Ionic liquid as a recyclable and efficient medium for lipase-catalyzed asymmetric cross aldol reaction

Article abstract of DOI:10.1016/j.molcatb.2014.10.008

PPL (lipase from porcine pancreas) was found to catalyze asymmetric cross aldol reactions of aromatic and heteroaromatic aldehydes with various ketones in ionic liquid ([BMIM][PF₆]) for the first time. Interestingly, PPL exhibited high catalyti

Full text of DOI:10.1016/j.molcatb.2014.10.008

Journal of Molecular Catalysis B: Enzymatic 110 (2014) 100–110
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Ionic liquid as a recyclable and efficient medium for lipase-catalyzed
asymmetric cross aldol reaction
Yang Zhang, Na Wang∗, Zong-Bo Xie, Long-Hua Zhou, Xiao-Qi Yu∗
Key Laboratory of Green Chemistry & Technology, Ministry of Education, College of Chemistry, Sichuan University, Chengdu 610064, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
PPL (lipase from porcine pancreas) was found to catalyze asymmetric cross aldol reactions of aromatic and
heteroaromatic aldehydes with various ketones in ionic liquid ([BMIM][PF6]) for the first time. Interest-
ingly, PPL exhibited high catalytic activity and excellent stereoselectivity in this efficient and recyclable
room temperature ionic liquid in the presence of moderate water, similar to the results obtained in
organic solvents. High yields of up to 99%, excellent enantioselectivities of up to 90% ee, and good
diastereoselectivities of up to >99:1 dr were achieved.
Received 11 June 2014
Received in revised form
23 September 2014
Accepted 16 October 2014
Available online 24 October 2014
© 2014 Elsevier B.V. All rights reserved.
Keywords:
Biocatalytic promiscuity
Lipase
Aldol reaction
Stereoselectivity
Ionic liquids
1. Introduction
enzyme activity and stability [19]. Moreover, ILs have been used for
the synthesis of sugar derivatives and other special compounds,
Room temperature ionic liquids (ILs) have been regarded as
potentially attractive solvents in modern organic synthesis [1,2].
ILs have low vapour pressure, low flammability, high ionic conduc-
tivity and excellent chemical and thermal stability, and they are
easily recycled [3,4]. Moreover, the unique and attractive physico-
chemical properties of ILs, such as viscosity, polarity and solubility,
can be designed by selecting the appropriate cation and anion.
Thus, ILs have been widely used in synthesis and catalysis owing
to these unique properties [5,6]. Many excellent reviews have
also been published in the past decade [7,8]. In biocatalysis, ILs
have been used as solvents or co-solvents for enzyme-catalyzed
reactions in the past decade [9,10], and several biocatalytic reac-
tions, such as oxidation [11], hydrolysis [12], polymerization [13]
and transesterification [14,15], have been achieved in ILs. Russell
and co-workers first reported the enzyme-catalyzed reaction in IL
[BMIM][PF6] for the synthesis of Z-aspartame in 2000 [16]. With
the rapid development of biocatalysis in ILs, Rantwijk and Sheldon
published an excellent comprehensive review on the various issues
associated with biocatalysis in ILs in 2007 [17]. Recently, Goto and
colleagues reviewed the activation and stabilization of enzymes
in ILs [18]. Yang et al. researched the specific ion effects of ILs on
owing to the strong dissolution power of ILs [20]. In addition to
their uses as reaction solvents, ILs also have many other significant
applications in biocatalysis and enzyme engineering. ILs can also be
utilized as extraction solvents to stabilize and extract enzymes [21]
and can incorporate and covalent modify some enzymes or modify
the immobilized material of enzymes [22,23]. In fact, as excellent
solvents for biocatalysis, ILs provide many advantages over con-
ventional organic solvents, including high activity and selectivity
and better stability as well as better recoverability and recyclability
[24]. However, few reports regarding the biocatalytic promiscuity
of ILs as solvents compare to conventional biocatalysis reactions in
ILs exist [25]. Thus, the combination of ILs with more biocatalysis
reactions remains an important challenge.
Biocatalysis, as an eco-friendly and sustainable methodology for
organic synthesis due to its high efficiency, excellent selectivity
and mild reaction conditions, is widely recognized as a practi-
cal alternative to traditional organic synthesis [26,27]. However,
biocatalysts exhibit specific catalytic activity for distinctly differ-
ent chemical transformation of natural or non-natural substrates
[28,29]. This kind of catalytic promiscuity immensely broadens
the application of biocatalysts and provides an available tool
for organic synthesis. Recently, several elegant studies on enzy-
matic promiscuity have been reported, especially with regard
to hydrolases that catalyze unconventional reactions, such as
aldol reaction [30,31], Mannich reaction [32], Henry reaction [33],
∗
Corresponding authors. Tel.: +86 28 85415886; fax: +86 28 85415886.
E-mail addresses: wnchem@scu.edu.cn (N. Wang), xqyu@scu.edu.cn (X.-Q. Yu).
http://dx.doi.org/10.1016/j.molcatb.2014.10.008
1381-1177/© 2014 Elsevier B.V. All rights reserved.

Y. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 110 (2014) 100–110
101
Michael additions [34,35], Ugi reaction [36] and three-component
2. Results and discussion
aza-Diels–Alder reaction [37]. Among these reactions, the power-
ful and atom economical asymmetric aldol reaction was frequently
reported. Berglund and co-workers first demonstrated that wild-
type lipase B from Candida antarctica (CAL-B) and Ser105Ala mutant
CAL-B possess catalytic activity for aldol addition in cyclohex-
ane [30]. Guan and co-workers exhibited several asymmetric aldol
reactions catalyzed by different hydrolyases [38,39]. Lin and co-
workers also reported an aldol condensation reaction catalyzed
by acylase [40]. In our previous work, we demonstrated the first
lipase-catalyzed asymmetric aldol reaction between acetone and
4-nitrobenzaldehyde greatly promoted by water [31]. However,
almost all lipase-catalyzed aldol reactions were performed in the
traditional organic solvents in which the enzymes were found to
preserve their activities [41,42]. Hence, the search for more effi-
cient media for biocatalytic asymmetric aldol reactions and other
chemical transformations has become a considerable challenge.
Recently, we found that the 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 sufficiently cost-efficient [43]. An enzymatic
aldol reaction in phosphate–citrate buffer was then attempted,
only achieving an optimal enantioselectivity of 66% [44]. Based on
these previous works, we continued to seek a more efficient bio-
catalytic route for asymmetric aldol reactions. We selected lipase
from porcine pancreas (PPL) as a catalyst and utilized different
ILs as the reaction medium in the presence of water. Surpris-
ing, we obtained similar results to those achieved in organic
solvents. To the best of our knowledge, no other reports have
demonstrated lipase-catalyzed asymmetric cross aldol reaction in
ILs.
It is well known that enzyme activity is strongly affected by
reaction medium [42,45]. The feasibility of using ILs as efficient
solvents for biocatalysis is well documented. However, ILs are
rarely used in enzyme-catalyzed promiscuous reactions, such as
aldol reaction. Therefore, we endeavored to find a more efficient
medium combination with biocatalysis to meet the criteria of
sustainable conscious development. Recently, we reported a lipase-
catalyzed asymmetric aldol reaction in cyclohexanone, which is
one of the substrates [43]. Although the reaction produced good
yield and high selectivity, the excessive ketones were not suffi-
ciently cost-efficient. Furthermore, we continued to attempt this
enzymatic aldol reaction in phosphate–citrate buffer considering
that lipase could maintain good activity in aqueous solvents [44].
