Resolution of 2-bromo-o-tolyl-carboxylic acid by transesterification using lipases from Rhizomucor miehei and Pseudomonas cepacia

Article abstract of DOI:10.1016/S0957-4166(01)00432-3

Several lipases were screened for their ability to catalyze the enantioselective transesterification of 2-bromo-o-tolyl acetic acid. Amongst the preparations tested, the lipases from Rhizomucor miehei and Pseudomonas cepacia were selected. The best enantioselectivity was obtained with Rhizomucor miehei lipase immobilized on polypropylene (E = 11.3), which was more stereoselective than the free form. Hydrophobic solvents with log P higher than 2.5 were the most suitable giving the highest E-values. In addition, factors such as the water activity and the reaction temperature had little effect on the resolution of the racemic mixture. The selectivity of the enzymes with respect to the substrate was also only weakly affected by the structure of the leaving alcohol except in the case of the iso-propyl group, which causes high steric hindrance. Operating conditions under reduced pressure were defined to resolve the racemic mixture with immobilized Rhizomucor miehei lipase.

Full text of DOI:10.1016/S0957-4166(01)00432-3

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 12 (2001) 2473–2480
Resolution of 2-bromo-o-tolyl-carboxylic acid by
transesterification using lipases from Rhizomucor miehei and
Pseudomonas cepacia
David Guieysse,a Christophe Salagnad,b Pierre Monsana and Magali Remaud-Simeona,*
aCentre de Bioinge´nierie Gilbert Durand, De´partement de Ge´nie Biochimique et Alimentaire, UMR CNRS 5504,
UMR INRA 792, INSA, 135 Avenue de Rangueil, F-31077 Toulouse Ce´dex 4, France
bAventis Pharma, Process Development Biotechnology, 9, quai Jules Guesde, F-94400 Vitry sur Seine, France
Received 12 September 2001; accepted 4 October 2001
Abstract—Several lipases were screened for their ability to catalyze the enantioselective transesterification of 2-bromo-o-tolyl
acetic acid. Amongst the preparations tested, the lipases from Rhizomucor miehei and Pseudomonas cepacia were selected. The best
enantioselectivity was obtained with Rhizomucor miehei lipase immobilized on polypropylene (E=11.3), which was more
stereoselective than the free form. Hydrophobic solvents with log P higher than 2.5 were the most suitable giving the highest
E-values. In addition, factors such as the water activity and the reaction temperature had little effect on the resolution of the
racemic mixture. The selectivity of the enzymes with respect to the substrate was also only weakly affected by the structure of the
leaving alcohol except in the case of the iso-propyl group, which causes high steric hindrance. Operating conditions under reduced
pressure were defined to resolve the racemic mixture with immobilized Rhizomucor miehei lipase. © 2001 Elsevier Science Ltd. All
rights reserved.
1. Introduction
modify the stereoselectivity of lipases for a given reac-
tion. However, no rules have been established to enable
the prediction of the enantioselectivity of a particular
enzyme towards a specific substrate.
Today many organic chemists employ lipases as cata-
lysts for the synthesis of enantiomercally pure com-
pounds, because of their remarkable stability in organic
media and their high stereoselectivity.1,2 Enantiopure
carboxylic acids are important building blocks for the
synthesis of many pharmaceuticals, pesticides, and nat-
ural compounds such as pheromones.3,4 Lipases have
been successfully employed for the resolution of various
racemic alcohols via hydrolysis, esterification or trans-
esterification but only a limited amount of information
exists on the chiral resolution of 2-substituted acids,5–10
especially 2-halogeno-carboxylic acids.11–15 Most stud-
ies concern the resolution of 2-arylpropionic acids, an
important group of non-steroid anti-inflammatory
drugs,16–20 or 2-hydroxy carboxylic acids that are also
important building blocks.21 The lipases of Candida
antarctica B,5 Candida rugosa22 and Rhizomucor
miehei16 have been successfully employed for the reso-
lution of 2-arylpropionic acids and Pseudomonas cepa-
cia lipase was used for the resolution of 2-hydroxy
acids.21 These studies report that many factors can
The 2-bromo-o-tolyl-acetic acid, methyl and ethyl esters
derivatives, are used as precursor for the synthesis of
drugs including benzylpenicillins,23 analgesics24 and
non-peptide angiotensin II-receptor antagonists.25,26
Here, the stereoselectivity of five commercial lipases for
the resolution of 2-bromo-o-tolyl-acetic acid was inves-
tigated. 2-Bromo-o-tolyl-acetic acid was resolved by
enzymatic transesterification of its esters (RS)-1 to give
the corresponding octyl esters (Scheme 1) and the influ-
ence of the solvent, the water activity and the tempera-
ture on the enzyme selectivity were examined.
