Application of Deep Eutectic Solvents in Promiscuous Lipase-Catalysed Aldol Reactions

Article abstract of DOI:10.1002/ejoc.201501553

The application of deep eutectic solvents has been demonstrated for the first time in promiscuous lipase-catalysed aldol reactions. The model reaction between 4-nitrobenzaldehyde and acetone was examined in depth, with excellent compatibility being found between porcine pancreas lipase and choline chloride/glycerol mixtures for the formation of the aldol product in high yields. The system was compatible with a series of aromatic aldehydes and ketones including acetone, cyclopentanone and cyclohexanone. In some cases, the corresponding α,β-unsaturated carbonyl compounds were found as minor products. Control experiments demonstrate that the enzymatic preparation was also responsible for a collateral dehydration reaction once the aldol product is formed. Deep eutectic solvents are used for the first time in promiscuous lipase-catalysed aldol reactions. The reaction between substituted benzaldehydes and aliphatic ketones has excellent compatibility with porcine pancreas lipase (PPL) and choline chloride/glycerol mixtures and gives the aldol product in high yields. PPL was also responsible for a collateral dehydration reaction.

Full text of DOI:10.1002/ejoc.201501553

DOI: 10.1002/ejoc.201501553
Full Paper
Biocatalytic Promiscuity
Application of Deep Eutectic Solvents in Promiscuous Lipase-
Catalysed Aldol Reactions
Daniel González-Martínez,[a] Vicente Gotor,[a] and Vicente Gotor-Fernández*[a]
Abstract: The application of deep eutectic solvents has been of aromatic aldehydes and ketones including acetone, cyclo-
demonstrated for the first time in promiscuous lipase-catalysed pentanone and cyclohexanone. In some cases, the correspond-
aldol reactions. The model reaction between 4-nitrobenzalde- ing α,ꢀ-unsaturated carbonyl compounds were found as minor
hyde and acetone was examined in depth, with excellent com- products. Control experiments demonstrate that the enzymatic
patibility being found between porcine pancreas lipase and preparation was also responsible for a collateral dehydration
choline chloride/glycerol mixtures for the formation of the aldol reaction once the aldol product is formed.
product in high yields. The system was compatible with a series
Introduction
talysis of multiple traditional organic[11] and organometallic re-
actions,[12] but the application of such solvents in biocatalysed
transformations remains rare.[13] Until now, the activities of
hydrolases,[14] oxidoreductases[15] and lyases[16] have been
demonstrated in high-yielding transformations using DES as
medium. DES can also be used to run tandem enzyme–organo-
catalyst aldol reactions;[17] however, to our knowledge, only
Shukla and co-workers have reported the use of DES as reaction
media in strictly enzyme promiscuous catalytic processes such
as the Biginelli reaction for the synthesis of novel dihydropyrim-
idin-2(1H)-ones derivatives.[18]
Herein, we wish to report the benefits of applying DES as
reaction media for nonconventional reactions. As a proof of
concept, a series of hydrolases have been screened in crossed
aldol reactions, searching for suitable and mild reaction condi-
tions for the development of carbon–carbon bond-formation
reactions. The benefits of an efficient and simple isolation of
the resulting aldol products have been explored, and recycling
of the catalytic system has been investigated.
