A. Pꢀlegrin, E. Vivꢀs et al.
chains of arginine and aspartic acid were protected by 2,2,5,7,8-
pentamethylchroma-6-sulfonyl (Pbf) and t-butyl ester (OtBu), re-
spectively. The side chain of the lysine residue was protected by
using allyloxycarbonyl (Alloc). Coupling reactions were performed
by using N-a-Fmoc-protected amino acid (2 equiv) relative to the
resin loading, activated in situ with TBTU (2 equiv), HOBt (2 equiv),
and DIEA (4 equiv) in DMF (10 mL per g resin) for 1 h. Coupling
completion was controlled by Kaiser tests. N-a-Fmoc protecting
groups were removed by treatment with a piperidine/DMF solu-
tion (1:4; 10 mL per g resin) for 5 min. The process was repeated
twice, and the completion of deprotection was checked by the UV
absorption of the piperidine/DMF washing at 299 nm. Deprotec-
tion of the Alloc protecting group was carried out by using a solu-
tion of Pd(PPh3)4 (0.2 equiv), PhSiH3 (24 equiv) in CH Cl for 2 h.
Cell lines and culture conditions: Human umbilical vein endothe-
lial cells (HUVEC), were cultured in endothelial growth medium
(EGM-2), containing 2% fetal bovine serum (FBS), growth factors
and supplements, and maintained at 378C, 5% CO , according to
2
the manufacturer’s instructions. Only cells from passages 3 to 7
were used in our experiments. A549 human lung carcinoma, MCF7
human breast adenocarcinoma and SK-MEL-28 human melanoma
were cultured in RPMI medium and maintained at 378C in 5% CO2
atmosphere. B16 melanoma cells were cultured in DMEM medium
and maintained at 378C in 5% CO atmosphere.
2
Evaluation of the expression of a b integrins by flow cytome-
v
3
try: The expression of a b integrins on the used cell lines was as-
v
3
sessed by flow cytometry. Briefly, cells were detached with versene
2
2
5
and counted. Cells (10 ) were incubated with PBS/FCS (100 mL) con-
Crude protected peptides were obtained by direct cleavage of the
resin with a solution of AcOH/TFE/CH Cl (2:2:6) for 2 h. Peptides
À1
taining 20 mgmL monoclonal anti-a b integrin antibody (LM-609
v
3
2
2
or anti CD51/61, Chemicon International, Temecula, USA, used in
accord with the manufacturer’s instructions) for 2 h at 48C. Cells
were precipitated with addition of anhyd. diethyl ether, filtered off,
dissolved in H O, and then lyophilized. The product was collected
2
were then washed twice with PBS/FCS and incubated for 30 min at
as a white powder without further purification (95% overall yield
from the first loading of the resin).
À1
4
8C with 20 mgmL secondary antibody (antimouse) conjugated
to FITC. After centrifugation and two washes with PBS, cells were
resuspended in PBS/FCS (600 mL) and analyzed by flow cytometry
General procedure for the cyclization of peptides: 1,1-Carbonyl-
diimidazole (110 mmol) dissolved in CH Cl (40 mL) was added over
(FACSCalibur, Becton-Dickinson, Le Pont de Claix, France). Nonspe-
2
2
cific binding was determined by incubating cells with the secon-
dary antibody only.
1
0 min to a solution containing the linear peptide (100 mmol) in
DMF/CH Cl (40 mL; 1:1) adjusted to pH 8–9 with DIEA. The pep-
2
2
tide solution (1.25 mm) was stirred for 2 h at 508C and then con-
centrated under reduced pressure. The cyclization was easily moni-
tored by HPLC. The crude product was isolated by precipitation
with diethyl ether/acetone (100 mL; 1:1). The precipitation was
repeated three times and the desired product was dissolved in
H O/CH CN (5 mL; 1:1) and lyophilized to obtain a white powder
In vitro analysis of peptide affinity for a b receptor: The affinity
v
3
of the different peptides for a b was assessed by flow cytometry.
v
3
HUVEC, SK-MEL-28, MCF7, or A549 cells were seeded in 24-well
4
plates (5ꢃ10 cells per well) and incubated overnight at 378C in
their respective medium. Cells were then rinsed twice with PBS
and incubated for 24 h at 378C in medium containing the fluores-
cent peptide (600 mL) at a final concentration of 1 or 5 mm. Cells
were washed twice with PBS/FCS, detached with trypsin, collected,
and centrifuged for 5 min at 2000 rpm. Supernatants were discard-
ed, and cells were then resuspended in PBS (600 mL) and analyzed
by FACS.