However, the best enantioselectivity achieved was only 66% ee.
Thus, we employed efficient ILs as the medium for the asymmet-
ric aldol reaction catalyzed by PPL. The aldol reaction between
4-nitrobenzaldehyde and cyclohexanone was used as a model reac-
tion. The initial experiments were performed in various pure ILs,
including five tetrafluoroborate ILs (Table 1, entries 1–5), three
hexafluorophosphate ILs (Table 1, entries 6–8) and two chiral ILs
(Table 1, entries 9 and 10). Unfortunately, PPL showed extremely
poor catalytic activity in all of the tested RTILs, as the products
were difficult to detect by thin-layer chromatography (TLC) or
high-performance liquid chromatography (HPLC). The most likely
reason for such low yields was that the high viscosity of the IL
systems might be limiting the mass transfer of substrates and
products to and from the active site of the enzyme [46]. In prin-
ciple, high viscosity could be overcome through the addition of
small amounts of other solvents as additives to the IL medium.
According to our previous research, water plays an important role
in lipase-catalyzed promiscuous aldol reaction. For most reaction
systems, a water content of approximately 10% has produced the
best results. Thus, we added 10% water to the ILs to investigate
the optimal IL for this reaction. To our surprise, when 10% water
was added to the ILs, we detected the aldol product in several
ILs. As shown in Table 1, PPL exhibited good catalytic activity in
all tested ILs. For the most utilized ILs, both hexafluorophosphate
and tetrafluoroborate ILs showed that ILs with shorter alkyl chains
on their cations promoted better enzyme activity. The two chiral
ILs also induced high activity, and 1-butyl-3-methylimidazolium
As a part of our continuing research on enzyme promiscu-
ity, we wish to report a lipase-catalyzed the asymmetric cross
aldol reaction between aromatic aldehydes and cyclic ketones
under mild conditions using an IL medium. Interestingly, we
also observed that the presence of water greatly promoted the
catalytic activity and selectivity of PPL, which was consistent
with our previous report [31]. High yields and good enantios-
electivities and diastereoselectivities were also obtained while
simultaneously investigating a wide variety of substrates. Mean-
while, the good reusability of ILs as the medium for PPL was also
observed.
Table 1
The effect of RTILs on the PPL-catalyzed asymmetric cross aldol reaction.a
Entry
RTILs
Catalyst
Yield%b
dr(anti/syn)b
ee%(anti)b
1
2
3
4
5
6
7
8
9
10
11
12
13
[EMIM][BF4]
[BMIM][BF4]
[HMIM][BF4]
[OMIM][BF4]
[EOMIM][BF4]
[BMIM][PF6]
[HMIM][PF6]
[OMIM][PF6]
[BMIM][LTar]
[BMIM][LLac]
[BMIM][PF6]
[BMIM][PF6]
[BMIM][PF6]
PPL
PPL
PPL
PPL
PPL
PPL
PPL
PPL
80
77
71
68
55
63
45
39
84
76
5
80:20
83:17
79:21
79:21
78:22
86:14
64:36
85:15
48:52
47:53
–
57
58
53
53
68
80
38
77
8
3
–
–
–
PPL
PPL
Bovine serum albumin (BSA)
No enzyme
1
1
–
–
Denatured PPLc
a
Conditions: 4-nitrobenzaldehyde (0.1 mmol), cyclohexanone (1.0 mmol), and PPL (10 mg) in RTILs with deionized water (10%, water/([BMIM][PF6] + water), v/v) at 50 ◦
C
for 24 h.
b
Yield, ee, and dr were determined by HPLC using AD-H chiral column.
Pretreated with guanidine hydrochloride solution (6 mol/L).
c

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Y. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 110 (2014) 100–110
l-tartrate ([BMIM][LTar]) showed the best catalytic activity with
yield of 84%. The reason for these yields may be that the ILs are
highly polar, and the cations with shorter alkyl chains have higher
polarity, which increased the solubility of the substrates and led
to more efficient and faster reactions [47]. However, 1-butyl-3-
methylimidazolium hexafluorophosphate ([BMIM][PF6]) exhibited
the highest selectivity (80% ee) and a good yield at 63% (Table 1,
entry 6). The biphasic (water content of 10% in [BMIM][PF6] is
two phase system) [BMIM][PF6]/H2O most likely maintained PPL’s
conformational flexibility, promoting the reaction. Thus, we chose
[BMIM][PF6]/H2O as the reaction medium for further reaction
investigations.
To demonstrate the specific catalytic effect of PPL on the aldol
reaction in the special media, some control experiments were con-
ducted in the same IL system (Table 1, entries 11–13). No product
could be detected in the absence of lipase. The non-enzyme pro-
teins bovine serum albumin (BSA) [48,49], which has a surface
amino acid distribution similar to that of many soluble enzymes,
also demonstrated very low activity with only a trace of product
obtained. We obtained a similar result to that of the blank reaction
after denatured PPL was employed as the reaction catalyst. Thus,
the possibility that the catalysis mainly arose from amino acids of
the protein was absolutely excluded. All these facts suggest that
the special spatial conformation of the enzyme and the specific
catalytic site of PPL are essential in catalyzing the aldol reaction.
The good results obtained with the initial use of [BMIM][PF6]
as an efficient reaction medium for lipase-catalyzed aldol reac-
tion encouraged us to expect better results after optimizing
the various reaction conditions. Thus, experiments were per-
formed to screen for the optimal biocatalyst for the aldol reaction
between 4-nitrobenzaldehyde and cyclohexanone in the biphasic
[BMIM][PF6]/H2O medium (Table S1). The results showed that PPL
promoted the highest biocatalyst activity among the tested seven
lipases, including lipase from porcine pancreas (PPL), lipase from C.
antarctica B (CAL-B), lipase from Mucor miehei (MML), lipase from
Candida cylindracea (CCL), lipase from Mucor javanicus (MJL), lipase
from Penicillium camemberti (PCL) and lipase from Rhizopus oryzae
(ROL). The other lipases exhibited very low activities with little
yield. The results are consistent with our previous works obtained
in organic solvents or buffer. Thus PPL appears to be the best bio-
catalyst for aldol reaction in the unique [BMIM][PF6]/H2O medium.
Water content dramatically influences the activity, stability
and probably tertiary structure of the enzymes [50]. Thus, the
water content of reaction system was investigated further. Water
contents ranging from 0% to 100% (water/([BMIM][PF6] + water),
v/v) in [BMIM][PF6] were screened, and the results are shown
in Fig. 1. The water content was found to greatly influence
the activity and selectivity of PPL for the aldol reaction. Both
good yields and stereoselectivities were obtained when the water
content ranged from 6% to 10%. PPL exhibited the best enan-
tioselectivity and diastereoselectivity with a water content of 8%
(water/([BMIM][PF6] + water), v/v), which produced a 71% yield
with 87:13 dr and 80% ee. As shown in Fig. 1, in the absence
of water, very little products were obtained. Interestingly, when
the water content was greater than 15%, the ee and dr values
declined sharply, and the product yield decreased to a low level.