Operating conditions optimizing the production of
enantiomerically pure (R)-1b are proposed.
2. Results and discussion
2.1. Lipase selection
The biocatalytic activity and enantioselectivity of com-
mercial lipases from P. cepacia, R. miehei, C. antarctica,
C. rugosa and H. lanuginosa were tested in transesterifi-
* Corresponding author. Tel.: +33 561559446; fax: +33 561559400;
e-mail: remaud@insa-tlse.fr
0957-4166/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(01)00432-3

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D. Guieysse et al. / Tetrahedron: Asymmetry 12 (2001) 2473–2480
Scheme 1.
cation reactions between 2-bromo-o-tolyl-ethyl acetate
and n-octanol in octane at 30°C (Scheme 1).
polypropylene was much less active than the free
enzyme, with less than 2% of the substrate being con-
sumed in 120 h. This illustrates how immobilization
influences (and in some cases improves) the selectivity
and activity of a catalyst. These effects can be
attributed to conformational changes, which would be
highly dependent on the catalyst and on the immobi-
lization methods involved. Edlund et al.32 observed
similar effects with C. rugosa lipase, which was found
to be more enantioselective after immobilization on
polypropylene for the esterification of ( )-2-methyl-
decanoic acid with long chain alcohols. When immo-
bilized CRL was used, E-values were higher (E=28a
or E=120–150b) than E-values of reactions using
crude lipase (E=14a or E=23–24b) with 1-hexa-
decanola or 1-octadecanolb, respectively. More recently
Fernandez-Lorente et al.33,34 showed that immobiliza-
tion of Pseudomonas fluorescens on different supports
is a valuable method for modulation of the activity and
selectivity. Indeed, the enantioselectivity of the free
enzyme and of some immobilized derivatives was
increased by more than one order of magnitude (from
E=7 to 80) by using lipase immobilized on hydropho-
bically-modified matrices such as octyl agarose or
decaoctyl Sepabeads.
As shown in Table 1, among the five lipases tested,
only the lipases from R. miehei (RML) and P. cepa-
cia (PCL) were selected for the transesterification of
the racemic acid. The reactions catalyzed by both
enzymes proceed with E-values of 2.8–11.3. But, the
enzymes display an opposite enantiopreference.
Indeed, the (S)-enantiomer is preferred by RML
whereas the (R)-enantiomer is preferred by PCL. The
opposing preferences observed for these lipases is
probably related to the structure of their active sites.
Although the amino acids involved in catalysis are
similar for both enzymes, the topology of their active
sites is very different.29–31 The binding pocket of
RML at the surface of the protein is obstructed by
the ‘so-called’ lid, whereas the binding pocket of PCL
forms an elliptical funnel which is more accessible
than the binding pocket of RML.
The enantioselectivity of immobilized forms of PCL
and RML was also investigated as shown in Table 1.
The E-values increased from 2.8 to 11.3 when RML
was immobilized on a hydrophobic support such as
polypropylene. In contrast, PCL immobilized on
Table 1. Lipase-catalyzed transesterification between a-bromo-o-tolyl-ethyl acetate 1b and n-octanol in octane in the pres-
ence of different free or immobilized lipases
c
Lipase
Support
Conversion (%)
e.e.p (%)
Ed
f.e.e
R. miehei
R. miehei
R. miehei
P. cepacia
No
22a
25a
34a
15b
B2b
7b
41
74
65
57
n.d.
23
n.d.
