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crc press - E-Lib FK UWKS

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156 Cell-Penetrating Peptides: Processes and Applications<br />

Mean Fluorescence<br />

7000<br />

6000<br />

5000<br />

4000<br />

3000<br />

2000<br />

1000<br />

0<br />

FIGURE 7.10 Peptides with greater arginine content exhibit faster rates of cellular uptake.<br />

Comparison of the cellular uptake of fluorescent peptides composed of either five, six, or<br />

seven arginines interspaced with 6-aminocaproic acid residues. Uptake was measured in<br />

triplicate at concentrations varying from 400 nM to 50 µM, but only the differential uptake<br />

at 12.5 µM is shown.<br />

7.4 DISCUSSION<br />

1666.0<br />

3145.9<br />

5739.1<br />

(Raca)4R (Raca)5R (Raca)6R<br />

The fundamental goal of this research was to determine structural requirements for<br />

cellular uptake of guanidine-rich transporters and to use this information to develop<br />

simpler and more effective molecular transporters. Given the importance of the<br />

guanidino headgroup and the apparent insensitivity of the oligomer chirality revealed<br />

in our peptide studies, a novel series of polyguanidine peptoids was designed and<br />

synthesized. Incorporating the arginine side chain, the peptoids N-arg 5, 7, and 9<br />

exhibited comparable cellular uptake to the corresponding d-arginine peptides r5, 7,<br />

and 9, indicating that the hydrogen bonding along the peptide backbone and backbone<br />

chirality are not essential for cellular uptake. This observation was consistent<br />

with molecular models of these peptoids, arginine oligomers, and Tat 49-57, all of<br />

which have a deeply embedded backbone and a guanidinium-dominated surface.<br />

Molecular models further reveal that these structural characteristics are retained<br />

in varying degrees in oligomers with different alkyl spacers between the peptoid<br />

backbone and guanidino headgroups. Accordingly, a series of peptoids incorporating<br />

2- (N-ethyl), 4- (N-butyl), and 6-atom (N-hexyl) spacers between the backbone and<br />

side chain were prepared and compared for cellular uptake with the N-arg peptoids<br />

(3-atom spacers) and d-arginine oligomers. The length of the side chains had a<br />

dramatic effect on cellular entry. The amount of cellular uptake was proportional to<br />

the length of the side chain, with N-hexyl > N-butyl > N-arg > N-ethyl. Cellular

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