Design of aromatic-containing cell-penetrating peptide mimics with structurally modified π electronics

Article abstract of DOI:10.1002/chem.201405381

Cell-penetrating peptides (CPPs) and their synthetic mimics (CPPMs) represent a class of molecules that facilitate the intracellular delivery of various cargo. Previous studies indicated that the presence of aromatic functionalities improved CPPM activity. Given that aromatic functionalities play prominent roles in membrane biology and participate in various π interactions, we explored whether these interactions could be optimized for improved CPPM activity. CPPMs were synthesized by ring-opening metathesis polymerization by using monomers that contained aromatic rings substituted with electron-donating and electron-withdrawing groups and covered an electrostatic potential range from -29.69 to + 15.57 kcalmol^-1. These groups altered the quadrupole moments of the aromatic systems and were used to test if such structural modifications changed CPPM activity. CPPMs were added to dye-loaded vesicles and the release of carboxyfluorescein was monitored as a function of polymer concentration. Changes in the effective polymer concentration to release 50% of the dye (effective concentration, EC₅₀) were monitored. Results from this assay showed that the strength of the electron-donating and electron-withdrawing groups incorporated in the CPPMs did not alter polymer EC₅₀ values or activity. This suggests that other design parameters may have a stronger impact on CPPM activity. In addition, these results indicate that a wide range of aromatic groups can be incorporated without negatively impacting polymer activity.

Full text of DOI:10.1002/chem.201405381

DOI: 10.1002/chem.201405381
Full Paper
&
Pi Interactions
Design of Aromatic-Containing Cell-Penetrating Peptide Mimics
with Structurally Modified p Electronics
Brittany M. deRonde,[a] Alexander Birke,[a] and Gregory N. Tew*[a, b]
Abstract: Cell-penetrating peptides (CPPs) and their synthet-
ic mimics (CPPMs) represent a class of molecules that facili-
tate the intracellular delivery of various cargo. Previous stud-
ies indicated that the presence of aromatic functionalities
improved CPPM activity. Given that aromatic functionalities
play prominent roles in membrane biology and participate
in various p interactions, we explored whether these interac-
tions could be optimized for improved CPPM activity. CPPMs
were synthesized by ring-opening metathesis polymerization
by using monomers that contained aromatic rings substitut-
ed with electron-donating and electron-withdrawing groups
and covered an electrostatic potential range from ꢀ29.69 to
+15.57 kcalmolꢀ1. These groups altered the quadrupole mo-
ments of the aromatic systems and were used to test if such
structural modifications changed CPPM activity. CPPMs were
added to dye-loaded vesicles and the release of carboxy-
fluorescein was monitored as a function of polymer concen-
tration. Changes in the effective polymer concentration to
release 50% of the dye (effective concentration, EC50) were
monitored. Results from this assay showed that the strength
of the electron-donating and electron-withdrawing groups
incorporated in the CPPMs did not alter polymer EC50 values
or activity. This suggests that other design parameters may
have a stronger impact on CPPM activity. In addition, these
results indicate that a wide range of aromatic groups can be
incorporated without negatively impacting polymer activity.
Introduction
tosis, protein-dependent translocation, and energy-independ-
ent translocation are involved in the internalization process.[5]
In efforts to elucidate the mechanisms of CPP(M) uptake and
assess the structural components of CPP(M)s necessary for
uptake, model vesicle membrane studies have frequently been
used.[6] Vesicle experiments represent a simpler system for eval-
uating energy-independent methods of transduction than
using cells, in which it is difficult to decouple various methods
of cellular uptake. Previously, Matile and co-workers have used
model vesicle systems to show that polyarginine, a widely
used CPP, requires hydrophobic counterions to efficiently cross
lipid membranes.[6b,7] For these studies, lipids were swollen in
a solution of carboxyfluorescein, which is a hydrophilic, anionic
dye that self-quenches at high concentrations, and dye release
Cell-penetrating peptides (CPPs) and their synthetic mimics
(CPPMs) represent a unique class of molecules that is capable
of crossing biological membranes.[1] The peptides are generally
short, cationic sequences rich in arginine and/or lysine resi-
dues, with some containing hydrophobic residues such as leu-
cine, phenylalanine, or tryptophan.[1b,g,i,j] They derive inspiration
from proteins with translocation abilities, such as HIV-1 Tat and
antennapedia homeodomain protein.[2] It has been shown that
the cation-rich domains of these proteins, referred to as pro-
tein-transduction domains (PTDs), are primarily responsible for
their uptake abilities.[2a,3] Many studies have highlighted the
ability of CPP(M)s to facilitate the intracellular delivery of vari-
ous cargo, including, but not limited to, small molecules,
siRNA, pDNA, and proteins by covalent or non-covalent inter-
actions.[1b,c,e,g–j,4] Although their mechanism of uptake is debat-
ed in the literature, various forms of endocytosis, macropinocy-
was monitored as
a function of peptide concentration.
