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Arginine-Rich Molecular Transporters for Drugs 147
Varying amounts of the peptides were added to 3 × 10 5 cells in a total of 200 µl, in
wells of microtiter plates, and incubated for 3 min at 23ºC. The cells were spun,
washed three times with cold PBS and resuspended in PBS containing 0.1% propidium
iodide. The cells were analyzed by fluorescent flow cytometry (FACScan,
Becton Dickinson, Milpitas, CA). Cells staining with propidium iodide were
excluded from the analysis.
Data presented are the mean fluorescent signal for the 5000 cells collected. To
prevent cellular uptake of the peptides, the cells were treated with sodium azide
(1.0%) for 25 min prior to exposure to the peptides. Intracellular fluorescence was
calculated by subtracting the fluorescence from cells treated with sodium azide from
that observed from untreated cells.
7.3 RESULTS
7.3.1 GUANIDINO PEPTOID DESIGN AND CELLULAR UPTAKE
A series of novel polyguanidine peptoid derivatives were designed that preserved
the 1,4-backbone spacing of side chains of arginine oligomers, but had an oligoglycine
backbone devoid of stereogenic centers. These peptoids, incorporating side
chains of arginine but attached to the amide nitrogen, were selected because of their
expected resistance to proteolysis 10 and potential ease of synthesis. 3,6 Furthermore,
epimerization, frequently encountered in peptide synthesis, is not a problem in
peptoid synthesis and the submonomer 3 approach to peptoid synthesis allows for
facile introduction of other side chains. Although the preparation of an oligourea 11
and peptoid–peptide hybrid 12 derivatives of Tat 49-57 have been previously reported,
their cellular uptake was not explicitly studied.
The desired peptoids were prepared using the submonomer 3 approach to peptoids,
followed by attachment of a fluorescein moiety through an aminohexanoic
acid spacer onto the amine termini. After cleavage from the solid-phase resin, the
fluorescently labeled polyamine peptoids thus obtained were converted in good
yields (60 to 70%) into polyguanidine peptoids by treatment with excess pyrazole-
1-carboxamidine 13 (Scheme 7.1). Previously reported syntheses of peptoids containing
isolated N-Arg units have relied on the synthesis of N-Arg monomers (5 to 7
steps) prior to peptoid synthesis and the use of specialized and expensive guanidine
protecting groups (Pmc, Pbf). 14,15
The compounds reported here represent the first examples of polyguanidinylated
peptoids prepared using a perguanidinylation step. This method provides easy access
to polyguanidinylated compounds from the corresponding polyamines and is especially
useful for the synthesis of perguanidinylated homooligomers. Furthermore,
this procedure eliminates use of expensive protecting groups (Pbf, Pmc). An additional
example of a perguanidinylation of a peptide substrate using a novel triflylsubstituted
guanidinylating agent has been reported recently. 16
The cellular uptake into the human T cell line, Jurkat, of the set of fluorescently
labeled polyguanidine peptoids of varying length, N-arg 5, 7, and 9, was analyzed
by flow cytometry and compared to the corresponding d-arginine peptides r5, 7, and
9. The amount of measured fluorescence inside the cells treated with N-arg 5, 7, and