MHz, CDCl
3
): 2.11 (s, 6), 2.90 (dd, 1, J ) 2.6, 5.3), 3.24
3
MHz, CDCl ): 2.12 (s, 6), 2.85 (d, 1, J ) 3.4), 3.7 (dd, 1,
(
7
)
dd, 1, J ) 4.0, 5.3), 3.96 (dd, 1, J ) 2.5, 4.0), 5.89 (s, 2),
J ) 8.6, 11.3), 3.83 (dd, 1, J ) 3.6, 11.3), 5.02 (ddd, 1, J )
3.4, 3.4, 8.3), 5.89 (s, 2), 7.23 (d, 1, J ) 9.2), 7.88 (dd, 1,
J ) 2.6, 8.3), 8.60 (d, 1, J ) 2.4). C NMR (100 MHz,
.20 (d, 1, J ) 8.1), 7.66 (dd, 1, J ) 2.5, 8.3), 8.56 (d, 1, J
2.5).
3 (from 12). To a crude solution of 12 (74.6 g mixture,
1
3
1
CDCl ): 13.11, 50.00, 71.06, 107.16, 121.92, 128.68, 135.18,
3
synthesized from 0.163 mol of 10) in DMSO (140 mL) was
added 2-[4-(2-amino-ethoxy)-phenyl]-N-methyl-acetamide
136.37, 147.34, 151.65. AP+ ) 251.1. Anal. Calcd for
C H N OCl: C, 62.28; H, 6.03; N, 11.17. Found: C, 62.09;
13
15
2
20
(5) (68.35 g, 0.3282 mol). The mixture was heated in an
H, 6.15; N, 11.28.
8
5 °C bath for 8.5 h. Heating was discontinued and the
11 (Bioreduction of 10 in Fermentor Cultures of
Zygosaccharomyces bailii ATCC 38924). Cultures of Z.
bailii ATCC 38924 were obtained from the American Type
Culture Collection (ATCC, Manassas, Virginia) and stored
as glycerol suspensions of vegetative cells at -80 °C. A
three-stage protocol was used to prepare cultures for the
bioreduction of 10. The medium used for all three stages
contained 2% glucose, 0.5% soy flour, 0.5% NaCl, and 0.5%
solution allowed to cool and stir at room temperature
overnight. The mixture was added to 0.5 M HCl (2.8 L),
which caused a gum to separate out. The solution was
extracted with diethyl ether (3 × 1 L), and the ether washes
were discarded. The aqueous layer was decanted from the
gum that settled out, and 1 M NaOH (2.1 L) was added.
The aqueous layer was extracted with EtOAc (2 × 1.4 L),
and the combined organic layers were extracted with brine
2 4
K HPO (pH adjusted to 7.0 before autoclaving). Addition-
2 3
(500 mL) and dried over K CO . The solution was filtered
ally, the medium for stages one and three contained 0.5%
TWEEN 80. The initial-stage cultures were grown in 250-
mL Erlenmeyer flasks containing 30 mL of growth medium.
These flasks were inoculated with 0.2 mL of the glycerol
suspensions of Z. bailii and agitated on a rotary shaker (210
rpm) for 24 h at 29 °C. Second-stage cultures were grown
in two 2.8-L Fernbach flasks (500 mL of medium) and were
each inoculated with the contents of a single first-stage
culture and agitated (210 rpm) for 48 h at 29 °C. Final-stage
cultures were grown in fermentor jars containing 8 L of
medium. Two fermentors were each inoculated with one of
the second-stage cultures and incubated at 29 °C with
and concentrated to an oil, which solidified to a tacky solid
upon storage to provide 43.38 g (63% from 9) of 13 which
was sufficiently pure to carry forward in the synthesis. A
small portion was purified by SiO
EtOAc/NEt ) to provide an analytically pure sample. H
NMR (300 MHz, CDCl ): 2.14 (s, 6), 2.77 (d, 3, J ) 4.8),
.84 (dd, 1, J ) 9.5, 12.2), 3.07-3.21 (m, 3), 3.54 (s, 2),
2
chromatography (MeOH/
1
3
3
2
4
5
1
.13 (t, 2, J ) 5.0), 4.84 (dd, 1, J ) 3.3, 9.3), 5.47 (br s, 1),
.92 (s, 2), 6.93 (d, 2, J ) 8.7), 7.20 (d, 2, J ) 8.7), 7.24 (d,
, J ) 8.1), 7.91 (dd, 1, J ) 2.3, 8.1), 8.60 (d, 1, J ) 2.2).
