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Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2004
ChemBioChem 2004
Supporting Information
for
Enhancing Membrane Permeability by Fatty Acylation of
Oligoarginine Peptides
Wellington Pham, Moritz F. Kircher, Ralph Weissleder, and Ching-Hsuan Tung
Peptide synthesis. Peptides were synthesized by conventional Fmoc solid
phase chemistry with β-alanine and lysine at both ends for conjugation with fatty
acid and fluorescein isothiocyanate (FITC) moieties respectively. The automatic
synthesizer (433A, Applied Biosystems, Foster City, CA) was loaded with 0.1
mmol of Rink amide MBHA resin (0.72 mmol/g) and 2-(1H-benzotriazole-1-yl)1,1,3,3-tetramethyluronium
hexafluorophostphate
(HBTU)/N-hydroxybenzo-
triazole (HOBt) as the activating and coupling reagents. Amino acids, FmocArg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Dde)-OH, Fmoc -Gly-OH, Fmoc-βAla-OH, Boc-β-Ala-OH, Fmoc-Gln(Trt)-OH and Fmoc -Phe-OH, were purchased
from Nova-biochem (San Diego, CA). The Fmoc moiety was deprotected by 20%
piperidine/DMF
and
the
activation
was
morpholine/DMF for each cycle.
1
catalyzed
with
0.4
M
methyl
Fatty acylated peptides. Freshly distilled N,N-diisopropylethylamine (DIEA) (1.0
mL) was added drop wise to slurry of fatty acylchloride (0.4 mmol) and peptidecontaining resin (0.1 mmol) in anhydrous methylene chloride (5.0 mL). The
mixture was stirred slowly at room temperature under a positive flow of argon for
two hours. The product was collected by filtering through a sinter glass filter and
washed with methylene chlo ride (3 x 10 mL) and methanol (3 x 10 mL).
Labeling
with
Fluorescein
isothiocyanate
(FITC).
2-(4,4 -dimethyl-2,6-
dioxocyclohexylidene)ethyl (Dde) group was deprotected using 2% hydrazine in
N,N-dimethylformamide (DMF) (2 x 10 mL) in 3 minutes with occasional swirling
following by washing with DMF (3 x 10 mL) and methanol (3 x 10 mL). The free
lysine-containing peptide was reacted with FITC (0.4 mmol, 155.8 mg) in
DIEA/DMF (1:4), and the slurry was stirred overnight at room temperature under
argon in dark.
Cleavage and purification. The bright yellow resin from the previous step was
filtered and washed with DMF (3 x 10 mL) and MeOH (3 x 10 mL) following by
treatment with deprotection cocktails (90:5:3:2 TFA/thioanisole/ethanedithiol/
anisole) and purified by C18 reversed-phase HPLC using 0.1% TFA and
acetonitrile as elution buffers to afford yellow cotton-like solid peptide after
lyophilization. The molecular weight was confirmed by MALDI- TOF Mass
Spectrometry at Tufts University Core Facility.
2
Cell Cultures and fluorescence microscopy. Hela cells were grown in
Dulbecco’s modified Eagle’s medium (DMEM, w/o L-glutamine, w/o phenol red;
Cat#: 17-205-CV, Cellgro, Mediatech, Washington DC) containing 10% fetal
bovine serum (FBS, Cellgro) and 1% Penicillin/Streptomycin at 37 °C in a
humidified 5% CO2 atmosphere. The medium was changed every 3 days until
cells reached near-confluency. After 2 passages, the cells were trypsinized and
subcultivated in Lab-Tek II coverglassed microscopy chambers (Nalge Nunc
International, Rochester, NY). Per well, 105 cells were seeded in 1 mL medium
and cultured for 24 h, washed with Hank’s balanced salt solution (HBSS) and
incubated with 10 µM of probe. Cells were washed three times with HBSS at
different time points before microscopy. For epi-fluorescence microscopy, cells
were viewed on a Zeiss Axiovert inverted microscope using appropriate filters;
Dual-channel laser scanning confocal microscopy was performed using a Zeiss
LSM 5 PASCAL (Zeiss, Thornwood, NY). Images were digitally captured using a
cooled charge-coupled device (CCD) camera, signal intensities between
epifluorescence images normalized in IPLab software and compiled using Adobe
Photoshop™ software (v7.0).
Costaining C14-R7 with R6 dye. Hela cells were treated in the same manner as
described above followed by incubation with 10 µM of C14-R7 for 1.5 h. Media
was removed and cells were washed extensively with HBSS (3x), followed by
incubation with R6 dye (Molecular Probes, Eugene, OR) at 0.5 µg mL-1 for 5 min
at room temperature.
3
FACS analysis. Freshly prepared peptide solutions in HBSS (10 µM ) were added
to FACS polystyrene tubes, each containing 10 6 Hela cells per mL, which had
been trypsinized as described above. The cells were incubated for 5 min at 37°C
in a humidified 5% CO2 atmosphere followed by three washing steps with HBSS.
Results were analyzed using a FACSCalibur and CellQuest software™ (Becton
Dickinson, San Jose, CA).
Cytotoxicity. To determine cytotoxic effects of fatty acylated peptide on Hela
cells, a Cyto Tox 96 cytotoxicity assay kit (Promega, Madision, WI) was used.
The experiments were performed according to the instructions of the
manufacturer. In order to avoid interference from phenol red contained in the
culture medium, medium was changed to phenol red-free DMEM containing 10%
FBS. The experiments were performed in triplicate; where applicable, 50 µL of
culture suspension containing 20000 cells were dispensed into 96-well plate and
the volume was adjusted to an appropriate concentration with a fi nal volume of
100 µL by adding peptide solution in HBSS. Peptide-induced toxicity was
evaluated by measuring lactate dehydrogenase (LDH) activity released in the
culture supernatant half an hour after exposure to peptides. The plate was
removed from the incubation chamber and centrifuged at room temperature for 4
min. 50 µL of the supernatant from each well were then transferred to a 96-well
plate followed by addition of substrate solution (50 µL) and incubation of the
reaction mixture at room temperature for 30 min, protected from light.
4
Quantitative evaluation of cell death was performed on a plate reader at 490 nm
and calculated using the formula:
% cytotoxicity = [(experimental – target sponta neous) / (target maximum – target
spontaneous)] x 100.
5