19
2.8 (90.1)
11.3 (90.7)
9.5 (90.2)
4.3 (90.5)
n.d.
S
S
S
R
n.d.
S
n.d.
S
Polypropylene PP 100
Duolite A568
No
Polypropylene PP 100
No
Polypropylene PP 100
No
No
P. cepacia
C. antarctica B
C. antarctica B
C. rugosa
n.d.
n.d.
n.d.
n.d.
B2b
5b
H. lanuginosa
B2b
n.d.
n.d.
a After 72 h.
b After 120 h.
c Enantiomeric excess of product determined by chiral HPLC.
d E (ratio of initial rates of product appearance (viR/viS)).
e Fast reacting enantiomer. The absolute configuration was determined by circular dichroism (Exciton method); as explained in Refs. 27 and 28
n.d.: not determined.

D. Guieysse et al. / Tetrahedron: Asymmetry 12 (2001) 2473–2480
2475
2.2. Influence of the structure of the leaving alcohol
enantioselectivity of 11.3 0.7 was measured with an
ethyl substituent but there was no correlation
between the size of the leaving alcohol and the
enantioselectivity measured.
To study the influence of the structure and size of the
leaving alcohol of the ester substrate on the enantiose-
lectivity of the studied lipases (RML and PCL), the
transesterification reaction was carried out using vari-
ous esters and n-octanol as substrate. Substituents
(Scheme 1) of increasing size were tested: methyl, ethyl,
propyl, iso-propyl and benzyl esters. The results of the
transesterification with n-octanol in octane are summa-
rized in Table 2.
“ Low E-values ranging from 3.7 to 6 were obtained
with the free PCL, benzyl ester being better resolved
than all the other esters.
The best substrate for RML, 1b and the best substrate
for PCL, 1e, were used for the remainder of the study.
“ The first observation is that the iso-propyl ester
reacts very slowly with both lipases. This is probably
due to the steric hindrance induced by this
substituent.
“ Except for the iso-propyl ester, increasing the size of
the -OR substituent did not drastically modify the
activity or the enantioselectivity of free RML. The
immobilized enzyme was more sensitive to the varia-
tion of structure of the acyl moiety. A moderate
2.3. Effect of the organic solvent
In order to investigate solvent effects, transesterification
reactions were carried out in a wide variety of solvents
with log P values of 1.3 to 4.5. For each reaction, the E
value was measured. Table 3 shows the results obtained
with immobilized RML and free PCL for the trans-
esterification of substrates 1b and 1e with n-octanol.
Table 2. Enantioselectivity of R. miehei and P. cepacia lipase in transesterification reaction in n-octane with different leaving
alcohols (-OR) (Scheme 1)
Acyl donor
R. miehei lipase
Immobilized on polypropylene
P. cepacia lipase
Free
Free
Ea
Conversionb (%)
Ea
Conversionb (%)
Ea
Conversionb (%)
1a
1b
1c
1d
1e
2.8 (90.1)
2.8 (90.1)
2.2 (90.1)
n.d.
18
32
26
B4
26
6.2 (90.1)
11.3 (90.7)
6.3 (90.5)
n.d.
23
34
30
B2
25
5.2 (90.3)
4.3 (90.5)
3.7 (90.2)
n.d.
17
15
21
B4
33
1.5 (90.1)
1.1 (90.1)
6.0 (90.2)
a E=(viR/viS).
b After 216 h n.d.: not determined.
Table 3. Effect of organic solvent on enantioselectivity of RML- and PCL-catalyzed transesterification of 1b and 1e,
respectively with n-octanol
Solvents
Log P
R. miehei lipase immobilized on polypropylene
Free P. cepacia lipase
Ea
Conversion
(%)
e.e.s (%)e
e.e.p (%)e
Ea
Conversion
(%)
e.e.s (%)e
e.e.p (%)e
Octane
4.5
4.5
4.5
4.0
3.5
3.2
2.5
1.9
11.3
9.5
8.6
10.3
10.6
8.9
12.6
3.4
37b
43b
44b
36b
43b
49b
30b
46c
45
48
49
41
52
57
34
33
73
63
62
73
69
59
79
39
6.0
8.0
8.2
5.7
5.7
7.4
8.0
7.1
39b
43b
49b
45b
45b
41b
14b
51d
43
49
62
47
45
50
10
62
63
65
61
56
54
67
73
60
Isooctane
Cyclooctane
Heptane
Hexane
Cyclohexane
Toluene
Diiso-propyl
ether
MTBE
MIBK
2M2B
1.9
1.3
1.3
5.6
2.2
n.d.
