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Arginine-Rich Molecular Transporters for Drugs 151
Mean Fluorescence
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r9 N-cyclohex 9 N-ethyl 9 N-arg 9 N-butyl 9 N-hexyl 9
FIGURE 7.4 Enhanced cellular uptake correlates with increased conformational freedom and
not hydrophobic content of the side chains of the transporter. Comparison of the cellular
uptake of a set of guanidino peptoids containing nine subunits, but differing in their side
chain composition. N-ethyl 9 contains two methylenes, N-arg, three, N-butyl, four, N-hexyl,
six, and cyclohex, a cyclohexyl ring, approximately equivalent to N-hexyl in hydrophobic
nature, but not conformational flexibility. 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.
receptor). Consequently, arginines not in contact with the common surface could in
principle be replaced with other residues without affecting transport.
Molecular modeling was used to generate secondary structures of the transporters
and to determine the theoretical number of guanidine headgroups capable of
contacting a common surface. Using NAMD2, a scalable molecular mechanics
engine, the solution phase structures of a decamer of arginine were evaluated. 7 One
of the low energy conformations found at 300 K positioned residues 2, 5, and 8
away from a surface plane making contact with the other residues (Figure 7.5). In
this conformation, the maximum contacts with a common surface were made with
the guanidine headgroups of residues 1, 3, 4, 6, 7, 9, and 10. The guanidine headgroups
of the arginines at positions 2, 5, and 8 appeared unable to contact the surface
for this family of conformers.
To test this hypothesis, a set of 17 peptides, substituting each of the naturally
occurring amino acids, except arginine, cysteine, and tryptophan, into the three
positions (2, 5, and 8) believed not in contact, were synthesized, conjugated with
fluorescein, and assayed for cellular uptake (Figure 7.6).
All peptides were decamers containing seven equivalently spaced arginines. Not
surprisingly, the peptides exhibited a wide range of abilities to enter the cells,
depending on the substituted amino acid. Substitution with aspartic and glutamic
acid resulted in analogues that entered cells relatively poorly. Previous experiments