CHEMICAL SYNTHESIS WITH ENZYMATIC METHODS
3
(400 MHz, CDCl3): δ 7.40 (2H, d, J = 7.3 Hz),
7.36–7.23 (3H, m), 4.75 (2H, s), 4.26 (4H, q, J
= 7.1 Hz), 2.56–2.49 (1H, m), 1.29 (6H, t, J
= 7.1 Hz), 1.05, (6H, d, J = 6.9 Hz).
of a reaction medium [13–18]. The temperature and
pH are also important parameters affecting enzyme
activity [19]. Therefore, all above mentioned factors
that may affect the enzymatic desymmetrization pro-
cedure were investigated in order to obtain the opti-
mum results.
General procedure for lipase-catalyzed hydrolysis
of diethyl 2-(benzyloxy)-2-isopropylmalonate (7)
Synthesis of diethyl 2-(benzyloxy)-
2-isopropylmalonate (7)
7 (0.2 mmol) was dissolved in 0.3 ml co-solvent and
then this solution was added to 0.7 ml phosphate buffer
with 20 mg lipase. The mixtures were continuously
stirred at 20–60 °C for 24 h. After the reactions were
stopped, adjusting the pH to 2.0 with 1 M HCl and then
the mixtures was extracted with ethyl acetate (3 × 2 ml).
The extracts were dried over anhydrous magnesium
sulfate and analyzed by chiral HPLC. 1H NMR
(400 MHz, CDCl3): δ 7.42–7.26 (5H, m), 4.76 (1H, d, J
= 10.9 Hz), 4.57 (1H, d, J = 10.9 Hz), 4.32 (2H, q, J
= 7.2 Hz), 2.56–2.42 (1H, m), 1.32 (3H, t, J = 7.2 Hz),
1.08, (6H, dd, J = 3.3, 6.9 Hz); HRMS (ESI) m/z: [M
+ Na]+ calcd for C15H20O5Na 303.1208, found
303.1203.
For the preparation of 7, commercially available 4
was chosen as starting material, which was oxidated
by dibenzoyl peroxide to give 5. Then the 6 was
obtained by alcoholysis of 5. 7 was synthesized by
the reaction of 6 and benzyl bromide. We found that
the temperature and the content of H2O were the
crucial factor of the synthesis of 5 and 6.
Enzyme screening
After the substrate in hand, we started the enzymatic
hydrolysis study from enzyme screening. The lipases
were used to catalyze the hydrolysis of 7 in phosphate
buffer (0.1 M, pH 8.0), and acetone was used as co-
solvent in experiments. To our delight, these lipases
gave satisfactory results except TL IM and lipases PS
(Table 1). Lipase AS, Lipase G, Lipase M and RM IM
led to moderate activity. However, Novozym 435 showed
the excellent enantioselectivity and good yield, with
enantiomer being preferentially formed at room tem-
perature. Novozym 435 indeed has a wide range of
applications in biocatalytic organic synthesis [20–22].
Thus, Novozym 435 was consequently used throughout
the further studies.
High performance liquid chromatography (HPLC)
analysis
The determination of enantiomeric excess (ee) of 2
was performed by chiral HPLC analysis carried out
on a HPLC system equipped with UV wavelength
detector and autosampler, using a Chiralcel IA-3
column (25 × 4.6 mm) thermostated at a temperature
of 40 °C. The mobile phase was a mixture of hexane,
isopropyl alcohol, ethanol and trifluoroacetic acid
(70:15:15:0.1, v/v/v/v) at a flow rate of 1.0 ml/min
and UV detection was performed at 293 nm. The
yield of 2 was measured by using the following equa-
tion: yield (%) = Mp/MS, Mp and MS are the final
mole of 2 and the original mole of the substrate,
respectively. The ee were obtained by the ratio of
peak areas of 2 (A1) and (S)-enantiomer (A2) as
follows: ee (%) = (A1 − A2)/(A1 + A2) × 100.
Choice of organic co-solvent
In order to resolve the poor solubility of prochiral sub-
strate 7 in aqueous medium, organic solvent was used as
co-solvent to improve the dissolubility and dissolution
rate. Besides, the activity and enantioselectivity of
Novozym 435 are also influenced by different organic co-
solvent. Therefore, we examined various organic co-
solvents on the reaction process. As shown in Table 2,
dimethyl sulfoxide (DMSO) gave the best results, so that
DMSO was chosen as co-solvent in the consequent
experiments.
Results and discussion
7 as enzyme catalyzed substrate was successfully
synthesized from commercially available 4 with
three-step chemical transformations (Scheme 2). We
subsequently focused on the synthesis of 2 by an
enzyme-catalyzed desymmetrization procedure start-
ing from the prochiral ester 7. Various lipases were
employed for this transformation. More importantly,
the use of organic co-solvent has proven to be an
effective strategy to overcome the low solubility of
hydrophobic substrates in an aqueous medium.
Besides, it was reported that enantioselectivity and
catalytic activity of enzymes can be improved by
organic co-solvent which can optimize the properties
Table 1. Various lipases for hydrolysis of 7.
No.
Lipase
Yield (%)
ee (%)
1
2
3
4
5
6
7
Lipase AS
Lipase G
Lipase M
RM IM
TL IM
Lipase PS
Novozym 435
18
23
15
19
10
5
64
54
69
61
29
6
30
90
Reaction conditions: Novozym 435 15 mg in 0.9 mL phosphate buffer
(0.1 M, pH 8.0), 0.2 mmol 7 in 0.1 mL acetone, 30 °C, 24 h.