Biocatalytic promiscuity is gaining considerable attention as a
practical tool for the discovery of new enzyme activities.[1] From
the biocatalyst tool-box, the application of hydrolases has
quickly gained attention in the development of promiscuous
reactions[2] because of their easy accessibility, lack of cofactor
dependency, and tolerance to different reaction media, in-
cluding water, organic solvents, ionic liquids and supercritical
fluids.[3]
Hydrolases, and particularly lipases, display significant levels
of activity in a variety of nonconventional reactions such as
carbon–carbon and carbon–heteroatom bond-formation reac-
tions and oxidative processes, amongst others.[2,4] However, the
development of efficient nonconventional reactions remains a
challenge, and many research groups are attempting to de-
velop a broader scope for hydrolase transformations. In the
search for sustainable conditions for the development of chemi-
cal reactions, deep eutectic solvents (DES) have appeared in
recent years as promising biodegradable and environmentally
friendly solvents.[5] DES are generally obtained as homogene-
ous solutions by simply mixing inexpensive hydrogen-bond ac-
ceptors and donors, usually under heating. Their high boiling Results and Discussion
points and polarity allow the complete solubilisation of many
different families of products, making their application as green
Hydrolase-Catalysed Aldol Reaction between 4-
Nitrobenzaldehyde and Acetone
solvents possible.[6]
Eutectic mixtures have attracted increasing attention in a va-
riety of fields such as lubricants,[7] nanomaterials,[8] pharmaceu-
ticals[9] and separation techniques.[10] With respect to organic
synthetic applications, DES have been used for the efficient ca-
The reaction between 4-nitrobenzaldehyde (1a) and acetone
(2) was tested as the model biotransformation to explore the
suitability of DES as reaction medium for hydrolase-catalysed
promiscuous aldol processes (Scheme 1). A mixture composed
of choline chloride and glycerol (ChCl:Gly, 1:1.5 mol/mol) was
selected as an initial starting point based on the suitability of
this hydrogen-bond donor and acceptor in previous studied
biotransformations. Three enzymes were initially selected based
on their previously demonstrated activity as efficient catalysts
in the formation of aldol products: Candida antarctica lipase
[a] Departamento de Química Orgánica e Inorgánica, Instituto Universitario
de Biotecnología de Asturias, Universidad de Oviedo,
33006 Oviedo, Spain
E-mail: vicgotfer@uniovi.es
http://bioorganica.grupos.uniovi.es
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/ejoc.201501553.
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type B (CAL-B),[19] protease from Bacillus licheniformis (Alcal- reaction with 4-nitrobenzaldehyde at 1
M
(entry 3). Interestingly,
ase-CLEA®),[20] and porcine pancreas lipase (PPL).[21]
no reaction was observed under these conditions in the ab-
sence of catalyst, so the catalytic potential of PPL was demon-
strated for this transformation (entry 4). Reactions at 0.1
M alde-
hyde concentration were also performed in other choline chlor-
ide-based DES. On one hand, the use of ChCl/urea (1:2 mol/
mol) allowed quantitative conversion after 24 h at 60 °C, al-
though low product recovery was observed after the extraction
purification. On the other hand, the use of ChCl/ethylene glycol
(1:2 mol/mol) provided a mixture of the expected products, but
some ethylene glycol was also extracted from the DES, which
made us decide to continue with the use of glycerol-based sol-
vents for further optimisation.
Scheme 1. Hydrolase-catalysed aldol reaction between 4-nitrobenzaldehyde
and acetone in ChCl:Gly (1:1.5 mol/mol) after 24 h at 250 rpm.
Attempts to follow the biotransformations by GC analysis
were unsuccessful because the retro-aldol reaction occurred at
the high temperatures used in the analysis. We thus fixed a 1 d
1
reaction time and measured the conversions by using H NMR
Table 1. PPL-catalysed aldol reaction between 4-nitrobenzaldehyde and acet-
one in ChCl:Gly (1:1.5 mol/mol) after 24 h at 60 °C and 250 rpm.
analysis. A simple extraction procedure with ethyl acetate was
performed to separate the DES and the biocatalyst from aldol
3a; the results are summarised in Figure 1.
Entry
PPL/1a[a]
[1a] [M]
c [%][b]
Ratio 3a/4a[b]
1
2
3
4
1:1
1:1
1:1
0:1
0.1
0.5
1.0
1.0
36
71
90
<3
5:1
6:1
4.5:1
–
[a] Ratio PPL/1a (w/w). [b] Conversion values and ratio of products 3a/4a
were determined by 1H NMR spectroscopic analysis of the crude reaction
mixture.
Figure 1. Enzyme-catalysed aldol reaction between 4-nitrobenzaldehyde (1a;
0.1
M) and acetone (2; 5 equiv.) in ChCl:Gly (1:1.5 mol/mol) after 24 h at
250 rpm. CAL-B and PPL were used in a 1:1 ratio of enzyme/1a (w/w),
whereas for Alcalase-CLEA the ratio was 2:1.
