Production of l-menthyl acetate through kinetic resolution by?Candida cylindracea lipase: effects of alkaloids as additives

Article abstract of DOI:10.1007/s11164-018-3525-7

Abstract: Enzymatic transesterification of dl-menthol with vinyl acetate in tert-Butyl methyl ether (TBME) catalyzed by Candida cylindracea lipase (CCL) was carried out in the presence of cinchona alkaloid as additive. The effects of various reaction parameters, such as lipase nature and loading, acylating agent, molecular sieves, solvents and various additives, on the reactivity as well as on the enantioselectivity were investigated. A significant improvement of CCL reactivity has been recorded after using cinchona alkaloid as additive in TBME. A high enantiomeric ratio (E = 80) was achieved when 30?mol% of quinidine was added, and l-(-)-menthyl acetate was obtained with 93% optical purity and 49% conversion. This process was easily applied to gram-scale quantities, using commercially inexpensive lipase, providing high yield optically active menthol under mild experimental conditions. Graphical abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s11164-018-3525-7

Research on Chemical Intermediates
https://doi.org/10.1007/s11164-018-3525-7(0123456789(). -, vo Vl
)
(
0
1
2
3
4
5
6
7
8
9
(
)
.
-
,
v
o
l
V
)
Production of l-menthyl acetate through kinetic resolution
by Candida cylindracea lipase: effects of alkaloids
as additives
Fatima Zohra Belkacemi1 • Mounia Merabet-Khelassi
1
•
1
2
Louisa Aribi-Zouioueche
•
Olivier Riant
Received: 11 May 2018 / Accepted: 2 July 2018
Ó Springer Nature B.V. 2018
Abstract
Enzymatic transesterification of dl-menthol with vinyl acetate in tert-Butyl methyl
ether (TBME) catalyzed by Candida cylindracea lipase (CCL) was carried out in
the presence of cinchona alkaloid as additive. The effects of various reaction
parameters, such as lipase nature and loading, acylating agent, molecular sieves,
solvents and various additives, on the reactivity as well as on the enantioselectivity
were investigated. A significant improvement of CCL reactivity has been recorded
after using cinchona alkaloid as additive in TBME. A high enantiomeric ratio
(
E = 80) was achieved when 30 mol% of quinidine was added, and l-(-)-menthyl
acetate was obtained with 93% optical purity and 49% conversion. This process was
easily applied to gram-scale quantities, using commercially inexpensive lipase,
providing high yield optically active menthol under mild experimental conditions.
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11164-
018-3525-7) contains supplementary material, which is available to authorized users.
&
Louisa Aribi-Zouioueche
louisa.zouioueche@univ-annaba.dz; louisa.zouioueche@gmail.com
Extended author information available on the last page of the article
123

F. Z. Belkacemi et al.
Graphical abstract
Keywords l-Menthyl acetate Á Kinetic resolution Á Candida cylindracea
lipase Á Alkaloids Á Additives
Introduction
Menthol is the world’s most-sold flavor ingredient and can be found in countless
products in common use. The worldwide demand of 25,000–30,000 metric tons
per year already exceeds the available supply and is constantly growing [1]. l-(-)-
Menthol has the best organoleptic performance, since it is widely used in flavor
and fragrance, food, cosmetics and pharmaceutics industries due to its refreshing
flavor and cooling effects, whereas d-(?) menthol has an undesirable taste [2–4].
The demand for the pure form of l-(-)-menthol is an important preoccupation at an
industrial level, which explains the research efforts to establish efficient routes for
yielding it at high purity [5–7]. One of the efficient routes to obtain it as optically
active is the biocatalytic process [8–15]. The enzymatic kinetic resolution of
racemates is one of the practical modes used for the separation of the two
enantiomers, and lipases (E.C.3.1.1.3) are the most used biocatalysts with
considerable industrial potential. Lipases can act under mild conditions, are
cheap, stable, and efficient, and present a remarkable chemo-, regio- and
enantioselectivity to a large panel of substrates, in particular secondary alcohols
[
16–19].
In kinetic resolution involving lipases, various parameters have to be taken into
account for controlling both reactivity and enantioselectivity. For instance, in the
kinetic resolution of racemic alcohols with lipases, several parameters such as the
nature of the enzyme [20, 21], the solvent [22–24], the effect of additives [25, 26],
the residual water in the reaction media [27, 28], the amount of lipase [29, 30] and
the nature of the acetylating agent [31–34] are often examined for the
optimization of selectivity and conversion. Some research is still dedicated to
the study of the kinetic resolution of dl-menthol using lipases. In all the reported
literature data, the use of free lipases exhibits no good reactivity or selectivity,
1
23

