Stereoselective acylation of DL-menthol in organic solvents by an immobilized lipase from Pseudomonas cepacia with vinyl propionate

Article abstract of DOI:10.1007/s11746-997-0102-4

An effective lipase-catalyzed stereoselective transesterification of (±)-menthol in organic solvent with vinyl propionate as acylating agent is described. Immobilization by adsorption and the presence of molecular sieves improved the formation of (±)-menthyl propionate by lipase (PS-30) from Pseudomonas cepacia. The reaction time course, mole ratio of substrates, temperature, amount of enzyme, as well as the effect of various organic solvents, were examined for their influence on the enzymatic stereoselective formation of (-)-menthyl propionate. Among the parameters studied, the stereospecificity toward (-)-menthol decreased significantly as temperature increased but the yields of both enantiomers increased. Organic solvents with log P(partition coefficient) values above 3.5 gave higher yield and stereoselectivity than solvents with lower log P values.

Full text of DOI:10.1007/s11746-997-0102-4

Stereoselective Acylation of DL-Menthol in Organic
Solvents by an Immobilized Lipase
from Pseudomonas cepacia with Vinyl Propionate
a
a,
b
Wen-Hsin Wu , Casimir C. Akoh *, and Robert S. Phillips
a
b
b
Departꢀents of Food Pcience and Tecꢁnoloꢂy, Cꢁeꢀistry, and Biocꢁeꢀistry
and Molecular Bioloꢂy, Tꢁe University of Georꢂia, Atꢁens, Georꢂia ꢅ3632-7613
ABSTRACT: An effective lipase-catalyzed stereoselective we chose lipase-catalyzed irreversible transesterification with
transesterification of (± ±-ꢀentꢁol in orꢂanic solvent ꢃitꢁ vinyl vinyl esters in place of acid anhydrides because of the diffi-
propionate as acylatinꢂ aꢂent is described. Iꢀꢀobilization by
adsorption and tꢁe presence of ꢀolecular sieves iꢀproved tꢁe
forꢀation of (± ±-ꢀentꢁyl propionate by lipase (ꢄP-ꢅ3± froꢀ
Pseudomonas cepacia. Tꢁe reaction tiꢀe course, ꢀole ratio of
substrates, teꢀperature, aꢀount of enzyꢀe, as ꢃell as tꢁe effect
of various orꢂanic solvents, ꢃere exaꢀined for tꢁeir influence
on tꢁe enzyꢀatic stereoselective forꢀation of (−±-ꢀentꢁyl pro-
pionate. Aꢀonꢂ tꢁe paraꢀeters studied, tꢁe stereospecificity to-
ꢃard (−±-ꢀentꢁol decreased siꢂnificantly as teꢀperature in-
culty we encountered in eliminating the background chemical
acylation that resulted from acid anhydrides. Our preliminary
screening indicated that lipase PS-30 appeared to be the most
promising biocatalyst under the experimental conditions ex-
amined, and that high enantioselectivity could be achieved
with this lipase when we used vinyl propionate for acylation
of (±)-menthol. The (−)-menthol and its esters are more im-
portant from an industrial point of view than (±)-menthol. Be-
creased but tꢁe yields of botꢁ enantioꢀers increased. Orꢂanic cause of its cooling and refreshing effects, (−)-menthol is an
solvents ꢃitꢁ loꢂ P (partition coefficient± values above ꢅ.5 ꢂave important fragrance and flavor compound that is used largely
ꢁ
iꢂꢁer yield and stereoselectivity tꢁan solvents ꢃitꢁ loꢃer loꢂ P in cosmetics, toothpastes, chewing gum, cigarettes, sweets,
values.
and medicines. It also possesses the characteristic peppermint
odor, which is lacking in other isomers. The (±)-menthol
cooling effect is not so distinct as that of (−)-menthol, and
therefore, it is not highly valued. However, it can be used in
medicine and liniments.
In this study, we report the use of an immobilized lipase
for the stereoselective transesterification of (±)-menthol with
vinyl propionate as acyl donor. Reaction parameters were also
JAOCS 74, 4ꢅ5–4ꢅ9 (1997±.
