Enzymatic transesterification of racemic lavandulol: Preparation of the two enantiomeric alcohols and of the two enantiomers of lavandulyl senecioate

Article abstract of DOI:10.1016/j.tetasy.2004.06.015

(R) and (S)-lavandulol are important compounds in the cosmetics industry and in pheromone research. The senecioyl ester of (S)-lavandulyl has recently been identified as the sex pheromone of the vine mealybug, a significant pest in vineyards. We herein report the preparation of the two enantiomers of lavandulol and lavandulyl senecioate, starting from racemic lavandulol. The preparation is based on a two-cycle enzymatic transesterification of racemic lavandulol with vinyl acetate using Porcine pancreas lipase. High enantioselectivity was achieved while the preparation yielded (R)-lavandulol with 96.7% ee and (S)-lavandulol with 92.6% ee.

Full text of DOI:10.1016/j.tetasy.2004.06.015

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 2339–2343
Enzymatic transesterification of racemic lavandulol: preparation
of the two enantiomeric alcohols and of the two
enantiomers of lavandulyl senecioate
Anat Zada* and Miriam Harel
Institute of Plant Protection, The Volcani Center, ARO, Bet Dagan 50250, Israel
Received 6 May 2004; accepted 15 June 2004
Abstract—(R) and (S)-lavandulol are important compounds in the cosmetics industry and in pheromone research. The senecioyl
ester of (S)-lavandulyl has recently been identified as the sex pheromone of the vine mealybug, a significant pest in vineyards. We
herein report the preparation of the two enantiomers of lavandulol and lavandulyl senecioate, starting from racemic lavandulol. The
preparation is based on a two-cycle enzymatic transesterification of racemic lavandulol with vinyl acetate using Porcine pancreas
lipase. High enantioselectivity was achieved while the preparation yielded (R)-lavandulol with 96.7% ee and (S)-lavandulol with
92.6% ee.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
of lavandulol,7;8 but so far only low enantioselectivity
has been achieved.8
Lavandulol is an important terpene constituent in plants
and has alsobeen found in insects. The ( R)-form is a
constituent of French lavender oil, which is used in the
perfume chemistry. (R)-Lavandulol has recently been
found to be a component of the aggregation pheromone
of the strawberry blossom weevil Anthonomus rubi
Herbst.1 (S)-Lavandulol, as the senecioyl ester, has also
been recently identified as the sex pheromone of the vine
mealybug, Planococcus ficus,2–4 which is a serious pest in
vineyards. Lavandulol, with unknown configuration, is
also a defensive pheromone of the carrion beetle,
Necrodes surinamensis.5
We studied, both the lipase-mediated transesterification
of lavandulol and the hydrolysis of a number of lavan-
dulyl esters. The best results were achieved by a two-
cycle transesterification with vinyl acetate that used
Porcine pancreas lipase. Both enantiomers of lavandulol
were obtained, with high chemical yield and enantio-
meric excess.
2. Results and discussion
Eleven lipases were investigated for transesterification of
lavandulol with vinyl acetate in t-butylmethyl ether
(Scheme 1), but only P. pancreas lipase exhibited
promising activity and selectivity (Table 1, entry 6). The
reaction was monitored by GC analysis on a chiral
column. Both lavandulol and lavandulyl acetate were
base-line separated on the Hydrodex-b-6-TBDM chiral
column; 25 m·0.25 mm (Macherey-Nagel); kept at 90 ꢁC
for 15 min, and then programmed at 5 ꢁC/min to170 ꢁC
and held for 10 min. The retention times of (R)-lavan-
dulyl acetate and (S)-lavandulyl acetate were 20.6 and
20.8 min and of (S)-lavandulol and (R)-lavandulol were
21.6 and 22.3 min, respectively. The activity of this
lipase was studied in two additional organic solvents
that are regularly used in enzymatic transesterification,
Due to the importance of lavandulol as an additive in
the perfume industry, much effort has been made in the
synthesis of racemic and chiral lavandulol.6 Most of the
syntheses involve several steps or use expensive chiral
reagents. We were interested in developing a simple
procedure to prepare the two enantiomers. Racemic
lavandulol is a commercial compound; therefore we
decided to pursue the enzymatic methodology for its
resolution. It should be noted that this is not the first
attempt to apply a biocatalytic route for the separation
* Corresponding author. E-mail: anatzada@volcani.agri.gov.il
0957-4166/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.06.015

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A. Zada, M. Harel / Tetrahedron: Asymmetry 15 (2004) 2339–2343
OH
lipase/organic solvent
OH
vinyl acetate
(R)-lavandulol
Racemic lavandulol
OH
OAc
hydrolysis
(S)-lavandulyl acetate
Scheme 1. Enzymatic separation of lavandulol using lipase in organic medium.
