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P. G. M. Wuts et al.
LETTER
Acetate Deprotection
Diol 16b
Flavobacterium dehydrogenans ATCC 13930 was tested against
3b,7b,11a-triacetyl DHEA. Cells were grown using a two-stage
seed protocol. Frozen vegetative cells of F. dehydrogenans were
thawed, transferred to nutrient agar (Difco) plates, and incubated at
28 °C for 72 h. A single colony of F. dehydrogenans was used to
inoculate a 500 mL shake flask containing 100 mL primary seed
medium. The primary seed medium consisted of the following, per
liter of RO water: nutrient broth, 8 g; glycerol, 4 mL; yeast extract,
1 g; KH2PO4, 2.72 g; polysorbate 80, 2 mL; final pH, 6.8. Shake
flasks, containing 100 mL primary seed medium, were sterilized for
30 min at 121 °C. Flavobacterium dehydrogenans was incubated
for 48 h at 28 °C, using a controlled-environment incubator–shaker
incubated at 270 rpm (2¢¢ orbital stroke).
13C NMR (100 MHz, CDCl3): d = 176.7, 173.4, 145.4, 118.3, 95.2,
71.4, 68.7, 51.7, 50.0, 46.1, 44.6, 43.8, 42.9 ppm. HRMS: m/z calcd
for C24H35O6 (M + H): 419.2434; found: 419.2435.
Enone 17
13C NMR (100 MHz, CDCl3): d = 199.6, 176.6, 172.8, 167.9, 126.1,
95.0, 69.1, 52.5, 51.6, 46.2, 45.2, 43.3, 42.4, 39.4, 37.3, 37.0, 36.3,
35.4, 34.1, 31.2, 29.1, 22.5, 18.4, 15.4 ppm. HRMS: m/z calcd for
C24H33O6 (M + H): 417.2277; found: 417.2296.
Acknowledgment
We thank William D. McGhee for doing some catalyst screening
that lead to the use of PdBr2 in the carbonylation. We thank Mark
Mowery, Diane Strother, Stephen Grode, and Dave Russell for their
spectroscopic help.
The secondary seed medium consisted of the following, per liter of
RO water: cerelose, 20 g; HySoy, 6 g; yeast extract, 6 g; SAG 471,
0.5 mL; final pH, 6.8. Shake flasks, containing 100 mL secondary
seed medium, were sterilized for 30 min at 121 °C. Secondary seed
medium was inoculated with 0.12 mL of primary culture and incu-
bated as above.
References
Bioconversions were performed in medium described by Boeren et
al.15 This consisted of the following, per liter of RO water: yeast ex-
tract, 15 g; (NH4)2SO4, 1 g; KH2PO4, 1 g; cerelose, 5 g; polysorbate
80, 1 mL; final pH was adjusted to 6.8. Prior to sterilizing the me-
dium, 3,7,11-triacetoxy DHEA (20a) dissolved in a minimal vol-
ume of acetone was added to vigorously stirring medium to a final
concentration of 1 g/L. Shake flasks, containing 50 mL medium,
were sterilized for 30 min.
(1) Information update: Drugs Future 2000, 25, 516.
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Mucciariello, G.; Vanzanella, F.; Kunda, S. A.; Letendre, L.
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(3) McMahon, E. G. Curr. Opin. Pharmacol. 2001, 1, 190.
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2004.
Cultures were sampled periodically. Culture (2 mL) were extracted
with MeOH (10 mL) for 30 min, and then centrifuged to remove
cells. The supernatant was transferred to a clean vial and dried under
nitrogen, then redissolved in MeOH (0.5 mL). Then, 2 mL were ap-
plied to a TLC plate and developed in cyclohexane–EtOAc–
MeOH–AcOH (90:60:30:1). The plate was photographed under UV
illumination, then sprayed with 50% H2SO4, and charred at 200 °C
for 10 min.
(5) Fu, X.; Tann, C.-H.; Thiruvengadam, T. K.; Lee, J.; Colon,
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(6) Mackey, S. S.; Matison, M. E.; Wu, H. unpublished results.
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Brown, J. M. Adv. Synth. Catal. 2002, 344, 104. (b) Takas,
J. M.; Lawson, E. C.; Clement, F. J. Am. Chem. Soc. 1997,
119, 5956. (c) Andersson, P. G.; Schab, S. Organometallics
1995, 14, 1.
Four more derivatives of DHEA were studied as substrates for F.
dehydrogenans, the tricarbonate 20b, a second with a 7a-furan
group at position 7, and acetate groups at positions 3 and 11 (24, 22,
and 15b). The tricarbonate 20b remained virtually untouched after
6 d. Lactone 15b was converted to a predominant product within 96
h. Structural analysis revealed that both acetate groups had been re-
moved while the carbomethoxy moiety and lactone ring remained
intact. A faint band visible under UV light was also isolated and
identified as 17. This second product was found to have the 3-hy-
droxyl further oxidized to a ketone group, and the 5,6-double bond
shifted to the 4,5-position. The harvested beer from the incomplete
Flavobacterium oxidation was pasteurized, and then supplemented
with nutrients to support the growth a proprietary strain of Myco-
bacterium fortuitum. Supplemented nutrients were, per liter of spent
beer: soyflour, 6 g; glycerol, 20 mL; KH2PO4, 20.4 g; (NH4)2SO4,
10 g; MgSO4·7H2O, 2 g; final pH, 7.0. This organism was able to
completely convert the deacetylated product of the Flavobacterium
bioconversion to compound 17.
(8) Wuts, P. G. M.; Ritter, A. R. J. Org. Chem. 1989, 54, 5180.
(9) Wuts, P. G. M.; Ashford, S. W.; Anderson, A. M.; Atkins, J.
R. Org. Lett. 2003, 5, 1483.
(10) Takahashi, Y.; Tsukiyama, K.; Sakai, S.; Ishii, Y.
Tetrahedron Lett. 1970, 1913.
(11) Murahashi, S.-I.; Imada, Y.; Taniguchi, Y.; Higashiura, S.
J. Org. Chem. 1993, 58, 1538.
(12) Microbial Transformations of Steroids: A Hand-
book,Charney W., Herzog H. L.; Academic Press: New
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(13) Kieslich, K.; Kapp, J. F. German Patent DE 2656575, 1978.
(14) Kieslich, K. German Patent DE 2746298, 1979.
(15) Boeren, S.; Laane, C. Biotech. Bioeng 1987, 29, 305.
(16) White, M. J.; Beck, D. M.; Wuts, P. G. M.; Gilbert, I. G. US
265948, 2004.
Synlett 2008, No. 3, 418–422 © Thieme Stuttgart · New York