Journal of Agricultural and Food Chemistry
Article
13.97 (CH3-13), 21.83 (CH2-12), 23.68 (CH2-12′), 23.16 (CH2-11),
23.17 (CH2-11′), 25.86 (CH2-9), 26.07 (CH-6), 32.42 (CH2-10),
35.67 (CH-10′), 38.22 (CH-1), 39.67 (CH-5), 89.72 (C-4), 125.70
(CH-7), 126.01 (CH-8), 178.97 (C-2); IR (NaCl, cm−1) 3019 (s),
2400 (m), 1757 (s), 1522 (m), 1425 (m); GC-EIMS 251 (M + 1).
Synthesis of Lactams 9 and 11−13. cis-3-Azabicyclo[4.3.0]non-7-
en-2-one (9). Synthesis of lactam 9 was performed using a high-
pressure autoclave Berghof HR equipped with a Teflon liner. To the
solution of cis-3-oxabicyclo[4.3.0]nonan-2-one (6) (1.518 g, 0.011
mol) in 2.0 mL of DMF placed in the Teflon liner was added 60 mL of
25% ammonia. The Teflon liner was put in a tightly closed autoclave.
The reaction mixture was stirred with heating at 160 °C for 15 h
(pressure increases to 21 bar). Product was extracted with diethyl
ether (5 × 50 mL). The extract was dried over MgSO4, and the solvent
was evaporated off. Crude product was purified by column
chromatography methylene chloride/methanol (97:3). Lactam 9 was
obtained with 65% (0.98 g) yield. The physical and spectral data of
lactone 9 are as follows: mp 63−66 °C; 1H NMR (500 MHz, CDCl3)
δ 1.80−1.85 (m, 1, one of CH2-6), 2.11−2.17 (m, 1, one of CH2-9),
2.23−2.26 (m, 1, one of CH2-6), 2.38−2.41 (m, 1, CH-1), 2.48−2.50
(m, 2, one of CH2-9, CH-5), 2.94−2.95 (m, 1, one of CH2-4), 3.41−
3.44 (m, 1, one of CH2-4), 5.66−5.72 (m, 2, CH-7, CH-8), 6.14 (s, 1,
NH); 13C NMR (151 MHz) δ 21.82 (CH2-6), 25.91 (CH2-9), 31.71
(CH-5), 38.71 (CH-1), 47.65 (CH2-4), 125.46 (CH-7), 126.07 (CH-
8), 180.13 (C-2); IR (NaCl, cm−1) 3154 (s), 2253 (s), 1695 (s); GC-
EIMS 138 (M + 1).
2-Butyric-2-azabicyclo[2.2.1]hept-5-en-3-one (11). To 2-
azabicyclo[2.2.1]hept-5-en-3-one (10) (0.3 g, 2.47 × 10−3 mol) was
added anhydrous THF (5 mL), followed by the addition of NaH (8.3
× 10−3 mol). Then, butyric acid chloride at room temperature was
added dropwise (5.64 × 10−3 mol). The reaction was continued until
all of the substrate was reacted (checked by TLC, 48 h). The reaction
mixture was filtered and purified by column chromatography using
methylene chloride/methanol (98:2.5) as eluent to give 0.24 g of pure
product 11 and 0.07 g of contaminated product 11 (yield = 63%, Rf =
0.27). The physical and spectral data of lactone 11 are as follows: oil;
1H NMR (500 MHz, CDCl3) δ 0.92 (t, 3, J = 7.4 Hz, CH3-11), 1.56−
1.69 (m, 2, CH2-10), 2.17 (dt, 1, J = 8.6, 1.7 Hz, one of CH2-7), 2.27
(dt, 1, J = 8.6, 1.7 Hz, one of CH2-7), 2.67−2.74 (m, 2, CH2-9), 3.40−
3.41 (m, 1, CH-4), 5.26−5.27 (m, 1, CH-1), 6.62−6.64 (m, 1, CH-5),
6.87−6.88 (m, 1, CH-6); 13C NMR (151 MHz) δ 13.69 (CH3-11),
17.81 (CH2-10), 37.80 (CH2-9), 54.69 (CH2-7), 54.77 (CH-1), 60.24
(CH-4), 138.10 (CH-5), 140.50 (CH-6), 173.02 (C-8), 177.53 (C-3);
IR (NaCl, cm−1) 3019 (s), 2400 (m), 1700 (m), 1521 (m); GC-EIMS
180 (M + 1).
was purified by column chromatography using methylene chloride/
methanol (98:2.5) as eluent to give 0.265 g (yield = 77.3%; Rf = 0.29).