However, the enzyme exhibited the highest catalytic activity in
the pure water with an 87% yield and poor selectivity. To obtain
the best diastereoselectivity and enantioselectivity, 8% water con-
tent was chosen as the optimum addition for the PPL-catalyzed
aldol reaction. In previous reports concerning enzyme-catalyzed
aldol reactions, nearly all reactions were optimized for water
content. For example, we reported the first lipase-catalyzed asym-
metric aldol reaction between acetone and 4-nitrobenzaldehyde
greatly promoted by adding water with an optimum water content
of 20% [31]. Guan et al. also found that nuclease-catalyzed aldol
Fig. 1. The effect of water content on the PPL-catalyzed asymmetric cross
aldol reaction.a [a] Conditions: 4-nitrobenzaldehyde (0.1 mmol), cyclohexanone
(1.0 mmol), and PPL (10 mg) in [BMIM][PF6] with deionized water (0–100%,
water/([BMIM][PF6] + water), v/v) at 50 ◦C for 24 h. [b] Yield, ee, and dr were deter-
mined by HPLC using AD-H chiral column.
reactions required an optimized water content of 15% [38], and the
chymopapain-catalyzed direct asymmetric aldol reaction had an
optimum water content of 12% [39]. From the discussion above, we
may conclude that a reaction system with an optimized water con-
tent may maintain the best conformational flexibility of the enzyme
and thereby produce the best results.
Next, we examined the influence of enzyme loading on the
model reaction. As shown in Fig. 2, the yield and the stereoselectiv-
ity were significantly influenced by different enzyme loading that
ranged from 2 mg to 80 mg. The best yields were obtained at 25 mg
or 30 mg (74%). However, low stereoselectivity was observed. Thus,
10 mg of PPL was selected as the optimal quantity for the model
reaction of 4-nitrobenzaldehyde and cyclohexanone, having better
stereoselectivity and approximate yield, considering stereoselec-
tivity and cost-efficient amount of enzyme. As shown, a small
decline in yield and diastereoselectivity was observed, and the
enantioselectivity changed very little when the enzyme loading
Fig. 2. The effect of enzyme loading on the PPL-catalyzed asymmetric cross
aldol reaction.a [a] Conditions: 4-nitrobenzaldehyde (0.1 mmol), cyclohexanone
(1.0 mmol), and PPL (2, 5, 10, 15, 20, 25, 30, 40, 50, 80 mg/mL) in [BMIM][PF6]
(920 ␮L) with deionized water (80 ␮L) at 50 ◦C for 24 h. [b] Yield, ee, and dr were
determined by HPLC using AD-H chiral column. [c] dr = 10 × (anti/syn).

Y. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 110 (2014) 100–110
103
Table 2
The effect of molar ratio on the PPL-catalyzed asymmetric cross aldol reaction.a
Entry
Molar ratiob
Yield%c
dr(anti/syn)c
ee%(anti)c
1
2
3
4
5
6
7
8
9
1:1
1:5
10
42
70
81
90
89
91
92
91
87:13
87:13
87:13
87:13
87:13
87:13
87:13
87:13
87:13
79
80
80
80
80
79
80
78
78
1:10
1:15
1:20
1:25
1:30
1:40
1:50
a
Conditions:4-nitrobenzaldehyde(0.1 mmol), cyclohexanone (0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0 mmol), and PPL (10 mg) in [BMIM][PF6] (920 ␮L) with deionized water
(80 ␮L) at 50 ◦C for 24 h.
b
Molar ratio = 4-nitrobenzaldehyde/cyclohexanone.
Yield, ee, and dr were determined by HPLC using AD-H chiral column.
c
was largely increased. During the reaction process, when a large
amount of PPL was added to the system, the aggregation of some of
the enzyme was clearly observed. This aggregation behavior may
be due to the viscosity of the IL, preventing the large enzyme from
dispersing efficiently in the reaction system. On the other hand, due
to the IL’s viscosity, the enzyme activity was controlled by affect-
ing the mass-transfer limitations [46]. Consequently, more enzyme
loading did not favor the lipase-catalyzed reaction.
Encouraged by the above results, other influencing factors were
screened to obtain higher yields and stereoselectivities. The effect
of the molar ratio of the substrates on the PPL-catalyzed aldol
reaction was investigated. As shown in Table 2, the molar ratio
substantially influenced the reaction yield. When the molar ratio
of 4-nitrobenzaldehyde to cyclohexanone was varied from 1:1
to 1:20, the yield was enormously improved from 10% to 90%.
However, no obvious difference was observed in the yield after
increasing the mole ratio from 1:20 to 1:50. The diastereose-
lectivity and enantioselectivity were maintained throughout the
process of testing the effect of the substrate molar ratio on the
reaction between 4-nitrobenzaldehyde and cyclohexanone. The
probable reason for these observations is that the reaction reached
equilibrium when the molar ratio of 4-nitrobenzaldehyde to cyclo-
hexanone was 1:20, therefore, no obvious improvements were
observed when the molar ratio was increased. Thus, 1:20 was
selected as the optimal molar ratio for the aldol reaction.
To obtain better results from this lipase-catalyzed asymmetric
cross aldol reaction, we also investigated the reaction temperature
and duration combinations. We performed the model reaction at
five different temperatures that ranged from 20 ◦C to 70 ◦C for 6 h,
12 h, 24 h, 48 h, 72 h and 120 h. As shown in Table S2, the reac-
tion at 37 ◦C for 72 h offered the best result with yields of up to
93% and the highest enantioselectivity (85% ee) and diastereos-
electivity (91:9 dr). At the higher temperature (50 ◦C and 70 ◦C),
and by prolonging the reaction time, both the enzyme activity and
the stereoselectivity decreased, in particular, the stereoselectivity
dropped sharply. It was most likely that the optimum conformation
of the enzyme appeared at the optimum temperature, and unfavor-
able conformation occurred at higher temperature. However, at the
lower temperature (20 ◦C and 30 ◦C), the enzyme activity was too
low. To obtain the best yield, diastereoselectivity and enantiose-
lectivity, the reaction temperature of 37 ◦C and time of 72 h were
chosen for the PPL-catalyzed aldol reaction.
enzyme was not easy to recycle and recover because a portion of
the enzyme usually collected on the wall of the round-bottom flask
or aggregated or dispersed into the IL. Thus, recycling and recover-
ing free PPL was difficult. However, the further research on using
immobilized PPL for recycling is in progress. We then recovered and
recycled only the IL [BMIM][PF6] using the model reaction. Amaz-
ingly, an unexpected result was observed. As shown in Table 3,
the recovered IL was recycled for five runs with no loss in enzyme
activity or stereoselectivity under the same reaction conditions.
[BMIM][PF6], itself is a widely used IL in biocatalysis, can undoubt-
edly broaden its applications in many aspects due to its excellent
recyclability and recoverability. Over the past decade, the field of
ionic liquids has grown at an unpredictable rate. Due to their unique
and tunable physicochemical properties, many applications of ILs
have been reported, such as the application in chemical industry
and pharmaceutical industry [7,51]. At the same time, we are con-
fident that ILs will enable more novel applications that are not
possible in conventional solvents in the future and will be widely
used in numerous fields including biocatalysis, electrochemistry,
physical chemistry, engineering and so on.