(S)-lavandulol
Table 1. Enzymes tested for the lipase-mediated transesterification of lavandulol with vinyl acetate in t-butylmethyl ether
Eb
a
a
Lipase
Time
Conversion (%)
Substrate alcohol ees (%)
Product acetate eep (%)
A. niger
17 d
4 d
0
18
32
37
9
––
––
––
––
c
c
A. oryzae
C. antarctica
C. cylindracea
C. rugosa
8 d
6 h
S, 54
R, 54
R, 60
S, 60
5.2
5.6
––
c
c
10 d
3 h
P. pancreas
P. cepacia
P. fluorescens
P. stutzeri
R. oryzae
53
20
58
43
17
0
R, 74
c
S, 84
c
41.7
––
22 h
3 h
R, 67
R, 63
S, 69
S, 72
19.8
10.5
––
20 d
22 h
2 d
c
c
Wheat germ
––
––
––
a Enantiomeric excess determined by chiral GC.
b Enantiomeric ratio, E ¼ ln½1 À Cð1 þ eepÞꢀ= ln½1 À Cð1 À eepÞꢀ.
c Nopreference.
namely hexane and tetrahydrofuran (THF) (Table 2).
Hexane gave the highest E value for the conversion and
was selected for all further experiments.
(Scheme 2). Six esters of lavandulol were prepared and
tested; they were lavandulyl acetate, lavandulyl prop-
ionate, lavandulyl 2-phenylbutyrate, lavandulyl piva-
late, lavandulyl trifluoro acetate and lavandulyl valerate.
Except for lavandulyl acetate and propionate, attempts
to resolve these esters on different chiral GC columns
failed. Also, the enantioselectivities of the hydrolyses
were all low (Table 3).
Table 2. P. pancreas lipase-catalyzed acetylation of lavandulol with
vinyl acetate as the acyl donor in organic solvents
Solvent
Time Conversion
Ees (%)a Eep (%)a Eb
Hexane
t-Butylmethyl
ether
3 h
5 h
49.6
47.8
75
73
86
85
37.8
28.9
Lipase-mediated reactions depend mainly on the type
and origin of the enzyme. Among all the lipases that
were screened for the separation of racemic lavandulol,
the best results were achieved by transesterification with
P. pancreas. However, the highest ee values were in the
range of 74–84%, and therefore not sufficient for further
biological work, particularly for the preparation of the
THF
48 h
45.9
70
83
22.6
a Enantiomeric excess determined by chiral GC.
b Enantiomeric ratio.
The lipase-mediated hydrolysis of various lavandulyl
esters, with P. pancreas as the lipase, was alsoexamined
OH
O
Lipase/buffer pH7
OCR
(S)-lavandulol
Racemic lavandulyl ester
O
OCR
hydrolysis
OH
(R)-lavandulyl ester
Scheme 2. Lipase-mediated hydrolysis of racemic lavandulyl esters in a phosphate buffer.