Production of l-menthyl acetate through kinetic…
and it is the use of immobilization techniques that generally improve the
efficiency of the various commercially available free lipases. The majority of the
described kinetic resolutions exploit Candida rugosa lipase (CRL) immobilized
on several supports in esterification, transesterification or hydrolysis reactions.
The recombinant CRL was used for the enantioselective hydrolysis of dl-menthyl
benzoate with high selectivity as opposed to the free lipase which showed a low
selectivity [35]. Another immobilization of Candida rugosa lipase (AYL) was
found to be the most appropriate in transesterification of dl-menthol with vinyl
acetate; the conversion recorded was of at least 34% [36]. The lipase from
Candida cylindracea (CCL), from the same source as CRL, was also investigated
in an immobilized form: in kinetic resolution of dl-menthol via esterification [37]
and transesterification [38], more recently for esterification reaction between
1
-octanol and three butyric acid derivatives in low-water solvent-free systems
39]. Few studies have been carried out on the resolution of dl-menthol with free
[
CCL and the enantio-discrimination displayed by lipases from Candida
cylindracea (Sigma Type VII) and Candida rugosa (Sigma Type VII and Amano
AY) was generally moderate [40]. Moreover, these yeast hydrolytic lipases are the
most commonly used due to their high activity and non-genotoxic or cancerogenic
effects on human health [41]. Hence, the species is generally regarded as safe (it
has GRAS status), and classified as a biologically safe level [42, 43]. The above-
mentioned lipases are most popular lipases and but, to date, no studies have
focused on the effects of some additives on the reactivity and selectivity of these
lipases.
In this paper, we report the optimization of the reactivity and selectivity of
free commercially available lipases by modulation of several parameters
influencing the catalytic process during the enzymatic kinetic resolution via
acylation of dl-menthol. The reaction parameters, such as the amount and
hydrolytic activity of the lipase sources, the nature of the acyl donors, solvent
hydrophobicity, lipase amount and introduction of several additives, were
optimized to achieve the ideal conversions and enantioselectivities during the
enzymatic acylation of dl-menthol (Scheme 1). The use of additives to improve
the selectivity of the lipases for the kinetic resolution of menthol is the
innovative aspect in this work.
Scheme 1 Enzymatic acylation reactions
123

F. Z. Belkacemi et al.
Materials and methods
Chemicals and materials
All reagents and solvents were of analytical grade and were used as purchased from
Sigma-Aldrich. Lipases of different sources were used as purchased without any
pre-treatment. CRL type VII (specific activity = 1170 U/mg), lipase from Porcine
pancreatic type II (PPL) (specific activity & 100–500 U/mg) and Candida
antarctica lipase immobilized on acrylic resin (CAL-B) (specific activity [ 10,000
U/g) were purchased from Sigma-Aldrich. The Pseudomonas cepacia lipase (PCL)
was purchased from Amano (specific activity [ 30,000 U/g). The CCL was
purchased from Fluka (specific activity = 2.8 U/ g). CRL is an extra-purified form
of CCL, but these forms have very distinctive hydrolytic activities. The outcome of
the reactions was monitored using TLC on Silica gel 60F254 plates (type
MERCK5179), 250 mesh. After stirring for the appropriate time, the lipase was
removed by simple filtration. The separation of the resulting acetates and the
remaining alcohols was performed by column chromatography using silica gel
˚
6
eluent.
0 A, 70–230 mesh, 63–200 lm using petroleum ether/ethyl acetate (v/v: 80/20) as
Instrumentation
The spectroscopic characterisation was performed with Br u¨ ker spectrometers
13
1
(
tetramethylsilane with solvent resonance as an internal standard for H NMR and
300 MHz for H, 75 MHz for C). Chemical shifts were reported in d ppm from
1
1
3
chloroform-d (d 77.0 ppm) for C NMR. Enantiomeric excesses were measured by
gas chromatography on a ThermoFinnigan Trace GC, equipped with an automatic
auto sampler and using a CHIRALSIL-DEX CB column (25 m; 0.25 mm;
0.25 lm). Retention times are reported in minutes. Optical rotations were
determined using a Perkin-Elmer 241 Polarimeter at room temperature using a
cell of 1 dm length and k = 589 nm.
Experimental setup
Synthesis of racemic 2-isopropyl-5-methylcyclohexyl acetate (1a)
The menthyl acetate (1a) was synthesized by classical chemical acylation via the
corresponding racemic menthol (1 equiv.), using 1.5 equiv. of acetic anhydride, 1.2
equiv. of Et N, and a catalytic amount of 4-dimethylaminopyridine (0.2 equiv.) in
3
5
in excellent yield. The structure was confirmed by H and 1 C NMR spectra.
mL of diethyl ether. The final products were obtained pure after standard work-up
1
3
Molecular formula: C H O . Crude oil. yield = 78%. R = 0.72. Eluent (v,v):
f
1
2
22
2
petroleum ether/ethyl acetate (90/10).
1
H NMR (300 MHz, CDCl ): d (ppm) = 0.74–0.76 (m, 3H, cycl-CH ),
3
3
0
.79–0.83 (m, 1H, CH cyclic), 0.87–0.90 (m, 6H, 2CH ), 0.91–0.98 (m, 1H,
2 3
1
23