The physical and spectral data of lactone 13 are as follows: mp 95−
100 °C; 1H NMR (500 MHz, CDCl3) δ 1.62−1.64 (m, 1, one of CH2-
7), 1.80−1.82 (m, 1, one of CH2-7), 2.84−2.90 (m, 1, CH-4), 3.56−
3.58 (m, 1, CH-5), 3.64−3.69 (m, 1, CH-6), 3.87−3.90 (m, 1, CH-1),
6.24−6.26 (s, 1, NH); 13C NMR (151 MHz) δ 31.12 (CH2-7), 46.88
(CH-4), 51.46 (CH-1), 55.61 (CH-5), 55.63 (CH-6), 180.43 (C-3);
IR (NaCl, cm−1) 3019 (s), 2400 (m), 1720 (s), 1518 (m), 1422 (m);
GC-EIMS 126 (M + 1).
Bioresolution of ( )-2-Azabicyclo[2.2.1]hept-5-en-3-one
(( )-10). Microbial Transformations of ( )-2-Azabicyclo[2.2.1]hept-5-
en-3-one (( )-10). (a) Screening Scale Biotransformation. Micro-
organisms were cultivated in Erlenmeyer flasks (300 mL) containing
75 mL of the cultivation medium S (a solution of peptone 1% and
glucose 3%) or C (an aqueous solution of NaNO3 0.3%, KH2PO4
0.1%, KCl 0.05%, MgSO4·7H2O 0.005%, FeSO4·7H2O 0.001 g, and
saccharose 3%). After 3−5 days of growth, 0.02 g of substrate ( )-10
in 0.5 mL of acetone was added to the shaken cultivation mixture. The
transformation was continued for 24, 72, 120, and 144 h. The products
were extracted with diethyl ether or chloroform and analyzed using
TLC and GC techniques. Enantiomeric excesses were determined by
GC using chiral column Cyclosil-B (30 m × 0.25 mm × 0.25 μm with
temperature program 80 °C (1 min), 130 °C (1 °C/min), and 250 °C
(40 °C/min). For selected microorganisms optimization of bio-
transformation conditions was performed with regard to increased
enantioselectivity and efficiency of a biocatalyst.
(b) Preparative Scale Biotransformation in Bench-Bioreactor. Bench-
scale batch runs were carried out in 7 L vessels of the bioreactor
(Brunswick, USA) in the selected conditions established on the basis
of screening experiments. Growth of P. thomi AM91 was performed
with the following parameters under control: medium volume (3 L),
aeration rate (1 v/m), stirring speed (200−300 rpm), temperature (26
°C), pH. After 4 days of growth on mineral medium (NaNO3 0.3%,
KH2PO4 0.1%, KCl 0.05%, MgSO4·7H2O 0.005%, FeSO4·7H2O 0.001
g, and saccharose 3%) (pH 4.2), 30 g of peptone was added (pH 8.3).
After 4 h, 1.0 g of ( )-10 in 2 mL of acetone was added. The
biotransformation was followed by GC with chiral column, once the
substrate ( )-10 was not detectable; the reaction mixture was
overnight extracted in the same manner as in the screening procedure.
The crude product was purified by column chromatography
(methylene chloride/methanol, 97:3). The yield of the biotransforma-
tion calculated only for lactam 10 was 70% and the enantiomeric
20
excess, 93% (GC, chiral column); [α] = −565° (c 2.0, CHCl3)
589
([α] = −525.9° (c 1.0, CH3OH)).35 The physical and spectral data
20
589
of lactone 10 are as follows: mp 55 °C; 1H NMR (500 MHz, CDCl3)
2-Butyl-2-azabicyclo[2.2.1]hept-5-en-3-one (12). To 2-azabicyclo[2.2.1]-
hept-5-en-3-one (10) (0.3 g, 2.47 × 10−3 mol) was added anhydrous
THF (5 mL), followed by the addition of NaH (8.3 × 10−3 mol).
