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J.-P. Fu et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 1–4
Table 1
Solvent screening and control experiments of ficin-catalyzed direct asymmetric aldol reaction.a
.
Entry
Solvent
Yield (%)b
dr (anti:syn)c
% ee (anti)d
1
2
3
4
5
6
7
8
9
10
11
12
13
14
MeCN
THF
i-PrOH
DMSO
DMF
Isopropyl ether
1,4-Dioxane
Butyl acetate
28
34
11
26
40:60
34:66
32:68
35:65
39:61
—
—
—
—
—
57
29
29
5
23
3
Trace
Trace
Trace
Trace
No reaction
43
—
—
—
—
—
3
1
—
0
CHCl3
MeCN (no enzyme)
MeCN (bovine serum albumin)
MeCN (ficin denatured with ureae)
MeCN (ficin inhibited with MMTSf)
37:63
35:65
—
5
Trace
48
g
NaHCO3
31:69
a
Reaction conditions: ketone 1a (0.25 mmol), 4-nitrobenzaldehyde 2a (0.50 mmol), and ficin (50 mg) in solvent (0.90 mL) and deionized water (0.10 mL) at 25 ◦C for 120 h.
Isolated yield after silica gel chromatography.
Determined by chiral HPLC.
Determined by chiral HPLC, and the absolute configuration was assigned by comparison with literatures.
Pretreated with urea (0.83 M, 50 mg urea in 1 mL water) at 100 ◦C for 24 h, water was removed before use.
Pretreated with MMTS (0.40 M, 50 mg MMTS in 1 mL MeCN) at 25 ◦C for 24 h, MeCN was removed before use.
The reaction was performed by employing ketone 1a (0.25 mmol), 4-nitrobenzaldehyde 2a (0.25 mmol), NaHCO3 (20 mg) in EtOH (1 mL) for 48 h.
b
c
d
e
f
g
from different commercial suppliers and were used without further
purification.
reaction media. The reaction gave product in the best yield of 34%
with a poor enantioselectivity of 29% ee in THF (Table 1, entry 2),
while the best enantioselectivity of 57% ee with a yield of 28% was
received in MeCN (Table 1, entry 1). In the tested polar solvents
including i-PrOH, DMSO, and DMF, ficin showed lower activity
and enantioselectivity (Table 1, entries 3–5). The reaction in other
the solvent for the ficin-catalyzed aldol reaction.
Besides, some control experiments were performed in order to
verify the specific catalytic effect of ficin on the aldol reaction. As
MeCN at 25 ◦C even after 5 days (Table 1, entry 10), indicating that
catalyst was essential for the reaction. The urea-denatured ficin
almost lost its activity in the model aldol reaction, and only 5% yield
was obtained with 1% ee (Table 1, entry 12), suggesting that the ter-
tiary structure of ficin is responsible for its activity and selectivity.
In addition, a complete inhibition of the catalytic activity of ficin in
entry 13), indicating that the active site of ficin contributed to this
enzymatic promiscuity. The reaction catalyzed by non-enzyme pro-
tein bovine serum albumin gave product in 43% yield with 3% ee
(Table 1, entry 11), which showed that a non-enzyme protein also
had the ability to catalyze the aldol reaction, but almost did not
exhibit enantioselectivity for aldol product. This experiment sug-
gested that the catalytic activity of ficin in aldol reaction did not
simply arise from the amino acid sequence of the enzyme. Thus,
fashion on the catalytic site of ficin.
2.2. General methods
Routine monitoring of reaction was performed by TLC using
precoated Haiyang GF254 silica gel TLC plates. All the column chro-
matography separations were done by using silica gel (100–200
mesh) at increased pressure. 1H NMR (300 MHz) and 13C NMR
(75 MHz) spectra were recorded on a Bruker AMX-300 MHz spec-
trometer. TMS (1H) and CDCl3 (13C) were used as internal standards.
Chemical shifts (ı) are reported in ppm and coupling constants (J)
in Hz.
2.3. General experimental procedure for the ficin-catalyzed aldol
reactions
A mixture of aldehyde (0.50 mmol), ficin (75 mg) and ketone
(0.25 mmol) in MeCN (0.85 mL) and deionized water (0.15 mL) was
stirred for the specified time at 30 ◦C. The reaction was terminated
by filtration to remove the enzyme. CH2Cl2 was used to wash the fil-
ter paper to assure that products obtained were all dissolved in the
filtrate. Then the filtrate was washed three times with water. The
organic phase was dried over anhydrous Na2SO4, and the solvents
were removed under reduced pressure. The crude products were
purified by column chromatography with petroleum ether/ethyl
acetate as eluent.
3. Results and discussion
nitrobenzaldehyde and N-Boc-piperidone was used as a model
reaction. Since reaction medium has been recognized to be one of
the most important factors influencing the enzymatic reactions
[29,30], the catalytic activity of ficin in the model aldol reaction
was evaluated in different solvents (Table 1). The catalytic activity
and stereoselectivity of ficin were significantly influenced by the
Water content has been considered as a very important fac-
tor in enzymatic reactions for it affects both the enantioselectivity
and activity of enzymes [32–34]. The control of this parameter was
proven to be vital. Thus, we designed some experiments to optimize
the percentageof water for this reaction. The effect of water concen-
tration on the ficin-catalyzed model aldol reaction was illustrated in