Several parameters were analysed in the PPL-catalysed proc-
ess between 4-nitrobenzaldehyde and acetone to identify opti-
mal reaction conditions for quantitative conversion. Thus, we
studied the influence of the loading of enzyme, the use of other
DES mixtures, and percentages of water in the solvent system.
The reaction time and temperature were fixed at 24 h and 60 °C,
respectively, and the results are summarised in Table 2.
Taking into account the decisive role of water for the devel-
opment of efficient promiscuous hydrolase-catalysed reac-
tions,[22] a small percentage of water (20 % v/v) was initially
added to the DES. Complete conversion and a similar ratio were
found in comparison with the process run in the absence of
water (Table 2, entries 1 and 2). The use of a DES with higher
glycerol content was also tested, which revealed a similar con-
version of the starting 4-nitrobenzaldehyde (entry 3); the reac-
tion did not occur in the absence of biocatalyst (entry 4).
At this point the loading of enzyme was reduced by half;
however, under these conditions, conversions around 80 %
No reaction was observed at 30, 45 or 60 °C in the absence
of enzyme, thus the possibility of a nonenzymatic reaction can
be discarded. Likewise, no reaction took place when CAL-B was
used as biocatalyst, and poor conversion was reached for the
Bacillus licheniformis protease (Alcalase-CLEA)-catalysed reac-
tion at high temperatures. Satisfyingly, PPL was found to be an
active enzyme under these conditions, with the formation of
the aldol-dehydration product 4a also being observed as a mi-
nor product (ratio 7:1) albeit only at 60 °C. These conditions
served as a good starting point for optimization.
Optimisation of Reaction Parameters for the Biocatalytic
Aldol Reaction between 4-Nitrobenzaldehyde and Acetone
To achieve higher conversions, biotransformations were at- were found with both DES (Table 2, entries 5 and 6), although
tempted at higher substrate concentrations (Table 1). In this a remarkable higher proportion of the aldol was observed with
case, attention was paid to the distribution of products, be- less enzyme. This observation was confirmed by an experiment
cause a mixture of aldol 3a and aldol-dehydration adduct 4a in which the aldol product 3a was incubated in DES at 60 °C
was obtained. The formation of 3a was favoured in all cases and in the presence of PPL; under these conditions, the de-
(entries 1–3), achieving a noteworthy 90 % conversion in the hydration product was formed in 43 % conversion after 24 h,
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Table 2. PPL-catalysed aldol reaction between 4-nitrobenzaldehyde (1 M) and acetone in ChCl:Gly mixtures after 24 h at 60 °C and 250 rpm.
Entry
PPL/1a[a]
Solvent[b]
2 [equiv.]
H2O [%][c]
c [%][b]
Ratio 3a/4a[d]
1
2
3
4
5
6
7
8
1:1
1:1
1:1
0:1
1:2
1:2
1:2
1.5:2
1:1
1:2
ChCl:Gly (1:1.5)
ChCl:Gly (1:1.5)
ChCl:Gly (1:2)
ChCl:Gly (1:2)
ChCl:Gly (1:1.5)
ChCl:Gly (1:2)
ChCl:Gly (1:2)
ChCl:Gly (1:2)
ChCl:Gly (1:2)
ChCl:Gly (1:2)
ChCl:Gly (1:2)
ChCl:Gly (1:2)
toluene
5
5
5
5
5
5
5
5
5
0
20
20
20
20
20
5
5
5
5
5
90
>97
>97
<3
80
82
80
89
>97
54
25
9
4.5:1
6:1
6:1
–
10:1
11:1
10:1
6:1
6:1
6.5:1
6:1
9
10
11
12
13
2.5
1
5
1:2
1:1[e]
1:2
5
0
3:1
5.5:1
5
8
[a] Ratio enzyme/substrate (w/w). [b] Ratio ChCl:Gly (mol/mol). [c] Percentage in volume of water in the reaction medium. [d] Conversion values and ratio of
products 3a/4a determined by 1H NMR spectroscopic analysis of the crude reaction mixtures. [e] Enzyme pretreated with PMSF for 16 h at 30 °C.
whereas conducting the reaction in the absence of enzyme pro- substrate concentration reactions without varying significantly
ceeded to a minimum extent (4 % conversion). An additional the ratio of products. Notably, just 54 % conversion was
experiment was made using inhibited PPL,[23] which revealed reached in the absence of DES, which highlights the use of DES
the formation of only 12 % yield of the dehydration product for synthetic purposes.