2
3
(
85 mmol, 85%) that can be used without further purification.
General procedure for the labelling of cyclic peptides with fluo-
rescein-maleimide: A solution containing NH (CH ) SNpys (2.6 mg,
2
2 2
1
1 mmol; see part 4 of the Supporting Information for the synthesis
of this compound), BOP (4.8 mg, 11 mmol), and DIEA (3.4 mL,
0 mmol) in DMF (100 mL) were added to a solution containing the
2
Fluorescence microscopy: HUVEC cells were seeded on sterilized
protected cyclic peptide (10 mmol) in DMF (100 mL). The reaction
was stirred for 2 h at RT. The resulting product was obtained after
8
-well Lab-Tek coverslips (200000 cells per well in 400 mL of
medium) and incubated overnight at 378C. Afterwards, cells were
washed with PBS and incubated at 378C for 30 min with fresh
medium in the presence of fluorescein-labeled peptides c(RGDfE)-
Fl, c(RGDK)e-Fl, c(RGEK)e-Fl at final concentrations of 5 mm. They
were then washed twice with PBS. The glass coverslips were then
inverted onto LABTEK wells by using Vectashield mounting
medium containing DAPI for staining of nuclei. Slides are allowed
to dry for 30 min. The slides were observed with a microscope
precipitation by adding H O (5 mL) followed by centrifugation. This
2
process was repeated four times to afford the desired product as a
yellow powder (9.6 mmol, 96%). RP-HPLC analysis, t =21.6 min
R
(
C , 214 nm, 30–70% B in 20 min); MS: m/z calcd for
18
+
C H N O O : 1022.2 [M+H] ; found: 1022.1. The peptide
4
3
64 11 12
3
(
9.6 mmol) was then deprotected on its side chains with a solution
containing 92.5% TFA, 2.5% H O, and 5% TIPS (500 mL) for 4 h at
2
RT. The peptide was precipitated by addition of diethyl ether
(Leica) and the following filter sets: excitation, BP340–380 nm, and
(
10 mL). After centrifugation the peptide was obtained as a yellow
emission, LP430 nm (for Hoechst staining); excitation, BP450–
powder (9 mmol, 94%). The selective deprotection of the Npys
4
90 nm, and emission LP520 nm (for fluorescein).
group was achieved by dissolving the peptide (9 mmol) in CH CN/
3
1
25
H O (100 mL), and Bu P (2 mL, 8 mmol) was added. The reaction was
Radiolabeling of echistatin with
I by the lactoperoxidase
2
3
À1
stirred for 30 min at RT. Then the crude product was freeze-dried
and triturated several times with diethyl ether (1.4 mL) to obtain
the fully deprotected peptide as a white powder (8.8 mmol, 97%).
The product was kept dry or in solution at À808C to avoid oxyda-
tion of the cystein residue and disulphide bridge formation. DMSO
method: Lactoperoxidase (10 mL, 1 UmL in PBS) was added to a
solution of echistatin (10 mL, 1 mgmL ) in PBS. Then Na
À1
125
I
(3.6 mCi) was delivered into the tube. H O (1 mL diluted at 1:20000
2
2
in PBS) was added to start the reaction. Additional H O (1 mL) was
2
2
added each minute during 4 min. The reaction was stopped by
(
20 mL) containing fluorescein-maleimide (4.25 mg, 10 mmol; see
adding PBS (25 mL) containing 1.5 mm NaN . The mixture was
3
part 4 of the Supporting Information for the synthesis of this latter
compound) was added to a solution of the deprotected peptide
eluted on a Sephadex G10 gel filtration column (Amersham Biosci-
ences) equilibrated with PBS–BSA (1 mgmL ) at pH 7.4. Twelve
À1
(
8.8 mmol) in Tris–HCl buffer (pH 7, 100 mm), . The reaction was
fractions (500 mL each) were collected, and the radioactivity was
counted by using a gamma counter (Hewlett–Packard). The three
fractions exhibiting the highest activity were pooled and the result-
ing mixture was gamma-counted. Specific activity was determined.
stirred at RT for 2 h and the desired product was isolated immedi-
ately after purification by RP-HPLC followed by lyophilization as an
orange powder (4.4 mg, 4.4 mmol, 50%).
1
090
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ChemBioChem 2010, 11, 1083 – 1092