Enantioselective enzymatic resolution of racemic alcohols by lipases in green organic solvents

Article abstract of DOI:10.1016/j.tetasy.2017.02.004

The effects of two eco-friendly solvents, 2-methyltetrahydrofuran (MeTHF) and cyclopentyl methyl ether (CPME), on the enzyme activity and enantioselectivity of Novozym 435, Candida rugosa lipase (CRL), Porcine pancreas lipase (PPL), Lipase AK, Lipase PS, and Lipozyme, a series of commercial lipases, in the enantioselective transesterfications of racemic menthol, racemic sulcatol and racemic α-cyclogeraniol were studied. Vinyl acetate was chosen as the acyl donor and the reactions were carried out at water activity 0.06. The activity of lipases in CPME was similar to that observed in other largely employed organic solvents [toluene and tert-butyl methyl ether (MTBE)], and was slightly lower in MeTHF. However, for most of the lipases tested, the enantioselectivity was higher in the eco-friendly solvents. Lipase AK exhibited a high enantioselectivity (E?=?232) for the resolution of racemic menthol but the reaction rate was low. Lipase formulation (the enzyme was frozen and lyophilized in potassium phosphate buffer without and with 5% (w/v) of sucrose, D-mannitol, or methoxy poly(ethylene glycol)) was tested with this lipase in order to improve its activity, which increased up to 4.5 times, compared to the untreated enzyme. CALB was found to be a useful biocatalyst for the resolution of racemic sulcatol, where high activity and enantioselectivity were obtained (E?≥?1000). For the resolution of the racemic primary alcohol α-cyclogeraniol, most of the lipases tested were active but not enantioselective, except lipase PS which displayed a moderate enantioselectivity (E?=?19). The effect of the presence of a low percentage of two ionic liquids (ILs) 1-Butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([BMIM][TFSI]) (5% (v/v)) and 1-Butyl-3-methylimidazoliumtetrafluoroborate ([BMIM][BF4]) (1% (v/v)) in the medium was also investigated. Only in the case of CRL the ILs slightly increased the enantioselectivity from E?=?91 to E?=?103 and E?=?120 for [BMIM][TFSI] and [BMIM][BF4], respectively. However, in all cases ILs caused a decrease of enzyme activity.

Full text of DOI:10.1016/j.tetasy.2017.02.004

Tetrahedron: Asymmetry xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Enantioselective enzymatic resolution of racemic alcohols by lipases
in green organic solvents
b
a
Abderahmane Belafriekh a, Francesco Secundo b, , Stefano Serra , Zeineddine Djeghaba
⇑
a Laboratory of Applied Organic Chemistry, Sciences Faculty, Chemistry Department, University of Badji Mokhtar, P.O. Box 12, 23000 Annaba, Algeria
b Istituto di Chimica del Riconoscimento Molecolare, CNR, via Mario Bianco 9, 20131 Milan, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The effects of two eco-friendly solvents, 2-methyltetrahydrofuran (MeTHF) and cyclopentyl methyl ether
(CPME), on the enzyme activity and enantioselectivity of Novozym 435, Candida rugosa lipase (CRL),
Porcine pancreas lipase (PPL), Lipase AK, Lipase PS, and Lipozyme, a series of commercial lipases, in
Received 18 January 2017
Revised 27 January 2017
Accepted 8 February 2017
Available online xxxx
the enantioselective transesterfications of racemic menthol, racemic sulcatol and racemic a-cyclogeran-
iol were studied. Vinyl acetate was chosen as the acyl donor and the reactions were carried out at water
activity 0.06. The activity of lipases in CPME was similar to that observed in other largely employed
organic solvents [toluene and tert-butyl methyl ether (MTBE)], and was slightly lower in MeTHF.
However, for most of the lipases tested, the enantioselectivity was higher in the eco-friendly solvents.