4a, a fact that suggest that a specific catalysis is occurring for
the transformation of 3a into 4a. These control experiments
highlight the role of the enzymatic preparation in the concomi-
tant dehydration process.
A similar result was also observed when just 5 % water was
used in combination with the ChCl:Gly (1:2) as reaction medium
(Table 2, entries 7 and 8), which means that minimum amounts
of water are required to design an efficient protocol towards
the formation of the aldol product 3a. The process seems to be
more effective at higher enzyme loadings, and enabled com-
plete conversion after 24 h (entry 9). The use of different equiv-
alents of acetone was also explored, and the results clearly
demonstrate the required use of a considerable excess of the
donor ketone to obtain good conversion values (entries 7, 10
and 11).
Remarkably, a control experiment with the inhibited PPL
Figure 2. PPL-catalysed aldol reaction between 1a (enzyme/1a ratio 1:2 w/w)
and acetone (5 equiv.) in ChCl:Gly (1:2 mol/mol) containing a 5 % water after
24 h at different aldehyde concentrations.
after treatment with phenylmethanesulfonyl fluoride (PMSF), a
commonly used serine protease inhibitor,[23] again demon-
strated the catalytic activity of the enzyme (Table 2, entry 12).
Interestingly, when a nonpolar organic solvent such as toluene,
which is commonly employed in lipase-catalysed reactions, was
used in the optimised conditions the conversion reached just
8 %, giving extra value to the process developed in DES systems
(entry 13).
Before extending the optimal conditions to a representative
number of substrates, we focused in the type of agitation, con-
sidering different speeds for orbital shaking or alternatively the
use of a magnetic stirrer. Overall, no significant differences were
observed in the distribution of aldol 3a and aldol-dehydration
4a products obtained under different shaking conditions. All
the reactions were carried out for 24 h at 60 °C using acetone
The use of different substrate concentration in ChCl:Gly (1:2) (5 equiv.) and 5 % water in different DES; the results are summa-
was then explored (Figure 2); it was thereby established that 1 rised in Figure 3. The use of an orbital shaker at intermediate
and 2
tical applications, and that much higher substrate concentra- highest conversion value was obtained with ChCl:Gly (1:2 mol/
tions (3–6 ) led to a slight decrease in the conversion. None- mol) at 300 rpm, which gave 87 % conversion with very high
theless, conversions of 75–80 % were still found for these high selectivity towards the formation of the aldol product 3a (8:1).
M
concentrations of 1a were the best conditions for prac- speeds provided the best results (250–300 rpm), and the
M
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(benzaldehydes with different pattern substitutions in the aro-
matic ring 1a–e) and donors (acetone, cyclopentanone and
cyclohexanone) for the PPL-catalysed aldol reaction (Table 3,
Table 4 and Table 5). Benzaldehydes 1a–e were first tested in
the reaction with acetone (5 equiv.); the results are summarised
in Table 3.
Table 4. PPL-catalysed aldol reaction between substituted nitrobenzalde-
hydes (1a–c, 1
M) and cyclohexanone (6) in ChCl:Gly (1:2 mol/mol) after 24 h
at 60 °C and 250 rpm.
Figure 3. PPL-catalysed aldol reaction between 1a (1:2 enzyme/aldehyde w/
w) and acetone (5 equiv.) in ChCl:Gly mixtures containing a 5 % water after
24 h using different types of agitation.
Entry
PPL/1[a]
[6] [M]
H2O [%][b]
c [%][c,d]
1
2
3
4
5
6
7
8
0:1 (1a)
1:2 (1a)
1:2 (1a)
1:2 (1a)
1:2 (1a)
1:2 (1a)
1:1 (1a)
1:1 (1b)
1:1 (1c)
1:1 (1a)
1.1
1.1
1.1
1.1
1.1
1.1
1.1
1.1
1.1
2.0
0
0
5
10
15
20
5
5
5
5
<3
Hydrolase-Catalysed Aldol Reaction between Substituted
Benzaldehydes and Acetone
82 (56:44)
86 (54:46)
87 (54:46)
87 (53:47)
87 (53:47)
92 (57:43)
88 (53:47)
70 (52:48)
>97 (55:45)
With the best results in hand (entry 9, Table 2), we explored
the suitability of these DES systems with a family of acceptors
Table 3. PPL-catalysed aldol reaction between substituted benzaldehydes 1a–
e and acetone (5 equiv.) using PPL (1:1 enzyme/aldehyde w/w) in ChCl:Gly
(1:2 mol/mol) and 5 % water after 24 h at 60 °C and 250 rpm.