KEY WORDS: Acylatinꢂ aꢂent, lipase, (± ±-ꢀentꢁol, (± ±-ꢀen-
tꢁyl propionate, Pseudomonas cepacia, stereoselective trans-
esterification, vinyl propionate.
In recent years, the use of lipases as biocatalysts for enzy- studied.
matic resolution of racemic substances has increasingly at-
tracted research interest because of their demonstrated high
EXPERIMENTAL PROCEDURES
enantioselectivity compared to chemical catalysts (1). In ad-
dition, lipases are readily available, relatively inexpensive, Enzymes and chemicals. Lipase “PS-30” (specific activity 34
and have no cofactor requirement (2).
IU/mg solid) from Pseudomonas cepacia was kindly supplied
Among the wide range of lipase-catalyzed reactions that by Amano Enzyme Co. (Troy, VA). The racemic secondary
have been studied, certain types of acylating agents, such as alcohol dl-menthol, i.e., (±)-menthol, and (−)-menthol were
acid anhydrides and enol esters, have been recommended be- obtained from Eastman Kodak Company (Rochester, NY)
cause they make the acyltransfer completely irreversible and Sigma Chemical Co. (St. Louis, MO), respectively. Vinyl
(
3–7). Previously, we have successfully achieved the enan- propionate was purchased from Aldrich Chemical Company,
tioselective resolution of (±)-menthol by a lipase (“AY-30,” Inc. (Milwaukee, WI). All solvents were high-performance
from Candida cylindracea) with acid anhydrides as acylating liquid chromatography-grade and were from either Fisher
agents (8). Menthol is a secondary terpene alcohol obtained Scientific (Norcross, GA) or J.T. Baker Chemical Co.
from Mentha piperita and M. arvenisis. In the present report, (Phillipsburg, NJ). Molecular sieves, 4Å, were from Davison
Chemical (Baltimore, MD) and were activated in a vacuum
oven prior to use.
*
To whom correspondence should be addressed at Department of Food Sci-
Enzymatic transesterification reactions. Transesterifica-
tion was carried out in 20 × 125-mm screw-capped culture
ence and Technology, Food Science Building, The University of Georgia,
Athens, GA 30602-7610.
Copyriꢂꢁt © 1997 by AOCP ꢄress
4ꢅ5
JAOCP, Vol. 74, no. 4 (1997±

4ꢅ6
W.-H. WU ET AL.
tubes with Teflon liners in a gyratory waterbath shaker (New menthyl propionates. A Varian 3300 gas chromatograph (Sun-
Brunswick Scientific Co., Inc., Edison, NJ) at 30°C, 200 rpm, nyvale, CA), equipped with a flame-ionization detector and a
TM
for 48 h in 2 mL of an organic solvent. Lipase PS-30 in the chiral Beta-DEX 120 fused-silica capillary column (30 m
amount of approximately 680 units (w/w, soluble × 0.25 mm i.d., 0.25 µm film thickness; Supelco Inc., Belle-
enzyme/total reactants) was used as the biocatalyst and added fonte, PA), was used to separate and identify the optically ac-
immediately before the incubation started. A control without tive menthyl propionate produced by enzymatic reaction. The
the presence of a lipase was simultaneously incubated to injector and detector temperatures were 200 and 225°C, re-
monitor any chemical background reaction. No product was spectively. The temperature program used to separate (+)- and
generated by the control reaction. Molecular sieves (approxi- (−)-menthyl propionates was held at 110°C for 12 min before
mately 0.1 g) were added 2 h after the incubation started. The being elevated to 150°C at 4°C/min and then held for 5 min.
time course study was conducted by taking samples at 1, 2, 3, The flow rate of the carrier gas, helium, was 0.8 mL/min. The
4
, 5, 6, 12, 24, 36, and 48 h. The effect of various organic sol- amount of each enantiomer was estimated from the peak area
vents, including n-hexane, isooctane, n-heptane, cyclohexane, recorded and integrated by an LDC/Milton Roy CI-10B inte-
benzene, toluene, methylene chloride, and chloroform, on the grator (Riviera Beach, FL). The retention times for (+)-men-
lipase-catalyzed enantioselective synthesis of menthyl propi- thol, (−)-menthol, (−)-menthyl propionate, and (+)-menthyl
onate was examined by mixing 1 mmole of (±)-menthol and propionate were 20.4, 20.5, 23.8, and 24.1 min, respectively.