With the optimal reaction conditions in hand, we further
studied the scope and the generality of this biocatalytic promis-
cuity. Several substrates were used to expand the PPL-catalyzed
aldol reaction. As shown in Table 4, both aromatic and het-
eroaromatic aldehydes with cyclic or heterocyclic ketones or with
acetone gave moderate to high yields, good enantioselectivities and
diastereoselectivities in the biphasic [BMIM][PF6]/H2O medium
(H2O/([BMIM][PF6] + H2O) = 8%, v/v). A maximum yield of 99%, opti-
mal diastereoselectivity of >99:1 dr and best enantioselectivity of
90% ee were achieved using this medium. Generally, benzaldehy-
des with strong electron-withdrawing substituents served as better
Table 3
Recycling studies for the aldol reaction of the model reaction.a
Number of cycles
Yield%b
dr(anti/syn)c
ee%c
1
2
3
4
5
92
93
93
93
92
92:8
91:9
91:9
90:10
92:8
86
85
85
84
86
a
At the beginning, the reaction was carried out using 4-nitrobenzaldehyde
(0.3 mmol), cyclohexanone (6.0 mmol), and PPL (30 mg) in [BMIM][PF6] (2760 ␮L)
with deionized water (240 ␮L) at 37 ◦C for 72 h.
To further demonstrate the advantage of ILs as the medium for
this enzymatic reaction, we recycled the biocatalyst and the media
together. After some recycling experiments, we found that the free
b
Yield of the isolated product after chromatography on silica gel.
ee, and dr were determined by HPLC using AD-H chiral column.
c

104
Y. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 110 (2014) 100–110
Table 4
Substrate scope of the PPL-catalyzed asymmetric cross aldol reaction.a
Entry
1
Product
Time (h)
240
Yield%b
37
dr(anti:syn)c
76:24
ee%(anti)c
80
2
3
240
192
45
38
84:16
83:17
77
89
4
5
72
99
84
94:6
69
87
168
87:13
6
7
8
9
192
96
60
72
96
48
86:14
99:1
82
88
67
90
100
84
>99:1
87:13
10
192
74
87:13
84

Y. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 110 (2014) 100–110
105
Table 4 (Continued)
Entry
Product
Time (h)
168
Yield%b
88
dr(anti:syn)c
85:15
ee%(anti)c
11
80
12
13
14
120
72
83
83
93
93:7
91:9
91:9
80
88
85
72
15
16
192
89
92
84:16
85:15
85
66
72
17
18
72
85
35
70:30
50:50
75
240
25/8d
19
20
21
288
170
192
41
88
85
–
35
74
74
71:29
72:28

106
Y. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 110 (2014) 100–110
Table 4 (Continued)
Entry
Product
Time (h)
192
Yield%b
80
dr(anti:syn)c
80:20
ee%(anti)c
22
79
a
Reaction conditions: aromatic aldehyde (0.3 mmol), ketone (6 mmol) and PPL (30 mg) in [BMIM][PF6] (2760 ␮L) with deionized water (240 ␮L) at 37 ◦C.
Yield of the isolated product after chromatography on silica gel.
dr and ee were determined by HPLC using a chiral column.
ee of anti/ee of syn.
b
c
d
acceptors, producing better yields (Table 4, entries 3–14). The
2,4-dichlorobenzaldehyde promoted the highest enantioselectiv-
ity of 88% with high diastereoselectivity of 99:1 dr (Table 4,
entry 7). While, the least sterically hindered compound 4-
chlorobenzaldehyde did not promote good results (Table 4, entry
6). In addition, to further examine the generality of this catalytic
system, a heteroaromatic aldehyde, 3-pyridylbenzaldehyde was
accepted by PPL as a substrate giving a good yield of 89% with
moderate diastereoselectivity and enantioselectivity (Table 4, entry
15). Heterocyclic ketones were also accepted by PPL as substrates,
Affording good yields and moderate stereoselectivities (Table 4,
entries 20–22). Furthermore, in order to demonstrate advantages of
this approach we have scaled up this reaction to the industrial scale.
The reaction was scaled up using 1.057 g of 4-nitrobenzaldehyde
(7 mmol), obtaining a fine results with yields of up to 80% and
almost no changed enantioselectivity (83% ee) and diastereoselec-
tivity (90:10 dr) determined by HPLC using a chiral column. All of
these results may illustrate that PPL in [BMIM][PF6] with 8% water
content exhibited outstanding activity and stereoselectivity.
Finally, we proposed a possible mechanism for the lipase-
catalyzed asymmetric cross aldol reaction using the biphasic
[BMIM][PF6]/H2O medium. PPL is a small globular protein com-
posed of a single chain of 449 amino acids and contains a catalytic
triad of Ser153, Asp177 and His264 in its active site [52]. On
the basis of the previously proposed mechanism of promiscuous
hydrolase-catalyzed aldol reaction [30,31,43], we hypothesized a
possible mechanism for the PPL-catalyzed aldol reaction, as shown
in Scheme 1. First, substrate cyclohexanone is stabilized by the
reactions of aromatic aldehydes with strong electron-withdrawing
substituents provided products in the highest yield of 99%, the
best diastereoselectivity of >99:1 dr and the best enantioselec-
tivity of 90% ee (Table 4, entries 4, 8 and 9). However, for
aromatic aldehydes with electron-donating substituents or no
substituents, such as 4-methyl benzaldehyde and benzaldehyde,
the yields were very low (Table 4, entries 1 and 2). Further-
more, we examined the effect of various ketones. Cyclopentanone
afforded the products in good yields and moderate stereoselec-
tivity (Table 4, entries 16 and 17). However, both cycloheptanone
and acetone produced poor yields and stereoselectivities (Table 4,
entries 18 and 19). In terms of yield and stereoselectivity, cyclo-
hexanone promoted the best results. Moreover, the substituent
positions on the benzene on benzaldehydes had a great impact
on the yield of the reaction. For instance, the aldol reaction
of 4-nitrobenzaldehyde with cyclohexanone generated the aldol
product with a higher yield than that of 2-nitrobenzaldehyde
and 3-nitrobenzaldehyde. However, 3-nitrobenzaldehyde had
the best enantioselectivity of 88% ee (Table 4, entries 12–14).
Meanwhile, the steric hindrance of substituents on benzalde-
hyde had a great influence on the yield and stereoselectivity
of the reaction. For example, among 2,6-dichlorobenzaldehyde,
2,4-dichlorobenzaldehyde, 4-chlorobenzaldehyde, the most hin-
dered substrate 2,6-dichlorobenzaldehyde promoted the highest
diastereoselectivity (>99:1 dr) and yield (96%) with a moder-
ate enantioselectivity of 67% ee (Table 4, entry 8). Additionally,
Scheme 1. Proposed mechanism for PPL-catalyzed aldol reaction.

Y. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 110 (2014) 100–110
107
Asp-His dyad and the oxyanion. Second,
a
proton is trans-
4.2. General procedure for aldol reaction
ferred from cyclohexanone to the His residue, forming an
enolate ion. Third, another substrate aldehyde accepts the pro-
ton from the His264 imidazolyl and forms a new C C bond
with cyclohexanone. Finally, the aldol adduct is released from
the oxyanion hole and separates from the active site. A fur-
ther mechanism study about this reaction is currently in
progress.
Aldehyde (0.3 mmol), PPL (30 mg), ketone (6.0 mmol),
[BMIM][PF6] (2.76 mL) and deionized water (0.24 mL) were
added in a 25 mL round-bottom flask. The mixture was stirred at
1000 rpm at 37 ◦C. After completion of the reaction, the product
was directly analyzed by HPLC in comparison with a standard
sample. Then, the solution was extracted with diethyl ether
(10 mL) for eight times. The organic phase was combined and
evaporated in vacuum. The residue was purified by flash column
chromatography using ethyl acetate–petroleum ether as mobile
phase.
