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Arginine-Rich Molecular Transporters for Drugs 157
uptake is improved progressively as the number of alkyl spacer units between the
guanidine headgroup and the backbone is increased. Significantly, N-hexyl 9 was
superior to r9, with the latter 100-fold better than Tat 49-57. This result led us to prepare
peptoid derivatives containing longer octyl spacers (N-octyl) between the guanidino
groups and the backbone. The limited solubility of this compound in aqueous buffers
prevented evaluation of cellular uptake.
Because both perguanidinylated peptides and perguanidinylated peptoids efficiently
enter cells, the guanidine headgroup (independent of backbone) is apparently
a critical structural determinant of cellular uptake. However, the presence of several
(over six) guanidine moieties on a molecular scaffold is not sufficient for active
transport into cells, as the N-cyclohex peptoids did not efficiently translocate into
cells. Thus, in addition to the guanidine headgroup, the conformational mobility of
side chains plays a role in cellular uptake.
These studies identified a series of novel peptoids, of which 17 members were
synthesized and assayed for cellular uptake. Significantly, the N-hexyl 9 transporter
was found to be superior in cell uptake to r9 that was comparable to N-butyl 9. This
research established that the peptide backbone and hydrogen bonding along that
backbone are not required for cellular uptake and, most significantly, that an extension
of the alkyl chain between the backbone and the headgroup provides superior
transporters. In addition to better uptake performance, these novel peptoids offer
several advantages over peptides , including lower cost of goods, ease of synthesis
of analogs, and protease stability. Such features, along with their significant water
solubility (>100 mg/ml), indicate that these novel peptoids could serve as effective
transporters for molecular delivery of drugs, drug candidates, and other agents into
cells.
The second goal of these studies was to determine whether all, or only a subset,
of the headgroups in an oligomer were necessary for optimal activity, as modeling
suggests that only a subset of side chains would contact most biological surfaces.
The data shown in Figure 7.6 demonstrate that several decamers consisting of seven
arginines interspersed at positions 2, 5, and 8 performed as well as the unsubstituted
decamer of arginine. These data support the hypothesis that only a subset of the
headgroups in the peptide transporter is necessary for uptake.
Significantly, with the exception of the analogs containing substitutions with
aspartic and glutamic acid, all of the decamers containing seven arginines were more
potent than heptaarginine homopolymers. These data indicate that, independent of
the characteristics of the side chain, an increase in spacing between the arginine
residues also leads to an increase in uptake. This second hypothesis is supported by
the demonstration that, when non-α-amino acids were substituted into the second,
fifth, and eighth positions, the rate of uptake increased as the number of methylenes
in the non-α amino acids increased. However, this substitution pattern is only one
of 63 different possible permutations of nonconsecutive spacer residues that can be
placed into a heptamer of arginine. To determine the optimal spacing pattern for
substituting up to six nonconsecutive 6-aminocaproic acid into a heptamer of arginine,
substituted peptides were synthesized in parallel and then assayed for cellular
uptake.