.
Angewandte
Communications
DOI: 10.1002/anie.201104624
Peptidomimetics
Protein Transduction Domain Mimics: The Role of Aromatic
Functionality**
Abhigyan Som, Anika Reuter, and Gregory N. Tew*
Cell-penetrating peptides (CPPs), or protein transduction
domains (PTDs), are a special class of membrane-active
proteins that can cross the cell membrane with unusual
efficiency.[1] They have attracted considerable attention
because of their ability to readily cross biological membranes,
in spite of their highly charged nature.[2] While the exact
mechanism of this transport remains under intense investiga-
tion, energy-independent pathways are known.[2a,3] Perhaps
the clearest example is the ability of CPPs, and their synthetic
mimics, to cross model phospholipid bilayer vesicle mem-
branes.[4] One suggested mechanism implies that, in fact,
CPPs like polyarginine (pR) need assistance to cross the
membrane.[1d,4a–c,5] It suggests that hydrophobic counterions
complex around the guanidinium-rich backbone, thus “coat-
ing” the highly cationic structure with lipophilic moieties. This
process has been termed “activation”, in which the lipophilic
anion acts as an activator. In a series of detailed studies it was
shown that aromatic activators outperform aliphatic
ones.[4a–c,5] For example, sodium 4-(pyren-1-yl)butane-1-sul-
fonate gave an EC50 (effective concentration to obtain 50%
activity) of 6.7 mm whereas the value for sodium dodecane-1-
sulfonate was 16 mm.[5] Among other activators studied, the
larger aromatic counterion, coronene, was not better than
pyrene; however, a fullerene analogue was surprisingly
effective.[5] While this work beautifully demonstrated the
role of various counterions for pR activation, it was not clear
if this better activation was due to general hydrophobicity or
to the aromatic nature of these activators.
rules out a dominant effect of hydrogen bonding.[7] It was
suggested that the flat-rigid shape, p-electronic structure, and
associated quadrupolar moments provide unique and highly
favorable interactions with the bilayer interface.[6b] Specific
interactions that have been proposed include p-cation,
electrostatic, dipole–dipole, and entropic factors related to
bilayer perturbation.[6,7,9] Even HIV-TAT, the original protein
that initiated the field of small PTDs, requires tryptophan
(Trp11) for translocation.[10] Moreover, an oligoarginine con-
sisting of seven arginine residues with a C-terminal trypto-
phan (R7W) and a TAT48–60 peptide with residue 59 substi-
tuted with a tryptophan (TAT48–60P59W) exhibit cellular
internalization through energy-independent pathways.[3b,11]
Another classical CPP, penetratin (Pen), contains two trypto-
phan residues. Substitution of tryptophan by phenylalanine
(Pen2W2F) did not significantly impact cell uptake.[11] Among
the aromatic amino acids, phenylalanine has the unique
ability to partition at the interface and in the membrane
core.[9f] In fact, aromatic residues, especially phenylalanine,
are most effective at anchoring proteins in the membrane due
to their “special ability” to form and stabilize essential
interactions with the polar elements of the bilayer.[12] As a
result, aromatic functionality could be a critical element
facilitating the interactions between CPPs and the bilayer
during transduction.
In the past few years, we and others have reported
polymers designed to mimic the transduction activity of
PTDs.[4d,13] More recently, we demonstrated that these protein
transduction domain mimics (PTDMs) have “self-activation”
properties when hydrophobic alkyl side chains were built into
the copolymers.[14] Here, a new series of PTDMs was designed
to determine if an aromatic functionality provides better
transduction efficiency than aliphatic ones, at the same
relative hydrophobicity. Given the importance of aromatic
amino acids in membrane proteins and their interactions with
the bilayer, it was proposed that aromatic side chains would
make better activators, given equal relative hydrophobicity.
Although aromatic groups have been studied in peptide-
based CPPs,[3b,11,15] demonstration of the importance of
aromatic functionality in these synthetic analogues is critical
to establishing them as appropriate mimics, or PTDMs. By
using reversed-phase HPLC to determine side-chain hydro-
phobicity and EC50 values in a classic transduction experi-
ment, it is demonstrated here that it was possible to differ-
entiate between side-chain hydrophobicity and aromaticity.
As shown in Table 1, a series of new PTDM polymers was
prepared by ring-opening metathesis polymerization (see the
Supporting Information for detailed synthesis and character-
ization of monomers and polymers). Reversed-phase HPLC,
commonly used to evaluate relative hydrophobicity,[16] was
There is good reason to think that aromatic functional
groups may play a special role, beyond their general hydro-
phobicity. It is well recognized that membrane proteins are
enriched in aromatic amino acids at the membrane surface.[6]
Their central hydrophobic core, composed mostly of aliphatic
residues, is flanked on both sides by “aromatic belts”.[6a,7]
Although this belt is predominantly composed of tryptophan
and tyrosine, as opposed to phenylalanine, it was shown that
aromatic residues, including N-methylindole, have favorable
free energies of insertion into the bilayer interface.[6b,8] This
[*] Dr. A. Som, A. Reuter, Prof. G. N. Tew
Polymer Science & Engineering Department
University of Massachusetts
120 Governors Drive, Amherst, MA 01003 (USA)
E-mail: tew@mail.pse.umass.edu
[**] Generous support was primarily provided by the NSF (CHE-
0910963). Dr. Semra Colak is acknowledged for her contribution in
octyl monomer synthesis.
Supporting information for this article is available on the WWW
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ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2012, 51, 980 –983
Som, Abhigyan