3. Conclusions
In summary, we develop a new efficient biocatalytic route
for the PPL-catalyzed asymmetric aldol reaction in the biphasic
[BMIM][PF6]/H2O medium for the first time. PPL exhibited high
catalytic activity and excellent stereoselectivity in this efficient
and recyclable room temperature ionic liquid in the presence of
moderate water, similar to the results obtained in organic sol-
vents. To optimize the PPL-catalyzed asymmetric aldol reaction,
some important factors, including solvent, enzyme, water con-
tent, temperature and reaction duration were investigated. This
catalytic system provides a wide reaction scope and is general.
Compared to benzaldehydes with electron-donating groups or
no groups, benzaldehydes with electron-withdrawing groups dis-
played better activity and stereoselectivity. Cyclohexanone could
act as a more suitable donor accepted by the enzyme to react with
different aromatic or heteroaromatic aldehydes. Heteroaromatic
aldehyde with cyclohexanone and the heterocyclic ketones with
aromatic aldehydes also promoted good reaction results. Excitingly,
[BMIM][PF6] exhibited excellent recyclability and recoverability,
as the recovered IL was recycled for five runs with no loss in
PPL’s activity or stereoselectivity. As an effective and efficient syn-
thetic method, the PPL-catalyzed asymmetric aldol reaction in
[BMIM][PF6]/H2O provides a novel case of catalytic promiscuity and
might be a useful strategy for organic synthesis and even industrial
applications.
4.3. General procedure for recovering and recycling [BMIM][PF6]
4-Nitrobenzaldehyde (0.3 mmol), PPL (30 mg), cyclohexanone
(6.0 mmol), [BMIM][PF6] (2.76 mL) and deionized water (0.24 mL)
were combined in a 25 mL round-bottom flask. The mixture was
stirred at 1000 rpm at 37 ◦C for 72 h. The solution was then
extracted eight times with diethyl ether (10 mL) until the reac-
tion substrates and products had been completely extracted, as
monitored via TLC and visualized using UV light or phosphomolyb-
dic acid ethanol solution. Afterwards, the IL layer was dissolved in
dichloromethane, filtered and evaporated under vacuum. After 12 h
in the vacuum drying oven, the recovered and recycled ionic liquid
was used in the next reaction.
4.3.1. 2-(Hydroxy(phenyl)methyl)cyclohexanone (a)
White solid; mp: 100–101 ◦C; 1H NMR (400 MHz, CDCl3): ı
7.4–7.24 (m, 5H), 5.41 (d, J = 2.2 Hz, 0.24H), 4.81 (d, J = 8.8 Hz, 0.74H),
4.00 (s, 1H), 2.65–2.61 (m, 1H), 2.52–2.49 (m, 1H), 2.42–2.34 (m,
1H), 2.15–2.05 (m, 1H), 1.84–1.76 (m, 1H), 1.72–1.51 (m, 3H),
1.36–1.26 (m, 1H); 13C NMR (100 MHz, CDCl3): ı 215.55, 214.80,
140.94, 128.38, 127.90, 127.03, 74.76, 57.44, 42.68, 30.86, 27.82,
24.73. Enantiomeric excess was determined by HPLC with a CHI-
RALCEL OD-H column (90:10 hexane:2-propanol), 25 ◦C, 220 nm,
0.5 mL/min; major enantiomer tr = 16.4 min, minor enantiomer
tr = 22.7 min.
4. Experimental
4.3.2. 2-(Hydroxy(4-tolyl)methyl)cyclohexanone (b)
Yellow solid; mp: 75–77 ◦C; 1H NMR (400 MHz, CDCl3): ı 7.20
(d, J = 7.5 Hz, 2H), 7.15 (d, J = 7.5 Hz, 2H), 5.35 (s, 0.15H), 4.75 (d,
J = 8.8 Hz, 0.95H), 3.91 (s, 1H), 2.64–2.57 (m, 1H), 2.50–2.46 (m, 1H),
2.42–2.30 (m, 4H), 2.10–2.06 (m, 1H), 1.80–1.49 (m, 5H); 13C NMR
(100 MHz, CDCl3): ı 215.62, 214.85, 138.49, 137.98, 137.55, 136.54,
129.05, 126.93, 74.54, 70.56, 57.45, 42.69, 30.89, 27.84, 24.74, 21.16.
Enantiomeric excess was determined by HPLC with a CHIRALPAK
AS-H column (95:5 hexane:2-propanol), 25 ◦C, 221 nm, 0.8 mL/min;
major enantiomer tr = 23.4 min, minor enantiomer tr = 30.3 min.
4.1. Materials and analytical methods
All enzymes were purchased from Sigma–Aldrich Co. LLC (US),
BSA was purchased from Aladdin Co., Ltd. (Shanghai, China).
1-Butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4]), 1-
hexyl-3-methylimidazolium hexafluorophosphate ([HMIM][PF6]),
1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM]
[PF6]), 1-hexyl-3-methylimidazolium tetrafluoroborate ([HMIM]
[BF4]),
([OMIM][PF6]), 1-ethyl-3-methylimidazolium tetrafluoroborate
([EMIM][BF4]), 1-octyl-3-methylimidazolium tetrafluorobo-
rate ([OMIM][BF4]), 1-butyl-3-methylimidazolim l-tartrate
([BMIM][LTar]), 1-butyl-3-methylimidazolium l-lactate
1-octyl-3-methylimidazolium
hexafluorophosphate
4.3.3. 2-((4-Fluorophenyl)(hydroxy)methyl)cyclohexanone (c)
White solid; mp: 74–76 ◦C; 1H NMR (400 MHz, CDCl3): ı
7.34–7.25 (m, 2H), 7.05 (t, J = 8.7 Hz, 2H), 5.37 (s, 0.18H), 4.79 (d,
J = 8.8 Hz, 0.85H), 4.04 (s, 1H), 2.62–2.58 (m, 1H), 2.52–2.48 (m, 1H),
2.43–2.32 (m, 1H), 2.13–2.09 (m, 1H), 1.83–1.80 (m, 1H), 1.73–1.51
(m, 3H), 1.35–1.27 (m, 1H); 13C NMR (100 MHz, CDCl3): ı 215.46,
214.76, 163.59, 161.15, 136.75, 128.63, 115.34, 115.13, 74.12, 57.46,
42.67, 30.76, 27.75, 24.71. Enantiomeric excess was determined by
HPLC with a CHIRALPAK AD-H column (95:5 hexane:2-propanol),
25 ◦C, 210 nm, 1.0 mL/min; major enantiomer tr = 22.2 min, minor
enantiomer tr = 19.7 min.
([BMIM][LLac]), 1-hydroxyethyl-3-methylimidazolium tetrafluo-
roborate ([EOMIM][BF4]) were purchased from ShangHai Cheng
Jie Chemical Co. Ltd. (China). Unless otherwise noted, all reagents
were obtained from commercial suppliers and were used without
further purification.
The NMR spectra were recorded on a Bruker 400 MHz instru-
ment using CDCl3 as solvent. Chemical shifts (ı) were expressed in
ppm with TMS as internal standard, and coupling 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). The melting points were measured by Microcomputer
melting point meter (WRS-2) which was produced by ShangHai
Shenguang Instrument Co. Ltd. (China).