Changes in peptide activity were assessed by calculating the
effective concentrations to release 50% of the dye (effective
concentration, EC50). Similar assays have also been used by Al-
meida and co-workers to explore CPP internalization mecha-
nisms.[8] The hydrophobic counterions selected for Matile and
co-workers studies were said to help mask the overall cationic
charge of the peptides to aid in transduction, a process re-
ferred to as activation.[6b,7] Although these studies showed that
bulky aromatic activators, such as pyrene butyrate, outper-
formed aliphatic activators, the roles of hydrophobicity and ar-
omaticity were not fully understood.
[a] B. M. deRonde, A. Birke, Prof. G. N. Tew
Department of Polymer Science & Engineering
University of Massachusetts, Amherst, MA 01003 (USA)
Fax: (+1)413-545-0082
E-mail: tew@mail.pse.umass.edu
[b] Prof. G. N. Tew
Department of Veterinary and Animal Sciences
Molecular and Cellular Biology Program
University of Massachusetts, Amherst, MA 01003 (USA)
Motivated by these studies, our lab previously developed
a series of oxanorbornene imide-based CPPMs to assess the
effect of hydrophobicity on CPPM activity.[6d,e] Instead of using
external activators, the hydrophobic components were chemi-
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201405381. It contains all experimental de-
tails.
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cally incorporated into the polymeric structures to give self-ac-
tivating polymers.[6d,e] These polymers were correctly predicted
to outperform their counterparts that only contained cationic
residues.[6a,d,e] Initially, various aliphatic chains were incorporat-
ed into the CPPMs to assess the effect of chain length on acti-
vity.[6e] These results were evaluated by assessing differences in
reported EC50 values from vesicle dye release assays.[7b] Al-
though polymer activity improved by increasing the alkyl
chain lengths from one carbon to four carbons, longer alkyl
chains were less water soluble and thus led to poorer perform-
ance.[6e]
membrane interactions.[10a,13] Because the quadrupole moment
collects the electron density on the face of these planar, aro-
matic rings, it was hypothesized that by strengthening or
weakening this phenomenon, the corresponding p membrane
interactions would provide additional handles for tuning of
CPPM activity. Specifically, this was attempted by incorporating
electron-donating and electron-withdrawing groups into the
aromatic systems as a way to alter the electron density of the
ring system. Although nature offers an electrostatic potential
range for its aromatic amino acids between ꢀ31.41 (Trp) and
ꢀ23.48 kcalmolꢀ1 (Tyr), by using synthetic systems, it was pos-
sible to examine a much wider electrostatic potential window
of ꢀ29.69 kcalmolꢀ1 (Scheme 1, R=b=CH3) to +15.57 kcal
molꢀ1 (Scheme 2, R’=c=NO2). All values are summarized in
Table S1–2 in the Supporting Information.