55c
26b
58
2
n.d.
47
6
n.d.
8.7
2.0
6.4
54b
19b
16b
68
8
16
57
10
79
B5b
a Ratio of initial rates.
b After 216 h.
c After 72 h.
d After 120 h. n.d.: not determined.
e Enantiomeric excess of substrate (e.e.s) and product (e.e.p) determined by chiral HPLC. MTBE=methyl tert-butyl ether. MIBK=methyl
isobutyl ketone. 2M2B=2-methyl-2-butanol

2476
D. Guieysse et al. / Tetrahedron: Asymmetry 12 (2001) 2473–2480
after 216 h. They are undoubtedly the most suitable
solvents for the resolution of 1b as they represent a
good compromise between reaction rates and E-
values.
2.4. Effect of water activity
It is well established that the hydration level of the
reaction medium influences the properties of enzymes,
especially their catalytic activity, but also in some cir-
cumstances the enantiomeric ratio.35,36 The influence of
water activity on the resolution of 1b by RML immobi-
lized on polypropylene was investigated. The initial rate
of production of both the (R)- and (S)-enantiomers
decreased with increasing activity, but no significant
effect was observed on the resulting enantioselectivity
as shown in Fig. 1.
Figure 1. Enantioselectivity of RML immobilized on
polypropylene as a function of water activity in transesterifi-
cation between 1b and n-octanol in octane.
2.5. Effect of temperature
“ Whatever the solvent used, lipases from RML and
PCL always showed the same preference for an
enantiomer, (the (S)-enantiomer for RML and the
(R)-enantiomer for PCL).
Temperature is another factor, which, in certain cases
affects the enantioselectivity of enzymatic reactions.
Consequently, the effect of temperature on the RML-
and PCL-catalyzed transesterification of 1b and 1e,
respectively with n-octanol in octane was investigated.
As shown in Fig. 2, the increase in temperature reduced
the enantioselectivity of RML, whereas it had no effect
on the enantioselectivity of the reactions using PCL.
“ Enzyme activities varied with the solvent used. How-
ever, no straightforward correlation can be estab-
lished between the activity measured and the
hydrophobicity of the solvent. From the results
shown in Table 3, the solvents can be classified into
three categories. The first category includes
hydrophilic solvents with log P<1.9. With these sol-
vents, both conversion and enantioselectivity were
low. 2-Methylbutan-2-ol seems to denature RML
because the conversion was less than five percent
after reaction of 216 h duration. The second cate-
gory of solvents, ethers, have intermediate log P
values ranging from 1.3 to 2.5. In their presence the
lipases were more active, but the enantioselectivity
was low. These solvents may change the enzyme/sub-
strate interaction, by modifying for example the
organization of water molecules in the active site,
thus increasing or decreasing the preference for one
of the enantiomers. The last category of solvents
includes more hydrophobic solvents with log P
higher than 2.5. The overall reaction rate decreased
in their presence as indicated by the values obtained
2.6. Operating conditions for the resolution of 1b
The best conditions for enantioselective transesterifica-
tion of 1b were obtained with immobilized RML in the
presence of n-octanol at 30°C. In these conditions,
(R)-1b cannot be separated from the (S)-1b because the
reaction equilibrium, is reached when the enantiomeric
excess of substrate is only about 45% and it is not
possible to convert more (S)-enantiomer at that point.
