A. Nakagawa et al. / Tetrahedron: Asymmetry 18 (2007) 2394–2398
2397
20
20
20
4.2. Chemicals
½aꢁD ¼ þ18:0 (neat): 4a, ½aꢁD ¼ ꢀ17:7 (neat): 1c, ½aꢁD
¼
20
ꢀ10:4 (c 10, H2O): 3c, ½aꢁD ¼ ꢀ14:1 (c 10, H2O).
The chemicals used in this study were of reagent grade.
(RS)-1a–3a and 1b–4b were purchased from Tokyo Kasei
(Tokyo, Japan). (RS)-4a was prepared in our laboratory
as follows. Sulfuric acid (6 mL) was slowly added to a stir-
red solution of (RS)-4b (150 g) and methanol (418 mL).
After reflux for 10 h at 70 °C, the crude product was
extracted with 550 mL of dichloromethane. The organic
layer was washed with 5% NaHCO3 solution until the
pH of the water layer reached 7.0. The organic layer was
washed with water, and dried over anhydrous Na2SO4.
The crude product was then purified (81% yield) by
distillation under vacuum condition (bp 80 °C/20 mmHg);
1H NMR d 1.88–2.09 (m, 3H), 2.22–2.32 (m, 1H), 3.74
(s, 3H), 3.90–4.06 (m, 2H), 4.47 (dd, J = 5.1, 8.4 Hz, 1H).
4.5. Analytical methods
The concentrations and enantiomeric purities of the car-
boxylic esters and the corresponding carboxylic acids were
determined by gas chromatography (Model GC-14A, Shi-
madzu, Kyoto, Japan). For analysis of the concentration,
the GC was equipped with a PEG 20M-HP (5%,
60/80 mesh, GL Science, Tokyo, Japan) column (3.2 mm
diameter and 1 m length). The pH values of the carboxylic
acid samples were adjusted to 4.0 with 80% phosphoric
acid. The conditions were as follows: sample size, 1 lL;
injection and detector temperature, 240 °C; flow rate,
50 mL minꢀ1; detector, a hydrogen flame ionization detec-
tor (FID). The column temperature programme was as
follows: 5 min at 150 °C, increased to 200 °C over 10 min.
For analysis of the enantiomeric purity, each carboxylic
ester sample (with the exception of 2a and 3a) was sub-
4.3. The reaction of microbial resolution
For the examination of the hydrolytic reaction, 5 g of
CaCO3 as a pH neutralizer was added to 100 mL of culture
broth in 500 mL Erlenmeyer flasks. The hydrolytic reac-
tions were started by the addition of 8 g of 1a or 3a, and
2 g of 2a or 4a to the substrate at 30 °C on a rotary shaker
(130 rpm), respectively. After the cells were removed by
centrifugation, the amount and the enantiomeric purity
of the residual carboxylic esters and carboxylic acids
formed were analyzed by gas chromatography, as
described below.
jected to
a
capillary column CHIRALDEXR-G-TA
(0.25 mm diameter, 30 m length, Astec Inc., NJ). Com-
pounds 2a and 3a were trifluorinated by treatment with
5% (v/v) trifluoroacetic anhydride in dichloromethane.
The evaporated syrups were dissolved in ethanol before
injection. The carboxylic acids 1b–4b were converted to
the corresponding esters with 4-dimethylaminopyridine
(DMAP). The conditions were as follows: sample size,
0.2 lL; injection and detector temperature, 200 °C; column
temperature, 90°; flow rate, 50 mL minꢀ1; split ratio,
1:100; detector, FID.
4.4. The production of carboxylic esters and acids
References
For the production, 300 mL of culture broth was added to
a 5-L jar fermenter Model KMJ-5B (Mitsuwa Rikagaku,
Osaka, Japan) with 2.7 L of distilled water. Each 529 g
(15%) of (RS)-3a or each 261 g (8%) of the other racemate
was added as a substrate. The reaction was performed
under the following conditions: agitation, 500 rpm; tempera-
ture, 30 °C; the pH was controlled at 6.9 with 25% (w/v)
NaOH as for 1a and 3a or 14% (w/w) ammonium aqueous
solution as for 2a and 4a. The reaction solution was
analyzed as described in the analytical methods.
1. Prelog, V.; Wilheim, M.; Bright, D. B. Helv. Chim. Acta 1954,
37, 221–224.
2. Lee, J. B.; Downie, I. M. Tetrahedron 1967, 23, 359–363.
3. Mori, K.; Takigawa, T.; Matsuo, T. Tetrahedron 1979, 35,
933–940.
4. Hale, K. J.; Cai, J.; Manaviazar, S.; Peak, S. A. Tetrahedron
Lett. 1995, 36, 6965–6968.
5. Fukushima, K.; Furuhashi, Y.; Sogo, K.; Miura, S.; Kimura,
Y. Macromol. Biosci. 2005, 5, 21–29.
6. Tsuji, H.; Hyon, S. H.; Ikada, Y. Macromolecules 1991, 24,
5651–5656.
After the reaction using DH5a(pKK-E3), the cells were
removed using a UF membrane (MiniKros Tangential Flow
Separation Module PS/50K 8000 cm2, SPECTRUMLABS,
CA, USA). Compounds 1a, 3a and 4a were extracted three
times with equal volumes of ethyl acetate. As for the
extraction of 2a, dichloromethane was used. Each organic
layer was evaporated and the crude esters remaining dis-
tilled under the following conditions: 1a or 3a, bp 60 °C/
15 mmHg: 2a, bp 40 °C/20 mmHg: 4a, bp 80 °C/20 mmHg.
The aqueous layer including 1b or 3b was washed twice
with ethyl acetate and evaporated. The first syrup obtained
was dissolved in 2.3 L of methanol and filtered under
reduced pressure. The solution was then evaporated. The
second syrup was resolved in 1 L of 2-propanol and left
at 4 ° for 24 h. The resulting precipitate was harvested as
a sodium salt (1c,3c) of either 1b or 3b by filtration under
educed pressure and freeze dried. These specific rotations
7. Ikada, Y.; Jamshidi, K.; Tsuji, H.; Hyon, S. H. Macromole-
cules 1987, 20, 904–906.
8. Evans, D. A.; Morrissey, M. M.; Dorow, R. L. J. Am. Chem.
Soc. 1985, 107, 4346–4348.
9. Corey, E. J.; Link, J. O.; Shao, Y. Tetrahedron Lett. 1992, 33,
3435–3438.
10. Yu, H.; Ballard, C. E.; Boyle, P. D.; Wang, B. Tetrahedron
2002, 58, 7663–7679.
11. Adam, W.; Lazarus, M.; Saha-Moller, C. R.; Schreier, P.
Tetrahedron: Asymmetry 1998, 9, 351–355.
12. Oikawa, T.; Mukoyama, S.; Soda, K. Biotechnol. Bioeng.
2001, 73, 80–82.
13. Inoue, K.; Makino, Y.; Itoh, N. Tetrahedron: Asymmetry
2005, 16, 2539–2549.
14. Zhou, S.; Yomano, L. P.; Shanmugam, K. T.; Ingram, L. O.
Biotechnol. Lett. 2005, 27, 1891–1896.
15. Ishida, N.; Suzuki, T.; Tokuhiro, K.; Nagamori, E.; Onishi,
T.; Saitoh, S.; Kitamoto, K.; Takahashi, H. J. Biosci. Bioeng.
2006, 101, 172–177.
20
20
were: 1a, ½aꢁD ¼ þ2:45 (neat): 2a, ½aꢁD ¼ þ11:2 (neat): 3a,