(R)-lavandulol

A. Zada, M. Harel / Tetrahedron: Asymmetry 15 (2004) 2339–2343
2341
Table 3. P. pancreas lipase-catalyzed hydrolysis of different lavandulyl esters in phosphate buffer (pH 7) at 25 ꢁC
a
a
Lavandulyl ester
Time
Conversion (%)
Substrate ester ees (%)
Product alcohol eep (%)
Eb
Lavandulyl acetate
Lavandulyl 2-phenylbutyrate
Lavandulyl pivalate
0.5 h
4 d
29
0
R, 62
––
S, 61
––
5.2
––
c
42 h
1 h
5.8
56.4
54.5
24.3
S, 58
S, 57
R, 51
S, 57
8.9
7.5
5.5
4.4
Lavandulyl propionate
Lavandulyl trifluoroacetate
Lavandulyl valerate
R, 55
c
1 h
1 h
c
a Enantiomeric excess determined by chiral GC.
b Enantiomeric ratio.
c Not separated by GC analysis on a chiral column.
€
enantiomers of the vine mealybug sex pheromone.
Therefore, a second cycle of transesterification was ap-
plied to the partially separated lavandulol enantiomers.
This two-step enzymatic process yielded (R)-lavandulol
with 96.7% ee and (S)-lavandulol with 92.6% ee. The
two lavandulol enantiomers were esterified with sene-
cioyl chloride to provide (R)-lavandulyl senecioate and
(S)-lavandulyl senecioate, which is the sex pheromone of
the vine mealybug. Aliquots of these two esters were
hydrolyzed to the corresponding lavandulol enantio-
mers in order to verify that no racemization had oc-
curred during the esterification. Direct chiral analysis of
the enantiomeric senecoyl esters failed because they
could not be separated on the available chiral GC col-
umns. The recovered lavandulol enantiomers had the
same enantiomeric excess as before the esterification.
The identities of the lavandulol enantiomers and the
senecioyl esters were confirmed by GC and GC–MS
comparison with authentic samples from our previous
work on the identification of the sex pheromone of the
vine mealybug.4
column (Macherey-Nagel, Duren, Germany), which was
held at 90 ꢁC for 15 min and then programmed to 170 ꢁC
at a rate of 5ꢁ/min. Analysis of the racemic esters and
monitoring of the reactions were conducted on a
30 m·0.25 mm·250 lm HP5 column (Hewlett–Packard,
USA), which was held at 60 ꢁC for 2 min, programmed
to170 ꢁC at 15ꢁ/min, held at 170 ꢁC for 3 min and then
programmed to 220 ꢁC at the same rate. Helium was
used as the carrier gas and the temperature of the
injector and detector held at 220 ꢁC. Analysis on the
chiral column was conducted in the split mode at a ratio
of 10:1 while the analysis on the HP5 column was con-
ducted in the splitless mode with the purge valve being
opened after 1 min. Optical rotations were determined
on a Jasco P-1010 polarimeter at a wavelength 589 nm,
cell length 0.5 dm.
4.1.2. Chemicals and enzymes. Lavandulol was obtained
from Fluka (Buchs, Switzerland), hexane from Merck
(Darmstadt, Germany), anhydrous t-butylmethyl ether
(t-BuOMe) from Aldrich (Milwaukee, WI), THF from
Fluka, senecoyl chloride, 2-phenylbutyryl chloride,
propionyl chloride, valeryl chloride and trifluoroaceti-
canhydride from Aldrich, pivaloyl chloride from Fluka,
acetic anhydride from Merck, anhydrous pyridine from
Aldrich and KH2PO4/Na2HPO4 buffer pH 7.00 from
3. Conclusion
Screening of 11 lipases for the transesterification of
racemic lavandulol with vinyl acetate indicated that the
best enantioselectivity could be obtained with P. pan-
creas. Approximately the same enantiomeric separation
was achieved in hexane, t-butylmethyl ether and THF.
Hexane was used for further work, which yielded the
twoenantiomers at 74–84% ee in gram quantities. A
second cycle of transesterification with the partially
separated lavandulol enantiomers yielded (R)- and (S)-
lavandulol in high chemical and enantiomeric excess.
The reverse reaction of the lipase-mediated hydrolysis of
several lavandulyl esters gave very low enantioselectivity
and hence is not suitable for the chiral separation of
racemic lavandulol.
€
Riedel-de Haen (Seelze, Germany); all were used with-
out further purification. Molecular sieves type 4 A from
ꢀ
BDH (Dorset, England) were oven-dried overnight at
150 ꢁC. Vinyl acetate (Aldrich) was distilled before use.