One way to improve the resolution involves shifting the
reaction equilibrium in order to ensure complete con-
version of the (S)-enantiomer. The reaction equilibrium
was thus shifted by carrying out the reaction under
reduced pressure. In this way, the ethanol produced
was continuously removed and the equilibrium shifted
Figure 2. Effect of temperature on the enantioselectivity of RML (immobilized on polypropylene) and PCL (free lipase)
lipase-catalyzed transesterification of 1b and 1e, respectively with n-octanol in octane.

D. Guieysse et al. / Tetrahedron: Asymmetry 12 (2001) 2473–2480
2477
Table 4. Resolution of racemic 1b under reduced pressure
catalyzed by RML immobilized on polypropylene
lanuginosa). Chirazyme L-3, lyo. (free lipase from C.
rugosa).
Conditions
Ea
e.e.s%c
e.e.p%c
% Conversion
4.2. Chemical reagents
30°C, 1 atmb
11.3
9.5
45
66
73
63
37 (216 h)
51 (139 h)
All reagents were of commercial quality and were pur-
30°C, 260 mBarb
chased from Sigma/Aldrich. All solvents were dried
a Ratio of initial rates.
,
over molecular sieve (3 A) before use.
b [ester]=50 mM, [octanol]=150 mM, 10 mg/mL of immobilized
RML in octane.
c Enantiomeric excess of substrate (e.e.s) and product (e.e.p) deter-
mined by chiral HPLC.
4.3. General procedure for the preparation of 2-bromo-
o-tolyl acetic acid alkyl ester o-tolylacetic acid
The bromoacetic acid was synthesized according to a
method previously described which was slightly
modified as follows.37–39
towards the product side. The results are summarized in
Table 4.
After incubation for 360 h (>75% conversion) under
260 mBar pressure at 30°C, unreacted substrate (R)-1b
was obtained with an e.e.s of >97 and 20% yield after
separation by column chromatography on silica gel.
(N.B. The preparation must be carried out in an
efficient fume cupboard).
A saturated solution of sodium metabisulphite was
prepared by stirring finely powdered sodium metabisul-
phite (207.5 g) with water (280 mL) for 0.5 h and then
filtering to remove the excess salt. In a 1 L beaker,
sodium cyanide (20.4 g, 0.41 mol) in water (82.5 mL)
was mixed with o-tolualdehyde (50 g, 0.41 mol). The
sodium metabisulphite solution was added from a
dropping funnel, slowly at first and then more rapidly
(addition time: 10–15 min). During the initial stage of
the addition, crushed ice (125 g) was added to the
reaction mixture in several portions. The two-phase
liquid mixture was transferred to a separating funnel.
The organic layer was removed and placed in a large
evaporating dish and concentrated hydrochloric acid
(62.5 mL) was added immediately with stirring. Hydrol-
ysis was allowed to proceed at rt for 12 h. The solution
was then evaporated to dryness on a steam bath and
stirred from time to time to break up the deposit of
ammonium chloride and o-tolyl-acetic acid. The residue
was washed with cold toluene (2–3×100 mL). Inorganic
salts were separated from o-tolylacetic acid by toluene
extraction using a Soxhlet apparatus. The acid crystal-
lized at rt and was collected on a Buchner funnel and
dried in air to afford the product as a white solid (25.90
g, 38% yield).
3. Conclusions
In conclusion the stereoselectivity of RML and PCL
towards a-bromo-o-tolyl acetic esters is poorly influ-
enced by the solvent used, the water activity and the
reaction temperature.
The best enantioselectivity E=11.3 was obtained with
an immobilized form of RML, whereas the value of
E=2.8 was obtained with free RML for 1b as substrate
showing that immobilization can improve the selectivity
of the catalyst.
Even with moderate enantioselectivity, controlling the
operating conditions enables the shift of reaction equi-
librium towards the products to prepare enantiopure
(R)-1b in 20% yield with e.e.s of >97. To improve the
yield the effect of enzyme immobilization and substrate
structure on the enantioselectivity and activity of the
enzyme will be further investigated.
