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63
7. DeVinney, I.; Steele-Mortmer, I.; Finlay, B. B. Trends
Microbiol. 2000, 8, 29.
addition and subsequent oxidation afforded the indolizines
in a clean reaction and good yields.
8. Koul, A.; Choidas, A.; Treder, M.; Tyagi, A. K.; Drlica,
K.; Singh, Y.; Ullrich, A. J. Bacteriol. 2000, 182, 5425.
9. Obaya, A. J.; Sedivy, J. M. Cell Mol. Life Sci. 2002, 59,
126.
10. (a) Galaktionov, K.; Lee, A. K.; Eckstein, J.; Draetta, G.;
Meckler, J.; Loda, M.; Beach, D. Science 1995, 269, 1575;
(b) Dixon, D.; Moyana, T.; King, M. J. Exp. Cell Res.
1998, 240, 236.
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1996, 118, 10337.
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19. All enzyme assays were performed by means of an
automated system consisting of a Zymark SciClone
ALH 500 in conjunction with a Twister II and a Bio-
Tek Power Wave 340 reader. The reaction volume was
10 lL. The reaction was started by the addition of 5 lL p-
nitrophenyl phosphate to 5 lL of a solution containing the
respective enzymes which had been pre-incubated for 10–
15 min with different concentrations from twofold dilution
series of inhibitors. Reaction velocity was determined
from the slope of the absorbance change at 405 nm and
related to control values in absence of the inhibitor. IC50
values were calculated from linear extrapolations of
reaction velocity as a function of the logarithm of
concentration. This non-biased approach did not allow
for the determination of IC50 values larger than 100 lM.
The error indicated in Table 1 is the standard deviation.
The overall experimental error, including the water
content of DMSO stock solutions and weighing errors,
is approximately 50% of the respective IC50 values. All
buffered solutions contained 2 mM DTE (1,4-dithio-D,L-
threitol added on the day of the experiment from 100 mM
stock) and 0.025% (v/v) of the detergent NP-40 (Calbio-
chem 492015). The buffers consisted of 50 mM Tris,
50 mM NaCl, 0.1 mM EDTA in the case of CDC25A, or
25 mM HEPES, 50 mM NaCl, 2.5 mM EDTA in the case
of PTP1b, MPTPA, and MPTPB, or 25 mM MOPS,
5 mM EDTA in the case of VHR. The twofold dilution
series were obtained from 10 lL of a buffered enzyme
solution containing 200 lM of inhibitor. Of this, 5 lL was
removed and mixed with 5 lL buffered enzyme solution
resulting in a twofold dilution. This step was repeated 5
times. Five microlitres of the final dilution was removed,
so that each well consisted of 5 lL buffered enzyme
inhibitor mix. After addition of 5 lL p-nitrophenyl phos-
phate, the concentrations for the enzyme reaction were
50 mM in the case of CDC25A or 1 mM for all other
phosphatases. The inhibitor concentrations were 100, 50,
25, 12.5, 6.25, or 3.125 lM, respectively. For all enzymes,
their concentration was adjusted to an initial absorbance
change of 1–2 OD405/h. All reactions were performed as
quadruplets from identical manual dilutions (1:10 in buffer
from 10 mM stock solutions in DMSO).
11. (a) Alonso, A.; Saxena, M.; Williams, S.; Mustelin, T.
J. Biol. Chem. 2001, 276, 4766; (b) Todd, J. L.; Rigas,
J. D.; Rafty, L. A.; Denu, J. M. Oncogene 2002, 21,
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Langer, T.; Saxena, K.; Schwalbe, H.; Furstner, A.;
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Rademann, J.; Waldmann, H. ChemBioChem, accepted
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(b) Huisgen, R.; Temme, R. Eur. J. Org. Chem. 1998, 2,
387; (c) Druta, I. I.; Andrei, M. A.; Ganj, C. I.; Aburel, P.
S. Tetrahedron 1999, 55, 13063; (d) Goff, D. A. Tetrahe-
dron Lett. 1999, 40, 8741.
14. (a) Uchida, T.; Matsumoto, K. Synthesis 1976, 209; (b)
Swinbourne, F. T.; Hunt, J. H.; Klinkert, K. In Advances
in Heterocyclic Chemistry; Katrizky, A. R., Ed.; Academic
Press: New York, 1978; Vol. 32; (c) Molina, P.; Fresneda,
P. M.; Lajara, M. C. J. Heterocycl. Chem. 1985, 22, 113;
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Wei, X.; Hu, Y.; Li, T.; Hu, H. J. Chem. Soc., Perkin
Trans. 1 1993, 2487; (c) Zhang, X.; Cao, W.; Wei, X.; Hu,
H. Synth. Commun. 1997, 27, 1395.
16. Typical experimental procedure: the indolizine precursor
was coupled to the TCP resin via standard Fmoc-solid-
phase chemistry. After the coupling, the resin was washed
with DMF and dichloromethane, dried, and subsequently
treated with a solution of ethyl 2-bromoacetate (20 equiv)
in DMF for 2 h at 40 °C. The resin was washed with DMF
and DCM. Afterwards the resin was transferred into a
glass vial and treated with a solution of acroylnitrile
(20 equiv) and triethylamine (20 equiv) in DMF at 60 °C
for 2 h. Washing with DMF and dichloromethane fur-
nished the resin bound tetrahydroindolizine, which was
treated with TCPD (3.0 equiv) in DMF. The oxidation
was run for 6 h at 80 °C to afford the indolizine after
washing with DMF and DCM. The desired product was
cleaved with 1:10 TFA/DCM and the ethyl ester was
hydrolyzed under basic conditions to furnish the crude
product, which was purified by HPLC. The [3+2]-cyclo-