.01 (CH2),
31.96 (CH2), 29.70 (CH2), 29.56 (CH2), 29.52 (CH2), 29.44 (CH2),
29.37 (CH2), 29.45 (CH2), 25.02 (CH2), 22.78 (CH2(CH3)), 14.32
(CH3), 14.18 (CH3); FAB-MS (m/z): 256.20 (M, 4%), 200
(M+1ꢁC4H8, 7.5%), 157 (MꢁC7H15, 22%), 129 (MꢁC9H19, 22%), 88
(MꢁC12H24, 100%), 73 (MꢁC13H27); IR mmax (film): 2924, 2853,
4.5. General procedure for resolution of 1-phenylethanol1
At first, CAL B (Novozym 435Ò; 160 mg) and racemic 1-phenyl-
ethanol (0.502 g, 4.1 mmol) were added to a plastic test tube
(10 mL) inside a glass trap attached to a vacuum pump system,
where a fatty ester (4.1 mmol) was being stirred. The reaction mix-
ture was stirred under reduced pressure (100 mm Hg) in a thermo-
static water bath. The above-mentioned reaction mixture was
1739, 1467, 1372, 1349, 1302, 1247, 1179, 1115, 1037, 722 cmꢁ1
.

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C. M. Monteiro et al. / Tetrahedron: Asymmetry 21 (2010) 952–956
filtered and the enzyme was washed with hexane (3 ꢀ 10 mL). The
solvent was then evaporated and the reaction mixture was distilled
under reduced pressure (1 mm Hg, 60 °C) in order to obtain (S)-1-
phenylethanol. The enzyme was dried under reduced pressure
(20 mm Hg) for 2 h. After distillation, the recovered enzyme and
the collected reaction medium containing the other enantiomer
as an ester, and the ethyl tetradecanoate were transferred to a plas-
tic test tube (10 mL). Alcohol (absolute ethanol or 2,2,2-trifluoro-
ethanol; for specific amount see Table 1) was added and the
mixture was stirred in a thermostatic water bath. The reaction
mixture was filtered and the enzyme washed with hexane
(3 ꢀ 10 mL). The solvent was then evaporated and the reaction
mixture distilled under reduced pressure (1 mm Hg, 60 °C) to iso-
late (R)-1-phenylethanol. Both compounds obtained by distillation
were analyzed by GLC.
containing the other enantiomer as an ester and the ethyl tetrade-
canoate were transferred to a plastic test tube (50 mL) Absolute
ethanol (2.5 equiv) was added and the mixture was stirred for
24 h at 40 °C in a thermostatic water bath. The reaction mixture
was filtered and the enzyme washed with hexane (3 ꢀ 15 mL).
The solvent was then evaporated and the reaction mixture distilled
under reduced pressure (1 mm Hg, 60 °C) in order to obtain the
(R)-sec-alcohol. The regenerated ethyl tetradecanoate and the en-
zyme were reused for the next cycle. Both compounds obtained
by distillation were analyzed by GLC.
Acknowledgements
We would like to acknowledge Fundação para a Ciência e Tecn-
ologia (POCI 2010) and FEDER (POCI/QUI/60175/2004), SFRH/BPD/
41175/2007, SFRH/BD/48395/2008 and ACS Green Chemistry Insti-
tute (Ref. GCI-PRF#49150-GCI) for their financial support, Nov-
ozym Co. for their generous enzyme supply.
4.6. General procedure for resolution of 6-methylhept-5-en-2-ol
(sulcatol) 2
At first, CAL B (Novozym 435Ò; 160 mg) and racemic 6-methylh-
ept-5-en-2-ol (0.531 g, 4.1 mmol) were added to a plastic test tube
(10 mL)inside a glass trapattachedto a vacuumpump system where
the fatty ester (4.1 mmol) was being stirred. The reaction mixture
was stirred under reduced pressure (350 mm Hg) in a thermostatic
water bath. Then it was filtered and the enzyme washed with hexane
(3 ꢀ 10 mL). The solvent was evaporated and the reaction mixture
distilled under reduced pressure (1 mm Hg, 50 °C) in order to obtain
(S)-6-methylhept-5-en-2-ol. The enzyme was dried under reduced
pressure (20 mm Hg) for 2 h. After distillation, the recovered en-
zyme and the collected reaction medium containing the other enan-
tiomer as an ester and the ethyl tetradecanoate were transferred to a
plastic test tube (10 mL). The alcohol (absolute ethanol or 2,2,2-tri-
fluoroethanol; for specific amount see Table 1) was added and the
mixture was stirred in a thermostatic water bath. The reaction mix-
ture was filtered and the enzyme washed with hexane (3 ꢀ 10 mL).
The solvent was then evaporated and the reaction mixture distilled
under reduced pressure (1 mm Hg, 50 °C) to give the (R)-6-methylh-
ept-5-en-2-ol. Both compounds obtained by distillation were ana-
lyzed by GLC.
References
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At first, CAL B (Novozym 435Ò; 500 mg) and racemic 1-phenyl-
ethanol (1.32 g, 10.35 mmol) were added to a magnetically stirred
ethyl tetradecanoate (2.95 g, 10.35 mmol) in a plastic test tube
(50 mL) inside a glass trap attached to a vacuum pump system.
The reaction mixture was stirred for 24 h under reduced pressure
(100 mm Hg) at 40 °C in a thermostatic water bath. The above-
mentioned reaction mixture was filtered and the enzyme washed
with hexane (3 ꢀ 15 mL). The solvent was then evaporated and
the reaction mixture distilled under reduced pressure (1 mm Hg,
60 °C) in order to obtain the (S)-1-phenylethanol. The enzyme
was dried under reduced pressure (20 mm Hg) for 2 h. After distil-
lation, the recovered enzyme and the collected reaction medium