1
3
3
C NMR (100 MHz, CDCl ): 13.15, 26.46, 42.70, 48.34,
5
1
6.69, 67.36, 69.37, 106.87, 114.96, 121.67, 127.34, 128.59,
30.67, 135.76, 136.68, 147.22, 151.36, 157.96, 172.06.
-1
agitation at 300 rpm and aeration at 3 L min . After 48 h,
1
6 g (potency of ∼80%) of 10 (200 g/L solution in DMSO)
was added to each fermentor, and incubation continued for
0 h. At the end of this time, the broths from the two
Anal. Calcd for C24
Found: C, 67.82; H, 7.39; N, 13.03.
R)-5-(2-Chloro-1-hydroxyethyl)-2-(2,5-dimethylpyrrol-
-yl)-5-(2-chloro-1-hydroxyethyl)pyridine (11). To a solu-
tion of (1S,2R)-2-amino-1,2-diphenylethanol (0.2429 g, 1.139
mmol) in THF (75 mL) was added BH SMe
(∼10.0 M,
.80 mL). This solution was allowed to stir at room
30 4 3
H N O : C, 68.22; H, 7.16; N, 13.26.
2
(
fermentors were combined and stirred overnight with 750 g
of Amberlite XAD-4 resin. The resin was then collected on
an 80 mesh screen, washed with 8 L of water, and extracted
with 8 L of of EtOAc. The EtOAc extract was washed with
water, dried over anhydrous magnesium sulfate, filtered, and
concentrated under vacuum to yield the crude product. Flash
chromatography on silica gel using EtOAc/hexanes mixtures
followed by crystallization from EtOAc/hexanes afforded
1
3
2
3
temperature for 15 h, during which time hyrdogen was
evolved. To the catalyst solution was added 10 (5.5134 g,
22.168 mmol) in THF (10 mL) via syringe pump over 3 h.
After an additional 3 h, the reaction was quenched by the
slow addition of water (15 mL), causing slow hydrogen
evolution. One hour after the addition of water, the reaction
solution was added to diisopropyl ether (50 mL), EtOAc (50
mL), and 2 N HCl (200 mL) and stirred well for 15 min.
The phases were separated, and the aqueous phase was
extracted with EtOAc (150 mL). The combined organic
phases were further extracted with brine (125 mL) containing
1
4.7 g (57%) of 11 with an enantiomeric excess of 98%.
1 (Bioreduction of 10 with Washed Cells of Z. bailii
1
ATCC 38924). A two-stage protocol was used to prepare
Z. bailii cells for the bioreduction of 10. In the first stage, a
3
00-mL Erlenmeyer flask containing 100 mL of growth
medium (2% glucose, 2% peptone, 1.2% yeast extract, and
.2% malt extract; pH adjusted to 6.0 before autoclaving)
1
was inoculated with 1 mL of a glycerol suspension of Z.
bailii cells and incubated at 29 °C on a rotary shaker (210
rpm) for 48 h. Second-stage cultures were set up in a manner
identical to that of the first stage except that the glucose
content of the medium was increased to 12%. Two second-
stage cultures were each inoculated with 4 mL of the first
stage culture and incubated under the conditions described
for stage one. After 72 h, the contents of the two second-
stage cultures were combined and centrifuged. The pellet
containing the cells was washed with 50 mL of 0.1 M
1
(
2.5 M NaOH solution (1.5 mL). The solution was dried
MgSO ), filtered, and concentrated to provide 5.27 g (95%)
of crude product. Chiral HPLC showed this material to be a
1.8:8.2 mixture of desired (R) to undesired (S) enantiomers.
4
9
The crude was crystallized from EtOAc/hexanes providing
two crops of material, totalling 3.7098 g (67%) of material
1
that was a 97.5:2.5 mixture of enantiomers. H NMR (400
(20) DeVries, K. M.; Dow, R. L.; Wright, S. W. PCT Int. Appl. WO98/21184,
May 22, 1998.
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