1
mmole of vinyl propionate in 2 mL of solvent and incubat-
The stereoselectivity and optical purity of the enzymatic
ing at 30°C in the presence of lipase PS-30 for 48 h. The ef- reaction were expressed by the enantiomeric ratio E, defined
fects of enzyme load, mole ratio of substrates, and reaction (9) as (−)/(+), as well as the enantiomeric excess ee%, which
temperature on the transesterification were also examined. All is equal to the absolute value of percentage excess of one
reactions were performed in duplicate, and results are re- enantiomer over the other: ee% = {[−] − [+]/[−] + [+]} × 100
ported as means ± standard deviation.
(10).
Optical purity of substrate (±)-menthol. A 1% (wt/vol) so-
Immobilization of lipase PS onto glass beads by adsorption.
Approximately 1 g of powdered lipase PS was mixed well with lution of (±)-menthol or (−)-menthol in 95% ethanol was pre-
.5 mL of 10 mM phosphate buffer, pH 7, at room temperature pared for examination of optical purity. The optical rotation
2
®
to form a slurry. The enzyme slurry was mixed in a 50-mL of the substrate (±)-menthol was determined by an Autopol
Pyrex beaker with 5 g of glass beads (0.5-mm diameter, IV automatic polarimeter (Rudolph Research, Flanders, NJ)
BiospecProducts, Bartlesville, OK) which had been rinsed and proven to be racemic.
twice with 5 mL deionized water and dried at 110°C in an oven.
Preparation of standard menthyl propionates. The stan-
The mixture of enzyme and glass beads was then placed in a dard (±)-menthyl and (−)-menthyl propionates were prepared
desiccator filled with Drierite (W.A. Hammond Drierite Com- by refluxing (±)-menthol and propionic anhydride in pyridine
pany, Xenia, OH) and stirred thoroughly at 4-h intervals for 24 for 2 h to determine their retention time in capillary GC. The
h and then stirred after 18 and 24 additional hours. The activity reaction product was washed with water, extracted with pe-
of immobilized lipase was estimated based on the mass of en- troleum ether, and monitored by capillary GC to ensure com-
zyme adsorbed onto glass beads after water and excess enzyme plete reaction.
on the wall of the beaker had been removed.
Quantitation of menthyl esters by gas chromatography
GC). An aliquot of 50 µL of the reaction product in organic
RESULTS AND DISCUSSION
(
medium was pipetted and diluted in 1 mL hexane that con- The use of immobilized lipase PS-30 and presence of molec-
tained 0.875 mg ethyl caproate (Aldrich Chemical Company, ular sieves improved the overall yield of (±)-menthyl propi-
Inc.) as internal standard. The residual moisture in the solvent onate from 16 to 35% (Table 1). Immobilization exposes the
and the enzymes was removed by passing the hexane solution enzyme more effectively to the substrate (11) and therefore
through an anhydrous sodium sulfate (Fisher Chemical, Fair improves the formation of menthyl propionate. The presence
Lawn, NJ) column. Quantitation was achieved with a of molecular sieves in the system was first recommended by
Hewlett-Packard 5890 Series II gas chromatograph (Hewlett- Degueil-Castaing et al. (7) to remove water that is liberated
Packard, Avondale, PA), equipped with a flame-ionization de- from side reactions of acetaldehyde that tautomerizes from
tector and DB-5 fused-silica capillary column (30 m × 0.32 vinyl alcohol, a product of the lipase-catalyzed transesterifi-
mm i.d., film thickness 1 µm; J&W Scientific, Folsom, CA), cation. The immobilized lipase was used for reaction parame-
operated isothermally at 170°C. The injector and detector ter studies, and molecular sieves were always included in the
temperatures were 250 and 260°C, respectively. The carrier reaction.
