M. Yasuda et al. / Tetrahedron: Asymmetry 17 (2006) 1775–1779
1779
4.5. The isolation and purification of cyclic amino acids
with PLC and with ion-exchange column chromatography.
(S)-4g was crystallized from a methanol/acetone mixture.
For the isolation of CAA, two enzyme reactions
(NMAADH and LO) were combined and carried out in
5 mL volume with a 2% glucose solution and incubated
at 30 ꢁC for 20 h. The products formed were purified with
preparative thin-layer chromatography (PLC) on silica gel
(Merck ART13793), which was then developed with a mix-
ture of acetonitrile/methanol/water (4:1:1). The CAA were
recovered with methanol, after which they were purified by
ion-exchange column chromatography (SK1B: Mitsubishi
Chemical Corp.) and then crystallized. The purified com-
4.5.4. 3-Morpholinecarboxylic acid (S)-4g. Yield: 35%
LC–Mass; MS: m/z = 132.15; H NMR (400 MHz, D2O):
d = 4.04 (1H, dd, J = 11.6, 3.0 Hz), 3.82 (1H, dt,
J = 12.4, 3.6 Hz), 3.55–3.67 (3H, m), 3.13 (1H, dt,
J = 13.1, 3.0 Hz), 2.98 (1H, ddd, J = 13.4, 10.1, 0.9 Hz).
1
Acknowledgments
1
We would like to thank Dr. Murakami of the Fuji Film
Corp. for the preparation of (S)-aminopropylcystein and
homolysine, and Dr. Tatsuya Suzuki of the Mitsubishi
Chemical Group, Science and Technology Research Center
for the NMR analysis.
pounds were analyzed with LC–Mass and H NMR.
4.5.1. Preparation of [1,4]-thiazepane-2-carboxylic acid
(S)-4f. The reaction mixture containing 174.7 mg (S)-1f,
1 lmol FAD, 6.5 units LO, and 1230 units catalase in
8 mL of 100 mM Tris–HCl buffer (pH 8.2) was incubated
for 5 h at room temperature, following which 2.33 mol glu-
cose, 10 lmol NADP+, and crude NMAADH (81 units of
NMAADH) were added and incubated at 28 ꢁC. The pH
decreased during the reaction; therefore, it was adjusted
occasionally from 7 to 8. The conversion was monitored
by TLC analysis. After 3 days, the (S)-1f spot could not
be detected. The reaction was stopped by adding 10 mL
of methanol and 40 mL of acetonitrile. The medium was
centrifuged to remove any precipitates and then filtered.
The product was then purified with PLC and ion-exchange
column chromatography. (S)-4f was crystallized from a
methanol/acetone mixture.
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