Lipase AK exhibited a high enantioselectivity (E = 232) for the resolution of racemic menthol but the reac-
tion rate was low. Lipase formulation (the enzyme was frozen and lyophilized in potassium phosphate
buffer without and with 5% (w/v) of sucrose, D-mannitol, or methoxy poly(ethylene glycol)) was tested
with this lipase in order to improve its activity, which increased up to 4.5 times, compared to the
untreated enzyme. CALB was found to be a useful biocatalyst for the resolution of racemic sulcatol, where
high activity and enantioselectivity were obtained (E ꢀ 1000). For the resolution of the racemic primary
alcohol a-cyclogeraniol, most of the lipases tested were active but not enantioselective, except lipase PS
which displayed a moderate enantioselectivity (E = 19). The effect of the presence of a low percentage of
two ionic liquids (ILs) 1-Butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([BMIM][TFSI])
(5% (v/v)) and 1-Butyl-3-methylimidazoliumtetrafluoroborate ([BMIM][BF4]) (1% (v/v)) in the medium
was also investigated. Only in the case of CRL the ILs slightly increased the enantioselectivity from
E = 91 to E = 103 and E = 120 for [BMIM][TFSI] and [BMIM][BF4], respectively. However, in all cases ILs
caused a decrease of enzyme activity.
Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction
affect enzyme enantioselectivity, thus making the selection of sol-
vent of crucial importance.6,8
Among industrial enzymes, lipases are one of the most impor-
tant for the preparations of enantiomerically pure compounds.1,2
They have a remarkable ability to catalyze a variety of reactions
in organic solvents such as esterification, transesterification, and
aminolysis with high activity, chemo-, regio-, and enantioselectiv-
ity for various synthetic substrates.3–5 Moreover, they can operate
in mild and simple reaction conditions. The use of organic solvents
as reaction media offer many advantages such as the possibility to
solubilize water-insoluble substrates, to facilitate the recovery of
reaction products removing the biocatalysts by simple filtration,
and the possibility to increase the enzyme thermal stability.6,7
Furthermore, organic solvents for lipase catalysis can markedly
A prerequisite for the application of biocatalysts on the large
scale is the sustainability and low environmental impact, reducing
the detrimental effects of the developed industrial process and of
the chemicals used on the environment. From this perspective,
the use of solvents derived from renewable resources (bio-sol-
vents) and environmentally friendly (green) solvents is a neces-
sity.9,10 Recently, solvents such as 2-methyltetrahydrofuran
(MeTHF), a biomass-derived solvent, and cyclopentyl methyl ether
(CPME) have been reported as promising media for biocatalysis
reactions due to their favorable characteristics. MeTHF (boiling
point 80 °C) and CPME (boiling point 106 °C) may replace the com-
monly used THF with the advantage of a reduced solvent evapora-
tion during the reaction. Moreover, because of the limited
miscibility in water (4.1% and 1.1% w/w for MeTHF and CPME,
respectively, at 23 °C), these solvents can be used in two-phase
⇑
Corresponding author.
E-mail address: francesco.secundo@icrm.cnr.it (F. Secundo).
http://dx.doi.org/10.1016/j.tetasy.2017.02.004
0957-4166/Ó 2017 Elsevier Ltd. All rights reserved.
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2
A. Belafriekh et al. / Tetrahedron: Asymmetry xxx (2017) xxx–xxx
reaction systems, which favors product recovery.11–14 Further-
more, MeTHF is considered a green solvent because (i) it can be
produced from renewable sources such as furfural or levulinic acid,
in accordance with the 7th principle of Green Chemistry, and (ii) it
is environmentally degraded in air and by sunlight.15 Antonucci et
al. reported the toxicological response of MeTHF in rats, resulting
in a permitted daily exposure in humans of 6.2 mg/day, making
it an appropriate green solvent for pharmaceutical and chemical
purposes.16 Compared to the classical ether solvents CPME has pro-
ven to be quite useful as a process solvent in numerous organic
synthesis because it is free from drawbacks like low boiling point,
easy peroxide (formation, and solubility in water, thus resulting in
inefficient recovery.17
Moreover, with the intent to verify if commonly adopted strate-
gies useful to improve enzyme activity in organic solvents can also
be exploited with these eco-friendly solvents,25,26 the role of the
enzyme formulation27,28 and the addition of ionic liquids (ILs)29–
32
were also evaluated. Thanks to a low vapor pressure, ILs have
attracted significant attention as ‘‘green solvents” in biocataly-
sis.33,34 In fact, the use of lipases in ionic liquids as solvent or (co)-
solvent has presented many advantages, such as high conversion
rates, high enantioselectivity and better enzyme stability.35 More-
over, ionic liquids exhibit excellent physical characteristics (melt-
ing point, polarity, and miscibility with water or organic
solvents) including the ability to dissolve polar and nonpolar
organic, inorganic, and polymeric compounds.36
In spite of the interesting features of these environmentally
friendly solvents, to the best of our knowledge, there is a lack of
systematic studies on their influence on the enantioselectivity of
some frequently used commercial lipases. In the present study,
we investigated the enzymatic kinetic resolution of some industri-
ally interesting racemic alcohols in the above mentioned green sol-
vents, carried out with some widely used lipases, comparing the
outcome with that previously obtained using the most commonly
used organic solvents.