gas was helium at a flow of 5 mL/min. The percentage mole
Figure 1 shows the time course of formation of (−)-men-
yield of menthyl propionate was defined as (mmole of men- thyl propionate. At 30°C, nearly 50% of (−)-menthol was
thyl propionate/initial mmole of menthol present in the sys- converted to the ester form after 48 h incubation. The reac-
tem) × 100 and was estimated from peak areas that were inte- tion rate was comparable to enzymatic resolutions of (±)-
grated by an on-line computer.
menthol obtained previously with lipase from C. cylindracea
Chiral capillary GC separation of (±)-menthols and (±)- (1,12).
JAOCP, Vol. 74, no. 4 (1997±

PTEREOPELECTIVE PYNTHEPIP OF MENTHYL ꢄROꢄIONATE
4ꢅ7
TABLE 1
Percentage Yields of (± ±-Menthyl Propionate by Lipase PS-ꢀ3 at ꢀ30C
as Affected by Enzyme Immobilization and Addition of Molecular
a
Sieves
Molecular sieves
Uniꢀꢀobilized ꢄP
Iꢀꢀobilized ꢄP
No
Yes
16.2 ± 2.1
22.6 ± 1.8
22.6 ± 2.8
ꢅ4.5 ± 5.ꢅ
aReaction ꢃas perforꢀed in duplicate in ꢁexane at a 1:1 ꢀole ratio of (± ±-
ꢀ
entꢁol to vinyl propionate. Tꢁe aꢀount of enzyꢀe ꢃas constant. Values
are reported as ꢀeans ± standard deviation.
The effect of the mole ratio of menthol and vinyl propi-
onate was studied at levels from 1:1 to 1:3 (Fig. 2). There was
not much difference across the three levels examined in both
yield and stereoselectivity (i.e., E values ranged from 121 to
155). It appears that a 1:1 ratio of the substrates is in fact suf-
ficient for satisfactory resolution of (±)-menthol.
It is widely believed and supported by experimental obser-
vations that the stereoselectivity of enzymes is affected by re-
FIG. 2. Effect of (± ±-ꢀentꢁol to vinyl propionate ratio (1:1, 1:2, and 1:ꢅ±
on forꢀation of (−±-ꢀentꢁyl propionate by iꢀꢀobilized lipase ꢄP-ꢅ3 in
n-ꢁexane at ꢅ3°C. M = ꢀentꢁol and Vꢄ = vinyl propionate. Pee Fiꢂure
action temperature (9). The effect of temperature after 48 h 1 for explanation of bars.
incubation is summarized in Table 2. The mole percentage
yield of (−)-menthyl propionate increased from 50 to 86% as
reaction temperature increased. However, the differences
within the temperature ranges 30–40°C and 50–60°C were factor when future industrial application is kept in mind. As
not significant. The highest increase in yield occurred be- expected, the percentage yield of (−)-menthyl propionate in-
tween 40–50°C. The stereoselectivity, on the other hand, de- creased from 20 to 67% when the enzyme load was increased
creased as reaction temperature increased (Table 2). Appar- from 100 to 700 units (Fig. 3). The increase was rather slow
ently, a higher chemical yield could be obtained at elevated after reaching 300 units. If the cost of enzyme is a problem, a
temperature at the expense of stereochemical purity of the higher level of enzyme may not be necessary.
products. We decided not to go over 60°C to avoid possible
loss of terpene esters as reported previously (13).
