7342
T. Yamamoto et al. / Bioorg. Med. Chem. 17 (2009) 7337–7343
pH 5.0, followed by 20 mL of Amberlite IRA-68 anion-exchange re-
sin (pre-equilibrated with 1 M HCl and extensively washed with
distilled water), and the suspension stirred for 30 min until the
solution turned colorless and the resin turned yellow. The resin
was suction-filtered and the filtrate rotoevaporated to remove
most of the organic solvent. The remaining solution was concen-
trated on Sep-Pak C18 cartridge (10 g, Waters, Milford, MA), then
eluted with CH3CN. The obtained yellow solution was concentrated
under reduced pressure for the final purification by preparative RP-
HPLC, and then lyophilized. Preparative RP-HPLC was performed
on Waters Delta Prep 4000 with Waters XTerra C-18 column
5.3.2. In vitro stability of peptide derivatives in rat plasma42
Stock solution of compounds (50 mg/mL in DMSO) were diluted
1000-fold into rat plasma (Lot 24927, Pel-Freez Biologicals, Rogers,
AK) to give an incubation concentration of 50
were incubated at 37 °C and 200 L of aliquots were withdrawn
at 1, 2, 4, 6, and 24 h. Then 300 L of acetonitrile was added and
lg/mL. All samples
l
l
the proteins were removed by centrifugation. The supernatant
was analyzed for the amount of remaining parent compound by
HPLC (Hewlett Packard 1090 m with Vydac 218TP104 C-18 col-
umn; 4.6 ꢁ 250 mm, 10
lm, 300 Å). The samples were tested in
three independent experiments (n = 3) for 1 and 5, and in two inde-
pendent experiments (n = 2) for 2–4. The mean values were dis-
played. The statistical significances were evaluated for 1 and 5
with the Student t-test (displayed in the Graphical Abstract).
(19 ꢁ 250 mm, 10
lm, a linear gradient of 33–53% or 40–60% aced-
tonitrile/0.1% TFA at a flow rate of 15.0 mL/min).
5.2.3. Characterization of peptides
The purified peptides were characterized by HRMS, TLC, analyt-
ical HPLC and 1H NMR. Sequential assignment of proton reso-
nances was achieved by 2D-TOCSY NMR experiments.39 High-
resolution MS were taken in the positive ion mode using FAB
methods at the University of Arizona Mass Spectrometry Facility.
TLC was performed on aluminum sheets coated with a 0.2 mm
layer of Silica Gel 60 F254 Merck using the following solvent sys-
tems: (1) CHCl3/MeOH/AcOH = 90:10:3; (2) EtOAc/n-BuOH/water/
AcOH = 5:3:1:1; and (3) n-BuOH/water/AcOH = 4:1:1. TLC chro-
matograms were visualized by UV light and by ninhydrin spray fol-
lowed by heating (hot plate). Analytical HPLC was performed on a
Hewlett Packard 1100 or Hewlett Packard 1090 m with Waters
Acknowledgments
The work was supported by grants from the USDHS, National
Institute on Drug Abuse, DA-13449 and DA-06284. We thank Ms.
Magdalena Kaczmarska for culturing cells, and the University of
Arizona Mass Spectrometry Facility for the mass spectra measure-
ments. We express appreciation to Ms. Margie Colie and Ms. Brigid
Blazek for assistance with the manuscript.
Supplementary data
Supplementary data (1H NMR, HPLC, and MS data of the cyclic
peptide derivatives 2–5 and their intermediates) associated with
this article can be found, in the online version, at doi:10.1016/
NOVA-Pak C-18 column (3.9 ꢁ 150 mm, 5
l
m, 60 Å) or Vydac
218TP104 C-18 column (4.6 ꢁ 250 mm, 10
l
m, 300 Å). 1H-1D-
NMR spectra were obtained on Bruker DRX-500 or DRX-600 spec-
trometer. 2D-TOCSY NMR spectra were performed on a Bruker
DRX-600 spectrometer equipped with a 5 mm Nalorac triple-reso-
nance single-axis gradient probe. The NMR experiments were con-
ducted in DMSO-d6 solution at 298 K. Spectra were referenced to
residual solvent protons as 2.49 ppm. The processing of NMR data
was performed with the XWINNMR software (Bruker BioSpin, Fre-
mont, CA). In the TOCSY experiments, the TPPI mode40 with
MLEV-17 Mixing Sequence41 were used with a mixing time of
62.2 ms, at a spin-lock field of 8.33 kHz. TOCSY spectra were ac-
quired with 2k complex pairs in t2 and 750 FIDs using a 90°-shifted
sine-squared window function in both dimensions.
References and notes
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5.3. In vitro pharmacology
5.3.1. Radioligand labeled binding assay, [35S]GTP
assay, GPI and MVD in vitro bioassay
The methods were carried out according to that previously de-
scribed.9,10,12 Briefly, the evaluation of the binding affinities of the
synthesized bifunctional peptide derivatives at the human d-opioid
cS binding
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receptors and rat
(HN9.10) membranes that stably express these corresponding
receptors using [3H]-c[ -Pen2, -Pen5]-enkephalin ([3H]DPDPE)
and [3H]-[ -Ala2, NMePhe4, Gly5-ol]-enkephalin ([3H]DAMGO) as
the radioligands, respectively. The [35S]GTP
S binding assays were
used to estimate the functional activities for d and opioid agonist
l-opioid receptors were performed on the cell
D
D
D
c
13. Ripley, T. L.; Gadd, C. A.; De Felipe, C.; Hunt, S. P.; Stephens, D. N.
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l
efficacies on the same cell membrane. The isolated tissue-based
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Szyfelbein, S. K.; Lipkowski, A. W. Life Sci. 1994, 54, 939.
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19. Largent-Milnes, T. M.; Yamamoto, T.; Davis, P.; Ma, S. W.; Hruby, V. J.;
Yamamura, H. I.; Lai, J.; Porreca, F.; Vanderah, T. W. In Neuroscience: San Diego,
CA., 2007, Poster 725. Visceral Pain: Transmitters and Receptors.
20. Largent-Milnes, T. M.; Yamamoto, T.; Nair, P.; Navratrilova, E.; Davis, P.; Ma, S.-
W.; Hruby, V. J.; Yamamura, H. I.; Lai, J.; Porreca, F.; Vanderah, T. W. In
International Association for the Study of Pain/12th World Congress on Pain:
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(hNK1) receptors, binding assays utilized membranes from trans-
fected CHO cells that stably express hNK1 receptors, using [3H]-
substance P as the standard radioligand. The binding assay at the
rat NK1 (rNK1) receptors also were performed using transfected
CHO cells that stably express rNK1 receptors. To evaluate antago-
nistic activities against substance P stimulation, isolated tissue bio-
assays using GPI were performed in the presence of naloxone to
block any opioid activities.