In particular, we studied three model substrates that possess
different structural features regarding the alcohol functional
group: (i) a cyclic and sterically hindered secondary alcohol; (ii)
a linear secondary alcohol and (iii) a primary alcohol possessing
2. Results and discussion
2.1. Rates of lipase catalyzed transesterification of racemic
alcohols in organic solvents
In a first screening, lipases were tested in the transesterification
of racemic alcohols to see their efficiency with the two green sol-
vents (MeTHF and CPME) against two other conventional solvents
(toluene and MTBE) for comparison. The conversion of racemic
alcohols was calculated after 24 h as shown in Table 1. It was
observed that the conversion degree is dependent on the lipase
and on the organic solvent.
a sterically hindered stereocentre in the
a-position. More specifi-
Among all the lipases screened for transesterification with ( )-
menthol, CRL was found to be the best in terms of conversion,
where high values were observed in shorter times; however, the
enantioselectivity was low. Lipase AK exhibited the best enantios-
electivity but with low activity and reaction rate. CALB, lipase PS,
lipozyme, and PPL were inactive, with no product being detected
even after 72 h reaction. On the other hand, CRL and lipase AK were
more active in CPME, toluene, and MTBE compared to MeTHF.
CALB, CRL, and lipase AK showed a higher reaction rate with ( )-
sulcatol leading to a higher conversion. Lipozyme and lipase PS
showed moderate activity and PPL very low activity. For the reso-
lution of the primary alcohol all lipases (except PPL) were active in
all solvents tested, and the highest reaction rate was displayed by
CRL.
cally we singled out the cyclic secondary alcohol ( )-menthol,
the linear secondary alcohol ( )-sulcatol and the primary alcohol
( )-a-cyclogeraniol (Fig. 1).
(ꢁ)-Menthol is one of the most important flavor compounds
and it is used extensively as a food additive. Racemic menthol is
a cheap commodity produced by Haarman and Raimer process
but the desired organoleptic properties are related only to
(1R,3R,4S) isomer. Therefore a number of resolution processes,
including one based on lipase-mediated esterification, have been
developed.18 In spite of this, a study on the use of environmentally
friendly solvents in such a process is still lacking.
Sulcatol has been used as model compound for a number of
studies on enzyme-catalysis19 and its selection as a substrate for
the present research can afford experimental data that can be com-
pared with those previously obtained.
2.2. Enantioselectivity of lipase catalized transesterification of
racemic alcohols in organic solvents
a
-Cyclogeraniol
(namely
(2,6,6-trimethylcyclohex-2-enyl)
methanol) is a relevant chiral building block that has been
employed for the synthesis of carotenoids and carotenoid-deriving
natural products.20,21 To date, only one enzymatic resolution pro-
The use of organic solvents as media for reactions catalyzed by
lipases is a well-established procedure. Moreover, it is known that
by modifying solvent composition it is possible to modulate lipases
activity and enantioselectivity.6,27,28 However, no data have been
reported on the activity and enantioselectivity of lipases in MeTHF
and CPME with respect to other organic solvents. For a comparison,
in this study we selected toluene and MTBE, two solvents that are
extensively used, for the resolution of racemic menthol, sulcatol,
cedure for the preparation of enantioenriched
a-cyclogeraniol
has been described.22 We have already investigated the lipase-
mediated resolution of primary alcohols possessing a stereocentre
in the
a
-position.23,24 Since the esterification of the latter sub-
strates is highly dependent both on the enzyme used and on the
experimental conditions, we decided to investigate the influence
of the solvent on the enantioselectivity of the
resolution.
a-cyclogeraniol
Figure 1. The three model substrates investigated. Menthol, sulcatol and a-cyclogeraniol.
