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Arginine-Rich Molecular Transporters for Drugs 143
residues 49 to 57 of HIV tat and the 16 amino acid peptide from antennapedia, both
widely used molecular transporters. 2 Interestingly, the activity of the HIV tat peptide
closely mimicked that of a hexamer of arginine, which possesses the same arginine
content. 2
The goal of this review is to summarize previously published work on the design
and biological activity of a family of guanidinium peptoid transporters that differ in
the length of their side chains and to compare these results with a more recently
designed family of transporters containing non-α-amino acids, i.e., variations along
the backbone. The latter group of peptides differs by the spacing of the arginine
subunits along the peptide backbone. Previous experiments have established clearly
that the key structural unit necessary for transport was the guanidine headgroup and
that only homopolymers containing more than five arginines exhibit significant
biological activity. However, the importance of each headgroup and their spacing,
both the distance they extend from the polymeric backbone and the distance between
the headgroups along the backbone, has not been extensively investigated. Prior to
these studies only individual alanine substitutions into a nine amino acid sequence
in HIV tat were examined. These experiments demonstrated that the central
glutamine is not required for activity, while replacement of any of the arginines
results in a significant loss of uptake function. 2
The experiments described in this review represent a portion of a broad effort
to understand the fundamental structural requirements for transport across biological
membranes and have led to the design of simple and cost effective transporters that
could be used to deliver drugs and probe molecules into target tissues and cells as
required for therapeutic applications. Peptoids have several advantages over
peptides 3 and were selected for comparative study. Even though both are synthesized
in a stepwise fashion using solid phase synthesis, peptoids are constructed from
significantly cheaper starting materials and, due to absence of a chiral center, do not
epimerize and are more amenable to side chain modification. This latter feature was
utilized to construct a family of polyguanidine peptoids that differed in the length
and flexibility of their side chains. Comparison of their ability to enter cells led to
the realization that an increase in the length and, consequently, conformational
mobility of the side chains resulted in a higher rate of cellular uptake.
A possible rationalization of the increase in biological activity of the peptoids
with more flexible side chains is that this enabled a higher percentage of guanidines
to contact a biological membrane. Molecular modeling was used to determine
whether this hypothesis has merit by determining the theoretical number of guanidine
headgroups in an oligomer of arginine that would contact a common surface. To
investigate the results of this modeling experimentally, the residues believed to be
unimportant in the complex formation were substituted in turn with each of the
naturally occurring amino acids. Surprisingly, when these analogs were assayed for
cellular uptake, the most important feature was shown to be not the nature of the
substituted amino acid, but rather the distance between each of the arginines. This
was established by inserting non-α-amino acids between the arginine subunits, which
both supported the hypothesis and also allowed the spacing to be optimized.
When results of the two studies were compared, a very similar pattern was
observed. By increasing the conformational freedom of either the side chains of