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Arginine-Rich Molecular Transporters for Drugs 155 Mean Fluorescence 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 2243.5 3418.4 6884.8 6534.4 8752.3 2562.7 r13 (rG)6r (rbA)6r (rAbu)6r (rAca)6r (rAcl)6r FIGURE 7.9 Cellular uptake increased as the spacing between the arginines increased until the residues were separated by 8-aminocaprylic acid. Comparison of the cellular uptake of a set of peptides containing seven arginine and either six alternating glycine (CH 2 = 1), βalanine (CH 2 = 2), 4-aminobutyric acid (CH 2 = 3), 6-amino caproic acid (CH 2 = 5), or 8-aminocaprylic acid (CH 2 = 7) with both r13. 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. Data are shown at a single concentration for ease of presentation. To attempt to distinguish between these possibilities and also to determine the optimal linear spacer, the set of peptides containing seven arginines with either six alternating glycine (CH 2 = 1), β-alanine (CH 2 = 2), 4-aminobutyric acid (CH 2 = 3), 6-amino caproic acid (CH 2 = 5), or 8-aminocaprilic acid (CH 2 = 7) residues were synthesized and assayed for cellular uptake (Figure 7.9). In this set of peptides darginine was used and any possibility of differential proteolysis was eliminated. With the exception of the peptide containing β-alanine being superior to that containing 4-amino butyric acid, a general trend of a faster rate of uptake into cells was observed when the spacing between the arginines increased, until a limit was reached with the 6-aminocaproic acid. A possible explanation for the increased rate of uptake of substituted peptides is that they utilize a different mechanism for cell entry. A characteristic of argininerich transporters is that their ability to enter cells correlates with their arginine content. To test whether the spaced peptides share this characteristic, a set of spaced peptides with an arginine content ranging from five to seven were synthesized and assayed (Figure 7.10). Among a family of peptides with the arginine residues equally spaced, increasing the arginine content increased the rate of cellular uptake.
156 Cell-Penetrating Peptides: Processes and Applications Mean Fluorescence 7000 6000 5000 4000 3000 2000 1000 0 FIGURE 7.10 Peptides with greater arginine content exhibit faster rates of cellular uptake. Comparison of the cellular uptake of fluorescent peptides composed of either five, six, or seven arginines interspaced with 6-aminocaproic acid residues. Uptake was measured in triplicate at concentrations varying from 400 nM to 50 µM, but only the differential uptake at 12.5 µM is shown. 7.4 DISCUSSION 1666.0 3145.9 5739.1 (Raca)4R (Raca)5R (Raca)6R The fundamental goal of this research was to determine structural requirements for cellular uptake of guanidine-rich transporters and to use this information to develop simpler and more effective molecular transporters. Given the importance of the guanidino headgroup and the apparent insensitivity of the oligomer chirality revealed in our peptide studies, a novel series of polyguanidine peptoids was designed and synthesized. Incorporating the arginine side chain, the peptoids N-arg 5, 7, and 9 exhibited comparable cellular uptake to the corresponding d-arginine peptides r5, 7, and 9, indicating that the hydrogen bonding along the peptide backbone and backbone chirality are not essential for cellular uptake. This observation was consistent with molecular models of these peptoids, arginine oligomers, and Tat 49-57, all of which have a deeply embedded backbone and a guanidinium-dominated surface. Molecular models further reveal that these structural characteristics are retained in varying degrees in oligomers with different alkyl spacers between the peptoid backbone and guanidino headgroups. Accordingly, a series of peptoids incorporating 2- (N-ethyl), 4- (N-butyl), and 6-atom (N-hexyl) spacers between the backbone and side chain were prepared and compared for cellular uptake with the N-arg peptoids (3-atom spacers) and d-arginine oligomers. The length of the side chains had a dramatic effect on cellular entry. The amount of cellular uptake was proportional to the length of the side chain, with N-hexyl > N-butyl > N-arg > N-ethyl. Cellular
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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 126 and 127: 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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