2696 Tan et al.
Asian J. Chem.
product separation and enantioselectivity in the aqueous/
[Bmim]PF6 biphasic system.
The supernatant was extracted by ethyl acetate and mixed with
octane as the internal standard for GC determination.
Analytical methods: The concentrations of COBE
and CHBE and enantiomeric excess (e.e.) of (S)-CHBE were
determined by GC (Aglient GC_6890, USA) equipped with a
chiral column (CP-Chirasil Dex CB, 0.32 mm diameter, 25 m
length, Varian USA). For the determination of e.e., the CHBE
should be derivated. The ethyl acetate layer was dried by the
rotating evaporation and the remainder reacted with acetic
anhydride containing excessive amount of pyridine catalyzed
by DMAP at 100 ºC for 20 min.
EXPERIMENTAL
Baker's yeast was kindly provided by meishan-mali yeast
Co. Ltd. (Guangdong, China). Ethyl 4-chloro-3-oxobutanoate
(ECOB) (> 98 %, analytical grade) were purchased from Leqi
Chemical Co. Ltd. (Shangdong, China). Enantiopure standards,
(R)-(96 %) and (S)-ECHB (97 %) were procured from Sigma-
Aldrich, Inc., (Steinheim, Germany). Ionic liquids were made
in the laboratory. All other chemicals used in the work were
analytical pure and were used without further purification.
Preparation of ionic liquid: The SO3H-functionalized
ionic liquid was prepared in laboratory according to the proce-
dure outlined in the literature14,15. The ionic liquids [Bmim][BF4]/
[Bmim][PF6] were prepared in the laboratory following the
procedure outlined in literature16. In a typical ionic liquid
preparation procedure, N-methyl imidazole (0.1 mol) and 1-
chlorobutane (0.12 mol) were added into a 50 mL stainless
steel autoclave. N2 was charged several times to replace air
and the final pressure of N2 was 0.7 MPa. The synthesis reaction
was carried out at 393 K for 12 h.After reaction, the production
was washed three times with ethyl acetate and yellow viscous
liquid 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) was
obtained. [Bmim]Cl (0.1 mol), stoichiometric amount of NaBF4
or NaPF6 (0.1 mol) and acetone (100 mL) were then added
to the reactor. The mixture was then stirred at 363 K for 4 h
to obtain the ionic liquid. The catalyst was abbreviated as
[Bmim][BF4] or [Bmim][PF6].
RESULTS AND DISCUSSION
Effect of different aqueous/ionic liquid biphasic
system: The ionic liquid played a significant role in a high
product yield and enantioselectivity of the cells, if it had a low
toxicity to the microbe19. Therefore, the selection of the ionic
liquid was important in an aqueous/ionic liquid biphasic
system. Four ionic liquids ([HSO3-pTEA][HSO4], [HSO3-
pmim][HSO4], [Bmim][BF4] and [Bmim][PF6]) were tested
for their influence on the asymmetric reduction of ECOB by
Baker's yeast and the results were shown in Fig. 1. It was noted
that aqueous/[Bmim][PF6] and aqueous/[Bmim][BF4] biphasic
system had a higher product yield and enantioselec-tivity than
aqueous/[HSO3-pTEA][HSO4] and aqueous/[HSO3-
pmim][HSO4] biphasic system. Because the [Bmim][BF4] and
[Bmim][PF6] are neutral ionic liquids, whereas the other two
are acidic, which would inhibit the activity of reductase in
baker's yeast with a lower product yield and the e.e. (S). In the
presence of the [Bmim][BF4] and [Bmim][PF6], the latter with
a property of slightly soluble in water had a remarkable increase
in the products and e.e. (S) compared with aqueous/
[Bmim][BF4] of water-soluble (v/v, 0.13 %20). [Bmim][PF6]
is slightly soluble in water as medium which is more suitable
for biological catalytic reaction of dual phase or multiphase
system.
Characterization of ionic liquids: The ionic liquids(ILs)
were characterized by FT-IR absorption spectrometer(Nicolet
380) and 1H and 13C NMR spectroscopy (BrukerAV500 spectro-
meter). The spectrum data showed the structures were consis-
tent with those reported in literature1,17,18. The spectrum data
of [Bmim][BF4]/[Bmim][PF6] were listed as follows:
[Bmim][BF4]: IR (KBr,νmax, cm-1): ν = 3186, 3173, 3146,
3127, 2968, 2961, 2952, 2878, 2874, 1573, 1572, 1467, 1466,
1170, 1169, 830; 1H NMR (500 MHz, DMSO-d6), δ 0.90 (t,
3H), 1.26 (m, 2H), 1.77 (m, 2H), 3.86 (s, 3H), 4.19 (t, 2H),
7.73 (s, 1H), 7.80 (s, 1H), 9.21 (s, 1H) ppm; 13C NMR (500
MHz, DMSO-d6), δ 13.37, 19.62, 32.48, 36.53, 49.72, 122.98,
124.34, 137.44 ppm.
[Bmim][BF6]: IR (KBr,νmax, cm-1): ν = 3186, 3173, 3146,
3127, 2968, 2961, 2952, 2878, 2874, 1573, 1572, 1467, 1466,
1170, 1169, 1030; 1H NMR (500 MHz, DMSO-d6), d 0.91 (t,
3H), 1.27 (m, 2H), 1.77 (m, 2H), 3.84 (s, 3H), 4.16 (t, 2H),
7.68 (s, 1H), 7.75 (s, 1H), 9.08 (s, 1H) ppm; 13C NMR (500
MHz, DMSO-d6), δ 13.29, 19.58, 32.31, 36.20, 49.84, 123.00,
124.38, 137.02 ppm.
100
Yield of ECHB (%)
e.e. (S) (%)
80
60
40
20
0
[HSO3-pmim][PTSA] [HSO3-pTEA][HSO4]
[Bmim][BF6]
[Bmim][BF4]
Bioreduction assay: The reduction of ECOB in an
aqueous/ionic liquid biphasic system was conducted in a 50 mL
shaking flask. A certain amount of dried cells and the ionic
liquid were mixed in phosphate buffer (50 mL, 0.05 mol/L,
pH 7) with a certain amount of glucose. After being pre-
incubated with rotary shaking at 30 ºC and 180 rpm for 15 min,
0.1 mol/L ECOB was added to the medium and the incubation
was continued. A fixed amount of medium was sampled and
centrifuged (8000 rpm, 10 min) at 4 ºC to remove the cells.
Type of ionic liquid
Fig. 1. Selection of the type of aqueous/ionic liquid biphasic system
Effect of phase ratio aqueous/[Bmim][PF6] biphasic
system on the reduction: As biotransformation in an aqueous/
ionic liquid biphasic system, the volumetric phase ratio has
an influence on the reaction rate, the product yield and e.e.(S)21.
The effect of phase ratio on the reaction rate, the product yield
and e.e.(S) were shown in Fig. 2. 87.2 % of the product yield