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simulation was performed at a constant temperature of 300 K,
sampling the trajectory every picosecond. The last frame was taken
as the starting structure for a 150 ps free MD simulation at 300 K,
sampling the Φ and Ψ angles every picosecond. The last frame of
the free MD was subjected to a further minimization and compared
with an energy-minimized average structure and with those derived
from the restrained MD simulation, obtaining substantially the same
results (rms ) 0.2 Å). The simulations with explicit water were
performed by surrounding the system by a sphere of water
molecules with radius of 25 Å. Then 20 ps of restrained molecular
dynamics simulations were carried out at 300 K and the last frame
was taken as a starting structure for 20 ps of free MD simulations.
The last frame of the free MD was subjected to a further
minimization and compared with the energy-minimized average
structure of the 20 derived from the free MD (rms ) 0.2 Å),
obtaining the distances reported in Table 1.
Solid-Phase Receptor-Binding Assay. The receptor binding
assays were performed as described by Kumar et al.35 Purified RVâ3
and RVâ5 receptors (Chemicon International Inc., Temecula, CA)
were diluted to 500 and 1000 ng/mL, respectively, in coating buffer
[20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM CaCl2, 1 mM
MgCl2, and 1 mM MnCl2]. An aliquot of diluted receptors (100
µL/well) was added to 96-well microtiter plates and incubated
overnight at 4 °C. Coating solution was removed, and an amount
of 200 µL of blocking solution (coating buffer plus 1% BSA) was
added to the wells and incubated for an additional 2 h at room
temperature. After incubation, the plates were rinsed three times
with 200 µL of blocking binding solution and incubated for 3 h at
room temperature with 0.05 nM and 0.1 nM [125I]echistatin
(Amersham Pharmacia) for RVâ3 and RVâ5 receptor binding assays,
respectively. After incubation, the plates were sealed and counted
in the γ-counter (Packard). The IC50 values were calculated as the
concentrations of compounds required for 50% inhibition of
echistatin binding. Each data point is a result of the average of
triplicate wells and was analyzed by nonlinear regression analysis
with the Prism GraphPad program.
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substituierte ω-Carboxy-R-aminosa¨uren (An effective synthesis of
R-trifluoromethyl-substituted ω-carboxy-R-amino acids). Chem. Ztg.
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753.
Acknowledgment. This work was partially supported by
Sigma-Tau, Rome, Italy, and by the University of Milano and
MIUR (Funds COFIN-2003 and FIRB-2001).
(20) Dal Pozzo, A.; Bergonzi, R.; Ni, M. H. N-Protected amino acids
bromides: efficient reagents for the incorporation into peptides of
extremely hindered R,R-dialkyl- and R-fluoroalkyl-amino acids.
Tetrahedron Lett. 2001, 42, 3925-3927.
Supporting Information Available: Table S1 listing 1H
chemical shifts and Table S2 listing interproton distances. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(21) Meldal, M.; Juliano, M. A.; Jansson, A. M. Azido acids in a novel
method of solid-phase peptide synthesis. Tetrahedron Lett. 1997, 38,
2531-2534.
(22) Jost, M.; Greie, J. C.; Stemmer, N.; Wilking, S. D.; Altendorf, K.;
Sewald, N. The first total synthesis of efrapeptin C. Angew. Chem.,
Int. Ed. 2002, 41, 4267-4269.
(23) Dal Pozzo, A.; Ni, M. H.; Muzi, L.; Caporale, A.; de Castiglione,
R.; Kaptein, B.; Broxterman, Q. B.; Formaggio, F. Amino acid
bromides: Their N-protection and use in the synthesis of peptides
with extremely difficult sequences. J. Org. Chem. 2002, 67, 6372-
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