Then, butyl bromide at room temperature was added dropwise (4.96
× 10−3 mol). The reaction was continued until all of the substrate was
reacted (checked by TLC, 48 h). The reaction mixture was filtered and
purified by column chromatography using methylene chloride/
methanol (98:2.5) as eluent to give 0.15 g of pure product 12 and
0.097 g of contaminated product 12 (yield = 54.4%; Rf = 0.33). The
δ 2.19−2.20 (m, 1, one of CH2-7), 2.37 (dt, 1, J = 6.0, 1.7 Hz, one of
CH2-7), 3.19−3.23 (m, 1, CH-4), 4.31−4.35 (m, 1, CH-1), 5.85 (s, 1,
NH), 6.64 (dt, 1, J = 7.4, 5.0 Hz, CH-5), 6.76 (dd, 1, J = 7.4, 5.0 Hz,
CH-6); 13C NMR (151 MHz) δ 53.21 (CH2-7), 59.32 (CH-1), 60.29
(CH-4), 138.18 (CH-5), 141.12 (CH-6), 185.34 (C-3); IR (NaCl,
cm−1) 3019 (s), 2400 (m), 1709 (s), 1521 (m); GC-EIMS 110 (M +
1).
Biotransformations of ( )-2-Azabicyclo[2.2.1]hept-5-en-3-one (( )-10)
with the Use of Isolated Enzymes. (a) Screening Scale Biotransforma-
tion. Substrate ( )-10 (5 mg) was added to enzyme (the amount
equal to 20 enzyme units of activity), dissolved in 0.5 mL of the
phosphate buffer of pH 7.2. The reaction was stirred at room
temperature. At the end of the process the reaction mixture was
filtered through Celite, and the biotransformation products were
extracted with diethyl ether. The enantiomeric excess of substrate 10
was determined by means of GC with application of chiral columns.
(b) Preparative Scale Biotransformation. Substrate ( )-10 (0.2 g) was
added to enzyme (the amount equal to 800 enzyme units of activity of
Lipase B from Candida antarctica immobilized on Innobead 150,
recombinant from Aspergillus oryzea), dissolved in 40 mL of the
phosphate buffer of pH 7.2. The reaction was stirred at room
temperature. At the end of the process the reaction mixture was
filtered through Celite, and the biotransformation products were
extracted with diethyl ether. The enantiomeric excess of substrate 10
1
physical and spectral data of lactone 12 are as follows: oil; H NMR
(500 MHz, CDCl3) δ 0.90 (t, 3, J = 7.4 Hz, CH3-11), 1.22−1.27 (m, 4,
CH2-9, CH2-10), 2.11 (dt, 1, J = 7.5, 1.7 Hz, one of CH2-7), 2.28 (dt,
1, J = 5.8, 1.7 Hz, one of CH2-7), 2.84−2.88 (m, 1, one of CH2-8),
3.09−3.14 (m, 1, one of CH2-8), 3.30−3.31 (m, 1, CH-4), 4.14−4.15
(m, 1, CH-1), 6.62 (dt, 1, J = 7.3, 5.6 Hz, CH-5), 6.69 (dt, 1, J = 7.3,
4.8 Hz, CH-6); 13C NMR (151 MHz) δ 13.81 (CH3-11), 20.09 (CH2-
10), 29.92 (CH2-9), 43.43 (CH2-8), 54.00 (CH2-7), 59.10 (CH-4),
63.14 (CH-1), 138.16 (CH-5), 139.66 (CH-6), 180.52 (C-3); IR
(NaCl, cm−1) 3019 (s), 2400 (m), 1695 (m), 1518 (m); GC-EIMS
165 (M + 1).
cis-5,6-Epoxy-2-azabicyclo[2.2.1]heptan-3-one (13). To 2-
azabicyclo[2.2.1]hept-5-en-3-one 10 (0.3 g, 2.47 × 10−3 mol) was
added methylene chloride (15 mL), followed by the addition of m-
CPBA (4.14 × 10−3 mol). The reaction was continued until all of the
substrate was reacted (checked by TLC, 24 h). The reaction mixture
C
dx.doi.org/10.1021/jf502148h | J. Agric. Food Chem. XXXX, XXX, XXX−XXX