Production of l-menthyl acetate through kinetic…
CH cyclic), 1.01–1.10 (m, 1H, CH cyclic), 1.30–1.39 (m, 1H,CHcyclic), 1.41–1.53
2
2
(
m, 1H,CHcyclic), 1.62–1.64 (m, 1H, CH cyclic), 1.66–1.69 (m, 1H, CH cyclic),
2 2
1
.81–1.90 (m, 1H,CH), 1.94–2.00 (m, 1H, CH cyclic), 2.02 (s, 3H,O–C–CH ),
2 3
4
.62–4.71 (m, 1Hcycl, O-CHcycl).
C NMR (75 MHz, CDCl ) d 170.82, 74.2, 47.14, 41.06, 34.39, 31.50, 26.25,
3.63, 22.15, 21.47, 20.87, 16.52.
13
3
2
General procedures of enzymatic kinetic resolutions
Enzymatic acylation with enol esters
To 1 mmol of racemic alcohol (1), 3 mmol of the appropriate enol ester
isopropenyl acetate or vinyl acetate) and a catalytic amount of lipase were
(
dissolved in 2 mL of organic solvent. The suspension was stirred at room
temperature for the indicated time. The reaction mixture was filtered on Celite and
concentrated in vacuum. The remaining alcohol and the produced acetate were
separated by chromatography on silica gel (petroleum ether/ethyl acetate: 95/5) and
analyzed by chiral GC. The same procedure was followed for the reactions in the
presence of 30 mol% of additives.
Enzymatic acylation with succinic anhydride
A dry Schlenk tube was charged with rac-alcohol (1 equivalent) and succinic
anhydride (1 equiv.) dissolved in 2 mL of diethyl ether. The reaction was initiated
by the addition of 100 mg of CCL. The reaction mixture was shaken at room
temperature for 24 h. After removal of the lipase by filtration, the filtrate was shaken
with 1 M Na CO solution, and the remaining alcohol and the produced monoester
2
3
succinate were separated by liquid–liquid extraction. The remaining enantiomer was
obtained from the organic layer and the aqueous phase was washed with an organic
solvent and treated by adding 1 M NaOH solution to obtain the other enantiomer.
The enantiomeric excesses values were quantified by chiral GC analyses.
Chiral GC: Chiralsil-DEX CB: (Tcolumn = 120 °C. flow: 1,2 mL/min); dl-(± )-
menthyl acetate: td-(?) = 9.58 min; tl-(-) =l0.85 min. dl-(± )-menthol:
t
d-
=
?)
13.25 min; tl-(-) = 13.69 min.
(
Results and discussion
First, we have examined the influence of hydrolytic activity of some lipase’s source
commercially available, as well as the catalytic loading during the acylation of dl-
menthol 1 using isopropenyl acetate (IA) as acyl donor. All the experiments were
carried out in diethyl ether as organic solvent, both in the presence and the absence
˚
of molecular sieves 4 A, in order to regulate the water in the reaction medium. The
conversions and the enantiomeric excesses of obtained acetates 1a and the
remaining alcohols 1 were quantified by chiral gas chromatography after their
separations by flash chromatography. The results are summarized in Table 1.
123

F. Z. Belkacemi et al.
Table 1 Transesterification of dl-menthol with various lipases in the presence and the absence of
˚
molecular sieves 4 A
a
f
h
% (Yield %) C(%)
g
g
E
˚
MS4 A
(mg)
Entry Lipase
mg)
Acyl
donor
ee
f
s
%
(Yield %)
ee
p
h
(
b
1
2
3
PCL (80)
PPL (100)
CAL-B
Without
IA
–
–
–
–
NR
NR
NR
–
–
–
–
–
(
200)
CAL-B
150)
CAL-B
100)
4
5
–
–
–
–
NR
NR
–
–
(
(
b
b
b
b
b
b
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
CRL (200)
CRL (150)
CRL (100)
CCL (200)
CCL (150)
CCL (100)
Without
20 (69)
31 (61)
68 (42)
31 (62)
62 (45)
9 (ND)
54 (35)
56 (30)
44 (53)
43 (54)
39 (64)
66 (38)
39 (64)
51 (40)
64.4 (42)
63.7 (38)
25 (39)
[ 99 (10)
98 (18)
17
[ 200
[ 100
[ 200
89
c
With
24
Without
[ 99 (33)
97 (20)
41
c
With
24
0
1
2
3
4
5
6
7
8
9
0
1
2
Without
[ 99 (30)
[ 99 (ND)
98 (28)
38.5
8.3
35.5
36.4
31
[ 200
[ 200
[ 100
[ 100
[ 100
[ 100
[ 200
[ 200
[ 200
[ 200
80
c
With
Without
c
With
98 (28)
Without
98 (25)
c
With
98 (23)
31
Without
[ 99 (14)
[ 99 (32)
[ 99 (14)
[ 99 (19)
95.2 (32)
95.2 (30)
53 (19)
28
d
d
40
c
With
28
34
b
e
Without
VA
SA
40.3
40
c
With
80
e
Without
32
4
NR no reaction, ND not determined
Bold is to highlight the most important results
a
Pseudomonas cepacia lipase from Amano ([ 30,000 U/g), Candida rugosa lipase type VII (1170 U/mg),
Porcine pancreatic lipase type II (100–500 U/mg), Candida antarctica lipase immobilized on acrylic
resin ([ 10,000 U/g) from Sigma-Aldrich. Candida cylindracea lipase from Fluka (2.8U/g)
b
Reaction conditions : 1 mmoldl-menthol, 3 mmolenol ester (IA or VA), 2 mL diethyl ether with lipase
for 24 h at room temperature
c
˚
With molecular sieve (4 A) (20 mg)
d
48 h at room temperature
e
1
temperature
mmoldl-menthol, 1 mmol succinic anhydride, 2 mL diethyl ether with lipase for 24 h at room
f
Enantiomeric excess measured by chiral GC
g
s p
Conversion: C = ee /ee ? ees; Selectivity: E = Ln [(1 - C) (11 - ee(S))]/Ln [(11 - C) (1 ? ee(S))]
[
44, 45]
h
Isolated yield quantified after separation by flash chromatography
1
23

