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K. D. Revell et al. / Bioorg. Med. Chem. 15 (2007) 2453–2467
lactam 1 (10 lg/mL, 1 · MIC); (c) lactam 1 (20 lg/mL,
2 · MIC); (d) no drug added. Each tube was incubated
Acknowledgments
3
for 5 min. at 37 ꢁC, then 2 lL of H-acetate (10 lCi/uL
We are grateful for the expert assistance of Ms. Betty
Loraam with the electron microscopy experiments, and
Ms. Sonja Dickey with culture preparations and gel elec-
trophoresis. We sincerely thank Professor Al Claiborne
at Wake Forest Medical School for a sample of coen-
zyme A disulfide reductase and also for helpful discus-
sion. We thank the NIH for supporting these studies
through Grant R01 AI51351.
in ethanol solution, sodium salt) was added to each tube
(4 lCi/mL radioactivity per tube). At intervals of 15, 30,
45, and 60 min, 0.8 mL aliquots were taken from each
tube, homogenized, and diluted with chloroform
(1 mL) and methanol (2 mL). An additional 1 mL of
chloroform and 1 mL of distilled water were added,
and the solution was mixed. The organic solution was
then washed with distilled water (1 mL), then 2 M KCl
(3· 1 mL), and then 0.1 M sodium acetate (3· 1 mL).
The radioactivity in the organic phase was then analyzed
by scintillation counting.
References and notes
1. Turos, E.; Konaklieva, M. I.; Ren, R. X.-F.; Shi, H.;
Gonzalez, J.; Dickey, S.; Lim, D. Tetrahedron 2000, 56,
8193.
4.16. Preparation of resin-bound b-lactam 6
2. Turos, E.; Long, T. E.; Konaklieva, M. I.; Coates, C.;
Shim, J.-Y.; Dickey, S.; Lim, D. V.; Cannons, A. Bioorg.
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A mixture of Merrifield’s resin, thioacetic acid and tri-
ethylamine was stirred at room temperature, after which
the resin was filtered. The resin was then reacted with
sulfuryl chloride in carbon tetrachloride to produce
the resin-bound sulfenyl chloride in situ. To this mixture
was added a solution of phthalimide and Hunig’s base in
benzene. The mixture was allowed to stir at rt, then fil-
tered and washed to give resin-bound phthalimide, The
resin was then combined with N–H b-lactam precursor,
and heated to reflux in benzene for 24 h, under nitrogen.
The mixture was cooled, and the resin filtered and
washed. The presence of the resin-bound lactam was
verified by reaction of a small quantity of the resin with
DIBAL, and subsequent analysis by 1H NMR and
HPLC.
4.17. Procedure for the isolation of CoA from S. aureus
lysate using Lactam-bound resin 6
8. Smith, D. M.; Kazi, A.; Smith, L.; Long, T. E.; Heldreth,
B.; Turos, E.; Dou, Q. P. Mol. Pharm. 2002, 61, 1348.
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A quantity of 1 liter of S. aureus (ATCC 25923) in broth
was cultured at 37 ꢁC for 24 h. The culture was then cen-
trifuged and washed. The resultant pellet was washed
with phosphate buffer and then resuspended in 5 mL
of phosphate buffer. The cells were then sonicated in
an ice bath for 30 min, stopping every 5 min to avoid
overheating. The lysed cells were then centrifuged and
the lysate was centrifuged again, and the resulting lysate
was separated off from the solids. Filtering of the lysate
yielded a slightly opaque yellow solution. 0.252 g of res-
in 6 was swelled in 0.5 mL DMSO and added to the ly-
sate solution. This mixture was then centrifuged at
200 rpm for 24 h. Next, the lysate was filtered and the
solid was repeatedly washed with boiling water and boil-
ing ethanol. The solid was dried and 196 mg of material
was collected. The material was dissolved in 5 mL of dry
CH2Cl2, and 1 mL of diisobutylaluminum hydride
(1.0 M in hexanes) was added at 0 ꢁC. The reaction
was worked up with 0.1 M HCl followed by freeze-dry-
ing. The crude extract was analyzed by high-perfor-
mance liquid chromatography using a Shimadzu LC-
8A HPLC on an analytical reverse phase column. A
9:1 mixture of acetonitrile:water at a 2 mL/min flow rate
was used, and detection was done using a Shimadzu
SPD-10A UV–vis detector. Identification of CoA was
achieved by comparison of the retention time to a com-
mercial sample.
15. Tipper, D. J.; Strominger, J. L. Proc. Natl. Acad. Soc.
U.S.A. 1965, 54, 1133.
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17. The advantage of preparing the samples from agar
cultures as opposed to broth is that a concentration
gradient is produced as the antibiotics diffuse through the
solid media, allowing microscopic examination among a
range of exposure amounts.
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