T. Odagami et al. / Bioorg. Med. Chem. 17 (2009) 6742–6747
6747
0.1% acetic acid to 98% acetonitrile containing 0.1% acetic acid over
Acknowledgments
20 min at a 10.0 mL/min flow rate.
Cyclic peptide library was prepared on Sulfamylbutyryl AM re-
sin (purchased from NOVA Biochem, 1.06 mmol/g, 300 mg,
0.32 mmol) using standard solid-phase Fmoc based procedures.
In the first place, the resin was shaken for 2 ꢀ 16 h with a mixture
of Fmoc-glycine (4 equiv, 1.27 mmol)/HATU (4 equiv, 1.27 mmol)/
HOAt (4 equiv, 1.28 mmol)/DIEPA (8 equiv, 2.56 mmol) in DMF.
The coupling solution was drained and the resin was washed
(5 ꢀ DCM, 5 ꢀ DMF, 5 ꢀ DCM). Fmoc group was removed with
20%-piperidine in DMF. Then the resin was shaken for 16 h with
a mixture of Fmoc-proline (4 equiv, 1.27 mmol)/HBTU (4 equiv,
1.27 mmol)/HOBt (4 equiv, 1.28 mmol)/DIEPA (8 equiv, 2.56
mmol) in DMF. Further extension of the peptide was accomplished
using standard Fmoc strategy as described. After elongation of the
linear peptide, the N-terminal Fmoc group was replaced by Trt fol-
lowing treatment with 20% piperidine in DMF and shaken with Trt-
Cl (3.5 equiv, 1.1 mmol), DIEPA (6 equiv, 1.9 mmol) in DMF for
24 h. The cyclization was achieved by shaking with iodoacetonitri-
le (10 equiv, 3.18 mmol), DIEPA (10 equiv, 3.18 mmol) in DMF for
24 h, treatment with TFA and TIS in DCM (1:5:94) for 3 ꢀ 3 min,
and then treatment with DIEPA (3 equiv, 0.95 mmol) in DMF for
5 days. The side-chain protected cyclic peptides were dissolved
in DCM, and the organic layer was washed with 10% citric acid fol-
lowed by 5% sodium bicarbonate. The organic layer was concen-
trated in vacuo. The residue was then mixed with TIS, EDT, and
H2O in TFA (5:2.5:5:87.5) for 2 h. The crude peptide solution was
concentrated in vacuo and a crude peptide was obtained by precip-
itation from ether.
The authors acknowledge Dr. Lawrence H. Lazarus, NIEHS, NC,
USA for his advice during preparation of the manuscript. This work
was supported in part by NEXT ‘Academic Frontier’ Project (2006).
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Calcium-mobilization assay was used for the estimation of UT-II
agonistic activity of cyclic peptides in HEK293 cells stably transfec-
ted with human GPR-14. The cells were seeded into 96 well plates
pre-coated with poly-
der 5% CO2/95% oxygen atmosphere. The growth medium was aspi-
rated and replaced with 50 L of assay buffer (20 mM HEPES, pH
7.4, 115 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 0.8 mM CaCl2,
13.8 mM -glucose, and 0.1% BSA) with 5 M Fura-2 AM and
D
-lysine and incubated at 37 °C overnight un-
l
D
l
0.2% Pluronic F-127 (Molecular Probes, Inc.) in each well and incu-
bated at 37 °C for 30 min. The cells were subsequently washed
twice with same assay buffer and 80 lL left in each well. The
washed cells were placed in an FDSS 4000 (Hamamatsu Photonics
K.K.) and changes in cellular fluorescence using double excitation
wavelength (340 and 380 nm) after the addition of 20 lL test com-
pounds in assay buffer and recorded immediately. The peak height
of the ratio of fluorescence (340/380 nm) was evaluated as relative
agonistic activity.
5.3. NMR analysis
One- and two-dimensional NMR experiments were performed
at 400 MHz in a JEOL LA-400 spectrometer at 25 °C. The NMR sam-
ples were prepared by dissolving 1 mg of the compound in DMSO-
d6 (0.5 ml). COSY and NOESY were collected by the methods of
States et al.37 Resonance assignments were determined by COSY
spectrum using PFG technique.
5.4. Calculations
The three-dimensional structure of 1 was investigated by con-
formational analysis (MMFF94) using the BATCHMIN program in-
cluded in the MacroModel v6.5 package.38 A 100,000 step Monte-
Carlo search was performed with rmsd of 0.25 Å for all heavy
atoms. Energy minimizations were performed in vacuum.
37. States, D. J.; Haberkorn, R. A.; Ruben, D. J. J. Magn. Reson. 1982, 48, 286.