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under 1 bar of H2 atmosphere for ca. 2 h. The reaction was
stopped once it was complete (monitored by HPLC). The
mixture was ltered through Celite and washed with ethyl
acetate. The combined organic layer was evaporated under
reduced pressure (the water bath temperature was kept below
Experimental section
Chemistry
General. All reagents and solvents were obtained from
Sigma-Aldrich (St. Louis, MO) and Fisher Scientic (Hanover
Park, IL) and were used without further purication. Reactions
were monitored either by thin-layer chromatography (TLC) or by
reverse-phase HPLC with a Shimadzu LC-20A series HPLC
system. TLC was performed using glass plates pre-coated with
1
ꢁ
30 C) to give anziaic acid as a white solid (78.6 mg, 95%). H
NMR (400 MHz, CD3OD, ppm) d 6.62 (d, J ¼ 2.4 Hz, 1H), 6.56 (d,
J ¼ 2.0 Hz, 1H), 6.27 (d, J ¼ 2.0 Hz, 1H), 6.22 (d, J ¼ 2.4 Hz, 1H),
2.92 (t, J ¼ 7.2 Hz, 2H), 2.86 (t, J ¼ 7.6 Hz, 2H), 1.62–1.60 (m, 4H),
1.34–1.32 (m, 8H), 0.91–0.86 (m, 6H); 13C NMR (100.5 MHz,
CD3OD, ppm) d 173.89, 170.41, 166.05, 164.34, 164.14, 154.90,
149.36, 149.04, 116.22, 113.16, 112.23, 109.03, 105.22, 102.01,
37.73, 36.79, 33.21, 33.13, 32.99, 32.62, 23.60, 23.46, 14.44,
14.38; MS (ESIꢀ): m/z 429.3 [M ꢀ H]ꢀ; HRMS (ESI+) calcd for
˚
silica gel (0.25 mm, 60 A pore size, 230–400 mesh, Sorbent
Technologies, GA) impregnated with a uorescent indicator
(254 nm). TLC plates were visualized by exposure to ultraviolet
light (UV). Debenzylation reactions were done using a domnick
hunter NITROX UHP-60H hydrogen generator, USA. Flash
column chromatography was performed using a Biotage Isolera
One system and a Biotage SNAP cartridge. Proton and carbon
nuclear magnetic resonance (1H and 13C NMR) spectra were
recorded employing a Bruker AM-400 spectrometer. Chemical
shis were expressed in parts per million (ppm), and J values
were expressed in Hertz. Mass spectra were recorded on a Var-
ian 500-MS IT mass spectrometer using ESI. High-resolution
mass spectra (HRMS) were recorded with a BioTOF II ESI mass
spectrometer. The purity of compounds was determined by
C
24H30O7 (M + Na+): 453.1884, found: 453.1887; HPLC purity:
100% (254 nm), tR: 7.54 min; 100% (220 nm), tR: 7.54 min.
The spectroscopic data of synthetic anziaic acid are consis-
tent with those of the isolated anziaic acid natural product from
lichen Hypotrachyna sp.27
Synthesis of other structural analogues of anziaic acid is
described in the ESI.†
Topoisomerase inhibition assays. The topoisomerase assays
were performed as previously described.27 The IC50 values were
determined from an average of experiments repeated at least
twice. Briey, inhibition of relaxation activity of 10 ng of E. coli
topoisomerase I was assayed with 250 ng of supercoiled pBAD/
Thio plasmid DNA substrate puried by a CsCl gradient. The
relaxation reaction was carried out in 20 mL of 10 mM Tris–HCl,
pH 8.0, 50 mM NaCl, 0.1 mg mLꢀ1 gelatin with 0.5 mM MgCl2.
Aer 30 min at 37 ꢁC, the reactions were terminated and
analyzed by agarose gel electrophoresis.
˚
analytical HPLC using a Gemini, 3 mm, C18, 110 A column
(50 mm ꢂ 4.6 mm, Phenomenex) and a ow rate of 1.0 mL
minꢀ1. Gradient conditions: solvent A (0.1% triuoroacetic acid
in water) and solvent B (acetonitrile): 0–2.00 min 100% A, 2.00–
7.00 min 0–100% B (linear gradient), 7.00–8.00 min 100% B,
8.00–9.00 min 0–100% A (linear gradient), 9.00–10.00 min 100%
A, UV detection at 254 and 220 nm.
