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158 Cell-Penetrating Peptides: Processes and Applications
A simple pattern was observed. Greater cellular uptake was observed as the
number of aca units in the analog increased. The exact location was unimportant,
in that all analogs with a single aminocaproic acid are approximately equivalent and
less effective than those with two spacing amino acids. Greater cellular uptake was
seen as more aminocaproic acid residues were added until reaching the fully substituted
analog containing six spacer amino acids alternating in the sequence with
the seven arginines. As mentioned previously, these results are consistent with
peptides containing greater aminocaproic acid content being more resistant to proteolysis
and thereby increasing the effective concentration of the more highly substituted
analogs.
Alternatively, the data supported the hypothesis that increasing the length
between the arginines results in greater cellular uptake. Support for the latter hypothesis
was generated by demonstrating a set of peptides containing seven d-arginines
with either alternating glycine (CH 2 = 1), β-alanine (CH 2 = 2), 4-amino butyric acid
(CH 2 = 3), 6-amino caproic acid (CH 2 = 5), or 8-aminocaprylic acid (CH 2 = 7)
differentially entering cells. Uptake increased as the spacing between the arginines
increased from CH 2 = 1 until CH 2 = 5, with the exception that the β-alanine-substituted
peptide was slightly superior to the one containing 4-amino butyric acid. Uptake did
not continually increase; the analog with 8-amino caprylic acid was less potent than
the one containing aminocaproic acid.
The enhanced uptake when non α-amino acids were substituted into the peptide
backbone was very similar to the effects seen when the side chains were extended
in the peptoid series. 2 The fact that a similar pattern was seen when methylenes were
substituted into the backbone or the side chain is noteworthy, not only due to the
apparent symmetrical effect, but also because increasing flexibility into a ligand is
counterintuitive for generating more potent ligands. These results, combined with
earlier studies 1 demonstrating a lack of chiral recognition, emphasize that these
compounds most likely are not interacting with a highly defined binding site characteristic
of enzymes and protein receptors of most ligands. Typically, the biological
activity of most biological ligands, particularly those functioning as inhibitors,
increases with conformational restriction because preorganization in the form of the
bound conformer favors tighter binding. In contrast, the enhanced activity associated
with increased conformational mobility observed for molecular transporters is in
agreement with a dynamic transport process in which turnover is critical for function.
The transporters are more likely forming ionic complexes with negatively
charged entities on the surface of the biological membranes, most likely, the phosphates
of phospholipids. The neutralization of the phosphate headgroups of lipids
with polycations has been shown to have manifold effects due to neutralization of
the charge, ranging from disruption of the order of the bilayer to release of associated
membrane proteins into the media. 17
The increased flexibility of the transporter might result in greater efficacy by
allowing the molecule to adopt significantly different conformations necessary for
the different steps in translocation. This possibility is consistent with a study demonstrating
that a single, common secondary structure induced by model membrane
systems was not observed for three transport peptides based on the antennapedia
sequence. 18 Alternatively, the peptide might need to adopt single conformation for