4.3.4. 2-(Hydroxy(perfluorophenyl)methyl)cyclohexanone (d)
Yellow solid; mp: 81–82 ◦C; 1H NMR (400 MHz, CDCl3): ı 5.33
(d, J = 9.6, 2.9 Hz, 1H), 3.96 (d, J = 3.3 Hz, 1H), 3.05–2.98 (m, 1H),
2.56–2.51 (m, 1H), 2.44–2.39 (m, 1H), 2.18–2.13 (m, 1H), 1.90–1.86

108
Y. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 110 (2014) 100–110
(m, 1H), 1.71–1.60 (m, 3H), 1.39–1.33 (m, 1H); 13C NMR (100 MHz,
CDCl3): ı 214.12, 211.81, 146.50, 143.98, 142.05, 139.58, 138.78,
136.28, 65.96, 54.20, 42.35, 30.16, 27.49, 24.49. Enantiomeric
excess was determined by HPLC with a CHIRALPAK AD-H col-
umn (90:10 hexane:2-propanol), 25 ◦C, 215 nm, 0.5 mL/min; major
enantiomer tr = 14.0 min, minor enantiomer tr = 17.1 min.
HPLC with a CHIR-ALPAK AD-H column (98:2 hexane:2-propanol),
25 ◦C, 220 nm, 1.0 mL/min; major enantiomer tr = 28.6 min, minor
enantiomer tr = 32.8 min.
4.3.10. 2-((4-Bromophenyl)(hydroxy)methyl)cyclohexanone (j)
Yellow solid; mp: 114–117 ◦C; 1H NMR (400 MHz, CDCl3): ı
7.47 (d, J = 8.4 Hz, 2H), 7.20 (d, J = 8.3 Hz, 2H), 5.34 (s, 0.05H), 4.75
(dd, J = 8.7, 2.7 Hz, 0.99H), 3.99 (d, J = 2.7 Hz, 1H), 2.62–2.51 (m,
1H), 2.49–2.45 (m, 1H), 2.39–2.34 (m, 1H), 2.11–2.07 (m, 1H),
1.88–1.74 (m, 1H), 1.71–1.50 (m, 3H), 1.31–1.27 (m, 1H); 13C NMR
(101 MHz, CDCl3): ı 215.22, 214.52, 140.03, 131.47, 128.73, 121.71,
74.17, 57.32, 42.65, 30.75, 27.72, 24.70. Enantiomeric excess was
determined by HPLC with a CHIRALPAK AD-H column (90:10
hexane:2-propanol), 25 ◦C, 220 nm, 0.5 mL/min; major enantiomer
tr = 30.3 min, minor enantiomer tr = 26.1 min.
4.3.5.
2-(Hydroxy(4-(trifluoromethyl)phenyl)methyl)cyclohexanone (e)
White solid; mp: 79–81 ◦C; 1H NMR (400 MHz, CDCl3): ı 7.62
(d, J = 8.1 Hz, 2H), 7.46 (d, J = 8.0 Hz, 2H), 5.46 (s, 0.13H), 4.87 (d,
J = 8.6 Hz, 0.87H), 4.08 (s, 1H), 2.65–2.58 (m, 1H), 2.52–2.49 (m, 1H),
2.41–2.34 (m, 1H), 2.14–2.09 (m, 1H), 1.84–1.81 (m, 1H), 1.73–1.67
(m, 1H), 1.61–1.55 (m, 2H), 1.40–1.33 (m, 1H); 13C NMR (100 MHz,
CDCl3): ı 215.12, 214.41, 144.96, 130.22, 129.89, 127.37, 126.08,
125.31, 74.26, 57.26, 42.67, 30.76, 27.71, 24.70. Enantiomeric
excess was determined by HPLC with a CHIRALPAK AD-H column
(98:2 hexane:2-propanol), 25 ◦C, 220 nm, 1.0 mL/min; major enan-
tiomer tr = 39.2 min, minor enantiomer tr = 27.7 min.
4.3.11. 4-(Hydroxy(2-oxocyclohexyl)methyl)benzonitrile (k)
Yellow solid; mp: 69–70 ◦C; 1H NMR (400 MHz, CDCl3): ı 7.64 (d,
J = 6.9 Hz, 2H), 7.46–7.42 (m, 2H), 5.43 (s, 0.17H), 4.84 (d, J = 8.4 Hz,
0.84H), 4.07 (s, 1H), 2.61–2.54 (m, 1H), 2.51–2.47 (m, 1H), 2.40–2.34
(m, 1H), 2.13–2.10 (m, 1H), 1.82 (d, J = 12.9 Hz, 1H), 1.71–1.50
(m, 3H), 1.40–1.25 (m, 1H); 13C NMR (100 MHz, CDCl3): ı 214.75,
214.03, 146.40, 132.09, 127.77, 118.77, 111.68, 74.19, 57.13, 42.62,
30.72, 27.73, 24.72. Enantiomeric excess was determined by HPLC
with a CHIRALPAK AD-H column (80:20 hexane:2-propanol), 25 ◦C,
220 nm, 0.5 mL/min; major enantiomer tr = 26.2 min, minor enan-
tiomer tr = 21.7 min.
4.3.6. 2-((4-Chlorophenyl)(hydroxy)methyl)cyclohexanone (f)
Yellow solid; mp: 91–93 ◦C; 1H NMR (400 MHz, CDCl3):ı
7.35–7.32 (t, J = 6.2 Hz, 2H), 7.28 (d, J = 8.4 Hz, 2H), 5.38 (s, 0.19H),
4.79 (d, J = 8.7 Hz, 0.86H), 4.01 (s, 1H), 2.64–2.55 (m, 1H), 2.53–2.48
(m, 1H), 2.42–2.33 (m, 1H), 2.19–2.04 (m, 1H), 1.91–1.77 (m, 1H),
1.70–1.56 (m, 3H), 1.35–1.26 (m, 1H); 13C NMR (100 MHz, CDCl3): ı
215.23, 214.52, 139.51, 133.57, 128.52, 128.38, 74.11, 57.36, 42.65,
30.75, 27.72, 24.70. Enantiomeric excess was determined by HPLC
with a CHIRALPAK AD-H column (90:10 hexane:2-propanol), 25 ◦C,
221 nm, 0.5 mL/min; major enantiomer tr = 27.9 min, minor enan-
tiomer tr = 24.4 min.
4.3.12. 2-(Hydroxy(2-nitrophenyl)methyl)cyclohexanone (l)
Yellow solid; mp: 97–99 ◦C; 1H NMR (400 MHz, CDCl3): ı 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, 0.08H), 5.45 (d, J = 7.0 Hz, 0.94H), 4.19
(s, 1H), 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, 5H); 13C NMR (100 MHz,
CDCl3): ı 214.89, 213.95, 148.75, 136.60, 133.04, 129.00, 128.39,
124.06, 69.76, 57.31, 42.82, 31.11, 27.75, 24.97. Enantiomeric
excess was determined by HPLC with a CHIRALCEL OD-H column
(95:5 hexane:2-propanol), 25 ◦C, 254 nm, 0.5 mL/min; major enan-
tiomer tr = 30.6 min, minor enantiomer tr = 35.8 min.
