Lipase-Catalyzed Oligomerization
Biomacromolecules, Vol. 11, No. 8, 2010 2009
the reaction apparatus, a similar apparatus was used for hydrophilic
alcohols (MeOH, EtOH, PrOH, and sBuOH), whereas a Dean-Stark
apparatus for hydrophobic alcohols (BuOH, iBuOH, PeOH, HxOH,
HpOH, and OcOH). In all preparations, alkyl lactates were prepared
in 8-36% yields.
Characterization of alkyl lactates was performed by measuring H
NMR, ESI-TOF-MS, and GPC. Optical purity was determined by GC
analysis using an optical resolution column, according to the relationships
Inhibition Type Test. A typical run was as followed. EtLLa (0.25
mmol) was added to the mixture containing EtDLa (2.0 mmol), 1,4-
dioxane (0.50 mL), n-propylbenzene (200 µL, internal standard), and
Novozym 435 (25 mg). The reaction was performed at atmospheric
pressure at 50 °C with stirring. The consumption of EtDLa was followed
by the GC analysis of the sample taken out at 10 min intervals from
the reaction mixture.
Similar experiments were performed with varying the added amount
of EtLLa from 0.0 to 0.25 to 0.50 mmol and with varying the substrate
amount of EtDLa from 2.0, 3.0, 4.0, and 5.0 mmol, thus, for the total
1
D(%) )
L(%) )
[
D*/(D* + L*)
]
× 100
× 100
1
2 runs.
Hydrolysis of Alkyl Lactates. A typical run was as follows. To a
mixture solution of EtDLa (5.0 mmol, 0.59 g) and 1,4-dioxane (0.50
mL) containing n-propylbenzene (200 µL, as internal standard), distilled
water (5.0 mmol, 90 mg) and Novozym 435 (25 mg) were added. The
mixture was homogeneous, except for the catalyst, and reacted at 50
°C with stirring. The consumption of EtDLa was followed by the gas
chromatography (GC) analysis of the sample taken out at 10 min
intervals from the reaction mixture.
[
L*/(D* + L*)
]
where D* and L* denote the peak area value of D- and L-isomers in
the GC chart.
1H NMR data (CDCl
, 500 MHz, δ relative to TMS), the mass
values, and the optical purity values are given as follows: MeLa (D
and L): 1.40-1.44 (s, 3H, CH ), 2.70-2.95 (s, 1H, OH), 3.76-3.82
d, 3H, CH ), 4.26-4.32 (q, 1H, CH); ms ) 104; D ) 99.5%, L )
9.5%. EtLa (D and L): 1.28-1.32 (t, 3H, CH ), 1.40-1.43 (d, 3H,
), 2.82-2.90 (s, 1H, OH), 4.22-4.28 (m, 2H, CH ), 4.22-4.27
3
1
3
Analytical Methods. H NMR measurements were recorded on a
(
3
spectrometer ARX-500 (500 MHz, Bruker BioSpin GmbH, GER).
ESI-TOF-MS analysis was performed by using a micrOTOF
instrument (Bruker Daltonik GmbH, GER).
9
3
CH
3
3
(
m, 1H, CH); ms ) 118; D ) 99.5%, L ) 99.3%. PrLa (D and L):
GC apparatus was equipped with an Rt-ꢀDEXsm 0.25 mm × 30 m
× 0.25 µm column (Restek Co., PA, U.S.A.) which was maintained at
80 °C with an FID detector. The injection and the detector temperatures
were set to 200 °C.
Molecular weights were determined by gel permeation chromatog-
raphy (GPC) with a refractive index detector (HLC-8220, Tosoh Co.).
The analytical conditions were as follows: columns, TSK gel Super
HZ2000, HZ4000, and a guard column (Tosoh Co.); column temper-
0
.94-0.97 (t, 3H, CH
), 2.89-2.93 (s, 1H, OH), 4.10-4.20 (m, 2H, CH
q, 1H, CH); ms ) 132; D ) 99.7%, L ) 99.7%. BuLa (D and L):
.92-0.97 (t, 2H, CH ), 1.36-1.44 (m, 3H, CH ), 1.41-1.44 (d, 3H,
CH ), 1.62-1.68 (q, 2H, CH ), 2.81-2.83 (s, 1H, OH), 4.16-4.24 (d,
H, CH ), 4.26-4.31 (q, 1H CH); ms ) 146; D ) 99.6%, L ) 99.2%.
sBuLa (D and L): 0.89-0.92 (t, 3H, CH ), 1.22-1.26 (t, 3H, CH ),
.38-1.42 (t, 3H, CH ), 1.55-1.65 (q, 2H, CH ), 2.80-2.99 (s, 1H,
OH), 4.21-4.25 (q, 1H, CH), 4.91-4.95 (q, 1H, CH); ms ) 146; D )
3
), 1.41-1.44 (d, 3H, CH
3
), 1.66-1.73 (d, 2H,
CH
(
0
2
2
), 4.25-4.30
3
3
3
3
3
3
3
3
1
3
2
3
ature, 40 °C; solvent for elution, CHCl ; and flow rate, 0.35 mL/min.
Polystyrene standards were used for calibration.