9
10
[a] Ratio of enzyme/aldehyde 1a–c (w/w). [b] Percentage in volume of water
in the reaction medium. [c] Conversion values were determined by 1H NMR
spectroscopic analysis of the crude reaction mixtures. [d] Diastereomeric ratio
was also calculated by 1H NMR spectroscopic analysis; the syn/anti ratios are
given in parentheses.
Satisfyingly, in all cases, the final products were obtained
with almost quantitative conversion (Table 3, entries 1–5). Aldol
adducts 3a–e were identified for all the tested substrates as
the major products, although a significant amount of the aldol-
dehydration compounds 4a–e was attained, especially for the
3-nitrobenzaldehyde (entry 2) or when weaker electron-with-
drawing groups were present in the aromatic ring (entries 4
and 5). Unfortunately, the approach was not highly efficient
when 2-cyanobenzaldehyde or the parent benzaldehyde were
Entry
R
Conv. [%][a]
Ratio 3/4[a,b]
1
2
3
4
5
4-NO2 (1a)
3-NO2 (1b)
2-NO2 (1c)
4-CN (1d)
4-CF3 (1e)
>97
>97
>97
>97
96
6:1 (99)
3:1 (99)
6:1 (99)
3.5:1 (92)
2:1 (72)
[a] Conversion and ratio of products 3/4 were determined by 1H NMR spec-
troscopic analysis of the crude reaction mixtures. [b] Isolated yield after five
liquid–liquid extractions with EtOAc are given in parentheses.
Table 5. Enzyme-catalysed aldol reaction between benzaldehydes (1a–e, 1
M) and cycloalkanones (5 and 6, 2.0 equiv.) using a PPL/aldehyde (1:1 ratio w/w)
in ChCl:Gly (1:2) and 5 % water after 24 h at 60 °C and 250 rpm.
Entry
Aldehyde
Ketone
c [%][a]
Aldol 7/8 dr [%][a]
Aldol-dehydration product 9,10 [%][a]
1
2
3
4
5
6
7
8
1a (R = 4-NO2)
1a (R = 4-NO2)
1b (R = 3-NO2)
1b (R = 3-NO2)
1c (R = 2-NO2)
1c (R = 2-NO2)
1d (R = 4-CN)
1d (R = 4-CN)
1e (R = 4-CF3)
1e (R = 4-CF3)
5
6
5
6
5
6
5
6
5
6
>97 (93)
>97 (89)
>97 (99)
>97 (91)
55 (n.d.)
70 (n.d.)
>97 (99)
>97 (96)
>97 (98)
>97 (99)
55:45
55:45
57:43
62:38
53:47
55:45
61:39
52:48
66:34
55:45
3
<3
13
7
<3
<3
11
<3
20[b]
<3
9
10
[a] Conversion, diastereomeric ratio of the aldol product and percentage of the alcohol-dehydration product values were determined by 1H NMR spectroscopic
analysis of the crude reaction mixture. n.d.: not determined. [b] An additional 8 % of a polycondensation product was also observed.
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used because, respectively, undesired polymerization side reac-
tions and complex product mixtures were observed.
Hydrolase-Catalysed Aldol Reaction between Substituted
Benzaldehydes and Cyclic Aliphatic Ketones
Other ketones, in this case cyclic ketones such as cyclopentan-
one (5) and cyclohexanone (6), were used (Tables 4 and 5). The
reaction between 4-nitrobenzaldehyde and cyclohexanone was
first analysed in detail, studying the effect of using variable en-
zyme loadings, equivalents of cyclohexanone, and percentages
of water (Table 4).
