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146 Cell-Penetrating Peptides: Processes and Applications
in DMF for 1 h and this was repeated. The Fmoc moiety was then removed by
treatment with 20% piperidine/DMF (5 ml) for 30 min. This step was repeated and
the resin was washed with DMF (3 × 5 ml). The free amine resin was then treated
with fluorescein isothiocyanate (0.2 mmol) and DIEA (2.0 mmol) in DMF (5 ml)
for 12 h. The resin was then washed with DMF (3 × 5 ml) and dichloromethane (5 ×
5 ml). Cleavage from the resin was achieved using 95:5 TFA/triisopropylsilane (8 ml).
Removal of the solvent in vacuo gave a crude oil that was triturated with cold
ether (20 ml). The crude mixture thus obtained was centrifuged, the ether was
removed by decantation, and the resulting orange solid was purified by reverse-phase
HPLC (H 2O/CH 3CN in 0.1% TFA). The products were isolated by lyophilization
and characterized by electrospray mass spectrometry and, in selected cases, by 1 H
NMR spectroscopy.
7.2.5 PERGUANIDINYLATION OF PEPTOID POLYAMINES
A solution of peptoid amine (0.1 mmol) dissolved in deionized water (5 ml) was
treated with sodium carbonate (5 equivalents per amine residue) and pyrazole-
1-carboxamidine (5 equivalents per amine residue) and heated at 50°C for 24 to 48
h. The crude mixture was then acidified with TFA (0.5 ml) and directly purified by
reverse-phase HPLC (H 2O/CH 3CN in 0.1% TFA). The products were characterized
by electrospray mass spectrometry and isolated by lyophilization and further purified
by reversed-phase HPLC. The yield for the perguanidinylated peptoids was 60 to
70%, while their purity was >95%, as determined by analytical reversed-phase HPLC
(H 2O/CH 3CN in 0.1% TFA).
7.2.6 MOLECULAR MODELING
NAMD2, 7 developed at the University of Illinois, was used to evaluate the structure
of an acetylated decamer of arginine with a carboxamidine terminus. The psfgen
program was used to create the necessary psf and pdb input files. CHARMM
parameters 8 were used to minimize the heptamer for 2000 steps, using a dielectric
constant of 80. At this point, molecular dynamics were performed for 100 psec over
which the average kinetic energy of the system was increased from 300 to 1000 K
in 25° increments by reassignment every psec, holding at 1000 K once reached.
Following 20 psec of equilibration, the system was reequilibrated to 300 K over
100 psec in 10° increments every psec, holding at 300 K once reached. The annealed
structures were then analyzed using VMD. 9
7.2.7 CELLULAR UPTAKE ASSAYS
The peptides and guanidine-substituted peptoids were each dissolved in PBS buffer
(pH 7.2) and their concentration determined by absorption of fluorescein at 490 nm
(ε = 67,000). The accuracy of this method for determining transporter concentration
was established by weighing selected samples and dissolving them in a known
amount of PBS buffer. The concentrations determined by UV spectroscopy correlated
with the amounts weighed out manually. The human T cell line, Jurkat, grown in
10% fetal calf serum in DMEM, was used for all the cellular uptake experiments.