General Papers
ARKIVOC 2016 (vi) 172-183
internal standard. Enantiomeric excesses were determined by chiral-phase HPLC [Chiralpak AS,
Daicel; eluent: 2-propanol/hexane (30:70); flow rate: 1.0 mL/min; detection: UV 254 nm].
Isolation and purification of cyclohexadiene acids 2 and 4
(5S,6S)-6-Amino-5-hydroxycyclohexa-1,3-dienecarboxylic acid (2,3-trans-CHA, 2). The cell-
free fermentation broth (LB or SOC medium)33 was concentrated at 60 °C by rotary evaporation
(>10 g/L of 2), and further dried in vacuo. The brownish residue was washed with cold methanol
and crystallized from water at 4 °C. A yield of 80% (purity >95%) of the product 2 (mp 185–188
°C) was obtained. 1H NMR (CD3OD, 300 K, ppm): δ 4.28 (d, J = 5.7 Hz, 1H), 4.46 (dd, J = 5.7,
4.6 Hz, 1H), 6.22 (dd, J = 9.5, 4.6 Hz, 1H), 6.34 (dd, J = 9.5, 5.7 Hz, 1H), 7.01 (d, J = 5.7 Hz, 1H).
13C NMR (CD3OD, 300 K, ppm): δ 51.9, 66.0, 125.7, 128.1, 130.5, 132.8, 173.0.
(3R,4R)-4-Amino-3-hydroxycyclohexa-1,5-dienecarboxylic acid (3,4-trans-CHA, 4)
Cell-free fermentation broth (200 mL LB or SOC medium)33 was basified by addition of aqueous
sodium hydroxide (2 M, 10 mL). The turbid liquid was centrifuged, and the supernatant was passed
through a column with ion-exchange resin (Dowex 50WX8, H+ form). The column was washed
with water (300 mL) and 4 eluted with 0.5 M NH3. The product-containing solution was
lyophilized; 350 mg of a white powder containing 14% of compound 4 (by comparison with
internal standard in the 1H NMR spectrum) was obtained as the main NMR-active compound. 1H
NMR (D2O, 300 K, ppm): δ 4.13 (dt, J = 11.2, 2.7 Hz, 1H), 4.67 (dd, J = 11.2, 3.5 Hz, 1H), 5.96
(dd, J = 10.0, 3.2 Hz, 1H), 6.57 (d, J = 10.0 Hz, 1H), 6.83 (m, 1H). 13C NMR (D2O, 300 K, ppm):
δ 52.8, 67.8, 122.7, 125.0, 128.5, 138.2, 168.0.
Preparation of the zinc complexes 8–10
Zn(L-proline)2 complex (8). L-Proline (6; 499 mg, 4.34 mmol) was dissolved in MeOH (30 mL),
then TEA (0.6 mL, 4.34 mmol) was added. The mixture was stirred at rt for 10 min, then zinc
acetate (7; 476 mg, 2.17 mmol) was added. A colorless precipitate appeared immediately. After
the mixture was stirred for 1 h, the solid material was collected by filtration and dried under
vacuum to give Zn(L-proline)2 complex (8); yield: 532 mg (84%). 1H NMR (D2O, 300 K, ppm): δ
1.87 (bs, 3H), 2.29 (bm, 1H), 3.04 (bs, 1H), 3.19 (bm, 1H), 3.92 (bs, 1H).
Zn(2,3-trans-CHA)2 complex (9). 2,3-trans-CHA (2; 350 mg, 2.26 mmol) was dissolved in
MeOH/H2O (1:1, 30 mL), then TEA (312 L, 2.27 mmol) was added. The mixture was stirred at
rt for 10 min, then zinc acetate (7; 248 mg, 1.13 mmol) was added. After the mixture was stirred
for 1 h, the solid material was removed by filtration. After 3 h, a white solid precipitated from the
filtrate. The precipitate was separated and dried under vacuum to give Zn(2,3-trans-CHA)2
complex (9); yield: 244 mg (58%). 1H NMR (D2O, 300 K, ppm): δ 3.98 (bs, 1H), 4.33 (bs, 1H),
6.22 (bm, 1H), 6.34 (bm, 1H), 6.92 (bm, 1H).
Zn(3,4-trans-CHA)2 complex (10). 3,4-trans-CHA (4; 167 mg, 1.08 mmol) was dissolved in
MeOH (7 mL). The same amount of water was added to obtain a clear solution. This was followed
by addition of TEA (150 L, 1.08 mmol). The mixture was stirred at rt for 10 min, then zinc acetate
(7; 119 mg, 0.54 mmol) was added. After the mixture was stirred for 1 h, the solid material was
removed by filtration. From the filtrate, a white solid precipitated after addition of TEA (240 L).
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