9
7.4%, L ) 99.9%. iBuLa (D and L): 0.92-1.0 0(d, 3H, CH
3
),
1
.42-1.45 (d, 3H, CH ), 1.93-2.04 (m, 1H, CH), 2.85-2.91 (s, 1H,
3
Results and Discussion
OH), 3.93-3.97, 3.98-4.35(q,d, 1H, CH), 4.26-4.32 (q, 1H, CH);
ms ) 146; D ) 98.4%, L ) 98.5%. PeLa (D and L): 0.88-0.94 (t,
Lipase-Catalyzed Enantioselective Oligomerization. For
the purpose of screening the monomer reactivity and the catalyst
activity, lipase (Novozym 435)-catalyzed polycondensation of
3
H, CH
d,t, 2H, CH
.24-4.30(q, 1H, CH); ms 160; D ) 98.7%, L ) 98.9%. HxLa (D
and L): 0.87-0.92 (t, 3H, CH ), 1.26-1.38 (m, 2H, CH ), 1.40-1.44
d, 3H, CH ), 1.63-1.69 (d,t, 2H, CH ), 2.70-2.94(s, 1H, OH),
.14-4.22 (m, 2H, CH ), 4.24-4.30 (q, 1H, CH); ms ) 174, D )
8.9%, L ) 99.4%. HpLa (D and L): 0.86-0.94 (t, 3H, CH ),
.24-1.38 (m, 2H, CH ), 1.40-1.44 (d, 2H, CH ), 1.63-1.70 (d,t, 2H,
), 2.80-2.85 (s, 1H, OH), 4.14-4.22 (m, 2H, CH ), 4.24-4.30
q, 1H, CH); ms ) 188; D ) 98.9%, L ) 98.8%. OcLa (D and L):
.86-0.91 (t, 3H, CH ), 1.24-1.40 (m, 2H, CH ), 1.40-1.44 (d, 2H,
CH ), 1.63-1.70 (d, t, 2H, CH ), 2.79-2.82 (d, 1H, OH), 4.12-4.22
m, 2H, CH ), 4.24-4.30 (q, 1H, CH); ms )202; D ) 98.4%, L )
3
), 1.32-1.38 (m, 2H, CH
2
), 1.40-1.44 (d, 3H, CH
3
), 1.64-1.70
(
4
2
), 2.76-2.95(s, 1H, OH), 4.14-4.22(m, 2H, CH
2
),
1
8 alkyl D- and L-lactates (RDLa and RLLa) was examined as
shown Table 1. It is clear that the reaction of all the alkyl
D-lactates was induced to give oligo(D-lactic acid)s (oligoDLAs)
in good to high yields. There was a tendency that primary alkyl
lactates of Et-, Pr-, and Bu- showed a higher reactivity compared
with longer alkyl lactates like Pe-, Hx-, Hp-, and Oc-, and also
a secondary alkyl lactate of sBuDLa showed a decreased
reactivity. On the other hand, all alkyl L-lactates did not show
any reactivity regardless of the primary or secondary structure
of alkyl groups; nothing happened under the reaction conditions.
The condensation oligomerization is definitely enantioselective
3
2
(
3
2
4
9
1
CH
(
2
3
2
2
2
2
0
3
2
2
2
(
9
2
9.6%.
(
Scheme 1).
Figure 1 shows 500 MHz H NMR spectra of the reaction
Oligomerization of Alkyl Lactates. A typical procedure is as
1
follows. To EtDLa (5.0 mmol, 0.59 g) in a test tube containing a stirrer
bar, 25 mg of Novozym 435 was added, and the mixture was allowed
to react at 50 °C with stirring under a reduced pressure at 3.3 kPa. The
consumption of the substrate was followed by sampling 20 µL from
the reaction mixture occasionally and by GC analysis of the reaction
mixture sample using n-propylbenzene as the standard substance.
mixture of iBuDLa, in which (A) clearly indicates signals due
to the product oligomers and unreacted monomer; in an
expanded version in (B), specific methine proton of the
monomer appears at δ 4.28 (peak b), the terminal methine proton
of oligomers at δ 4.36 (peak a), and internal methine protons
of oligomers at δ 5.37 (peak a′). From the integrated ratio of
peaks a, a′, and b, conversion of monomer (70%) as well as an
average degree of oligomerization (n ) 2.72 in Scheme 1) could
be calculated. In contrast, the reaction of iso-butyl L-lactate
1
Characterization of the product oligoLAs was performed by H NMR
and ESI-TOF-MS analyses.
Co-Oligomerization between EtDLa and EtLLa. A mixture of
the substrates (total amount of EtDLa + EtLLa ) 5.0 mmol, with
varying the feed ratio), without or with 1,4-dioxane (0.50 mL) solvent,
n-propylbenzene (200 µL, internal standard), and Novozym 435 (75
mg or 25 mg) was allowed to react at 3.3 or 101 kPa at 50 °C with
stirring. The consumption of EtDLa and EtLLa was followed by the
GC analysis of the sample taken out at 2 h or 10 min intervals from
the reaction mixture.
(
iBuLLa) did not occur under the similar reaction conditions;
the monomer recovered unchanged as seen in (C). Data of
conversion values of all other lactates have been similarly
obtained and included in Table 1.
Figure 2 demonstrates the TOF-MS chart of the oligomers
obtained from the reaction of iBuDLa indicating the formation