Figure 4. Recycling experiments in the reaction between 4-nitrobenzaldehyde
(1a, 1 M) and cyclohexanone (6, 2.0 equiv.) using PPL (1:1 w/w ratio with 1a)
in ChCl:Gly (1:2) and 5 % water after 24 h at 60 °C and 250 rpm.
As expected, the reaction in the absence of PPL did not pro-
ceed, and a noteworthy 82 % conversion was achieved when
PPL was used in a 1:2 ratio by weight with respect to the sub-
strate (Table 4, entry 2). Slightly higher conversions could be
achieved in the presence of water, although the use of up to
20 % water did not provide significant changes (86–87 % con-
version, entries 3–6). Gratifyingly, other benzaldehydes reacted
to give the corresponding aldol products in good to high yield
(entries 7–9). Finally, the use of a higher loading of enzyme in
the reaction with 4-nitrobenzaldehyde (1a) with a larger excess
of cyclohexanone (2 equiv.) led to formation of the final product
with quantitative conversion (entry 10). It must be mentioned
that the corresponding aldol-dehydration product was not ob-
served in any case. In addition, just two equivalents were re-
quired to achieve full conversion in comparison to a large ex-
cess for the aldol reactions with acetone (5 equiv.).
conditions (high substrates concentration and 60 °C). This was
not surprising because crude enzyme was used without being
immobilised on a support, which highlights the possibility of
recycling the biocatalyst together with the DES medium. The
activity of the catalytic system decreased continuously after ev-
ery reuse, although the drop of activity was higher during the
first recycling in comparison with the second, third and fourth
cycles. The diastereomeric ratio of product 7a was not affected
in the repetitive experiments and the alcohol dehydration prod-
uct 9a was never observed.
Conclusions
This synthetic approach was extended to a series of alde-
hydes 1a–e using both cyclopentanone and cyclohexanone.
The aldol products were formed as major products with quanti-
tative conversion for most of the substrates (Table 5). Total con-
version was observed for all the benzaldehydes used without
substitutions in the ortho-position, with the aldol products 7a–
e and 8a–e isolated as major products albeit with poor dia-
stereoselectivities. The reactions with cyclohexanone highly fa-
voured the formation of aldols 7a–e, with the corresponding
products resulting from the dehydration process being ob-
served in a maximum of 7 % (even entries). For reactions with
cyclopentanone (odd entries) the 4-nitrobenzaldehyde seems
to be the perfect donor because the aldol product was ob-
tained in excellent yield (entry 1).
Deep eutectic solvents represent a promising alternative to con-
ventional organic volatile solvents. For the first time, a promis-
cuous lipase-catalysed aldol reaction has been performed in
this type of neoteric solvents. The use of porcine pancreas
lipase (PPL) has generally led to the aldol product in excellent
conversions after exhaustive optimization of the reaction condi-
tions, with the corresponding α,ꢀ-unsaturated carbonyl com-
pounds being found in low percentages as a result of the de-
hydration of the so-obtained alcohol product. Considering that
this research opens new possibilities in synthetic chemistry, the
study of new hydrolase-catalysed nonconventional reactions in
deep eutectic solvents is underway.
Experimental Section
Recycling Studies of the DES and the Enzyme
General: Porcine pancreas lipase (PPL) type II (30–90 U/mg of pro-
tein using triacetin) and protease from Bacillus licheniformis (Alcal-
ase-CLEA®, 5.81 U/g) were purchased from Sigma–Aldrich. Candida
antarctica lipase type B (CAL-B, Novozyme 435, 7300 PLU/g) was a
gift from Novozymes (Denmark). All other reagents and solvents
were used as received from commercial suppliers without further
purification.
To fully disclose the potential of DES, the recycling of DES and
the enzyme was studied for the reaction of 4-nitrobenzalde-
hyde and 2 equiv. cyclohexanone (Table 5, entry 2). The enzyme
and the DES were recovered after the biotransformations, prior
to extraction of the target products with ethyl acetate. Then
the catalytic system was immediately used, measuring the ac-
tivity after 24 h (Figure 4).