Aspergillus niger, Aspergillus oryzae, Candida antarctica
(immobilized in sol–gel-AK), Candida cylindracea,
Candida rugosa, Pseudomonas stutzeri, Rhizopus oryzae
and wheat germ lipases were obtained from Fluka. P.
pancreas lipase (type II, crude; Sigma). Pseudomonas
cepacia (lipase PS) and Pseudomonas fluorescens (lipase
AK) lipases were a gift from Amano Enzyme (Japan).
Flash chromatography was carried out with silica gel
ꢀ
70–230 mesh, 60 A (Aldrich). (R)-Lavandulol, that had
been isolated from lavandin oil,4 was used as a standard
for the determination of the absolute configuration of
the lavandulol enantiomers.
4. Experimental
4.1. General methods and materials
4.1.3. Calculation of enantioselectivity. Both enantiomers
of lavandulol (the substrate) and lavandulyl acetate (the
product) were baseline separated in the GC analysis on
the chiral column to obtain ees and eep. The tR values for
(R)- and (S)-lavandulyl acetate were 20.82 and
4.1.1. Analytical methods. GC analysis was performed
on an HP6890 gas chromatograph equipped with a
flame ionization detector. Enantiomeric purity was
determined on a 25 m·0.25 mm, Hydrodex-b-6-TBDM

2342
A. Zada, M. Harel / Tetrahedron: Asymmetry 15 (2004) 2339–2343
21.03 min, respectively, and those for (S)- and (R)-lav-
andulol were 21.84 and 22.52 min, respectively. The
conversion (C) was calculated from the equation
C ¼ ees/(eesþeep). Enantioselectivity was calculated by
means of the equation
(300 mL) was stirred at room temperature. After 3 h,
when the conversion had reached 50% (according to the
GC analysis), the reaction was terminated by filtration
and the solvent was evaporated. The residue was dis-
tilled under reduced pressure (75–107 ꢁC/12 mm) toyield
4.65 g of a mixture of lavandulol and lavandulyl acetate.
Flash chromatography of 2.25 g of the distilled mixture
on silica (45 g) gave (S)-lavandulyl acetate (750 mg,
ee ¼ 82.7%), which was eluted with hexane/diethyl ether
at a ratio of 98:2, followed by unreacted (R)-lavandulol
(1.25 g, ee ¼ 73%) and then with hexane/diethyl ether at
a ratioof 9:1.
E ¼ ln½1 À Cð1 þ eepÞꢀ= ln½1 À Cð1 À eepÞꢀ
according to Chen et al.9 In the enantioselective
hydrolysis of the various lavandulyl esters only lavan-
dulyl acetate and lavandulyl propionate were baseline
separated. Therefore, the conversion (C) of the reactions
of lavandulyl 2-phenylbutyrate, lavandulyl pivalate,
lavandulyl trifluoroacetate and lavandulyl valerate were
calculated from the relative amounts of the esters and
alcohol. Unsuccessful attempts were made, to separate
the higher esters on different chiral GC columns, such as
4.2.2. Enhancing the chiral purity of (S)-lavandulol.
4.2.2.1. Hydrolysis of (S)-lavandulyl acetate. (S)-Lav-
andulyl acetate (750 mg, 3.8 mmol, ee ¼ 82.7%) was
hydrolyzed by stirring in 15 mL of KOH/MeOH (0.5 M)
for 1.5 h. The hydrolysis was monitored by GC analysis
every 0.5 h. The reaction mixture was poured onto 10 g
of ice and extracted with hexane (3 · 5 mL). The organic
phase was washed with brine (5 mL), dried over MgSO4
and evaporated under reduced pressure to yield 567 mg
o f S()-lavandulol (ee ¼ 82.7%) (3.7 mmol, recovery of
96%).
€
25 m·0.25 mm LipodexG (Macherey-Nagel, Duren,
Germany), 30 m·0.25 mm·0.25 mm CDX-B (J & W
Scientific, Folson, CA, USA), 30 m·0.25 mm·0.25 mm,
Rt-bDEXsm (Restek, Bellefonte PA, USA).