Another series of polymers was designed to evaluate the
impact of various aromatic, cyclic non-aromatic, and alkyl hy-
drophobic moieties of similar hydrophobicity on polymer acti-
vity.[6d] This was done to gain a better understanding of the in-
terplay between hydrophobicity and aromaticity. Aromaticity
was the cornerstone of that report because of the significant
role it plays in protein–membrane interactions. The aromatic
amino acids tyrosine and tryptophan are present as part of ar-
omatic belts that flank either end of transmembrane proteins.[9]
These residues sit at the interface between the hydrophobic
core and the more hydrophilic external environment to en-
hance stability at those regions.[9] Although not typically pres-
ent in aromatic belts, phenylalanine has also been shown to
aid in anchoring proteins in the membrane.[10] All three of
these aromatic amino acids have been shown to provide favor-
able energies of insertion into membranes.[10] It was further
reasoned that aromatic moieties are ideal for incorporation
into CPPMs because such residues are found in many CPPs, in-
cluding Penetratin, Pep-1, and MPG, and have been shown in
some cases to be critical for uptake.[1e,11] Experiments, in which
aromatic residues in Tat and Penetratin were replaced with
non-aromatic hydrophobic residues, led to a reduction in cellu-
lar internalization efficiencies.[1e,11]
Scheme 1. Synthesis of diester monomers containing p-rich and p-poor aro-
matic rings. i) RꢀOH, DMAP, CH2Cl2,, RT, overnight; ii) 1,3-di-Boc-2-(2-hydrox-
yethyl)guanidine, EDC, DMAP, CH2Cl2, 08C to RT, overnight.
Using HPLC retention times to assess relative hydrophobicity
of the polymer side chains,[12]
these values were compared to
the polymer EC50 values to illus-
trate that the effects of hydro-
phobicity and aromaticity could
be distinguished. Through these
studies, it was suggested that ar-
omatic hydrophobic moieties
Scheme 2. Synthesis of imide monomers containing p-rich and p-poor aromatic rings. i) R1ꢀOH, PPh3, diisopropyl
were superior activators.[6d] Simi-
lar results were obtained by
Matile and co-workers when
azodicarboxylate (DIAD), THF, RT, 18 h.
they monitored dye release of polyarginine with various exter-
nal activators.[7d]
As part of this study, p-rich and p-poor CPPMs were de-
signed and synthesized based on both the diester and imide
ROMP scaffolds. CPPMs based on the diester system were syn-
thesized because the dual-functional monomers offer greater
potential for polymeric structure variations. Also, CPPMs based
on the imide scaffold were synthesized as a direct comparison
to polymers from previous hydrophobicity structure activity re-
lationships (SARs) with model membranes.[6d,e] These CPPMs
were designed to contain p-rich and p-poor aromatic function-
alities to assess the role of p interactions in tuning membrane
activity.
Given these observations and the different electronic prop-
erties of tyrosine, phenylalanine, and tryptophan (Figure S1
and Table S1 in the Supporting Information), the role of aroma-
ticity in CPP(M) activity was studied by exploring the effect
that changes in quadrupole moments have on these systems.
The flat, planar structures of these aromatic rings and their as-
sociated quadrupole moments are thought to enable various
p interactions, such as p–p, p cation, p anion, and p polar in-
teractions within the cellular environment that can aid in
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ble half esters. From this and a study by Nanjappan and Czar-
nik, it was concluded that electron-withdrawing groups desta-
bilized Diels–Alder adducts and accelerated the retro-Diels–
Alder reaction.[15]
Results and Discussion
Monomer synthesis
Diester monomers were synthesized by using a two-step pro-
cess (Scheme 1). These procedures were adapted from previ-
ously described methods with modifications.[4a,14] In brief, oxa-
norbornene anhydride (1) was ring opened by using various
aromatic alcohols (a–h) and 4-dimethylaminopyridine (DMAP)
to give the half-ester intermediates.
Compounds 2g, h were not pursued for monomer forma-
tion because the retro-Diels–Alder impurities 4g,h have the
same reactive functional groups (ꢀCOOH, C=C) as 2a–f. All
stable half-esters and monomers were characterized by
1H NMR, 13C NMR spectroscopy, and MS. In terms of electrostat-
ic potential values, the six stable monomers covered an elec-
trostatic potential range from ꢀ4.66 kcalmolꢀ1 (Scheme 1, R=
e=NO2) to +29.69 kcalmolꢀ1 (Scheme 1, R=b=CH3). Electro-
static potential values are summarized in Table S2 in the Sup-
porting Information; all characterization data is provided in the
Supporting Information.