4. Experimental
4.1. Biological reagents
4.3.1. a-Bromo-o-tolylacetic acid 1. Compound ( )-1
(10 g) and sulphuric acid (1.63 mL) were added succes-
sively to a stirred solution of 48% aqueous hydro-
bromic acid (12.5 g) and concentrated sulfuric acid (2
mL). The solution was stirred under reflux for 3 h,
cooled and added to water (50 mL). The organic phase
was isolated by extraction with ether and evaporated,
giving an oil, which was purified by silica-gel column
chromatography (n-hexane:ethyl acetate 98:2) to afford
the product as a white solid with a (5.39 g, 39%).
All lipases were purchased from Roche Diagnostics
(Germany). Chirazyme L-9, lyo. (free lipase from R.
miehei ), Chirazyme L-9, c-f, C2, lyo. (lipase from R.
miehei immobilized by adsorption on porous
polypropylene), Chirazyme L-9, c-f, dry, (lipase from
R. miehei immobilized by adsorption on a macroporous
anion exchange resin). Chirazyme L-1, lyo. (free lipase
from P. cepacia), Chirazyme L-1, c-f, C2, lyo. (lipase
from P. cepacia immobilized by adsorption on porous
polypropylene). Chirazyme L-2, lyo. (free lipase from
C. antarctica), Chirazyme L-2, c-f, C2, lyo. (lipase from
C. antarctica immobilized by adsorption onto an acrylic
resin), Chirazyme L-2, c-f, C3, lyo. (lipase from C.
antarctica immobilized by adsorption onto porous
polypropylene). Chirazyme L-8, lyo (free lipase from H.
4.3.2. 2-Bromo-o-tolyl-acetate esters 1a–1e
4.3.2.1. Methyl, ethyl, propyl and iso-propyl esters.
The bromo acid (43.6 mmol), alcohol (100 mL) and
p-toluenesulphonic acid (0.2 g) were stirred under reflux
(4–5 h). The reaction was followed by TLC using

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D. Guieysse et al. / Tetrahedron: Asymmetry 12 (2001) 2473–2480
n-hexane:ethyl acetate 9:1 as eluent. The alcohol was
evaporated at 35°C under water-pump vacuum. The
residual oil was dissolved in dichloromethane (25 mL)
and washed with saturated sodium bicarbonate solution
(3×10 mL) and finally with distilled water (10 mL). The
dichloromethane solution was dried over magnesium
sulphate, filtered and then evaporated to dryness at
35°C under water pump vacuum.
4.4.4. ( )-2-Bromo-o-tolyl propyl acetate. Yield: 9.06 g,
(77%). IR (neat), 1750 and 1725 (wCꢀO), 1600 and 1475
1
(wCꢀC), 1280 to 1140 (wC-O) cm−1. H NMR (CDCl3): l
0.86–0.94 (t, J=7.2 Hz, 3H, -CH2CH3
J=7.2 Hz, 2H, -OCH2CH2CH3): l 2.41 (s, 3H,
ArCH3): l 4.11–4.18 (td, J=1.2–7.2 Hz, 2H, -OCH2): l
5.64 (s, 1H, -CHBr): l 7.15–7.26 (m, 3H, ArH): l
7.60–7.64 (m, 1H, ArH
); 13C NMR (CDCl3): l 10.25
(-CH2CH3), 19.32 (ArCH3), 21.82 (-CH2-), 44.78
(-CHBr), 68.12 (-OCH2-), 126.83, 128.85, 129.16, 130.76,
134.47, 136.03, 166.34 (C
6 ): l 1.60–1.74 (m,
6
6
6
6
6
6
6
6
6
6
6
4.3.2.2. Benzyl and octyl esters. Benzyl or octyl alco-
hol (50 mmol), bromo acid (60 mmol), p-toluenesul-
phonic acid (0.2 g) and toluene (80 mL) were stirred
and heated under reflux. The water formed in the
reaction was continuously removed azeotropically using
a Dean–Stark apparatus. The toluene solution was
cooled to rt and washed with saturated sodium bicar-
bonate solution (3×25 mL) and finally with distilled
water (25 mL). The toluene solution was dried over
magnesium sulfate, filtered and evaporated to dryness
at 50°C under water pump vacuum.
+
’
6
OO). Mass: calcd M =
+
’
270.02554, found M =270.02493.