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A. Belafriekh et al. / Tetrahedron: Asymmetry xxx (2017) xxx–xxx
3
Table 1
Substrate conversion, initial rate and enantiomeric ratio (E) of lipase catalyzed transeterification reaction with ( )-menthol, ( )-sulcatol or ( )-
a
-cyclogeraniol as substrate and
MeTHF, CPME, toluene or MTBE as reaction mediaa
Substrate
Lipase
c (%)b; initial rate (nmol hꢁ1 mg-1); Ec
MeTHF
CPME
Toluene
MTBE
( )-menthol
CRLd
35; 36; 91
5; 2; 121
0.4
0.5
1.3
47; 144; 119
9; 3; 232
0.5
0.9
1.7
41; 146; 58
8; 2; 103
0.4
1.4
1.2
53; 158; 101
7; 4; 106
0.7
1.1
1.4
Lipase AKe
CALBf
Lipase PSe
Lipozymee
PPLd
0.1
0.2
0.3
0.2
( )-sulcatol
CRLd
51; 160; 4
38; 40; 28
66; 1690; 222
11; 7; 37
13; 10; 49
6; 1.6; 25
84; 350; 5
51; 40; 35
70; 1490; 422
26; 20; 25
20; 15; 58
12; 2; 15
99; 340; 6
53; 55; 21
83; 1800; 58
31; 19; 46
20; 12; 43
7; 1.8; 21
92; 344; 8
56; 40; 26
79; 1700; >1000
22; 16; 12
22; 16; 61
12; 3; 19
Lipase AKe
CALBf
Lipase PSe
Lipozymee
PPLd
( )-a-cyclogeraniol
CRLd
96; 161; 1
29; 6; 6
32; 14; 8
18; 4; 11
7; 1.5; 1
1; 0.4; 1
97; 496; 1
27; 10; 11
46; 50; 12
23; 10; 18
9; 5; 2
99; 632; 1
25; 10; 9
38; 90; 6
19; 5; 7
8; 5; 1
86; 484; 1
16; 10; 6
29; 34; 7
16; 10; 19
78; 5; 1
Lipase AKe
CALBf
Lipase PSe
Lipozymee
PPLd
2.5; 0.6; 2
8; 0.8; 1
4; 1.2; 2
Lipase amount was (d) 50 or (e) 20 or (f) 10 mg.
a
Conversion, initial rate and E are in normal, italic and bold character, respectively.
Conversion values after 24 h of reactions.
b
c
All the lipases tested transformed preferentially (ꢁ)-menthol, (R)-
a-cyclogeraniol and (R)-sulcatol, except CRL that had enantiopreference for (S)-sulcatol. E was cal-
culated at 20% conversion according the formula (1) and (2) using the enantiomeric excess of product in the case of racemic menthol and sulcatol and the enantiomeric excess
of substrate in the case of the primary alcohol.
and
biocatalysts.
The data of Table 1 show that although CRL exhibited with
( )-menthol the highest activity in MTBE, relatively good
activity was observed in CPME, which was similar to that found
in toluene, while a moderate activity was obtained in MeTHF.
However, the enantioselectivity of CRL in both CPME and
MeTHF, was similar to that observed in MTBE, but higher than
that in toluene. Lipase AK showed the highest enantioselectivity
in the kinetic resolution of ( )-menthol in CPME (E = 232). How-
ever, this enzyme had a lower activity than CRL in all the sol-
vents tested.