Production of l-menthyl acetate through kinetic…
Among all the lipases screened, CRL and CCL gave the best conversions and
high enantiomeric excesses as shown in Table 1. The transesterification of dl-
menthol with IA in diethyl ether by employing lipases from sources such as PCL,
PPL, CAL-B showed no progress even after prolonged reaction time (Table 1, entry
1
–5). However, the lipases from CRL and CCL provided moderate to good
enantioselectivity for l-menthyl acetate. Experiments carried out with and without
molecular sieves illustrated its effect on the catalytic process, and this effect is
particularly interesting. An increase in the amount of CRL was clearly in disfavor of
the lipase reactivity without any perturbation of selectivity factor (E [ 200)
(
Table 1, entries 6, 8 and 10), and the conversion decreased from c = 41% to
c = 17% when the loading amount of the CRL was 150 and 200 mg, respectively
Table 1, entry 8 vs. 6). The same observation was made in our previous
(
investigation on the influence of CCL amount during the acylation of ferro-
cenylethanol [29]. A drastic decrease of CRL reactivity by the introduction of the
molecular sieves was observed when amounts of 150 and 100 mg were used
(
Table 1, entry 9 vs. 8 and 11 vs. 10). This effect strongly declined at 200 mg of
CRL (Table 1, entry 7 vs. 9 and 11). In the case of CCL, no significant effects were
observed in the presence of the molecular sieves either on reactivity or on
selectivity. The decrease of the lipase amount exhibits optimization of the acylation
advancement without affecting the selectivity (Table 1, entries 12, 14, 16 vs. entries
1
3, 15, 18).
The CRL and CCL lipases used are commercial and acquired from two different
suppliers, and they are supposed to be differentiated solely by their hydrolytic
activity; however, we found that only CRL was strongly influenced by the addition
of the molecular sieves. This is probably due to the perturbation of the molecular
water partition as well as to the reduction of its presence in the organic media which
seems necessary for CRL. This result confirms the results of previous studies
[
46, 47] which note the need to add water to improve CRL conversion, and this even
in the case of immobilized lipase. Both lipases are specific to (1R,2S,5R)-1a; the
absolute configuration of these compounds is well known and l-menthyl acetate
enantiomer is obtained. Due to the sensitivity of the free CRL, we have selected
CCL as being less expensive and more stable as well as modestly studied for the
resolution of the dl-menthol [40], with the appropriate catalytic load of m = 100 mg
(
280U). All the enzymatic reactions dedicated to the study of several parameters
affecting the catalytic process were stirred for 48 h at room temperature (Table 1,
entry 17). Other acetylating agents were used under optimized conditions: vinyl
acetate (VA) and succinic anhydride (SA). With the first one, (-)-menthyl acetate 1a
was recovered with 95% ee at 40% of conversion with and without molecular
P
sieves, but with a slight diminution of selectivity in the first case (Table 1, entries
2
0–21). On the other hand, a drastic decrease in the selectivity was shown using
succinic anhydride as acetyl donor E = 4, regardless of the reactivity c = 32%
Table 1, entry 22). Since it is well known that the enantioselectivity of enzymes
(
depends essentially on the polarity of the solvents, we have envisaged studying the
dependence of both enol esters to the hydrophobicity of the employed organic
solvent, and its impact on the CCL-catalyzed resolution of this a-substituted
cycloalkanol. For that, the same optimum conditions were undertaken, using five
123

