S. Milker et al.
MolecularCatalysis466(2019)70–74
in a ratio of 1:3 sample:ethyl acetate (v/v). The fractions were com-
bined, diluted 1:10 (v/v) with ethyl acetate with 0.5 mM N-acetyl-p-
aminophenol as internal standard, filtered with 0.2 μm polyamide syr-
ingetop filters and stored at −20 °C until analysis.
All other DES samples were diluted 1:3 (v/v) with ethyl acetate,
then diluted 1:2 (v/v) with ethyl acetate with 1 mM N-acetyl-p-ami-
nophenol as internal standard and then further diluted 1:5 (v/v) with
ethyl acetate with 0.5 mM N-acetyl-p-aminophenol as internal standard.
The samples were filtered with 0.2 μm polyamide syringetop filters and
stored at −20 °C until analysis.
Scheme 1. Aldol addition of acetone with 4-nitrobenzaldehyde 1 to the main
aldol product 2 and the side product olefin 3.
publication with the same model reaction in choline chlor-
ide:glycerol DES [14] are mainly attributed to high concentrations
of acetone as co-substrate and co-solvent and not to the applied DES
system
2.5. HPLC analysis
The HPLC analysis was performed with a normal phase Shimadzu
HPLC with a SPD-M10AVP PDA detector equipped with a Chiralpak IB
column (5 μm particle size, 4.6 mm × 250 mm, Daicel chemical in-
2. Experimental
2.1. Materials
dustries, LTD). The mobile phase was
a mixture of 20:80 iso-
propanol:hexane with a flow rate of 1 mL min−1 at 35 °C. All analytes
and the internal standard acetophen were detected at 254 nm.
All chemicals used in this study were purchased by commercial
suppliers except noted otherwise. The lipase from porcine pancreas
(PPL, Type II, 100–500 units/mg protein), the bovine serum albumin
(BSA, > 98%) and 4-nitrobenzaldehyde (4-NBA, > 99%) were pur-
chased from Sigma-Aldrich. The aldol 2 (4-hydroxy-4-(4-nitrophenyl)
butan-2-one) and olefin 3 standards ((E)-4-(4-nitrophenyl)but-3-en-2-
one) were kindly synthesized by Jan Deska (Department of Chemistry,
Aalto University, Espoo). All chemicals were used without any addi-
tional purification or dehydration step.
2.6. Calculation of the initial velocity of the reaction
For the calculation of the initial velocity of the reaction, the aldol
concentration was plotted over time and the first 5 h of the reaction
were fit using linear regression with weighted least squares with the
software Origin2016 G, OriginLab.
2.2. Preparation of DES
3. Results and discussion
The four DES were mixed in the molar ratio depicted in Table 1 and
heated to the corresponding synthesis temperature with stirring at
200 rpm for approx. 1 h until a homogeneous liquid was obtained. The
density of the DES was measured by weighting 0.5 mL of each DES at
60 °C [Supplementary Table S1].
3.1. Comparison three different DES reaction systems with 250 mM and
BSA as control
As it has been shown in previous publications, BSA is able to cata-
lyze aldol additions in general [15,16] and the desired reaction with
low conversion rates in organic solvents [13]. However, our goal was to
investigate the impact of DES with differences in hydrophobicity on the
velocity and specificity of the reaction (Scheme 1) with the control
the PPL catalysis from the effects of BSA-catalyzed control reactions.
The aldol product (2) formation was higher in the hydrophilic
ChCl:Gly than in the more hydrophobic DES TOABr:EG and TOABr:PD
(Fig. 1). For a starting 4-NBA (1) concentration of 250 mM, the reaction
reached saturation after approximately 24 h. However, the undesired
byproduct olefin (3) formation was higher in ChCl:Gly for BSA as well
as PPL-catalyzed reactions. The deviations in the initial measured
concentration of the substrate 4-NBA from 250 mM could be explained
with an insufficiently homogeneous reaction medium for the first 1 h
reaction time, therefore making homogeneous sampling impractical.
Additionally, the extraction protocol for the ChCl:Gly system allowed
further deviations due to a seven step extraction procedure.
2.3. Screening reactions with BSA/Lipase
Every reaction was performed in 10 mL glas vials with screw caps
and stirring bar in a water bath at 60 °C reaction temperature in a 4 mL
reaction volume. After synthesis of the DES, the DES were incubated at
60 °C and first 4-nitrobenzaldehyde, then the enzyme powder was
added under stirring with 200 rpm. After a homogeneous mixture was
achieved, water (50 μL mL−1 DES) was added. The reaction was started
with addition of acetone. The acetone was added in a molar excess of
5:1 to the 4-nitrobenzaldehyde in the 4 mL DES. The first sample was
taken immediately after acetone addition. The concentration of lipase/
BSA in the screenings with initial concentrations of 250 mM 4-ni-
trobenzaldehyde was 38.8 mg mL-1. For further screening experiments,
the concentrations of the 4-nitrobenzaldehyde and the PPL were varied.
All concentrations in this work were related to the reaction volume of
the DES before acetone/water addition.
The ratio of the product aldol (2) to byproduct olefin (3) showed a
higher specificity for aldol production with PPL over BSA; especially
being evident in the hydrophobic DES (Table 2). Overall, the BSA-cat-
alyzed reactions were less specific towards either one of the products.
With BSA, only a slight excess of aldol in the more hydrophobic DES
(TOABr:PD and TOABr:EG) and no selectivity in the ChCl:Gly DES
2.4. Sampling process and sample preparation
The samples from reactions in ChCl:Gly were extracted 7 times with
ethyl acetate with 0.5 mM N-acetyl-p-aminophenol as internal standard
Table 1
Synthesis parameters for the DES applied in this study.
DES
Hydrogen bond acceptors
Hydrogen bond donors
Molar ratio
Synthesis temperature [°C]
ChCl:Gly
choline chloride
glycerol
1:1.5
1:3
50
70
70
80
TOABr:EG
TOABr:PD
TBACl:4-NBA
tetraoctylammonium bromide
tetraoctylammonium bromide
tetrabutylammonium chloride
ethylene glycol
1,5-pentadiol
4-nitrobenzaldehyde
1:3
2.2:1.5
71