1390
Y. Fujima et al. / Tetrahedron: Asymmetry 14 (2003) 1385–1391
mmol) was added, and the mixture was heated with
stirring at reflux for 2 h and 10 min. The mixture
was allowed to cool to room temperature, and sub-
jected to the same extractive workup as described in
4.5. N,N-Dicyclohexylammonium (R)-tetrahydrofuran-
2-carboxylate 4
The MEK solution of (R)-1 obtained in Section 4.4 was
concentrated in vacuo until the volume was reduced to
35 mL (16% of the original volume). To the residue was
added MeOH (7 mL) at room temperature. N,N-Dicy-
clohexylamine (DCHA, 9.06 g, 50 mmol) was added
dropwise at room temperature. The stirred mixture was
heated to reflux, and allowed to cool to 5°C. Precipi-
tated solids were collected by filtration, and dried in
vacuo at an oven temperature of 50°C to give 4 (8.95 g)
in 22% overall yield from ( )-3d: mp 164.4–165.5°C;
[h]2D0 +17.6 (c 1.0, MeOH); IR wmax (KBr) 3427, 2931,
2808, 2521, 2420, 1581, 1460, 1416, 1311, 1068 cm−1; 1H
NMR l 1.14–1.27 (m, 6H), 1.43–1.52 (m, 3H), 1.64 (m,
2H), 1.74–2.04 (m, 14H), 2.23 (m, 1H), 3.84 (m, 1H),
3.96 (m, 1H), 4.26 (t, J=6.4 Hz, 1H), 7.0 (br s, 2H).
Anal. found: C, 68.0; H, 10.4, N, 4.6. calcd for
C17H31NO3·0.2 CH4O (methanol): C, 67.99; H, 10.55,
N, 4.61. To a portion (50 mg) of the salt were added
H2O (0.5 mL) and (NH4)2SO4 (0.1 g). The pH of the
mixture was adjusted to 2 with 35% aqueous solution of
HCl. The mixture was extracted with MEK (1 mL×1),
and 10 mL of the MEK solution was injected to the
chromatograph running under the conditions described
in Section 4.3.2 to determine the enantiomeric purity of
(R)-1 contained in 4 to be 99.1% ee. From a portion of
4 was liberated free (R)-1 according to the usual
procedures3,7 to confirm its chemical and stereochemi-
cal integrity: [h]D20 +30.0 (c 0.34, CHCl3) {lit:3a [h]D
+30.4 (CHCl3)}; wmax (KBr) 2984, 1743, 1448, 1402,
1
Section 4.2.2 to give crude ( )-3e (11.1 g, 70.4%): H
NMR l 1.24 (d, J=6.0 Hz, 3H), 1.26 (d, J=6.0 Hz,
3H), 1.90–2.05 (m, 3H), 2.23 (m, 1H), 3.92 (m, 1H),
4.02 (m, 1H), 4.41 (dd, J=5.2, 8.4 Hz, 1H), 5.05 (m,
1H); MS (70 eV) m/z 158 (M+). This was employed
in the enzymatic reaction without further purification.
4.3. Screening of hydrolases and reaction parameters
A 2 mL eppendorf® microtube was charged with an
appropriate amount of ( )-3, a hydrolase, and potas-
sium phosphate buffer to set up a reaction specific to
the experimental purpose. The tube was agitated for a
specific period. The progress of the reaction was mon-
itored and the enantioselectivity assessed by the fol-
lowing procedures: From the reaction mixture was
taken a portion (0.2 mL), to which were added a 2.5
M aqueous solution of H2SO4 (0.1 mL), (NH4)2SO4
(0.1 g), and i-PrOH (1 mL). The mixture was agi-
tated, and centrifuged to separate layers. From the
upper layer was taken a portion (10 mL), which was
subjected to twofold chiral HPLC analyses, one
designed to quantify the enantiomeric composition of
3 (Section 4.3.1) and the other designed to quantify
that of 1 (Section 4.3.2).
4.3.1. Determination of the enantiomeric composition of
3. Column: Chiralcel OD (Daicel), 0.46 cmf×25 cm;
elution: n-hexane/i-PrOH (99.5:0.5), 1.5 mL/min;
detection: UV at 220 nm; (R)-3a, tR=17.4 min; (S)-
3a, tR=9.5 min; (R)-3b, tR=13.4 min; (S)-3b, tR=8.0
min; (R)-3c, tR=35.7 min; (S)-3c, tR=24.7 min; (R)-
3d, tR=14.2 min; (S)-3d, tR=7.7 min; (R)-3e, tR=
13.2 min; (S)-3e, tR=7.8 min.
1
1351, 1203, 1180, 1080, 1038, 928, 804 cm−1; H NMR
l 1.97 (m, 2H), 2.10 (m, 1H), 2.32 (m, 1H), 3.96 (m,
1H), 4.05 (m, 1H), 4.52 (dd, J=5.2, 8.8 Hz, 1H), 10.24
(br. s, 1H).
Acknowledgements
M.I. thanks Mr. Masafumi Moriwaki, Director of
Research and Development Center, Nagase & Co.,
Ltd., for his consistent encouragement throughout
this project.
4.3.2. Determination of the enantiomeric composition of
1. Column: Chiralpak WH (Daicel), 0.46 cmf×25 cm;
elution: 2 mM aqueous solution of CuSO4, 1 mL/
min; detection: UV at 254 nm; (R)-1, tR=15.0 min;
(S)-1, tR=13.4 min.
References
4.4. (R)-Tetrahydrofuran-2-carboxylic acid (THFC) 1
1. For a concise review on synthetic approaches to non-clas-
sical b-lactam antibiotics and case studies thereof, see:
Carbi, W.; Di Fabio, R. From Bench to Market: The
Evolution of Chemical Synthesis; Oxford University Press:
Oxford, 2000; pp. 25–119.
2. (a) Ishiguro, M.; Iwata, H.; Nakatsuka, T.; Tanaka, R.;
Maeda, Y.; Nishihara, T.; Noguchi, T. J. Antibiot. 1988,
41, 1685–1693; (b) Nishino, T.; Maeda, Y.; Ohtsu, E.;
Koizuka, S.; Nishihara, T.; Adachi, H.; Okamoto, K.;
Ishiguro, M. J. Antibiot. 1989, 42, 977; (c) Nakatsuka, T.;
Kaneko, A. FARUMASHIA (Japanese) 2002, 38, 219–
223 (The Pharmaceutical Society of Japan).
To a 1.5 M potassium phosphate buffer solution (pH
8, 68.5 mL) was added ( )-3d (19.7 g, 137 mmol)
followed by an A. melleus protease (137 mg). The
mixture was stirred at 10°C for 20 h. The pH of the
mixture was adjusted to 10.0 with 5 M aqueous solu-
tion of KOH. The mixture was extracted with n-hex-
ane (30 mL×3). To the aqueous layer were added
(NH4)2SO4 (10.3 g) and 35% aqueous solution of HCl
to adjust its pH to 2. The aqueous mixture was
extracted with MEK (50 mL×5). The combined MEK
extracts were analyzed for the content of (R)-1 and
its enantiomeric purity under the conditions described
in Section 4.3.2: 36.5% yield and 94.4% ee.
3. (a) Be´langer, P. C.; Williams, H. W. R. Can. J. Chem.
&
1983, 61, 1383–1386; (b) Cervinka, O.; Bajanzulyn, O.;