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Arginine-Rich Molecular Transporters for Drugs 155
Mean Fluorescence
10000
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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.