F. Z. Belkacemi et al.
organic solvents with different LogP values: tetrahydrofuran (THF), diethylether
(
Et O), diisopropylether (DIPE), tert-butylmethylether (TBME) and heptane. All
2
experiments were conducted without molecular sieves for 48 h. The obtained results
are summarized in Table 2.
The results in Table 2 show very good enantioselectivity for l-(-)-menthyl
acetate. The conversion and selectivity factors recorded depend on the nature of the
enol ester and the solvent used (Fig. 1). The CCL maintains its high selectivity in
the five solvents used using IA as acetyl donor (E [ 200), the best conversion being
achieved in diethylether c = 40% (Table 2, entry 7). Unfortunately, a denaturation
effect of this yeast was noted in THF, TBME and heptane, where the conversion rate
does not exceed 4% (Table 2, entries 6, 8 and 10), and a loss of reactivity is noted in
DIPE, c = 26% (Table 2, entry 9).
We can conclude here that in some cases the nature of the organic solvent acts as
an inhibitor factor of the CCL-catalyzed acylation using IA, whereas, a significant
effect of the solvent hydrophobicity on the CCL reactivity and selectivity was
revealed when vinyl acetate was used. In diethyl ether, TBME, DIPE and heptane,
the CCL reactivity was still stable and the conversion varied between
3
3% \ c \ 44% (Table 2, entries 2–5). Moderate advancement was noted in
THF c = 12% (Table 2, entry 1), while a drastic decrease of the selectivity factor
was obtained in DIPE, E = 44 (Table 2, entry 4). It is to be underlined that the best
results in terms of reactivity and selectivity are obtained with diethyl ether, TBME,
DIPE as solvents during the acylation of dl-menthol by means of the free CCL. In
Table 2 Influence of enol ester and solvent hydrophobicity on the CCL-catalyzed resolution of dl-
menthol
a
b
% (Yield %)
d
b
% (Yield %)
d
c
c
E
Entry Enol ester
Solvent (log P) ee
S
ee
P
C (%)
1
2
3
4
5
6
7
8
9
1
VA
THF (0.48)
Et O (0.85)
14 (55)
64 (42)
54 (36)
69 (46)
48 (40)
3 (ND)
66 (38)
4 (ND)
34 (ND)
4 (ND)
[ 99 (7)
12
40
35
43
33
3
[ 200
76
2
95 (32)
TBME (1.35)
DIPE (1.4)
Heptane (4)
THF (0.48)
[ 99 (29)
91 (36)
[ 200
44
[ 99 (25)
[ 99 (ND)
[ 99 (32)
[ 99 (ND)
[ 99 (ND)
[ 99 (ND)
[ 200
[ 200
[ 200
[ 200
[ 200
[ 200
IA
Et O (0.85)
2
40
4
TBME (1.35)
DIPE (1.4)
26
4
0
Heptane (4)
ND not determined
Bold is to highlight the most important results
a
Reaction conditions: 1 mmoldl-menthol, 3 mmolenol ester, 2 mL organic solvent with CCL (280U) for
48 h at room temperature
b
Enantiomeric excess are measured by chiral GC
c
Conversion : C = ee
44, 45]
S P
/ee ? ees; selectivity: E = Ln [(1 - C) (1 - ee(S))]/Ln [(1 - C) (1 ? ee(S))]
[
d
Isolated yield quantified after separation by flash chromatography
1
23

Production of l-menthyl acetate through kinetic…
200
150
100
eeP%
VA
Conversion (%)
E (Selecꢀvity)
5
0
0
THF (0,48) Et2O
TBME DIPE (1,4) Heptane
(0,85)
(1,35)
(4)
eeP%
Conversion (%)
E (Selecꢀvity)
200
150
100
IA
5
0
0
THF (0,48) Et2O (0,85) TBME (1,35) DIPE (1,4)
Fig. 1 Effect of the solvent on the CCL performance in kinetic resolution of dl-menthol
addition, it was found that the highest selectivity and reactivity were obtained with
vinyl acetate as the acylating agent. To the best of our knowledge, these results are
the first to have been described.
Finally, an attempt at optimization of the CCL-catalyzed resolution of menthol
was carried out by the introduction of some additives directly into the organic
medium. This alternative used in our previous works has shown that the activity of
some lipases in kinetic resolution could be highly influenced by the alkaloid
additives [25]. Therefore, we selected the acylation conditions, using vinyl acetate
as acetyl donor and as additives: crown ether (18-C-6), a mineral base (Na CO ) and
2
3
organic base (NEt ), as well as two chiral Lewis bases, alkaloids type (cinchonidine
3
and quinidine). A catalytic amount of those additives were introduced directly
following the previously described procedures [25, 26]. The obtained results are
summarized in Table 3.
Introduction of quinidine (30 mol%) in CCL-catalyzed acylation of dl-menthol in
TBME, exhibited a large activation of the catalytic process. The conversion rate
achieve the threshold of 49% and l-(-)-menthyl acetate 1a was obtained at high
enantiomeric excess, ee = 93% (Table 3, entry 3) and E = 80. In diethyl ether, the
P
recorded conversion was 56% with a drastic decrease of selectivity, E = 30, and the
remaining alcohol, d-(?)-menthol was recovered at ee = 97% (Table 3, entry 1).
S
The use of heptane as an organic solvent causes a significant enhancement of the
CCL reactivity during the acylation of dl-menthol, as the enantiodiscrimination of
CCL disappears and the reaction velocity of both menthol enantiomers are quasi-
123