Representative procedure for the synthesis of dimeric
precursors. To a stirred solution of benzyl 2,4-dihydroxy-6-pen-
tylbenzoate (5) (94.3 mg, 0.3 mmol) and 2,4-bis(benzyloxy)-6-
pentylbenzoic acid (3) (121.4 mg, 0.3 mmol) in dry toluene (3 mL)
was slowly added triuoroacetic acid anhydride (486 mL, 3.45
mmol) at room temperature. The mixture was stirred overnight.
The solvent was then removed under reduced pressure. The
residue was puried by ash column chromatography (hex-
ane : ethyl acetate ¼ 97 : 3) to give 6 as a solid (135 mg, 64%). 1H
NMR (400 MHz, CDCl3, ppm) d 11.45 (s, 1H), 7.44–7.23 (m, 15H),
6.63 (d, J ¼ 2.0 Hz, 1H), 6.50 (d, 2.4 Hz, 1H), 6.48 (d, J ¼ 2.0 Hz,
1H), 6.38 (d, J ¼ 2.4 Hz, 1H), 5.36 (s, 2H), 5.06 (s, 2H), 5.05 (s, 2H),
2.70–2.66 (m, 4H), 1.66–1.62 (m, 2H), 1.35–1.31 (m, 6H), 1.14–1.10
(m, 2H), 1.02–1.01 (m, 2H), 0.88 (t, J ¼ 7.2 Hz, 3H), 0.79 (t, J ¼ 7.2
Hz, 3H); 13C NMR (100.5 MHz, CDCl3, ppm) d 171.09, 166.06,
164.41, 161.02, 157.66, 155.27, 148.32, 143.94, 136.49, 136.35,
134.79, 129.10, 128.83, 128.78, 128.71, 128.57, 128.22, 128.11,
127.59, 127.54, 116.13, 115.62, 109.56, 108.78, 107.46, 98.24,
70.70, 70.20, 67.81, 36.84, 33.95, 31.92, 31.86, 31.71, 31.06, 22.60,
22.57, 14.08, 14.04; MS (ESIꢀ): m/z 699.6 [M ꢀ H]ꢀ; HRMS (ESI+)
calcd for C45H48O7 (M+): 701.3473, found: 701.3472; HPLC purity:
100% (254 nm), tR: 8.91 min; 100% (220 nm), tR: 8.91 min.
Representative procedure for debenzylation of dimers and
synthesis of anziaic acid. A solution of benzyl 4-((2,4-bis(ben-
Human topoisomerase IIa (from TopoGen) relaxation assay
and E. coli DNA gyrase (from New England BioLab) supercoiling
assay were carried out as recommended by the suppliers. A
relaxed plasmid DNA substrate for DNA gyrase was purchased
from New England BioLabs.
Antibacterial testing. The MICs of compounds against
different bacterial strains grown in cation-adjusted Mueller-
Hinton Broth were measured with standard microdilution
procedures.27 Complete growth inhibition was recorded aer
ꢁ
24 h in a 37 C incubator.
Acknowledgements
This work was supported in part by the National Institutes of
Health Grants P20RR016467, P20GM103466, and R15AI092315
(D.S.) as well as R01AI069313 (Y.T.). We also thank Dr Benjamin
Clark and Dr Robert Borris for the assistance with collecting the
HRMS data of the synthesized compounds.
Notes and references
1 J. C. Wang, Nat. Rev. Mol. Cell Biol., 2002, 3, 430–440.
2 S. M. Vos, E. M. Tretter, B. H. Schmidt and J. M. Berger, Nat.
Rev. Mol. Cell Biol., 2011, 12, 827–841.
zyloxy)-6-pentylbenzoyl)oxy)-2-hydroxy-6-pentylbenzoate
(6)
(135 mg, 0.19 mmol) in ethyl acetate (5 mL) was treated with
10% Pd/C (38 mg). The mixture was stirred at room temperature
3 J. E. Deweese and N. Osheroff, Nucleic Acids Res., 2009, 37,
738–748.
This journal is ª The Royal Society of Chemistry 2013
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