4.3.7. 2-((2,4-Dichlorophenyl)(hydroxy)methyl)cyclohexanone
(g)
White solid; mp: 89–91 ◦C; 1H NMR (400 MHz, CDCl3) ı 7.51
(d, J = 8.4 Hz, 1H), 7.37 (d, J = 2.1 Hz, 1H), 7.32–7.28 (m, 1H), 5.31
(dd, J = 8.0, 3.6 Hz, 1H), 4.08 (d, J = 3.9 Hz, 1H), 2.67–2.61 (m, 1H),
2.50–2.47 (m, 1H), 2.41–2.27 (m, 1H), 2.13–2.09 (m, 1H), 1.85 (d,
J = 6.0 Hz, 1H), 1.71–1.55 (m, 4H); 13C NMR (100 MHz, CDCl3): ı
215.10, 137.82, 133.79, 133.50, 129.26, 128.90, 127.63, 70.09, 57.51,
42.71, 30.37, 27.76, 24.89. Enantiomeric excess was determined by
HPLC with a CHIRALPAK AS-H column (90:10 hexane:2-propanol),
25 ◦C, 220 nm, 1.0 mL/min; major enantiomer tr = 11.0 min, minor
enantiomer tr = 9.4 min.
4.3.13. 2-(Hydroxy(3-nitrophenyl)methyl)cyclohexanone (m)
Yellow solid; mp: 110–111 ◦C; 1H NMR (400 MHz, CDCl3): ı
8.31–8.06 (m, 2H), 7.68 (d, J = 7.5 Hz, 1H), 7.53 (t, J = 7.9 Hz, 1H),
5.48 (s, 0.08H), 4.90 (d, J = 8.4 Hz, 0.93H), 4.13 (s, 1H), 2.72–2.57 (m,
1H), 2.51 (d, J = 13.8 Hz, 1H), 2.45–2.31 (m, 1H), 2.13 (d, J = 10.9 Hz,
1H), 1.84 (d, J = 12.7 Hz, 1H), 1.73–1.34 (m, 4H); 13C NMR (100 MHz,
CDCl3): ı 214.81, 214.03, 148.28, 143.30, 133.20, 129.21, 122.84,
122.03, 73.99, 57.12, 42.62, 30.73, 27.62„ 24.69. Enantiomeric
excess determined by HPLC with a CHIRALPAK AD-H column (95:5
hexane:2-propanol), 25 ◦C, 254 nm, 0.7 mL/min; major enantiomer
tr = 44.0 min, minor enantiomer tr = 56.0 min.
4.3.8. 2-((2,6-Dichlorophenyl)(hydroxy)methyl)cyclohexanone
(h)
Yellow solid; mp: 128–129 ◦C; 1H NMR (400 MHz, CDCl3): ı 7.33
(d, J = 8.0 Hz, 2H), 7.17 (t, J = 7.9 Hz, 1H), 5.86 (dd, J = 9.5, 2.9 Hz, 1H),
3.68 (d, J = 3.1 Hz, 1H), 3.60–3.44 (m, 1H), 2.54 (d, J = 13.7 Hz, 1H),
2.49–2.35 (m, 1H), 2.12 (d, J = 9.5 Hz, 1H), 1.85 (d, J = 11.5 Hz, 1H),
1.79–1.61 (m, 2H), 1.44–1.35 (m, 2H). 13C NMR (100 MHz, CDCl3):
ı 214.36, 135.68, 134.73, 129.33, 70.55, 53.65, 42.43, 29.87, 27.63,
24.69. Enantiomeric excess was determined by HPLC with a CHI-
RALPAK AS-H column (98:2 hexane:2-propanol), 25 ◦C, 220 nm,
0.5 mL/min; major enantiomer tr = 53.0 min, minor enantiomer
tr = 45.2 min.
4.3.14. 2-(Hydroxy(4-nitrophenyl)methyl)cyclohexanone (n)
Yellow solid; mp: 96–97 ◦C; 1H NMR (400 MHz, CDCl3): ı 8.23
(d, J = 7.6 Hz, 2H), 7.53 (d, J = 7.7 Hz, 2H), 5.51 (s, 0.19H), 4.92 (d,
J = 8.2 Hz, 0.88H), 4.09 (s, 1H), 2.75–2.57 (m, 1H), 2.52 (d, J = 13.7 Hz,
1H), 2.43–2.35 (m, 1H), 2.14 (d, J = 10.6 Hz, 1H), 1.85 (d, J = 12.4 Hz,
1H), 1.73–1.51 (m, 4H); 13C NMR (100 MHz, CDCl3): ı 214.68,
213.96, 148.40, 127.87, 123.48, 73.97, 57.17, 42.62, 30.75, 27.72,
25.90, 24.72. Enantiomeric excess was determined by HPLC with a
CHIRALPAK AD-H column (93:7 hexane:2-propanol), 25 ◦C, 254 nm,
1.0 mL/min; major enantiomer tr = 39.8 min, minor enantiomer
tr = 29.1 min.
4.3.9. 2-((2-Bromophenyl)(hydroxy)methyl)cyclohexanone (i)
Yellow solid; mp: 93–95 ◦C; 1H NMR (400 MHz, CDCl3): ı
7.56–7.52 (m, 2H), 7.39–7.35 (m, 1H), 7.17–7.06 (m, 1H), 5.67 (s,
0.04H), 5.32 (d, J = 7.9 Hz, 0.97H), 4.09 (s, 1H), 2.74–2.67 (m, 1H),
2.50–2.45 (m, 1H), 2.41–2.28 (m, 1H), 2.18–2.03 (m, 1H), 1.93–1.80
(m, 1H), 1.77–1.51 (m, 4H); 13C NMR (100 MHz, CDCl3): ı 215.25,
214.71, 140.73, 132.50, 129.11, 128.47, 127.87, 123.37, 72.85, 57.67,
42.75, 30.56, 27.83, 24.96. Enantiomeric excess was determined by

Y. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 110 (2014) 100–110
109
1.51–1.28 (m, 9H); 13C NMR (100 MHz, CDCl3): ı 210.57, 209.16,
154.32, 147.74, 147.47, 127.67, 123.82, 80.98, 71.81, 56.20, 45.55,
43.70, 41.54, 28.44 (3C). Enantiomeric excess was determined by
HPLC with a CHIRALCEL AD-H column (96:4 hexane:2-propanol),
25 ◦C, 254 nm, 1.0 mL/min; major enantiomer tr = 48.5 min, minor
enantiomer tr = 55.3 min.
4.3.15. 2-(Hydroxy(pyridin-3-yl)methyl)cyclohexanone (o)
Yellow liquid; 1H NMR (400 MHz, CDCl3): ı 8.51–8.45 (m, 2H),
7.69 (d, J = 6.3 Hz, 1H), 7.33–7.18 (m, 1H), 5.39 (s, 0.52H), 4.84 (d,
J = 8.6 Hz, 0.49H), 3.88 (s, 1H), 2.69–2.52 (m, 1H), 2.49–2.42 (m, 1H),
2.40–2.28 (m, 1H), 2.15–2.01 (m, 1H), 1.88–1.24 (m, 5H); 13C NMR
(100 MHz, CDCl3): ı 214.76, 213.79, 149.12, 148.67, 148.18, 147.49,
137.49, 136.68, 134.64, 133.98, 123.51, 123.18, 72.37, 57.15, 56.89,
42.54, 30.77, 27.68, 24.71. Enantiomeric excess was determined by
HPLC with a CHIRALPAK AD-H column (90:10 hexane:2-propanol),
25 ◦C, 254 nm, 0.8 mL/min; major enantiomer tr = 29.8 min, minor
enantiomer tr = 35.9 min.
