L. D. Jennings et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1427–1431
1431
6. Denny, W. A. Current Medicinal Chemistry 2002, 9, 1655.
7. For an account of an alternate approach to the optimiza-
tion of the indoloquinolizinone inhibitor lead, see:
Sutherland, A. G.; Alvarez, J.; Ding, W.; Foreman, K. W.;
Kenny, C. H.; Labthavikul, P.; Mosyak, L.; Petersen,
P. J.; Rush, T. S.; Ruzin, A.; Tsao, D. H. H.; Wheless,
K. L. Org. Biomol. Chem. 2003, 1, 4138.
8. Rosenmund, P.; Trommer, W.; Dorn-Zachertz, D.;
Ewerdwalbesloh, U. Liebigs Annalen der Chemie 1979, 11,
1643.
blank channel in which a mock thiol coupling with no
peptide had been performed and the signal from the blank
was subtracted from that with the peptide. Compounds
were added to ZipA, filtered, and then binding to the
peptide was determined. Percent inhibition is calculated
from the average resonance signal for the sensogram col-
lected with inhibitor present and the sensogram collected
in the absence of inhibitor.
13. ‘Methods for Dilution of Antimicrobial Susceptibility
Tests for Bacteria That Grow Aerobically’, Approved
Standards: M7-A5, Vol. 20, National Committee for
Clinical Laboratory Standards, Wayne, PA. Bacterial
species in the panel included S. aureus (ATCC 29213), E.
faecalis, S. pneumoniae, B. subtilis, H. influenzae (ATCC
49247), M. catarrhalis, and E. coli.
9. Grandi, T.; Sparatore, F.; Sparatore, A. Il Pharmaco
1999, 54, 479.
10. EmrysTM Microwave Synthesizer, Personal Chemistry
AB, Uppsala, Sweden.
11. Na2CO3 (0.034 g, 0.32 mmol) in water (0.3 mL) was
added to a solution of propylamine 9 (0.024 g, 0.08 mmol)
in 0.3 mL CH2Cl2. The reaction was shaken for 1 min.
Furoyl chloride (0.021 g, 0.16 mmol) was added and the
reaction was mixed at ambient temperature overnight.
The reaction was diluted with 2 mL CH2Cl2 and washed
with approximately 0.8 mL aqueous NaHCO3. The
organic phase was removed and the solvent was evapo-
rated. The residue was purified by RP-HPLC (Gilson
Semi-Preparative HPLC system with Unipoint Software
v. 1.71, Phenomenex C18 Luna column, 21.2Â100 mm, 5 m
particle size, water–acetonitrile solvent system with added
0.05% NH4OH buffer, at 22.5 mL/min) to give 14.3 mg
(36.5 mmol) of 10a [LC/MS (Hewlett Packard 1100 MSD
with ChemStation Software, Keystone Aquasil C18 50
mmÂ2 mm column, 5m particle size, at 40 ꢀC, 10 mM
NH4OAc–acetonitrile solvent system at 0.8 mL/min flow
rate, 254 nm DAD detection, API-ES scanning mode,
fragmentor 70 mV): m/z 392 (M+H); retention time
1.96 min].
14. 2 mM of 10b was added to ZipA (20 mg/mL), so that the
final mixture contained 2:1 compound versus the protein.
Co-crystals of approximate dimensions 0.5Â0.7Â0.15
mm3 were grown by vapor diffusion from 2 mL droplets
consisting of 1 mL of protein solution and 1 mL of pre-
cipitant (20% PEG 6000, 0.1 M MES pH 6.0). To produce
high quality crystals, streak seeding with the native crys-
tals as seeds was applied. Crystals belonging to space
group P21 (a=52.45 A, b=38.85 A, c=71.52 A, b=106.3
deg) containing two ZipA molecules per asymmetric unit
were obtained. Prior to data collection, the crystals were
flash cooled in liquid nitrogen at 100 K using 25% ethyl-
ene glycol as a cryoprotectant. The 2.1 A data were col-
lected in house using RAXIS IV imaging plate system.
The data were again integrated using DENZO and then
scaled and merged with SCALEPACK.17
15. Navaza, J. Acta Crystallogr. 1994, A50, 157.
16. Brunger, A. T.; Adams, P. D.; Clore, G. M.; DeLano,
W. L.; Gros, P.; Grosse-Kunstleve, R. W.; Jiang, J.-S.;
Kuszewski, J.; Nilges, M.; Pannu, N. S.; Read, R. J.; Rice,
L. M.; Simonson, T.; Warren, G. L. Acta Crystallogr.
1998, D54, 905.
12. E. coli ZipA(185-328) was passed over the immobilized
peptide in Biacore’s HBS-EP buffer (10 mM Hepes, pH
7.4, 150 mM NaCl, 3 mM EDTA, 0.005% P20) with
DMSO added to a final concentration of 5%. The run-
ning buffer was the same. The sample was also run over a
17. DENZO and SCALEPACK: Otwinowski, Z.; Minor, W.
Methods Enzymol. 1997, 276, 307.