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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
148 Cell-Penetrating Peptides: Processes and Applications H2N NHBoc X O N H BrCH2C O2H DIC, DMF (Step#1) N H n Br N N HN SCHEME 7.1 Preparation of guanidine-substituted peptoids. Mean Fluorescence 600 500 400 300 200 100 O N H NH 2 NHBoc BocNH-X-NH X O DMF (Step#2) N H N H Repeat Steps#1,2 FIGURE 7.1 The length dependence of cellular uptake is observed for both polyguanidino peptoids and oligomers of d-arginine. The mean fluorescence from 5000 cells of a human T cell line, Jurkat, is shown after incubation with 12.5 µM of each of the peptides for 5 min. Uptake was measured in triplicate at concentrations varying from 400 nM to 50 µM. The data are shown at a single concentration for ease of presentation. Details of the assay are described in Section 7.2. 9 was found to be related to the number of guanidine residues in the peptoid, with the nonamer entering cells more effectively than the heptamer, which in turn was more effective than the pentamer (N-arg9 > N-arg7 > N-arg5) (Figure 7.1). These results are analogous to previously reported behavior for the different oligomers of OH 1. FMOC aca OH DIC, DMF 2. piperidine, DMF 3. FITC-NCS O CO2H 4. 95:5 TFA/TIS S 5. Na2C O 3 , H2O, 50°C O N H 0 N-etg X=(CH 2 ) 2 N-arg N-btg N-hxg X=(CH 2 ) 3 X=(CH 2 ) 4 X=(CH 2 ) 6 N H N-chg X= n = 5-9 r5 r7 r9 N-arg5 N-arg7 N-arg9 HN X N O NH NH2 O NH2 n
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CELL- PENETRATING PEPTIDES Processe
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Pharmacology and Toxicology: Basic
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Library of Congress Cataloging-in-P
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to the handbook are prominent resea
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REFERENCES 1. Green, M. and Loewens
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Contributors Mats Andersson Microbi
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Erin T. Pelkey Department of Chemis
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Contents Section I Classes of Cell-
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Chapter 16 Cell-Penetrating Peptide
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The Tat-Derived Cell-Penetrating Pe
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24 Cell-Penetrating Peptides: Proce
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3 Transportans Margus Pooga, Mattia
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Transportans 55 Indeed, galparan is
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Transportans 57 especially the endo
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Transportans 59 In the penetration
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Transportans 61 A 21-mer antisense
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Transportans 63 Commonly, the prote
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Transportans 65 antibiotin antibodi
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Transportans 67 For cross-linking o
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Transportans 69 and still retain ef
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Model Amphipathic Peptides 73 its D
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Model Amphipathic Peptides 75 pmol/
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TABLE 4.1 Internalization of Peptid
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TABLE 4.2 Internalization of Peptid
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Model Amphipathic Peptides 81 relat
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Model Amphipathic Peptides 87 A cat
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Model Amphipathic Peptides 89 At th
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Model Amphipathic Peptides 91 16. H
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Signal Sequence-Based Cell-Penetrat
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Page 186 and 187: Interactions of Cell-Penetrating Pe
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Structure Prediction of CPPs and It
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FIGURE 9.7 (Color Figure 9.7 follow
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FIGURE 9.8 The center of the figure
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Biophysical Studies of Cell-Penetra
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Toxicity and Side Effects of Cell-P
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264 Cell-Penetrating Peptides: Proc
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Kinetics of Uptake of Cell-Penetrat
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Cell-Penetrating Peptide Conjugatio
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FIGURE 15.9 (Color Figure 15.9 foll
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Cell-Penetrating Peptides as Vector
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TABLE 16.1 Examples of Transport of
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CPP 2 Cargo or mRNA CAP Antisense A
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Microbial Membrane-Permeating Pepti
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Index A Abaecin, 129 Abz radiolabel
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Index 399 Diffraction, 168 Disulphi
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Index 401 structure prediction, 187
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Index 403 pRB proteins, Tat-E1A bin
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Index 405 lipid perturbation (secon
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