F. Z. Belkacemi et al.
Table 3 Influence of the additives on the outcome of the CCL-catalyzed resolution of dl-menthol
a
b
% (Yield %)
d
b
% (Yield %)
d
c
c
E
Entry Additive
Solvent (log P) ee
S
ee
P
C (%)
1
2
3
4
5
6
7
8
9
1
Quinidine
Et
2
O (0.85)
97 (42)
27 (20)
88 (38)
–
75 (48)
9 (55)
56
30
Heptane (4)
75
1
TBME (1.35)
93 (39)
–
49
80
Cinchonidine Et
2
O (0.85)
NR
30
–
Heptane (4)
TBME (1.35)
TBME (1.35)
40 (44)
45.5 (40)
42 (40)
24 (57)
61 (52)
24 (52)
[ 99 (20)
98 (22)
98 (20)
98 (12)
[ 99 (21)
98 (12)
[ 200
[ 100
[ 100
[ 100
[ 200
[ 100
32
Na
Et
18-C-6
2
CO
3
30
3
N
19.6
38
Et
2
O (0.85)
0
TBME (1.35)
20
Bold is to highlight the most important results
a
Reaction conditions: 1mmoldl-menthol, 3 mmol vinyl acetate, 2 mL organic solvent with CCL (280U),
30 mol% additive for 48 h at room temperature
b
Enantiomeric excesses are measured by chiral GC
c
Conversion : C = ee
44, 45]
S P
/ee ? ees; selectivity: E = Ln [(1 - C) (1 - ee(S))]/Ln [(1 - C) (1 ? ee(S))]
[
d
Isolated yield quantified after separation by flash chromatography
equal (c = 75% and E = 1) (Table 3, entry 2). On the other hand, no effect was
observed on the CCL performance when cinchonidine was added to both heptane
and TBME (Table 3, entry 5, 6), whereas the catalytic activity was totally inhibited
in diethyl ether (Table 3, entry 4). The addition of Na CO , a weak mineral base,
2
3
led to a slight diminution of the conversion from c = 35% without additive to
c = 30% with it (Table 3, entry 7), and this effect was amplified by the addition of
Et N, an organic base, c = 19% (Table 3, entry 8). Moreover, the introduction of an
3
ether crown-type additive, 18-C-6, led to a more drastic decrease of the conversion
rate with TBME as solvent, c = 20% than in diethylether, c = 38% (Table 3, entries
9
, 10). Thus, the direct introduction of a catalytic amount of a natural additive, such
as quinidine, significantly optimizes the catalytic performance of CCL during the
acylation of dl-menthol using vinyl acetate in TBME, so that this solvent is an
alternative to diethyl ether in terms of environmental exigencies [48, 49]. To the
best of our knowledge, this result is the first described using such an additive to
access l-menthol quantitatively at high enantiomeric purity. In order to valorize this
simple and easy methodology under mild conditions, we have applied it at a larger
scale (Scheme 2).
Optimal conditions were undertaken and applied to resolve 10 mmol (1.5 g) of
racemic menthol using 30 mmol of vinyl acetate and 1 g of CCL in the presence of
3
0 mol% (0.97 g) of quinidine as additive and dissolved in 20 mL of TBME. The
suspension was stirred at room temperature for 48 h. After filtration, the remaining
alcohol and the produced acetate were separated by chromatography on silica gel
(
petroleum ether/ethyl acetate: 95/5) and analyzed by chiral GC. The reaction
1
23

Production of l-menthyl acetate through kinetic…
Scheme 2 Multigram scale of acylation of dl-menthol with quinidine
CCL-catalyzed acylation of dl-
OAc
OH
menthol with quinidine.
l-(-)-1a: ee = 94%
dl-1a
dl-1
Fig. 2 Chromatograms of scale-up acylation of dl-menthol
results were reproduced, and the most important l-(-)-1a enantiomer was obtained
quantitatively with ee = 94% under mild conditions (Fig. 2).
p
123