It is widely believed that the nature and polarity of organic
media in which enzymatic reactions take place affect the ac-
The amount of enzyme used can be a crucial economical tivity of biocatalyst (14,15). The polarity of organic solvents
can be quantitatively measured by the value of log P, the log-
arithm of the partition coefficient of a given solvent between
water and 1-octanol. Organic solvents with log P values <2.0
are generally not considered good for biocatalysis (13). In this
study, a few commonly used organic solvents were examined
for their suitability for lipase-catalyzed stereoselective reso-
TABLE 2
Values of E, ee%, and Yields of Menthyl Propionate Synthesized
a
by Immobilized Lipase PS as Affected by Temperature
%
Yield
Teꢀperature (°C±
E
ee%
(−±-ꢀentꢁyl propionate
2
ꢅ3
3
ꢅ22 ± 13
156 ± 2ꢅ
97 ± 42
47 ± 18
ꢅ1 ± 6
99.4 ± 3.1
98.7 ± 3.2
97.7 ± 1.3
95.5 ± 1.9
9ꢅ.7 ± 1.1
53.2 ± 11.8
63.3 ± 13.6
58.5 ± 18.4
87.2 ± 4.9
86.4 ± ꢅ.7
43
53
63
aReactions ꢃere perforꢀed in ꢁexane at a 1:1 ꢀole ratio of (± ±-ꢀentꢁol to
vinyl propionate. E, enantioꢀeric ratio; ee%, enantioꢀeric excess. Tꢁe val-
ues are reported as ꢀeans ± standard deviation and ꢃere obtained by cꢁi-
ral ꢂas–liquid cꢁroꢀatoꢂrapꢁy ꢃitꢁ a Beta-DEX 123 fused-silica capil-
lary coluꢀn (Pupelco Inc., Bellefonte, ꢄA±. Pee tꢁe Experiꢀental ꢄrocedures
section for separation conditions.
FIG. 1. Tiꢀe course (3, 6, 12, 18, 24, ꢅ6, 48 ꢁ± of forꢀation of (−±-ꢀen-
tꢁyl propionate by iꢀꢀobilized lipase ꢄP-ꢅ3 at ꢅ3°C. Reactions ꢃere
perforꢀed in n-ꢁexane ꢃitꢁ a 1:1 ꢀole ratio of (± ±-ꢀentꢁol to vinyl pro-
pionate. Bars indicate standard deviation.
TM
JAOCP, Vol. 74, no. 4 (1997±

4ꢅ8
W.-H. WU ET AL.
it may still provide valuable information in choosing suitable
organic solvents for this type of reactions.
We have demonstrated an effective means for achieving
stereoselective resolution of (±)-menthol with vinyl propi-
onate as the acylating agent and immobilized lipase PS-30 as
the biocatalyst. For practical applications, a 50% overall con-
version of (±)-menthol to (−)-menthyl propionate at 30°C
may represent a suitable index where the reaction can be
stopped because the lipase-catalyzed reaction is highly stereo-
specific in nonpolar solvents.
ACKNOWLEDGMENTS
Contributed by the College of Agricultural and Environmental
Sciences, The University of Georgia. Research supported by the
University of Georgia Research Foundation, Biotechnology
Grant.
FIG. ꢀ. Forꢀation of (−±ꢀentꢁyl propionate by iꢀꢀobilized lipase ꢄP at
ꢅ3°C as affected by levels of enzyꢀe. Reactions ꢃere perforꢀed in n-
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ꢁ
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TABLE ꢀ
Percentage Yield, E value, and ee% of (−±-Menthyl Propionate
Synthesized from (± ±-Menthol and Vinyl Propionate at ꢀ30C
by Immobilized PS-ꢀ3 as Affected by Selected Organic Solvents
a
%
Yield
−±-ꢀentꢁyl
propionate
57.3 ± 14.9 177 ± ꢅ1 98.9 ± 3.2
(
Loꢂ P valueb
Polvent
E
ee%
Isooctane
n-Heptane
n-Hexane
Cycloꢁexane
Toluene
4.5
4.3
ꢅ.5
ꢅ.2
2.5
2.3
1
5ꢅ.4 ± 4.6
56.4 ± 9.3
215 ± 25 99.1 ± 3.1
191 ± 8ꢅ 98.8 ± 3.5
ꢅ9.7 ± 23.9 163 ± 16 98.8 ± 3.1
1
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27.7 ± 6.5
ꢅ3.5 ± ꢅ.4
15ꢅ ± 27 98.7 ± 3.2
119 ± 2 98.4 ± 3.1
Benzene
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b
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PTEREOPELECTIVE PYNTHEPIP OF MENTHYL ꢄROꢄIONATE
4ꢅ9
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JAOCP, Vol. 74, no. 4 (1997±