The recycling experiments were conducted up to four times,
with a significant loss of activity being observed after the first
cycle. This result made us consider a deactivation of the biocat-
Column chromatography was performed using silica gel 60 (230–
240 mesh) purchased from Merck. 1H, 13C and DEPT NMR experi-
ments were performed with AC-300 and DPX-300 Bruker spectrom-
eters at room temperature (1H, 300.13 MHz and 13C, 75.5 MHz).
alyst in the early stage of the process due to the severe reaction Conversion and diastereomeric ratio values were calculated by 1H
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Angew. Chem. 2004, 116, 6156; c) K. Hult, P. Berglund, Trends Biotechnol.
2007, 25, 231–238; d) I. Nobeli, A. D. Favia, J. M. Thornton, Nat. Biotech-
nol. 2009, 27, 157–167.
NMR analysis by comparing the CH signal adjacent to the hydroxyl
group (see the Supporting Information for further details).[24]
General Procedure for the ChCl:Gly (1:2) DES Preparation:
Choline chloride (50.0 mmol, 6.98 g) and glycerol (100.0 mmol,
7.30 mL) were mixed and stirred at 80 °C until a clear solution was
obtained (30 min). After cooling to room temperature, the formed
DES could be used directly in lipase-catalysed reaction without fur-
ther purification. For the preparation of ChCl:Gly (1:1.5) the protocol
was the same but using 75.0 mmol of glycerol (5.48 mL).
[2]
[3]
a) E. Busto, V. Gotor-Fernández, V. Gotor, Chem. Soc. Rev. 2010, 39, 4504–
4523; b) M. Kapoor, M. N. Gupta, Process Biochem. 2012, 47, 555–569; c)
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General Procedure for the Aldol Reaction between 4-Nitro-
benzaldehyde and Acetone in DES: A mixture of porcine pancreas
lipase (PPL, 151 mg), 4-nitrobenzaldehyde (1a, 151 mg, 1.0 mmol)
and acetone (2, 368 μL, 5.0 mmol) in choline chloride/glycerol
(1:2 mol/mol, 1 mL) containing 5 % water (50 μL) was shaken at
60 °C and 250 rpm. After 24 h, the mixture was extracted with ethyl
acetate (5 × 5 mL). The combined organic layers were washed with
brine (2 × 10 mL), dried with Na2SO4, filtered, and the solvent was
evaporated under reduced pressure. The mixture of aldol products
was isolated as an orange solid, and the composition was analysed
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General Procedure for the Aldol Reaction between Benzalde-
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(2 × 10 mL), dried with Na2SO4, filtered, and the solvent was evapo-
rated under reduced pressure. The mixture of aldol products was
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1
isolated, and the composition was analysed by H NMR analysis.
General Procedure for the Aldol Reaction between Benzalde-
hydes and Cyclic Aliphatic Ketones in DES: A mixture of porcine
pancreas lipase (PPL, 151 mg), 4-nitrobenzaldehyde (1a–e,
1.0 mmol), and cyclopentanone (5, 107 μL, 2.0 mmol) or cyclohex-
anone (6, 207 μL, 2.0 mmol) in choline chloride/glycerol (1:2 mol/
mol, 1 mL) containing 5 % water (50 μL) was shaken at 60 °C and
250 rpm. After 24 h, the mixture was extracted with ethyl acetate
(5 × 5 mL). The combined organic layers were washed with brine
(2 × 10 mL), dried with Na2SO4, filtered, and the solvent was evapo-
rated under reduced pressure. The mixture of aldol products was
1
isolated, and the composition was analysed by H NMR analysis.
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Acknowledgments
Financial support to this work was provided by the Spanish
Ministerio de Economía y Competitividad (MEC) (CTQ2013-
44153-P) and the Gobierno del Principado de Asturias (FC-15-
GRUPIN14-002). The authors thank Novo Nordisk Co., Denmark
for the generous gift of CAL-B (Novozyme 435). D. G.-M. thanks
the Fundación para el fomento en Asturias de la investigación
científica aplicada y tecnología (FICYT) for a predoctoral fellow-
ship.
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Keywords: Aldol reactions · Biocatalytic promiscuity · Enzyme
catalysis · Biotransformations · Deep eutectic solvents
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Received: December 10, 2015
Published Online: February 16, 2016
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© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim