Pepsin-Catalyzed Asymmetric Cross Aldol Reaction Promoted by Ionic Liquids and Deep Eutectic Solvents

Article abstract of DOI:10.1007/s10562-020-03176-1

Abstract: Pepsin was found to catalyze asymmetric cross aldol reactions of aromatic and polycyclicaromatic aldehydes with cyclic ketones in ionic liquids and deep eutectic solvents for the first time. Pepsin exhibited high catalytic activity and excellent

Full text of DOI:10.1007/s10562-020-03176-1

Catalysis Letters
https://doi.org/10.1007/s10562-020-03176-1
Pepsin‑Catalyzed Asymmetric Cross Aldol Reaction Promoted by Ionic
Liquids and Deep Eutectic Solvents
Yun Wang1 · Xin‑Yi Chen1 · Xin‑Yi Liang1 · Zhi‑Hui Liang1 · Hong Cheng1 · Xiang Li1 · Li‑Ling Li1
Received: 4 February 2020 / Accepted: 10 March 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
Pepsin was found to catalyze asymmetric cross aldol reactions of aromatic and polycyclicaromatic aldehydes with cyclic
ketones in ionic liquids and deep eutectic solvents for the frst time. Pepsin exhibited high catalytic activity and excellent
stereoselectivity in ionic liquids and deep eutectic solvents in the presence of moderate water. High yields of up to 93.1%,
excellent enantioselectivities of up to 96% ee, and good diastereoselectivities of up to 5:95 dr were achieved.
Graphic Abstract
Keywords Biocatalytic promiscuity · Pepsin · Aldol reaction · Ionic liquid · Deep eutectic solvent
1 Introduction
reaction [11, 12], Henry reaction [13, 14] and epoxidation
reaction [15]. Among these reactions, asymmetric aldol
condensation with strong atom economy is often reported.
Berglund and co-workers frstly reported that wild CAL-B
(lipase B from Candida antarctica) and Ser105Ala mutant
CAL-B have the catalytic activity for aldol reactions in
2003 [16]. Guan and co-workers used diferent hydrolyases
to catalyze several asymmetric aldol reactions [17]. Lin
and co-workers also reported an application of acylase in
aldol reaction [18]. Wang and co-workers demonstrated the
lipase-catalyzed asymmetric aldol reaction in organic sol-
vents greatly promoted by water [19]. These lipase catalyzed
aldol reactions were carried out in organic solvents, and it
is found that the enzyme can maintain its activity in organic
solvents [20, 21]. However, the harm of organic solvents to
the environment is inevitable. In addition, there are more
or less problems such as long reaction time, high reaction
temperature and low enzyme activity. Hence, the search for
environmentally friendly media for biocatalytic asymmet-
ric aldol reactions and other chemical transformations has
become a considerable challenge. In 2011 and 2013, Guan
group [22] and Wang group [23] respectively found that the
Biocatalysis has high selectivity, mild reaction conditions
and potential utilization of cheap renewable resources. It is
an efcient and green tool for modern organic synthesis [1].
However, biocatalysis has some disadvantages. For exam-
ple, the efectiveness of the enzyme is limited, and the sub-
strate specifcity of the enzyme is limited [2]. Therefore, it is
important to fnd new unnatural activities((i.e.promiscuity)
of existing enzymes to broaden the applicability of existing
enzymes [3]. Recently, the study of biocatalytic promiscuity
has attracted signifcant attention from chemists and bio-
chemists [4, 5]. These enzymes display signifcant levels
of activity in a variety of nonconventional reactions such
as Michael addition [6–8], Mannich reaction [9, 10], aldol
* Yun Wang
wangyun@lingnan.edu.cn
1
School of Chemistry and Chemical Engineering, Key
Laboratory of Clean Energy Materials Chemistry
of Guangdong Higher Education Institutes, LingNan Normal
University, Zhanjiang 524048, China
Vol.:(0123456789)
1 3

Y. Wang et al.
lipase-catalyzed stereoselective cross aldol reaction could
be performed under solvent-free conditions. However, the
enormously excessive ketones used as one of the substrates
were not sufciently cost-efcient or eco-friendly. In 2013,
an enzymatic aldol reaction was then attempted in phos-
phate-citrate bufer by Wang and co-workers, only achieving
an optimal enantioselectivity of 66% [24]. Therefore, it is
urgent to fnd better reaction media for enzyme catalysis.
In the search for sustainable conditions for the development
of chemical reactions, ionic liquids (ILs) and deep eutectic
solvents (DESs) have appeared in recent years as promising
biodegradable and environmentally friendly solvents.
Ionic liquids (ILs) are the frst non-traditional media
compatible with enzymes, developed from the concept of
green and sustainable (considering their low vapor pressure).
Many reactions, such as hydrolysis and redox reactions, and
the formation of C–C bonds, have been successfully carried
out in ILs-containing media [25]. Wang et al. reported lipase
from porcine pancreas (PPL) was used to catalyze asym-
metric cross aldol reactions in ionic liquid ([BMIM][PF6])
with deionized water for the frst time in 2014 [26]. PPL
exhibited high catalytic activity and good stereoselectivity in
this efcient and recyclable room temperature ionic liquid in
the presence of moderate water. Although the reaction yield
is high, the reaction time is long.
material synthesis, biotransformation, biocatalysis and other
felds [28–31]. Gotor-Fernández and co-workers reported the
lipase-catalysed aldol reaction has been performed for the
frst time in DESs in 2016 [32]. The aldol reaction between
4-nitrobenzaldehyde and acetone was examined in depth,
with excellent compatibility being found between PPL and
DESs(choline chloride/glycerol mixtures) for the formation
of the aldol product in high yields.The system was compat-
ible with a series of aromatic aldehydes and ketones includ-
ing acetone, cyclopentanone and cyclohexanone. Thus, the
application of DESs in other enzyme-catalyzed reactions
have attracted increasing research attention.
In this study, we hope to fnd a more green and efective bio-
catalytic pathway for asymmetric aldol reaction. We selected
Pepsin as a catalyst and utilized “green” solvent (water, ILs
and DESs) as the reaction medium. High yields, good enanti-
oselectivities and diastereoselectivities were also obtained in
ILs and DESs in the presence of moderate water when a wide
variety of substrates were simultaneously investigated.
2 Results and Discussion
It is well known that the catalytic activity of enzyme depends
mainly on the type and origin of the enzyme. In this study,
the aldol reaction of cyclohexanone and 4-nitrobenzalde-
hyde as a model reaction. Eight kinds of enzymes catalyz-
ing aldol reaction were screened out (Table 1). It could be
found that all the selected enzymes can catalyze the aldol
reaction, but they all displayed poor selectivity. Among the
tested enzymes, BPL showed the highest catalytic activ-
ity with a good yield of 69.8% with 8% ee(Table 1, entry
Deep eutectic solvents (DESs) are similar to ILs, with
low melting point, low volatility and high thermal stabil-
ity. In addition, DESs are biodegradable, non-toxic, cheap
and easy to prepare. Therefore, DESs are also called “green
solvent”, just like ionic liquid. DESs frst came to the pub-
lic vision in 2001 [27]. Since then, the research on deep
eutectic solvent has been developing rapidly in extraction,
Table 1 The catalytic activities of diferent enzymes
OH
O
O
CHO
enzymes
50oC
+
O2N
O2N
Entry
Enzyme
Yield %a
dr(anti/syn)a
ee%(syn)a
1
2
3
4
5
6
7
8
9
Blank
<1
–
-
8
3
25
20
27
2
15
5
Bovine pancreatic lipase(BPL)
Lipase from Mucormiehei(MML)
Pepsin
Trypsin from porcine pancreas
Trypsin from bovin pancreas
Lipase acrylic resin from Candida antarctia
Subtilisin
69.8
63.8
60.0
41.9
40.7
42.6
43.5
34.0
51/49
74/26
41/59
36/64
52/48
63/37
54/46
54/46
Lipase from Candida rugosa(CRL)
Conditions: 4-nitrobenzaldehyde (0.1 mmol), cyclohexanone (2.0 mmol), and Pepsin (5 mg)at 50 °C for 24 h
aYield, ee, and dr were determined by HPLC using AD-H chiral column
1 3

Pepsin-Catalyzed Asymmetric Cross Aldol Reaction Promoted by Ionic Liquids and Deep Eutectic…
2), indicating that BPL had the ability to catalyze the aldol
reaction, but it displayed poor selectivity under solvent-free
conditions. When Trypsin from bovin pancreas was used as
catalyst (Table 1, entry 6), the selectivity of the reaction was
the highest (27%), but the yield was relatively low (40.7%).
In contrast, when Pepsin was used as catalyst, the yield and
selectivity of the reaction were good, which providing the
aldol product in a good yield of 60.0% with 25% ee (Table 1,
entry 4). To verify the specifc catalytic efect of enzyme on
the aldol reaction, some control experiments were performed
under the same conditions. In the absence of enzyme, only
a trace amount of product was detected after 24 h (Table 1,
entry 1).Thus, Pepsin was chosen as the catalyst in the fol-
lowing experiments.
Reaction medium signifcantly infuences the activity,
stability and probably tertiary structure of the enzymes
[33]. We tried to fnd a green medium combined with bio-
catalysis to achieve sustainable and environmental devel-
opment standards. Thus, the infuence of “green” solvents
on the Pepsin-catalyzed aldol reaction was investigated in
this study. The “green” solvents are water, ILs and DESs.
The aldol reaction between 4-nitrobenzaldehyde and
cyclohexanone was used as a model reaction. The initial
experiments were performed in six pure ILs(Table 2, entries
Table 2 The efect of green solvent on the Pepsin-catalyzed asymmetric cross-aldol reaction
OH
O
O
CHO
Pepsin
+
solvent, 50oC
O2N
O2N
Entry
Solvent
Yield %a
dr(anti/syn)a
ee%(syn)a
1
2
3
4
5
6
7
8
[HOEtMIM]BF4
[HOEtMIM]PF6
[HOEtMIM]NO3
[BMIM]BF4
[BMIM]PF6
[BMIM]NO3
ChCl/Gly(1:2)
ChCl/EG(1:2)
deionized water
ChCl/Gly(1:2):deionized water=5:95
ChCl/Gly(1:2):deionized water=10:90
ChCl/Gly(1:2):deionized water=15:85
ChCl/Gly(1:2):deionized water=20:80
ChCl/Gly(1:2):deionized water=25:75
ChCl/Gly(1:2):deionized water=30:70
ChCl/Gly(1:2):deionized water=40:60
ChCl/Gly(1:2):deionized water=50:50
ChCl/Gly(1:2):deionized water=80:20
[HOEtMIM]BF4:deionized water=5:95
[HOEtMIM]BF4:deionized water=10:90
[HOEtMIM]BF4:deionized water=15:85
[HOEtMIM]BF4:deionized water=20:80
[HOEtMIM]BF4:deionized water=25:75
[HOEtMIM]BF4:deionized water=30:70
[HOEtMIM]BF4:deionized water=40:60
[HOEtMIM]BF4:deionized water=50:50
[HOEtMIM]BF4:deionized water=80:20
36.7
35.5
14.6
19.5
12.0
43.0
62.1
32.7
63.3
60.0
73.0
76.2
79.5
82.3
79.7
73.4
46.8
17.4
64.4
79.9
82.1
87.2
88.7
91.8
64.8
57.0
57.3
48/52
45/55
62/38
45/55
52/48
43/57
47/53
53/47
34/66
41/59
36/64
30/70
47/53
36/64
28/72
36/64
41/59
38/62
36/64
33/67
29/71
35/65
33/67
39/61
40/60
46/54
41/59
84
42
74
39
40
30
52
35
52
41
47
54
2
52
60
57
27
41
60
52
70
54
72
63
35
21
42
9
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Conditions: 4-nitrobenzaldehyde (0.1 mmol), cyclohexanone (2.0 mmol), and Pepsin (5 mg) in green solvent (0.3 mL), DES with deionized
water (0.3 mL, DES/water, v/v)or in ionic liquid with deionized water (0.3 mL, ionic liquid/water, v/v)at 50 °C for 24 h
aYield, ee, and dr were determined by HPLC using AD-H chiral column
1 3

Y. Wang et al.
1–6), two pure DESs (Table 2, entries 7–8) and deionized
water(Table 2, entries 9). Unfortunately, Pepsin showed
moderate to poor catalytic activity in all of the tested ILs.
For example, Pepsin exhibited the highest selectivity (84%
ee) in [HOEtMIM]BF4, but the yield is very low, only
36.7%(Table 2, entries 1). Thus, when DESs were used as
medium in the model reaction, the yields were moderate, but
higher than in ionic liquids. The most likely reason for such
low and moderate yields was that the high viscosity of the IL
and DES systems might be limiting the mass transfer of sub-
strates and products to and from the active site of the enzyme
[34]. In principle, high viscosity could be overcome through
the addition of small amounts of other solvents to the IL and
DES medium. According to our previous research, water
plays an important role in lipase-catalyzed aldol reaction,
and the addition of water can reduce the viscosity of IL and
DES. Thus, we added 20%, 50%, 60%, 70%, 75%, 80%, 85%,
90% and 95% water to the ILs and DESs respectively to
investigate the optimal ILs and DES for this reaction. To our
surprise, when 70% water was added to [HOEtMIM]BF4, the
yield of the reaction was increased from 36.7% to 91.8%, but
the selectivity was decreased from 84 to 63%. Meanwhile,
when 75% water was added to ChCl/Gly (1:2), the yield
of the reaction was increased from 62.1 to 82.3%, and the
selectivity(52%) was not afected. In order to prove the pro-
motion of Pepsin-catalyzed aldol reaction by ILs and DESs,
some control experiments were performed under the same
conditions. In the presence of water without ILs and DESs,
the yield is 63.3% with 52% ee after 24 h (Table 2, entries
9). As shown in Table 2, because in [HOEtMIM]BF4/water
(30/70), the increase of yield is relatively higher than that in
ChCl/Gly(1:2)/water, we chose [HOEtMIM]BF4/H2O(3/7)
as the reaction medium for further reaction investigations.
Then, other infuencing factors were screened to obtain
higher yields and stereoselectivities. The efect of the molar
ratio of the substrates on the Pepsin-catalyzed aldol reac-
tion was investigated. As shown in Table 3, when the molar
ratio of 4-nitrobenzaldehyde to cyclohexanone was varied
from 1:5 to 1:30, the yield was enormously improved from
60 to 90%, and the diastereoselectivity and enantioselec-
tivity were increased. The molar ratio of 4-nitrobenzalde-
hyde to cyclohexanone was 1: 25, the yield (93.1%) and
the enantioselectivity (63% ee) were highest. However,
the yield and selectivity of the reaction decreased to some
extent after increasing the mole ratio from 1:25 to 1:30. The
probable reason for these observations is that the reaction
reached equilibrium when the molar ratio of 4-nitrobenza-
ldehyde to cyclohexanone was 1:25. When the mole ratio
increased from 1:25 to 1:30 (i.e. the amount of cyclohex-
anone increased), the by-products increased, so the yield
decreased. Thus, 1:25 was selected as the optimal molar
ratio for the aldol reaction.
We investigated the infuence of reaction temperature on
the model reaction. The model reaction was performed at
four diferent temperatures that ranged from 30 °C to 60 °C
for 24 h. As shown in Table 4, the reaction at 50 °C for 24 h
ofered the best result with yields up to 93.1% and the enan-
tioselectivity (63% ee) and diastereoselectivity (46:54 dr).
At the higher temperature (60 °C), both the enzyme activity
and the stereoselectivity decreased. However, at the lower
temperature (30 °C), the enantioselectivity (80% ee) and
diastereoselectivity (28:71 dr) were highest, but the enzyme
activity was poor (55.4%). The optimal conformation of
Table 3 The efect of molar ratio on the Pepsin-catalyzed asymmetric cross aldol reaction
OH
O
O
CHO
Pepsin
+
[HOEtMIM]BF /H2O,50oC
O2N
4
O2N
Entry
Molar ratioa
Yield %b
dr(anti/syn)b
ee%(syn)b
1
2
3
4
5
6
1:5
60.7
69.3
66.7
91.8
93.1
80.0
43/57
43/57
35/64
39/61
46/54
41/59
37
33
54
63
63
61
1:10
1:15
1:20
1:25
1:30
Conditions:4-nitrobenzaldehyde(0.1 mmol), cyclohexanone (0.5, 1, 1.5, 2.0, 2.5, 3.0 mmol), and Pepsin (5 mg) in mixed solvents(0.3 mL,
[HOEtMIM]BF4/water, 3/7) at 50 °C for 24 h
aMolar ratio=4-nitrobenzaldehyde/cyclohexanone
bYield, ee, and dr were determined by HPLC using AD-H chiral column
1 3

Pepsin-Catalyzed Asymmetric Cross Aldol Reaction Promoted by Ionic Liquids and Deep Eutectic…
Table 4 The efect of temperature on the pepsin-catalyzed asymmetric cross aldol reaction
4
Entry
Temperature (°C)
Yield %a
dr(anti/syn)a
ee%(syn)a
1
2
3
4
30
40
50
60
55.4
71.7
93.1
87.6
28/71
26/74
46/54
51/49
80
68
63
56
Conditions:4-nitrobenzaldehyde(0.1 mmol), cyclohexanone (2.5 mmol), and Pepsin (5 mg) in mixed solvents(0.3 mL, [HOEtMIM]BF4/water,
3/7) at 50 °C for 24 h
aYield, ee, and dr were determined by HPLC using AD-H chiral column.
improvements were observed when the reaction time was
prolonged. So, 24 h was selected as the optimal reaction time
for the aldol reaction.
With the optimal reaction conditions in hand, we fur-
ther studied the scope and the generality of this biocata-
lytic promiscuity. Several substrates were used to expand
the Pepsin-catalyzed aldol reaction. The results are sum-
marized in Table 5. The highest yield of 93.1%, the best
diastereoselectivity of 5:95 dr and the best enantioselec-
tivity of 96% ee were achieved. Generally, benzaldehydes
with strong electron-withdrawing substituents were better
acceptors, producing better yields (Table 5, entries 1–4). The
reactions of aromatic aldehydes with strong electron-with-
drawing substituents provided products in the highest yield
of 93.1%. However, for aromatic aldehydes with electron-
donating substituents or no substituents, such as 4-methy-
oxybenzaldehyde and benzaldehyde, the yields were very
Fig. 1 The efect of reaction time on the Pepsin-catalyzed asymmetric
cross aldol reactiona,b. (aConditions:4-nitrobenzaldehyde(0.1 mmol),
cyclohexanone (2.5 mmol), and Pepsin (5 mg) in in mixed sol-
low with only a trace of product obtained (Table 5, entries
5–6). Furthermore, we examined the efect of two ketones.
Because the volume of cyclopentanone is smaller than that
of cyclohexanone, cyclopentanone is easier to attack aro-
matic aldehydes. Then, the aldol reaction of cyclopentanone
with aldehyde aforded the products in better yields than that
of cyclohexanone, except for p-nitrobenzaldehyde. This may
be because for p-nitrobenzaldehyde, the electronic efect
plays a leading role. Moreover, the substituent positions
on benzaldehydes had a great impact on the yield of the
reaction. For instance, the aldol reaction of 4-nitrobenzalde-
hyde with cyclohexanone generated the aldol product with a
higher yield than that of 2-nitrobenzaldehyde. This may be
because the steric hindrance of substituents on benzaldehyde
had a great infuence on the yield and stereoselectivity of
the reaction. All of these results may illustrate that Pepsin
in [HOEtMIM]BF4/H2O exhibited outstanding activity and
stereoselectivity.
b
vents(0.3 mL, [HOEtMIM]BF4/water, 3/7) at 50 °C for 24 h. Yield
was determined by HPLC using AD-H chiral column.)
enzyme is most likely to occur at the optimum temperature,
while the most unfavorable conformation is at the higher
temperature. To obtain the best yield, diastereoselectivity
and enantioselectivity, the reaction temperature of 50 °C was
chosen for the Pepsin-catalyzed aldol reaction.
Then, the infuence of reation time on the model reaction
was examined. As shown Fig. 1., the yield was increasing
with the extension of reaction time from 6 to 24 h. When
reaction time was 24 h, the yield was highest (93.1%). Then,
the reaction time continues to be extended to 48 h, 72 h and
96 h, the yield did not inreased. The probable reason for
these observations is also that the reaction reached equilib-
rium when the reaction time was 24 h. Therefore, no obvious
1 3

Y. Wang et al.
Table 5 Substrate scope of the pepsin-catalysed asymmetric cross aldol reaction
4
Entry
1
Product
Time(h)
48
Yield %a
21.5
dr(anti/syn)a
52/48
ee%(syn)a
62
2
3
4
5
6
48
24
24
96
96
79.5
54/46
46/54
31/69
-
66
63
60
-
93.1
57.5
Trace
Trace
-
-
9
48
48
49.2
58.8
5/95
65
96
10
49/51
Reaction conditions: aromatic aldehyde (0.1 mmol), cycloketone (2.5 mmol) and Pepsin (5 mg) in mixed solvents(0.3 mL, [HOEtMIM]BF4/
water, 3/7) at 50 °C
aYield, ee, and dr were determined by HPLC using AD-H chiral column.
1 3

Pepsin-Catalyzed Asymmetric Cross Aldol Reaction Promoted by Ionic Liquids and Deep Eutectic…
or ethylene glycol (100.0 mmol, 5.56 mL) were mixed and
3 Conclusions
stirred at 80 °C until a clear solution was obtained. The pre-
pared DESs were cooled and used for the enzyme-catalyzed
the aldol reaction without any purifcation.
To sum up, in the present work, the Pepsin-catalyzed cross-
aldol reaction between aromatic aldehydes and cyclic
ketones was promoted by ILs and DESs. It was more mean-
ingful to fnd that ILs/water and DESs/water system had
decisive infuence on the activity and stereoselectivity of
Pepsin. Excitingly, ILs and DESs exhibited excellent recy-
clability and recoverability, as the recovered ILs and DESs
were recycled for next reaction.The reactions could be per-
formed smoothly between a wide range of aromatic alde-
hydes and cyclic ketones in [HOEtMIM]BF4/water(3/7), and
good to excellent yields as well as better selectivity were
obtained. As a green and efcient synthetic method, it not
only expands the application felds of enzymatic promiscu-
ity, but might be a potential synthetic method for industrial
application.
4.3 General Procedure for Aldol Reaction
Aldehyde (0.1 mmol), Pepsin (5 mg), cyclic ketone
(2.5 mmol), [HOEtMIM]BF4 (0.09 mL) and deionized water
(0.21 mL) were added to an Erlenmeyer fask at 250 rpm at
50 °C. After completion of the reaction, the product was
directly analyzed by HPLC in comparison with a standard
sample. The organic phase was combined and evaporated
in vacuum. The residue was purifed by fash column chro-
matography using ethyl acetate-petroleum ether as mobile
phase.
4.4 General Procedure for Recovering and Recycling
IL and DES
4 Experimental
IL and DES were recovered according to the procedures
reported in the literature [32, 35]. The reaction on the reus-
ability of IL and DES was conducted under the optimal
reaction conditions. After the reaction had been completed,
enzyme and the product were fltered, and IL or DES was
washed with diethyl ether for many times in order to extract
the residual substrates. After 12 h in the vacuum drying
oven, the recycled IL or DES was reused in the next reac-
tion under the same conditions.
4.1 Materials and Analytical Methods
All enzymes were purchased from Sigma-Aldrich Co. LLC
(US), BPL was purchased from Aladdin Co., Ltd. (Shang-
hai, China). 1-butyl-3-methylimidazolium tetrafuoroborate
([BMIM][BF4]), 1-butyl-3-methylimidazolium hexafuoro-
phosphate ([BMIM][PF6]), 1-butyl-3-methylimidazolium
nitrate ([BMIM][NO3]), 1-hydroxyethyl-3-methylimidazo-
lium tetrafuoroborate ([HOEtMIM][BF4]), 1-hydroxyethyl-
3-methylimidazolium hexafuorophosphate ([HOEtMIM]
[PF6]), 1-hydroxyethyl-3-methyl-
4.5 2‑(hydroxy(2‑nitrophenyl)methyl)
cyclohexanone
imidazolium nitrate ([HOEtMIM][NO3]) were purchased
from ShangHai Cheng Jie Chemical Co. LTD. (China). Cho-
line chloride (ChCl) was purchased from Aladdin Co., Ltd.
(Shanghai, China). Glycerol( Gly) and ehylene glycol (EG)
were purchased from Tianjin Damao Chemical Reagent
Factory. Unless otherwise noted, all reagents were obtained
from commercial suppliers and were used without further
purifcation.
1H NMR (400 MHz, CDCl3): δ 7.95(d, J = 8.1 Hz, 1H),
7.85 (d, J = 7.9 Hz, 1H), 7.73 (t, J = 7.5 Hz, 1H), 7.50 (t,
J=7.6 Hz, 1H), 5.92 (s, 0.32H), 5.42(d, J=7.1 Hz, 0.67H),
4.09(s, 0.87H), 2.84–2.70 (m, 1H), 2.40–2.28 (m, 2H), 2.07
(s, 1H), 1.86–1.54 (m, 5H).
Enantiomeric excess was determined by HPLC with a
CHIRALPAK AD-H column (90:10 hexane:
2-propanol), 25 °C, 254 nm, 0.5 mL/min; major enanti-
omer tr=14.7 min, minor enantiomer tr=15.9 min.
The NMR spectra were recorded on a Bruker 400 MHz
instrument using CDCl3 as solvent. Chemical shifts (δ) were
expressed in ppm with TMS as internal standard, and cou-
pling constants (J) were reported in Hz. HPLC was carried
out on SHIMADZU instrument (LC-2010A HT, UV/VIS
Detector) using a chiral column (4.6 mm, 250 mm).
4.6 2‑(Hydroxy(2‑nitrophenyl)methyl)
cyclopentanone
1H NMR (400 MHz, CDCl3): δ 7.85 (d, J = 8.1 Hz, 1H),
7.77 (d, J = 7.8 Hz, 1H), 7.64 (t, J = 7.4 Hz, 1H), 7.43 (t,
J=7.7 Hz, 1H), 5.96 (s, 0.34H), 5.45 (d, J=7.0 Hz, 0.65H),
4.19 (s, 0.89H), 2.84–2.70 (m, 1H), 2.46 (d, J= 12.9 Hz,
1H), 2.40–2.28 (m, 1H), 2.11–2.05 (m, 1H), 1.86–1.58 (m,
3H).
4.2 General Procedure for DESs Preparation
In this study, DESs were prepared according to the pro-
cedures reported in the literature [32]. Choline chloride
(50.0 mmol, 6.98 g) and glycerol (100.0 mmol, 7.30 mL)
1 3

Y. Wang et al.
Acknowledgements We gratefully acknowledge the Natural Science
Foundation of Guangdong Province (Grant No. 2018A030307022),
Special Innovation Projects of Common Universities in Guangdong
Province (Grant No. 2018KTSCX126).
Enantiomeric excess was determined by HPLC with a
CHIRALPAK AD-H column (90:10 hexane: 2-propanol),
25 °C, 254 nm, 0.5 mL/min; major enantiomer tr=13.4 min,
minor enantiomer tr=14.3 min.
Compliance with Ethical Standards
4.7 2‑(Hydroxy(4‑nitrophenyl)methyl)
cyclohexanone
Conflict of interest All authors of this paper are aware of the submis-
sion and agree to its publication and declare no conficts of interest.
1H NMR (400 MHz, CDCCl3): δ 8.21 (d, J=8.1 Hz, 2H),
7.50 (d, J=7.4 Hz, 2H), 5.49 (s, 0.45H), 4.90 (d, J=8.4 Hz,
0.45H), 4.08(s, 0.55H), 3.17(s, 0.47H), 2.64–2.56 (m,
J = 13.8 Hz, 1H), 2.54–2.45 (m, 1H), 2.44–2.38 (m,
J=19.3 Hz, 1H), 2.15−2.10 (d, J=12.8 Hz, 1H), 1.83 (d,
J=12.7 Hz, 1H), 1.73−1.45 (m, 4H). Enantiomeric excess
was determined by HPLC with a CHIRALPAK AD-H col-
umn (90:10 hexane:
Research Involving Human and Animal Participants This study does
not cover human participants and/or animal studies.
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