Boyd et al.
597
dimethyl polysiloxane (0.33 m) as the bonded phase. The
injector port was held at 250°C and the oven was pro-
grammed at 100°C for 1 min and then ramped to 300°C at
10°C min–1. Helium was employed as carrier gas at a flow
rate of 0.8 mL min–1 and the sample (1 L) was injected at a
split ratio of 25:1. Under these conditions the cis-diols 29
and 30 and the trans-diol 31 were eluted at 7.34, 6.96, and
6.00 min, respectively. The mass-selective detector was op-
erated in the full-scan mode measuring ion currents between
m/z 30 and 450 amu. The relative yields of diols were calcu-
lated by comparing the relative peak areas of diols (29/31
and 30).
27. The yields obtained from the small and larger scale
biotransformations, respectively, are shown together.
(+)-cis-(3S,4S)-3,4-Dihydroxy-3,4-dihydro-2-quinolone (12)
Colourless crystals (0.164 g, 2.0% and 3.10 g, 5.7%); mp
177–179°C (MeOH–hexane). Rf = 0.14 (7% MeOH–CHCl3).
[ꢀ]D +6.0 (c 0.72, pyridine). CD ꢅ (nm): 285.20 ( ꢆ =
+0.683), 252.40 ( ꢆ = –2.237), 217.50 ( ꢆ = +1.760),
200.40 ( ꢆ = –3.258). IR ꢇmax (cm–1): 3220 (OH), 1674
1
(CONH). H NMR (500 MHz, (CD3)2CO) ꢁ: 4.05 (d, J =
3.0 Hz, 1H, OH), 4.13 (d, J = 3.0 Hz, 1H, OH), 4.34 (d,
J3,4 = 3.7 Hz, 1H, H-3), 4.77 (d, J4,3 = 3.7 Hz, 1H, H-4),
7.03 (m, 2H, H-6 and H-8), 7.30 (m, 1H, H-7), 7.36 (m, 1H,
H-5), 9.36 (br s, 1H, NH): NOE enhancement of H-3 (1%)
and H-5 (2%) from irradiation of H-4; NOE enhancement of
H-4 (1%) from irradiation of H-3. 13C NMR (125 MHz,
(CD3)2CO) ꢁ: 70.0, 71.1, 105.2, 116.1, 123.0, 129.8, 130.1,
137.8, 171.0. MS m/z (%): 179 (M+, 77), 161 ([M – H2O]+,
54), 122 (100). Anal. calcd. for C9H9NO3: C 60.3, H 5.1,
N 7.8; found: C 59.8, H 4.9, N 7.6.
Substrates 2, 4, 24, and 28 were obtained from commer-
cial sources. Substrate 3 (35) and (–)-(S)- and (+)-(R)-2-(1-
methoxyethyl)benzene boronic acids were synthesized and
used according to literature methods (23–25, 36).
Substrates were metabolized using growing cultures of P.
putida UV4, a constituent mutant strain devoid of cis-diol
dehydrogenase activity, according to the reported method
(30). P. putida 9816/11, an inducible mutant, without the
corresponding cis-diol dehydrogenase enzyme activity, was
grown on minimal-salts medium containing 0.05% sodium
succinate. The naphthalene dioxygenase (NDO) present was
induced by addition of 0.5% sodium salicylate at the late ex-
ponential phase of growth. S. yanoikuyae, also an inducible
mutant without the corresponding cis-diol dehydrogenase
enzyme activity, was grown on minimal-salts medium with
0.5% sodium pyruvate and 0.05% of yeast extract; the late
exponential phase of growth the biphenyl dioxygenase
(BPDO) was again induced by the addition of m-xylene
(0.05 mL L–1) every 1/2 h for 7 h. When the cells were
grown in a 100-L fermenter, continuous addition of the vola-
tile inducer (m-xylene) was necessary since it was constantly
depleted by the air supply. E. coli pKST11 biotrans-
formations were carried on cells grown on minimal-salts me-
dium supplemented with glucose (20 mM), thiamin (1 mM),
and ampicillin (100 g mL–1) at 37°C. TDO activity was in-
(+)-cis-(5R,6S)-2-Chloro-5,6-dihydroquinoline-5,6-diol (7)
Colourless crystals (0.66 g, 8% and 5.4 g, 9%); mp 120–
122°C (EtOAc–hexane). Rf = 0.30 (7% MeOH–CHCl3). [ꢀ]D
+140 (c 0.55, MeOH). CD ꢅ (nm): 294.60 ( ꢆ = –1.891),
254.40 ( ꢆ = +7.648), 226.00 ( ꢆ = +8.017), 195.60 ( ꢆ = –
1
3.821). IR ꢇmax (cm–1): 3402 (OH). H NMR (500 MHz,
(CD3)2CO) ꢁ: 4.00 (m, 1H, OH), 4.18 (dd, J6,5 = 4.9, J6,7
=
4.6 Hz, 1H, H-6), 4.24 (m, 1H, OH), 4.55 (d, J5,6 = 4.9 Hz,
1H, H-5), 6.27 (dd, J7,6 = 4.6, J7,8 = 9.9 Hz, 1H, H-7), 6.37
(d, J8,7 = 9.9 Hz, 1H, H-8), 7.13 (d, J3,4 = 8.1 Hz, 1H, H-3),
7.71 (d, J4,3 = 8.1 Hz, 1H, H-4); NOE enhancement of H-4
(1%) from irradiation of H-5. 13C NMR (125 MHz,
(CD3)2CO) ꢁ: 67.3, 70.5, 123.6, 130.0, 133.5, 136.8, 138.8,
150.5, 153.5. MS m/z (%): 197 (M+(35Cl), 34), 179 ([M –
H2O]+, 8), 168 (100). Anal. calcd. for C9H8ClNO2: C 54.7, H
4.1, N 7.1; found: C 54.2, H 3.8, N 6.9.