Half-esters 2a–f were then further reacted with 1,3-di-boc-2-
(2-hydroxyethyl)guanidine by using 1-ethyl-3-(3-dimethylami-
nopropyl)carbodiimide (EDC)-coupling conditions to give mon-
omers 3a–f. Half-esters 2g–h were not used for monomer syn-
thesis, because they proved to be unstable in solution at room
temperature. As shown in Figure 1A, half-esters 2g–h under-
went a spontaneous retro-Diels–Alder reaction to yield 4g–h
and furan (5). This was demonstrated by isolating the retro-
Imide monomers were synthesized by using a one-step pro-
cess adapted from Som et al, as illustrated in Scheme 2.[6d,e]
Unlike the diester system, there were no issues with stability
for the imide system, and
no retro-Diels–Alder products
were observed. All monomers
were characterized by 1H NMR,
13C NMR techniques, and MS. In
terms of electrostatic potential
values for the aromatic groups
incorporated, all stable mono-
mers covered an electrostatic
potential range from ꢀ18.10 kcal
molꢀ1 (Scheme 2, R1 =a=OCH3)
to 15.57 kcalmolꢀ1 (Scheme 2,
R1 =c=NO2). These monomers
expand the negative end of the
electrostatic potential range so
that in total the monomer
design
spans
ꢀ29.69
to
+15.57 kcalmolꢀ1 as summar-
ized in Table S2 in the Support-
ing Information.
Figure 1. Stability of p-poor monomers. A) Retro-Diels–Alder reaction that occurs for monomers 4g–h. B) Stable
and unstable p-poor monomer aromatic groups with their corresponding electrostatic potential maps. The range
for electrostatic potential was set between ꢀ30.00 and 30.00 kcalmolꢀ1. The color scale bar reflects this range
with red representing electron-rich surfaces, and blue representing electron-poor surfaces. Surfaces were calculat-
ed at the HF level by using the 3-21G* basis set.[17]
Polymer Synthesis
Polymers were synthesized by
using ROMP with Grubbs third-
Diels–Alder product, 4g, using column chromatography and
generation catalyst, as illustrated in Schemes 3, 4, and 5.
All tert-butyloxycarbonyl (Boc)-protected polymers (8a–f,
10a–c, 13a,c,e) were characterized by 1H NMR to assess chem-
ical composition and gel-permeation chromatography (GPC) to
assess relative molecular weights. GPC data are summarized in
Table 1.
1
verifying its chemical composition by using H NMR, 13C NMR,
and mass spectrometry (MS). Retro-Diels–Alder product 4h
proved to be more difficult to isolate because of additional
nitro-based impurities.
However, peaks for the retro-Diels-Alder product were ob-
1
served in the H NMR of 2h (see the Supporting Information).
Polymers were subsequently deprotected using trifluoroace-
tic acid (TFA) and CH2Cl2 (1:1) overnight.[4a,6d,e,14] TFA was re-
moved by azeotropic distillation with methanol. Diester poly-
mers were dialyzed for three days in water by using mem-
branes with a molecular weight cut-off of 2000 gmolꢀ1 for ho-
mopolymers and 1000 gmolꢀ1 for random copolymers. All
polymers were then dissolved in water and lyophilized to yield
dry 9a–f, 12a–c, and 15a,c,e.
Because 2a–f did not appear to undergo the retro-Diels–Alder
reaction, it was hypothesized that this reaction was related to
the electron-withdrawing substituents attached to the aromat-
ic rings. To investigate this, all p-poor aromatic rings were
modeled using Spartan molecular modeling software[17] as
shown in Figure 1B. The most electron-poor (most blue in
color) aromatic rings were the rings associated with the unsta-
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Biophysical Characterization
All polymers were tested by using a vesicle dye-re-
lease assay to assess relative polymer activity by
using a fluorescence plate reader.[6a,6c–e] This high-
throughput screening method enabled the testing of
all samples in a 12-well plate at the same time. Car-
boxyfluorescein (CF)-filled phosphatidylcholine (PC)
or PC/phosphatidylserine (PS) vesicles were prepared
as described in the Supporting Information and then
used for these experiments.