It can also be seen from Table 1 that with ( )-sulcatol in MeTHF
and CPME the lipases tested had similar activity and enantioselec-
tivity to that observed with toluene and MTBE. Interestingly, in the
two green solvents CALB had an enantioselectivity significantly
higher than that observed in toluene.
a
-cyclogeraniol at low water activity, using lipases as
indicates that enzyme dissolution in buffer at pH 8, followed by
lyophilization, increased 3.6 times the initial rates of the transes-
terification reaction in CPME, compared to the non-treated com-
mercial enzyme. The increase was slightly higher (up to 4.5-fold)
when lipase AK was lyophilized in the presence of MeOPEG or
a
sucrose or D-mannitol. The increase of activity could be due both
to the ‘‘pH-memory” effect,7,39 being the enzyme lyophilized from
a solution at the optimal pH for activity40 and to the lyoprotectant
effect of the additives, particularly MeOPEG, as previously reported
for lipases.37 Beyond these effects, the fact that with the additives
the enzyme molecules are more dispersed in the lyophilized
sample than in the commercial untreated powder, also contributes
to the increase of catalytic activity. Thus, besides formulation with
additives, from the perspective of further improving the catalytic
activity of lipase AK in organic solvents, enzyme immobilization
on suitable supports might be a valid approach.41 However,
lyophilization in the presence of this latter additive caused a
plunge of the enantioselectivity of lipase AK. A decrease of enan-
tioselectivity was also observed with lipase AK lyophilized with
The enantioselectivity of most lipases with ( )-a-cyclogeraniol
was low in all solvents except moderate values in the case of lipase
PS in CPME and MTBE E = 18 and E = 19, respectively (Table 1).
However, these lipases seem very active with this primary alcohol
where high conversions were obtained except with lipozyme and
PPL.
the sugars sucrose and D-mannitol, even though high values (102
and 100) were still obtained.
In the last decade, numerous studies have focused on the use of
ionic liquids (ILs) as green media for lipases. In addition, ILs have
also been suggested as additives, in biocatalyzed reactions, for
improving the performance of enzymes.30,42–44 Herein, the effect
of the presence of a low percentage of ionic liquid in the green sol-
vents was also investigated. In particular, we selected the two ionic
liquids [BMIM][TFSI] and [BMIM][BF4]. Analogously to organic sol-
vents, these ILs do not dissolve lipases. Therefore, their use does
not interfere with some of the advantages of using organic solvents
(e.g., easy recovery of the products and increase of the enzyme
thermal stability).45,46 The use of [BMIM][TFSI] (1%) and [BMIM]
[BF4] (5%) caused a decrease of the initial reaction rates for CRL
and lipase AK in transesterfication of ( )-menthol (Table 3). How-
ever, the enantioselectivity of CRL was slightly improved in the
MeTHF with [BMIM][TFSI] and [BMIM][BF4], and in CPME with
2.3. Effects of lipase formulation and ionic liquids on the
performance of lipases
Numerous studies have shown that lipase behavior may be
greatly altered by the formulation.28,37,38 Therefore we adopted
this strategy to increase the transesterification activity of lipases
in CPME. In particular, in the present study it has been shown that
Lipase AK has high enantioselectivity in the kinetic resolution of
( )-menthol, that is a prerequisite for the scaling up of this kinetic
resolution. Nevertheless, the low catalytic activity of the enzyme
might be a drawback. Therefore, in order to improve the perfor-
mance of the catalyst, the influence of the enzyme formulation
on the reaction rate and enantioselectivity was evaluated. Table 2
[BMIM][BF4]. Instead, Lipase AK showed
a
decrease of
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A. Belafriekh et al. / Tetrahedron: Asymmetry xxx (2017) xxx–xxx
Table 2
Transesterification activity of lipase AK lyophilized with different additives in transesterification of ( )-menthol
Catalyst
CPME
eep (%)
C (%)
Ea
v )
(nmol hꢁ1 mgꢁ1
Lipase AKb,e
98.9
93.3
95.1
87.2
97.0
91.0
23.0
44.1
46.3
43.3
31.1
29.6
232
64
102
29
100
31
3
Lipase AK lyophilized without additivesb,f
Lipase AK + Saccharosec,f
Lipase AK + MeOPEGc,f
11
13
13
7
Lipase AK +
D
-Mannitold,f
Lipase AK + MeOPEGd,f
7
Lipase amount was (b,c) 20 or (d) 10 mg.