4.3.21.
3-(Hydroxyl(4-nitrophenyl)methyl)dihydro-2H-pyran-4(3H)-one
(u)
Yellow solid; mp: 117–119 ◦C; 1H NMR (400 MHz, CDCl3) ı
8.25 (d, J = 8.8 Hz, 2H), 7.54 (d, J = 8.8 Hz, 2H), 5.57 (s, 0.64H),
5.01 (d, J = 11.1 Hz, 0.54H), 4.35–4.15 (m, 1H), 3.93–3.81 (m, 1H),
3.82–3.70 (m, 2H), 3.55–3.40 (m, 1H), 3.00–2.90 (m, 1H), 2.81–2.63
(m, 1H), 2.60–2.42 (m, 1H); 13C NMR (100 MHz, CDCl3): ı 209.24,
208.28, 148.17, 147.35, 127.48, 123.79, 71.33, 69.78, 68.33, 57.64,
42.85. Enantiomeric excess was determined by HPLC with a CHI-
RALCEL AD-H column (90:10 hexane:2-propanol), 25 ◦C, 254 nm,
1.0 mL/min; major enantiomer tr = 51.0 min, minor enantiomer tr
=42.2 min.
4.3.16. 2-(Hydroxyl(perfluorophenyl)methyl)cyclopentanone (p)
White solid; mp: 74–75 ◦C; 1H NMR (400 MHz, CDCl3): ı 5.38 (d,
J = 5.8 Hz, 0.30H), 5.15 (d, J = 10.1 Hz, 0.86H), 4.53 (s, 1H), 2.93–2.85
(m, 1H), 2.67–2.47 (m, 1H), 2.37–2.06 (m, 2H), 1.92–1.76 (m, 2H),
1.50–1.46 (m, 1H); 13C NMR (100 MHz, CDCl3): ı 217.44, 146.62,
144.10, 142.27, 139.74, 138.84, 136.39, 66.25, 52.02, 38.25, 26.48,
20.31. Enantiomeric excess was determined by HPLC with a CHI-
RALPAK AD-H column (90:10 hexane:2-propanol), 25 ◦C, 215 nm,
0.5 mL/min; major enantiomer tr = 17.0 min, minor enantiomer
tr = 16.4 min.
4.3.22. 3-(Hydroxyl(4-nitrophenyl)methyl)dihydro-2H-
thiopyran-4(3H)-one
4.3.17. 2-(Hydroxy(3-nitrophenyl)methyl)cyclopentanone (q)
Yellow solid; mp: 79–81 ◦C; 1H NMR (400 MHz, CDCl3): ı 8.23
(d, J = 1.6 Hz, 1H), 8.13 (dd, J = 17.5, 8.1 Hz, 1H), 7.69 (t, J = 8.1 Hz,
1H), 7.63–7.48 (m, 1H), 5.41 (s, 0.37H), 4.84 (m, 1.30H), 2.57–2.38
(m, 2H), 2.36–2.12 (m, 1H), 2.07–1.97 (m, 1H), 1.78–1.70 (m,
2H), 1.59–1.54 (m, 1H); 13C NMR (100 MHz, CDCl3): ı 148.35,
145.06, 143.68, 132.67, 131.63, 129.40, 122.98, 122.26, 121.60,
120.61, 74.41, 70.27, 56.07, 55.08, 38.97, 38.60, 29.64, 26.86, 22.43,
20.34. Enantiomeric excess was determined by HPLC with a CHI-
RALPAK AD-H column (95:5 hexane:2-propanol), 25 ◦C, 254 nm,
1.0 mL/min; major enantiomer tr = 32.7 min, minor enantiomer
tr = 49.0 min.
(v)
Yellow solid; mp: 120–121 ◦C; 1H NMR (400 MHz, CDCl3): ı 8.26
(d, J = 8.8 Hz, 2H), 7.57 (d, J = 8.7 Hz, 2H), 5.54 (s, 0.32H), 5.08 (dd,
J = 8.0, 4.0 Hz, 0.71H), 3.64 (d, J = 4.1 Hz, 1H), 3.09–2.97 (m, 3H),
2.90–2.76 (m, 2H), 2.70 (dd, J = 13.6, 10.9 Hz, 1H), 2.57–2.52 (m,
1H); 13C NMR (100 MHz, CDCl3): ı 211.14, 210.83, 147.72, 147.29,
127.80, 123.79, 73.15, 59.46, 44.70, 32.81, 30.80. Enantiomeric
excess was determined by HPLC with a CHIRALCEL AD-H col-
umn (90:10 hexane:2-propanol), 25 ◦C, 254 nm, 1.0 mL/min; major
enantiomer tr = 62.1 min, minor enantiomer tr = 34.3 min.
Acknowledgements
4.3.18. 2-(Hydroxy(4-nitrophenyl)methyl)cycloheptanone (r)
Yellow solid; mp: 89–91 ◦C; 1H NMR (400 MHz, CDCl3): ı 8.23
(d, J = 8.5 Hz, 2H), 7.54 (d, J = 8.5 Hz, 2H), 5.31 (s, 0.47H), 4.94 (d,
J = 7.5 Hz, 0.55H), 2.99–2.86 (m, 1H), 2.72–2.43 (m, 2H), 1.89–1.63
(m, 4H), 1.52–1.22 (m, 4H); 13C NMR (100 MHz, CDCl3): ı 217.55,
216.99, 149.48, 149.25, 147.47, 147.08, 127.76, 126.77, 123.56,
74.77, 72.41, 57.86, 57.12, 44.13, 43.89, 29.09, 28.63, 28.21, 23.91,
23.48. Enantiomeric excess was determined by HPLC with a CHI-
RALPAK AD-H column (90:10 hexane:2-propanol), 25 ◦C, 254 nm,
1.0 mL/min; anti-diastereomer: major enantiomer tr = 43.8 min,
minor enantiomer tr = 19.1 min; syn-diastereomer: major enan-
tiomer tr = 12.6 min, minor enantiomer tr = 15.5 min.
This work was financially supported by the National Pro-
gram on Key Basic Research Project of China (973 Program,
2013CB328900) and the National Natural Science Foundation of
China (nos. 21001077 and 21321061). We also thank the Sichuan
University Analytical & Testing Center for SEM analysis.
Appendix A. Supplementary data
Supplementary material related to this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.molcatb.
2014.10.008.
References
4.3.19. 4-Hydroxy-4-(3-nitrophenyl)butan-2-one (s)
Yellow solid; mp: 60–61 ◦C; 1H NMR (400 MHz, CDCl3): ı 8.24
(d, J = 8.8 Hz, 2H), 7.56 (d, J = 8.6 Hz, 2H), 5.32–5.25 (m, 1H), 3.59 (s,
1H), 2.94–2.81 (m, 2H), 2.25 (s, 3H); 13C NMR (100 MHz, CDCl3): ı
208.60, 148.39, 144.90, 131.84, 129.52, 122.57, 120.72, 68.79, 51.53,
30.72. Enantiomeric excess was determined by HPLC with a CHI-
RALCEL AS-H column (70:30 hexane:2-propanol), 25 ◦C, 254 nm,
1.0 mL/min; major enantiomer tr = 11.9 min, minor enantiomer tr
=14.2 min.
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