Scheme 3. Synthesis of diester homopolymers containing p-rich and p-poor aromatic
rings. i) Dichloro-di(3-bromopyridino)-N,N’-dimesitylenoimidazolino–Ru=CHPh (G3) cata-
lyst, CH2Cl2,, RT, 45 min; ii) ethyl vinyl ether, RT, overnight; iii) TFA/CH2Cl2 (1:1), RT, over-
night. Products 9a–f were further purified by dialysis with molecular weight cut-off
2000 gmolꢀ1. All polymers were synthesized with n=20; R is defined in Scheme 1.
For this dye release assay, the baseline fluores-
cence (F0) of 2-amino-2-hydroxy-
methyl-propane-1,3-diol
(Tris)
saline buffer with CF-filled vesi-
cles was determined. Then, poly-
mer solutions (in DMSO) of vary-
ing concentrations were added
to the vesicle-containing solu-
tions. After ten minutes, the
fluorescence intensity (Ff,0) was
measured again. Triton X-100
(5% in DMSO) was then added
to each experimental solution to
lyse the vesicles and release all
of the dye. The final fluorescence
measurement (Ft) was taken
after five minutes. Complete ex-
Scheme 4. Synthesis of imide random copolymers containing p-rich and p-poor aromatic rings. i) Dichloro-di(3-
bromopyridino)-N,N’-dimesitylenoimidazolino–Ru=CHPh (G3) catalyst, CH2Cl2, RT, 45 min; ii) ethyl vinyl ether, RT,
overnight; iii) TFA/CH2Cl2 (1:1), RT, overnight. All polymers were synthesized with n=20 and m=20; R1 is defined
in Scheme 2.
perimental details for these experiments can be found in the
Supporting Information, Figure S18. The results were normal-
ized according to the baseline and Triton controls to yield frac-
tional dye release (If) according to Equation (1):
Table 1. Molecular characteristics of p-rich and p-poor CPPMs.
Diester homopolymers
Imide and diester random copolymers
[a]
[a]
CPPM
Mn
ꢀ[a]
CPPM
n/m[b]
Mn
ꢀ[a]
8a
8b
8c
8d
8e
8 f
11600
11200
11300
12600
11400
11500
1.05
1.05
1.05
1.05
1.05
1.05
11 a
11 b
11 c
14a
14c
14e
56:44
55:45
58:42
38:62
40:60
39:61
16200
13700
17000
10600
12100
10700
1.06
1.07
1.06
1.10
1.08
1.14
If ¼ ðFf,0ꢀF0Þ=ðFtꢀF0Þ
ð1Þ
For Hill analysis, If was plotted against polymer concentra-
tion, c, and fit to the Hill equation, Equation (2), to give the
EC50, in which If,0 and If,max are the minimum and maximum
value of If obtained for each well, respectively:
[a] Number average molecular weight (Mn) and polydispersity indices
(ꢀ=Mw/Mn) determined by GPC by using polymethyl methacrylate
(PMMA) standards for diester polymers, and polystyrene standards for the
imide polymers using THF as the eluent and toluene as the flow marker.
[b] Ratio of residues, in which n represents the percentage of hydropho-
bic residues and m represents the percentage of guanidine-containing
residues.
n
If ¼ If,0 þ ðIf,maxꢀIf,0Þ=½ð1 þ c=EC50Þ ꢁ
ð2Þ
The first set of polymers tested were 9a–f and 12a–c, be-
cause both sets of polymers had comparable hydrophobic and
Scheme 5. Synthesis of diester random copolymers containing p-rich and p-poor aromatic rings. i) Dichloro-di(3-bromopyridino)-N,N’-dimesitylenoimidazoli-
no–Ru=CHPh (G3) catalyst, CH2Cl2, RT, 45 min; ii) ethyl vinyl ether, RT, overnight; iii) TFA/CH2Cl2 (1:1), RT, overnight. Products 15a,c, and e were further purified
by dialysis with molecular weight cut-off:1000 gmolꢀ1. All polymers were synthesized with n=8 and m=12; R is defined in Scheme 1.
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hydrophilic contents (ca. 1:1) but different backbone composi-
tions. A summary of the EC50, If,max, and n values obtained from
testing 9a–f and 12a–c with PC vesicles are displayed in
Table 2.
though many of our previous SAR studies were based on the
imide system, in this paper all further testing was conducted
with the diester system because backbone architecture had
little impact on results and this system offers more options for
structural tuning.