4.4.5. ( )-2-Bromo-o-tolyl iso-propyl acetate. Yield: 2.35
g, (20%). IR (neat) 1745 and 1720 (wCꢀO), 1600 and 1475
1
(wCꢀC), 1280 to 1100 (wC-O) cm−1. H NMR (CDCl3): l
1.19–1.23 (d, J=6.3 Hz, 3H, -CH(CH3
J=6.3 Hz, 3H, -CH(CH3): l 2.41 (s, 1H, ArCH3
5.04–5.12 (m, 1H, CH): l 5.59 (s, 1H, -CHBr),. l
7.16–7.26 (m, 3H, ArH): l 7.59–7.63 (m, 1H, ArH
); 13C
NMR (CDCl3): l 19.32 (ArCH3), 21.46 (-OCH(C
21.56 (-OCH(CH3)2), 45.14 (-CHBr),
(-OC
136.09, 167.74 (C
6
): l 1.27–1.30 (d,
6
6
): l
6
6
6
6
6
H3)2),
70.44,
6
6
4.4. Spectroscopic data
6
H(CH3)2 126.60, 128.66, 129.12, 130.79, 134.53,
+
’
OO). Mass: calcd M =270.02554,
6
Infrared spectra were recorded on a Perkin Elmer, 1310
+
1
infrared spectrophotometer. H and 13C NMR spectra
’
found M =270.02615.
were recorded on a Bruker AC-200.1 (1H, 200.1 MHz
and 13C, 50.3 MHz) spectrometer. Mass spectra were
recorded on a Micro Mass, auto-spect EBEQ (EI+, 70
ev).
4.4.6. ( )-2-Bromo-o-tolyl benzyl acetate. Yield: 14.16 g,
(74%). IR (neat) 1750 and 1730 (wCꢀO), 1600 and 1475
1
(wCꢀC), 1280 to 1140 (wC-O) cm−1. H NMR (CDCl3): l
2.4 (s, 3H, ArCH3
-OCH2ph): l 5.71 (s, 1H, -CH
ArH): l 7.34–7.36 (m, 5H, ArH
ArH
); 13C NMR (CDCl3): l 19.38 (ArC
(-CHBr), 68.21 (-OCH2ph), 126.92, 128.34(×2),
128.60(×2), 128.68, 128.95, 129.33, 130.91, 134.30,
6
): l 5.17–5.32 (2H, d, J=12.2 Hz,
Br): l 7.18–7.27 (m, 3H,
): l 7.60–7.64 (m, 1H,
H3), 44.71
4.4.1. ( )-2-Bromo-o-tolyl-acetic acid. Yield: 5.39 g,
(39%): IR (KBr, neat) 3300 to 2800 (wO-H), 1710 (wCꢀO),
1600 (wCꢀC), 1310 to 1240 (wC-O) cm−1. 1H NMR (d,
6
6
6
6
6
6
DMSO): l 2.35 (s, 3H, ArCH
l 7.20–7.51 (m, 4H, ArH): l 13.24 (s, 1H, COOH
NMR (d, DMSO): l 18.74 (ArCH3), 47.39 (-CHBr),
126.420, 128.59, 128.81, 130.66, 135.39, 138.16, 169.06
6
3): l 5.95 (s, 1H, -CH
6 Br):
6
6
6
6
); 13C
+
6
6
’
135.07, 136.18, 168.16 (C6 OO). Mass: calcd M =
+
’
318.02554, found M =318.02649.
+
+
’ ’