Lipase/additive ratio (w/w) was (c) 4/1 or (d) 2/1.
Reactions time was (e) 120 or (f) 72 h.
a
In all cases the enantiopreference was for (ꢁ)-menthol.
Table 3
Effect of ionic liquids on the activity and enantioselectivity of lipases in the transesterification of ( )-menthol
Lipase
Medium
Solvent
MeTHF
eep (%)
CPME
c (%)
Ea
91
103
120
121
87
v
(nmol hꢁ1 mg-1
)
eep (%)
c (%)
Ea
v
(nmol hꢁ1 mgꢁ1
)
CRLb
Without Ionic Liquid
[BMIM] [TFSI] (1%)
[BMIM] [BF4] (5%)
Without Ionic Liquid
[BMIM] [TFSI] (1%)
[BMIM] [BF4] (5%)
97.3
97.6
98.1
98.0
97.4
95.3
19.2
17.8
11.9
16.7
11.9
10.1
36
42
34
2
97.1
97.0
97.7
98.9
99.1
98.3
36.5
34.0
27.3
23.0
16.6
16.0
119
108
121
232
254
138
144
134
76
3
Lipase AKc
1.5
1.5
2
1.5
46
Reactions time was (b) 4 or (c) 120 h.
a
In all cases the enantiopreference was for (ꢁ)-menthol.
enantioselectivity in the presence of ionic liquids except in CPME
with [BMIM][TFSI]. These results differ from those reported by
other research groups with other lipases. In fact, Itoh et al.47,48
found that the addition of small amounts of different ILs in organic
solvents, caused a remarkable enhancement of enantioselectivity
of lipase PS-catalyzed acetylation of 1-phenylethanol, using vinyl
acetate as an acyl donor. Filice et al.49 showed that Rhizomucor
In the resolution of the primary alcohol, the lipases tested
showed a low or, at most, moderate enantioselectivity, as in the
case of lipase PS in CPME and MTBE. A very high reaction rate
was observed with CRL. The presence of a low percentage of ILs
in the reaction medium increased slightly the enantioselectivity
but decreased the reaction rate. The results highlight that both
CPME and MeTHF can be considered as excellent and greener sub-
stitutes of toluene and MTBE. We also observed that CPME gave
better results compared to MeTHF, both in terms of enzyme cat-
alytic activity and of enantioselectivity. It should be mentioned
that, as already shown, the enantioselectivity of a given enzyme
can change, sometimes dramatically, as a function of the nature
of the solvents and of the substrates.8 The results obtained in this
study strongly encourage the use of green solvents as media for the
kinetic resolution of secondary alcohols, especially in the perspec-
tive to develop sustainable large scale industrial processes.
miehei lipase activity was enhanced, maintaining
a high
regioselectivity, by addition of ILs in the hydrolysis of hexa-O-
acetyl lactal.
3. Conclusions
Herein, the eco-friendly solvents MeTHF and CPME were used
for the first time as reaction media for the lipase-catalyzed kinetic
resolution of ( )-menthol, ( )-sulcatol and ( )-a-cyclogeraniol.