To further probe the effect of p electronics on CPPM activity,
dye-swelled vesicles were prepared by adding negatively
charged PS lipids (20 mol%) to PC lipids. This lipid composition
was selected to exploit potentially favorable p-anion interac-
tions that can occur between anionic lipids and electron-defi-
cient aromatic systems, while also capitalizing on electron re-
pulsions between anionic lipids and electron-rich aromatic sys-
tems.[16] Based on the nature of p interactions, it was anticipat-
ed that CPPM activity would trend based on p-electron densi-
ty, with p-poor CPPMs exhibiting better activity due to
favorable p-anion interactions. In contrast, p-rich polymers
were expected to have weaker activity due to electron repul-
sion between the anionic lipids and the electron-rich aromatic
rings. For these studies, polymers 9a, c, and e were tested,
and the results were compared to those obtained for PC vesi-
cles. A summary of the EC50, If,max, and n values can be found in
Table S3 in the Supporting Information. In addition, representa-
tive overlays of p-rich and p-poor polymers tested with PC and
PC/PS vesicles can be found in Figure 3. EC50 values for poly-
Table 2. EC50, Ymax, and Hill coefficient n values for diester homopolymer
and imide random copolymer-based CPPM activity by using 100 nm PC
large unilamellar vesicles swelled with carboxyfluorescein.
[b]
CPPM
EC50[a] [nm]
Ymax
n[c]
9a
9b
9c
9d
9e
9 f
12a
12b
12c
8.27ꢂ0.84
7.36ꢂ1.87
9.07ꢂ0.79
7.48ꢂ0.50
9.93ꢂ0.50
8.63ꢂ0.52
10.09ꢂ1.96
11.85ꢂ0.19
10.64ꢂ0.21
0.89ꢂ0.00
0.92ꢂ0.02
0.90ꢂ0.01
0.91ꢂ0.01
0.92ꢂ0.02
0.85ꢂ0.02
0.87ꢂ0.01
0.84ꢂ0.05
0.72ꢂ0.02
2.01ꢂ0.04
1.59ꢂ0.04
1.93ꢂ0.02
1.43ꢂ0.01
1.50ꢂ0.06
1.79ꢂ0.01
1.59ꢂ0.07
1.63ꢂ0.14
1.93ꢂ0.15
[a] EC50 needed to reach Ymax/2. [b] Maximum fraction of carboxyfluores-
cein released compared to total dye released upon addition of Triton-
X 100. [c] Value n is the Hill coefficient. Standard deviation from three in-
dependent experiments is reported.
In addition, representative overlays of p-rich and p-poor
polymers from both sets of polymers are shown in Figure 2. All
EC50 values were similar (7–12 nm) and, with the exception of
the lower If,max for 12c, the Hill plots were also almost identical.
Only EC50 values that differ over several orders of magnitude
represent significant changes, as was observed in previous
studies, in which the aliphatic hydrophobic group incorporated
was changed from a methyl group (EC50 =6.4 mm) to a butyl
group (EC50 =0.003 mm).[6e]
Within each series, it was determined that the nature of the
p-rich or p-poor aromatic rings incorporated did not have a sig-
nificant impact on CPPM activity. By comparing CPPMs from
both series, it was determined that the nature of the CPPM
backbone also had a negligible effect on CPPM activity. Al-
Figure 3. Anionic versus zwitterionic vesicle Hill plots for p-rich polymer 9a
(red) and p-poor polymer 9e (blue) using two types of 100 nm large unila-
mellar vesicles swelled with carboxyfluorescein: PC (solid lines) and PC/PS
(80:20, dashed lines). Data was fit to the Hill Equation. If represents the frac-
tion of dye released.
mers 9a, c, and e tested with PC/PS vesicles were similar to
those obtained from studies with PC vesicles. There was also
little difference in the Hill plots for these polymers, regardless
of the nature of the p-rich or p-poor aromatic ring incorporat-
ed or type of vesicles used for the study.