(C6 OOH). Mass: calcd M =227.97859, found M =
227.97803. Mp 106–108°C.
4.4.7. ( )-2-Bromo-o-tolyl octyl acetate. Yield: 13.87 g,
(68%). IR (neat) 1750 and 1730 (wCꢀO), 1600 and 1460
(wCꢀC), 1280–1140 (wC-O) cm−1. 1H NMR (CDCl3): l
4.4.2. ( )-2-Bromo-o-tolyl methyl acetate. Yield: 8.12 g,
(77%). IR (neat) 1750 and 1730 (wCꢀO), 1600 and 1475
1
0.85–0.91 (t, J=6.8 Hz, 3H, -CH2CH3
CH2): l 1.63 (m, 2H, CH2): l 2.41 (s, 3H, ArCH3
4.15–4.22 (t, J=6.8 Hz, -OCH2CH2-): l 5.63 (s, 1H,
-CHBr): l 7.18–7.25 (m, 3H, ArH): l 7.60 (m, 1H,
ArH H3), 19.37
); 13C NMR (CDCl3): l 14.16 (-CH2C
(ArCH3), 22.69, 25.74, 28.42, 29.13, 29.17, 31.78, 44.85
(-CHBr), 66.75 (-OCH2-), 126.87, 128.88, 129.21,
130.82, 134.51, 136.05, 168.36 (COO). Mass: calcd
6
): l 1.25 (s, 10H,
(wCꢀC), 1280 to1140 (wC-O) cm−1. H NMR (CDCL3): l
6
6
6
): l
2.41 (s, 3H, ArCH3
1H, -CHBr): l 7.19–7.26 (m, 3H, ArH
1H, ArH
); 13C NMR (CDCL3): l 19.34 (ArC
(-CHBr), 53.53 (-OCH3), 126.95, 128.85, 129.34,
130.91, 134.37, 136.11, 168.80 (COO). Mass: calcd
6
): l 3.79 (s, 3H, -OCH3
): l 7.58–7.62 (m,
H3), 44.52
6 ): l 5.65 (s,
6
6
6
6
6
6
6
6
6
6
6
6
6
+
+
’
6
6
’
M =241.99424, found M =241.99324.
6
+
+
’
’
M =340.10379, found M =340.10298.
4.4.3. ( )-2-Bromo-o-tolyl ethyl acetate. Yield: 8.70 g,
(78%). IR (neat) 1750 and 1730 (wCꢀO), 1600 and 1475
1
(wCꢀC), 1280 to 1140 (wC-O) cm−1. H NMR (CDCl3):
4.5. Pre-equilibration of water activity (aw)
l 1.24–1.31 (t, J=7Hz, 3H, -OCH3
3H, ArCH3): l 4.19–4.31 (qd, J=3.2–7 Hz, 2H,
-OCH2CH3): l 5.63 (s, 1H, -CH
ArH): l 7.59–7.64 (m, 1H, ArH
14.05 (-CH2CH3), 19.36 (ArC
(-OCH2CH3), 126.90, 128.79, 129.25, 130.87, 134.48,
136.13, 168.30 (C
6 ): l 2.41 (s,
6
Enzyme preparations and organic solvent were equili-
brated with saturated salt solutions for at least four
days at 4°C in separate containers. The salts used were
LiBr (water activity, aw=0.06), LiCl (aw=0.11),
MgCl2·6H2O (aw=0.32), Mg(NO3)2·6H2O (aw=0.53),
NaCl (aw=0.75), KNO3 (aw=0.92), K2Cr2O7 (aw=
6
6
Br): l 7.19–7.26 (m, 3H,
6
); 13C NMR (CDCl3): l
6
6
6 H3), 44.83 (-CHBr), 62.68
6
+
’
6
OO). Mass: calcd M =256.00989,
+
’
,
found M =256.00961.
0.98). With molecular sieve (3 A) aw=0.04.

D. Guieysse et al. / Tetrahedron: Asymmetry 12 (2001) 2473–2480
2479
4.6. General procedure for the enzymatic
transesterification
Acknowledgements
A typical transesterification was carried out in solvent
(5 mL) containing the ester (0.25 mmol) (50 mM),
octanol (150 mM) (0.75 mmol) and lipase (5 mg/mL for
free lipase and 10 mg/mL for immobilized lipase). The
temperature was maintained at 30°C unless otherwise
stated in the text. The mixture was shaken at 250 rpm
for the time indicated in Tables 1–4. The progress of
the reaction was followed by taking samples at regular
intervals.
The authors thank Dr. Lorenzo Di Bari of the Diparti-
mento di Chimica e Chimica Industriale (Pisa, Italy) for
the determination of absolute configurations by the
Circular Dichroism Exciton method.
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