(+)-cis-(7S,8R)-2-Chloro-7,8-dihydroquinoline-7,8-diol (8)
Colourless crystals (1.63 g, 18% and 12.0 g, 20%); mp
110–111°C (EtOAc–hexane). Rf = 0.45 (7% MeOH–CHCl3).
[ꢀ]D +148° (c 0.55 in MeOH). CD ꢅ (nm): 309.80 ( ꢆ = –
0.932), 252.20 ( ꢆ = +3.750), 246.80 ( ꢆ = +3.665), 210.00
duced by the addition of
1
mM isopropyl-ꢄ-
thiogalactopyranoside (IPTG). The NDO in E. coli JM109
was similarly induced by the addition of IPTG. P. putida Q1
was grown on minimal-salts medium supplemented with
quinoline (0.3 mg mL–1) as the carbon source.
1
( ꢆ = +7.257). H NMR (500 MHz, (CD3)2CO) ꢁ: 4.01 (d, J
= 5.8 Hz, 1H, OH), 4.49 (dd, J7,8 = 5.0, J7,6 = 4.2 Hz, 1H,
H-7), 4.59 (m, 1H, OH), 4.61 (d, J8,7 = 5.0 Hz, 1H, H-8),
Biotransformation of substrates: isolation and
identification of metabolites
6.17 (dd, J6,7 = 4.2, J6,5 = 9.6 Hz, 1H, H-6), 6.57 (d, J5,6
=
9.6 Hz, 1H, H-5), 7.34 (d, J3,4 = 8.0 Hz, 1H, H-3), 7.61 (d,
J4,3 = 8.0 Hz, 1H, H-4); NOE enhancement of H-4 (3%) and
H-6 (3%) from irradiation of H-5. 13C NMR (125 MHz,
(CD3)2CO) ꢁ: 68.2, 72.4, 124.6, 126.2, 128.3, 133.3, 137.7,
149.6, 158.4. MS m/z (%): 197 (M+(35Cl), 37), 179 ([M –
H2O]+, 6), 168 (100). Anal. calcd. for C9H8ClNO2: C 54.7, H
4.1, N 7.1; found: C 54.2, H 3.8, N 6.7.
2-Chloroquinoline 2 with P. putida UV4
Biotransformation (10-L fermenter) of 2-chloroquinoline
(2) (7.5 g) by P. putida UV4, repeated extraction of the
aqueous biotransformed material with EtOAc, and removal
of solvent from the extract under reduced pressure (Proce-
dure 1), yielded a crude mixture of three cis-diols 12, 7, and
8; these were separated by a combination of column chroma-
tography (CHCl3 J 10% MeOH–CHCl3) and PLC (7%
MeOH–CHCl3). A larger-scale biotransformation (100-L
fermenter) of 2-chloroquinoline 2 (50 g) was also carried
out. The bioproducts were isolated by removal of water (re-
duced pressure) from the biotransformed material followed
by hot extraction (45°C) of the residual material with 20%
MeOH–EtOAc (Procedure 2). Column chromatography and
PLC separations yielded five metabolites: 12, 7, 8, 26, and
2-Chloro-3-hydroxyquinoline (26)
White crystalline solid (0.5 g, 1%); mp 311–312°C
(EtOAc–hexane) (lit. (37) mp > 210°C). Rf = 0.55 (EtOAc–
1
hexane, 1:1). H NMR (500 MHz, (CD3)2CO) ꢁ: 7.41 (ddd,
J7,6 = 8.3, J7,8 = 8.3, J7,5 = 1.4 Hz, 1H, H-7), 7.45 (ddd, J6,5
= 8.3, J6,7 = 8.3, J6,8 = 0.6 Hz, 1H, H-6), 7.60 (s, 1H, H-4),
7.68 (dd, J5,6 = 8.3, J5,7 = 1.4 Hz, 1H, H-5), 7.73 (dd, J8,7
=
8.3, J8,6 = 0.6 Hz, 1H, H-8). The physical and spectral data
© 2002 NRC Canada