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150 Cell-Penetrating Peptides: Processes and Applications
Mean Fluorescence
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r5 r6 r7 r8 r9 NNNNNhexyl 5 hexyl 6 hexyl 7 hexyl 8 hexyl 9
FIGURE 7.3 Guanidino peptoids with hexyl side chains enter cells more efficiently than
oligomers of d-arginine of equal length. The mean fluorescence from 5000 cells of a human
T cell line, Jurkat, is shown after incubation with 6.25 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.
the nonamers, the peptoid with four methylenes, N-butyl 9, is approximately equivalent
to a nonamer of d-arginine, r9, with the peptoid with six methylenes in the
side chain, N-hexyl 9, significantly more potent (Figure 7.2). The cellular uptake of
the corresponding heptamers and pentamers also showed the same relative trend. In
general, N-hexyl peptoids of n residues in length (where n = 5 to 9) enter cells as
well as or better than peptide of n+1 residues (Figure 7.3).
To determine whether the enhanced cellular uptake was due to the increased
hydrophobic nature or greater conformational freedom of the side chains, a set of
peptoids was synthesized containing cyclohexyl side chains, N-cyclohex 5, 7, and
9 peptoids. These contained the same number of side chain carbons as the N-hexyl
peptoids, but possessed significantly fewer degrees of conformational freedom.
When assayed for cellular uptake, the N-cyclohex peptoid showed much lower
cellular uptake activity than all of the previously assayed peptoids, including the Nethyl
peptoids (Figure 7.4). Therefore, the conformational flexibility and sterically
unencumbered nature of the straight chain alkyl spacing groups — not the increased
hydrophobic nature of the side chain — was the important factor in determining
efficiency of cellular uptake.
7.3.3 ROLE OF THE BACKBONE CONFORMATIONAL FREEDOM OF GUANIDINO
PEPTIDE TRANSPORTERS IN CELLULAR UPTAKE
For many secondary structures of the transporter, not all of the guanidino groups
are capable of contacting a common surface involved in transport (either cell or