F. Z. Belkacemi et al.
Conclusion
The kinetic resolution of dl-menthol by CCL-catalyzed acylation was optimized
with vinyl acetate and quinidine as additives in TBME as solvent. Optimal
conversion, c = 49% ,and high selectivity, E = 80, were obtained under optimized
conditions with quinidine as additive. The reaction parameters such as enzyme load,
the nature of enol esters and organic solvents and water activity were found to have
profound effects on the conversion and enantioselectivity. The comparison of the
behavior of CRL and CCL for the enantioselective acylation of dl-menthol showed a
greater stability of CCL activity and did not depend on the water content. The gram-
scale reaction was efficient and constitutes a new approach to the enantioselective
transesterification of dl-menthol. Another interesting aspect of these results is the
use of TBME as a substitute for diethyl ether, which is considered a highly
hazardous solvent. We can expect to find this alternative in a preparative production
of optically active l-menthol enantiomers which constitutes a simple protocol for the
production of the enantiomerically enriched l-menthyl acetate.
Acknowledgements Algerian Ministry of Higher Education and Scientific Research (FNR 2000 and
PNR), Catholic University of Louvain, and the Fund for Scientific Research (FNRS) are gratefully
acknowledged for financial support of this work. The Catholic University of Louvain is acknowledged for
a grant ‘‘Cooperation development’’ awarded to Fatma-Zahra BELKACEMI. The authors knowledge Dr.
N. GRINE for English reviewing of this manuscript.
Compliance with ethical standards
Conflicts of interest The authors declare no conflict of interest.
References
1. Chemistry Views, New Process for Menthol Production. (Wiley-VCH VerlagGmbH & Co. KGaA,
BASF SE Ludwigshafen, German. 30 June 2012)
. R. Eccles, J. Pharm. Pharmacol. 46(8), 618 (1994)
. N. Galeotti, L.D.C. Mannelli, G. Mazzanti, A. Bartolini, C. Ghelardini, Neurosci. Lett. 322(3), 145
2
3
(2002)
4
5
6
. E. Brenna, C. Fuganti, F.G. Gatti, S. Serra, Chem. Rev. 111(7), 4036 (2011)
. M. McCoy, Chem. Eng. News 88(35), 30 (2010)
. R.A. Sheldon, Chirotechnology: Industrial Synthesis of Optically Active Compounds (CRC Press,
Boca Raton, 1993)
. R. Noyori, Angew. Chem. Inter. Ed. 41(12), 2008 (2002)
7
8
9
. H. Itoh, H. Maeda, S. Yamada, Y. Hori, T. Mino, M. Sakamoto, Org. Chem. Front. 1(9), 1107 (2014)
. D. Brady, S. Reddy, B. Mboniswa, L.H. Steenkamp, A.L. Rousseau, C.J. Parkinson, J. Chaplin, R.K.
Mitra, T. Moutlana, S.F. Marais, N.S. Gardiner, J. Mol. Catal. B Enzym. 75, 1 (2012)
1
1
1
1
1
0. M. Li, L.R. Yang, G. Xu, J.P. Wu, Biochem. Eng. J. 109, 81 (2016)
1. S. Serra, E. Brenna, C. Fuganti, F. Maggioni, Tetrahedron Asymmetry 14(21), 3313 (2003)
2. L. Yu, Y. Xu, X. Wang, X. Yu, J. Mol. Catal. B Enzym. 47(3), 149 (2007)
3. J. Pan, N.-D. Dang, G.-W. Zheng, B. Cheng, Q. Ye, J.-H. Xu, Bioresour. Bioprocess. 1(1), 12 (2014)
4. G.-W. Zheng, J. Pan, H.-L. Yu, M.-T. Ngo-Thi, C.-X. Li, J.-H. Xu, J. Biotechnol. 150(1), 108 (2010)
1
23