Lipase AK was found to be the best catalyst for the resolution of
racemic menthol in terms of enantioselectivity. The reaction rate,
which was low, could be improved in CPME, at least 2.5-fold by
lipase formulation. CALB should be the best choice for the resolu-
tion of racemic sulcatol in MeTHF and CPME, thanks to its the
excellent enantioselectivity and reaction rate. However, it has to
be emphasized that although the increase of activity obtained is
valid only for the exact formulations utilized, at an industrial level
the goal is to develop biocatalytic processes always with formu-
lated and immobilized enzymes that can facilitate enzyme reuse
and improve their properties (activity, stability, selectivity). In fact,
just for lipases, the catalytic activity depends on the conformation
4. Experimental
4.1. Materials and reagents
Novozym 435 (immobilized lipase from Candida antarctica B)
was donated by Novozymes, CRL (lipase from Candida rugosa)
and PPL (lipase from porcine pancreas) were purchased from
Sigma, Lipase AK (lipase from Pseudomonas fluorescens), Lipase PS
(lipase from Burkholderia cepacia), Lipozyme IM from Novo. ( )-
Sulcatol (purity > 99%), ( )-menthol, vinyl acetate (>99%), vinyl
laurate, toluene, tert-butyl methyl ether (MTBE), 1-butyl-3-
methylimidazolium bis(trifluoromethylsulfonyl)imide ([BMIM]
[TFSI], ꢀ98%), and 1-butyl-3-methylimidazolium tetrafluoroborate
([BMIM][BF4], ꢀ98.5%) were purchased from Sigma–Aldrich. Cyclo-
pentyl methyl ether (>99%) and 2-methyltetrahydrofuran (99%)
of a a-helix fragment, named ‘‘lid’’, which regulates the entrance to
the catalytic active site (e.g., moving from a ‘‘close” to an ‘‘open”
position). Thus, the formulation and immobilization method are
crucial to favor and maintain the open position, which has been
correlated to the activation of lipases.50
were purchased from Alfa Aesar. Racemic
a-cyclogeraniol ((2,6,6-
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A. Belafriekh et al. / Tetrahedron: Asymmetry xxx (2017) xxx–xxx
5
Where
trimethylcyclohex-2-enyl)methanol) was prepared by LiAlH4
reduction of ethyl
a
-cyclogeraniate.23 (ꢁ)-menthol, (S)-sulcatol
A þ B
c ¼ 1 ꢁ
ð3Þ
ð4Þ
ð5Þ
were purchased from Sigma–Aldrich; (ꢁ)-(S)-
a-cyclogeraniol
A0 þ B0
{[a]
20 = ꢁ106.5 (c 2.1, EtOH), 95% ee} was prepared by resolution
D
of the racemic alcohol.22 Other chemicals were from commercial
sources and were of the highest purity available.
B ꢁ A
ees ¼
eeP ¼
A þ B
4.2. Lipase catalyzed transesterification of racemic alcohols
P ꢁ Q
P þ Q
For all reactions tested, all reagents, solvents, and lipases, before
use, were separately equilibrated for at least 24 h at water activity
(aw) value of 0.06 in sealed vessels in the presence of LiBr saturated
salt solution at 25 °C.51 The reaction mixture was prepared by add-
ing 1 mL of organic solvent (MeTHF, CPME, toluene, or MTBE) con-
taining racemic alcohols ( )-menthol (32 mM), ( )-sulcatol
where A0 and B0 represent the initial enantiomers concentration of
substrate, A and B represent the substrate enantiomers concentra-
tions after a certain time of reaction, P and Q represent the product
enantiomers concentrations after a certain time of reaction,54 in a
reaction where A is the faster reacting enantiomer that produces P.
(35 mM) or ( )-a-cyclogeraniol (32 mM) and vinyl acetate
Acknowledgements
(325 mM) to 10–50 mg of lipases in a 3 mL vial. Then the reaction
mixture was shaken at 150 rpm at 25 °C and at scheduled times a
sample from the supernatant was withdrawn and analyzed by GC.
When the effect of ionic liquids was tested, the reaction was car-
ried out by adding to the organic solvent 5% or 1% (v:v) of
[BMIM][TFSI] or [BMIM][BF4], respectively. Initial reaction rates
(nmol hꢁ1 mgꢁ1) were calculated only from conversion values
lower than 15%. In this study, the amount of lipases refers to crude
powder unless otherwise specified.
Absolute configuration of the transformed enantiomers was
assigned by comparison of the chiral chromatographic analyses
of the various lipase catalyzed reaction with literature data that
reported studies with the same substrate and lipase.8,22,52,53
Francesco Secundo acknowledges the funding of COST Action
CM1206 EXIL ‘‘Exchange on Ionic Liquids”, for supporting partici-
pation at meetings on ionic liquids and allowing discussion of
the results presented in this work.
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a
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ln½1 ꢁ cð1 þ eepÞꢃ
E ¼
E ¼
ð1Þ
ð2Þ
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ln½ð1 ꢁ cÞð1 ꢁ eesÞꢃ
ln½ð1 ꢁ cÞð1 þ eesÞꢃ
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