4.1.4. Lipase-mediated resolution of racemic lavandulol.
A mixture of the racemic lavandulol (20 mg), lipase
(20 mg), vinyl acetate (1 mL) and t-BuOMe (3 mL) (or
hexane or THF), with two or three pellets of molecular
sieve was stirred at room temperature. Aliquots of 1 lL
were withdrawn at intervals and analyzed by gas chro-
matography.
4.2.2.2. Second cycle of lipase-mediated transesterifi-
cation of (S)-lavandulol. Transesterification was per-
formed as described in Section 4.2.1, but without the
distillation step. The 567 mg of (S)-lavandulol
(ee ¼ 82.7%) was converted into (S)-lavandulyl acetate,
purified by chromatography and then hydrolyzed to give
4.1.5. General procedure for the synthesis of racemic
lavandulyl esters. To an ice-cooled, stirred solution of
lavandulol (0.2 mL, 1.1 mmol) and pyridine (0.5 mL) in
ether (3 mL), acyl chloride (2.2 mmol) was added drop-
wise. The reaction mixture was stirred until the disap-
pearance of lavandulol, as determined by GC analysis.
The reaction mixture was poured into cold HCl (1 M,
5 mL), and extracted twice with hexane (2 mL). The
organic phase was washed with saturated NaHCO3
solution (5 mL), brine (5 mL) and then dried over
MgSO4. After evaporation of the solvent under reduced
pressure, the crude residue was purified by flash chro-
matography on silica (hexane/diethyl ether 95:5).
331 mg (2.1 mmol) of (S)-lavandulol (ee ¼ 92.6%, by
25
GC). ½aꢀ ¼ þ10:1 (c 1.13, MeOH) [lit.10 ¼ þ9:94
D
(c ¼ 1, MeOH)].
4.2.3. Increasing the chiral purity of (R)-lavandulol. A
second cycle of enzymatic separation has been carried
out in the same way as described above without distil-
lation. (R)-Lavandulol (ee ¼ 73%) (1.25 g) yielded
971 mg (R)-lavandulol (ee ¼ 96.75%, by GC), recovery
25
of 77%. ½aꢀ ¼ À9:6 (c 1.0, MeOH) [lit.6 ¼ À10:05
D
(c ¼ 1:1, MeOH)].
4.1.6. Lipase-mediated resolution of racemic lavandulyl
esters. A mixture of racemic lavandulyl ester (20 mg)
and P. pancreas lipase (20 mg) was dissolved in 5 mL of
sodium phosphate buffer (50 mM, pH 7.00) and stirred
at room temperature. Aliquots of 20 lL were withdrawn
at intervals, dispersed intohexane (1 mL), dried over
MgSO4 and analyzed by gas chromatography.
4.3. Preparation of (R)-lavandulyl senecioate and (S)-
lavandulyl senecioate
The syntheses were performed in the same manner as
described in Section 4.1.5 to yield the (R)-enantio-
25
D
mer ½aꢀ ¼ À8:3 (c 1.2, hexane) and the (S)-enantiomer
25
½aꢀ ¼ þ9:1 (c 0.99, hexane).
D
4.2. Gram-scale lipase-catalyzed separation of lavandulol
enantiomers
Acknowledgements
4.2.1. Enzymatic separation of racemic lavandulol by
P. pancreas lipase. A mixture of lavandulol (5 g,
32 mmol), P. pancreas lipase (5 g), vinyl acetate (12 mL,
0.11 mol) and molecular sieve pellets (0.25 g) in hexane
We would like to thank Dr. Ezra Dunkelblum, Institute
of Plat Protection, ARO, for his enlightening comments
€
€
and Prof. Wilfried A. Konig, Institute fur Organische

A. Zada, M. Harel / Tetrahedron: Asymmetry 15 (2004) 2339–2343
2343
4. Zada, A.; Dunkelblum, E.; Assael, F.; Harel, M.; Cojo-
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55, 4047–4051.
€
Chemie, Universitat Hamburg, for his advice regarding
the selection of the chiral column.
6. Piva, O. J. Org. Chem. 1995, 60, 7879–7883, and references
cited therein.
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