Production of l-menthyl acetate through kinetic…
15. G.-W. Zheng, H.-L. Yu, C.-X. Li, J. Pan, J.-H. Xu, J. Mol. Catal. B Enzym. 70(3), 138 (2011)
16. G. Grogan, Ann. Rep. Prog. Chem. Sect. B: Org. Chem. 109, 15 (2013)
17. U.T. Bornscheuer, R.J. Kazlauskas, Hydrolases in Organic Synthesis: Regio- and Stereoselective
Biotransformations (Wiley, Weinheim, 2005)
1
1
2
2
2
2
2
2
2
8. K. Faber, Biotransformations in Organic Chemistry, 6th edn. (Springer, Berlin, 2011)
9. A. Mehta, U. Bodh, R. Gupta, J. Biotechnol. Res. 8, 58 (2017)
0. C. Bidjou, L. Aribi-Zouioueche, J.-Y. Legros, J.-C. Fiaud, J. Soc. Alg. Chim. 9(2), 261 (1999)
1. A. Ghanem, H.Y. Aboul-Enein, Chirality 17(1), 1 (2005)
2. A.M. Klibanov, Acc. Chem. Res. 23(20), 114 (1990)
3. P.A. Fitzpatrick, A.M. Klibanov, J. Am. Chem. Soc. 113(8), 3166 (1991)
4. Y. Kitamoto, Y. Kuruma, K. Suzuki, T. Hattori, J. Org. Chem. 80(1), 521 (2015)
5. M. Merabet-Khelassi, L. Aribi-Zouioueche, O. Riant, Tetrahedron Asymmetry 19, 2378 (2008)
6. M. Merabet-Khelassi, N. Mela ¨ı s, M. Boukachabia, J.-C. Fiaud, L. Aribi-Zouioueche, J. Soc. Alg.
Chim. 17(2), 185 (2007)
27. A. Zaks, A.M. Klibanov, Proc. Natl. Acad. Sci. USA 82, 3192 (1985)
28. L. Chua, S.M.R. Sarmidi, Enzyme Microb. Technol. 38, 551 (2006)
29. M. Merabet-Khelassi, N. Bouzemi, J.-C. Fiaud, O. Riant, L. Aribi-Zouioueche, C. R. Chimie. 14(11),
978 (2011)
3
3
3
3
0. N. Bouzemi, I. Grib, Z. Houiene, L. Aribi-Zouioueche, Catalysts 4, 215 (2014)
1. M. Kawasaki, M. Goto, S. Kawabata, T. Kometani, Tetrahedron Asymmetry 12, 585 (2001)
2. N. Bouzemi, H. Debbeche, L. Aribi-Zouioueche, J.-C. Fiaud, Tetrahedron Lett. 4(3), 627 (2004)
3. T. Miyazawa, E. Kaito, T. Yukawa, T. Murashima, T. Yamada, J. Mol. Catal. B:Enzym. 37, 63
(2005)
3
4. N. Melais, L. Aribi-Zouioueche, O. Riant, C. R. Chimie 19(8), 971 (2016)
5. S. Vorlov a´ , U.T. Bornscheuer, I. Gatfield, J.M. Hilmer, H.J. Bertram, R.D. Schmid, Adv. Synth.
Catal. 344(10), 1152 (2002)
3
36. J.C.D. Silva, M.D.G. Nascimento, J. Brazil. Chem. Soc. 27(12), 2226 (2016)
37. W.-H. Wu, C.C. Akoh, R.S. Phillips, Enzyme Microb. Technol. 18, 538 (1996)
38. C.J. Gray, J.S. Narang, S.A. Barker, Enzyme Microb. Technol. 12(10), 800 (1990)
39. T. Kuroiwa, K. Hamazaki, M. Katayama, S. Sato, T. Matsui, Process Biochem. 51(12), 2047 (2016)
4
0. Z. L u¨ , Y. Chu, Y. Han, Y. Wang, J. Liu, J. Chem. Technol. Biotechnol. 80(12), 1365 (2005)
4
1. J.N. Trbojevi c´ , A.S. Dimitrijevi c´ , D.V. Veli cˇ kovi c´ , M. Gavrovi c´ -Jankulovi c´ , N.B. Milosavi c´ , HEM.
IND. 67(5), 703 (2013)
4
4
4
4
2. S. Benjamin, A. Pandey, Yeast 14(12), 1069 (1998)
3. M.T. Flood, M. Kondo, Reg. Toxicol. Pharmacol. 33, 157 (2001)
4. C.S. Chen, Y. Fujimoto, G. Girdaukas, C.J. Sih, J. Am. Chem. Soc. 104(25), 7294 (1982)
5. H.B. Kagan, J-C. Fiaud, Kinetic Resolution: Topics in stereochemistry. ed. by E.L. Eliel, S.H. Wilen,
vol 42 (Wiley, New York, 1988), p. 249
4
4
4
6. D.L. Wang, A. Nag, G.C. Lee, J.F. Shaw, J. Agricul, Food Chem. 50(2), 262 (2002)
7. S. Bai, Z. Guo, W. Liu, Y. Sun, Food Chem. 96, 1 (2006)
8. F.P. Byrne, S. Jin, G. Paggiola, T.H.M. Petchey, J.H. Clark, T.J. Farmer, A.J. Hunt, C.R. McElroy, J.
Sherwood, Sustain. Chem. Process. 4, 7 (2016)
49. D. Prat, A. Wells, J. Hayler, H. Sneddon, C.R. McElroy, S. Abou-Shehada, P.J. Dunn, Green Chem.
18(1), 288 (2015)
Affiliations
Fatima Zohra Belkacemi1 • Mounia Merabet-Khelassi
1
•
1
2
Louisa Aribi-Zouioueche
•
Olivier Riant
Fatima Zohra Belkacemi
zahrachimorg@live.com; fatimazohra.belkacemi@univ-annaba.org;
http://www.sciencedirect.com/science/article/pii/S0957416617303877
123

F. Z. Belkacemi et al.
Mounia Merabet-Khelassi
mounia.merabet@univ-annaba.dz
Olivier Riant
olivier.riant@uclouvain.be
1
2
Ecocompatible Asymmetric Catalysis Laboratory (LCAE), Badji Mokhtar Annaba-University,
Annaba, Algeria
Institute of Condensed Matter and Nanosciences Molecules, Solids and Reactivity (IMCN/
MOST), Universit e´ Catholique de Louvain, Louvain La Neuve, Belgium
1
23