Based on these results, a set of diester random copolymers
was designed that had a more dilute hydrophobic content to
make sure that the results observed were not due to the
CPPM hydrophobic content being too high. The CPPMs de-
Figure 2. Diester versus imide Hill plots for p-rich CPPMs 9a and 12a (red)
and p-poor CPPMs 9e and 12c (blue) by using 100 nm PC large unilamellar
vesicles swelled with carboxyfluorescein. Data was fit to the Hill Equation,
and If represents the fraction of dye released. Solid lines represent diester-
based CPPMs, and dashed lines represent imide random copolymer-based
CPPMs.
signed are shown in Scheme 4. A summary of the EC50, If,max
,
and n values can be found in Table S4 for PC and Table S5 in
the Supporting Information for PC/PS vesicles. In addition, rep-
resentative overlays for the diester random copolymers as they
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these same assays previously illustrated that adding hydropho-
bicity improves activity, with aromatic groups outperforming
aliphatic groups.[6d,e] Even though we were able to synthesize
a series of polymers that contained aromatic groups with an
electrostatic potential range of ꢀ29.69 to 15.57 kcalmolꢀ1, it is
also possible that the structural modifications made to the
polymers may not have been significant enough to impact
CPPM activity. Although it is likely that overall CPPM hydropho-
bicity and cationic charge are more influential design parame-
ters than p-electronics, these results indicate that a wide range
of aromatic groups can be incorporated into the polymer
structures with limited impact on CPPM activity. From a design
standpoint, this opens up additional ways in which CPPMs can
be modified without inhibiting their performance.
Figure 4. Homopolymer versus random copolymer Hill plots for p-rich poly-
mers 9a and 15a (red) and p-poor polymers 9e and 15e (blue) using
100 nm PC large unilamellar vesicles swelled with carboxyfluorescein. Data
was fit to the Hill Equation, and If represents the fraction of dye released.
Solid lines represent diester homopolymers, and dashed lines represent di-
ester random copolymers.
Conclusion
For this study, aromatic groups containing electron-donating
and electron-withdrawing groups were chemically incorporat-
ed into CPPM structures as a way to tune p interactions. It was
hypothesized that tuning the quadrupole moments of the aro-
matic rings would provide additional control over CPPM activi-
ty. When synthesizing the monomers for these studies, it was
established through small-molecule synthesis that highly elec-
tron-withdrawing groups could not be chemically incorporated
into the diester versions of our oxanorbornene monomers, be-
cause it spontaneously induced a retro-Diels–Alder reaction.
Within the synthetically accessible series, vesicle-dye-release
experiments were performed as a way to determine the
CPPMs’ EC50 values and assess their relative activities. It was
shown by using PC vesicles that polymer backbone did not
impact activity for the p-rich/p-poor CPPMs and that the elec-
tron-donating or electron-withdrawing groups, as well as the
relative hydrophobic content did not impact activity either.
Diester CPPMs were also tested with PC vesicles containing
20% PS anionic lipids in efforts to more thoroughly under-
stand p-membrane interactions.
Figure 5. Homopolymer versus random copolymer Hill plots for p-rich poly-
mers 9a and 15a (red) and p-poor polymers 9e and 15e (blue) using
100 nm PC/PS (80:20) large unilamellar vesicles swelled with carboxyfluores-
cein. Data was fit to the Hill Equation, and If represents the fraction of dye
released. Solid lines represent diester homopolymers, and dashed lines rep-
resent random copolymers.
However, it would seem that only overall hydrophobicity
dictated polymer activity and not the incorporated electron-
donating or electron-withdrawing groups. Although it is possi-
ble that the assay used could not distinguish the subtleties of
p interactions, it is also likely that the structural modifications
made to the polymers were not significant enough to impact
CPPM activity, despite the fact that molecules containing aro-
matic rings with an electrostatic potential range of ꢀ29.69 to
+15.57 kcalmolꢀ1 were explored. This suggests that other
design parameters, such as overall hydrophobicity and cationic
charge, have a greater impact on CPPM activity. The results
also indicate that a wide range of aromatic groups can be in-
corporated into the polymer structures with limited impact on
CPPM activity. This is encouraging from a design standpoint,
because it expands potential functionality without impacting
activity. Understanding these design principles will help to
guide the development of future CPPMs.
compare to their corresponding diester homopolymers for PC
and PC/PS vesicles can be observed in Figures 4 and 5, respec-
tively.
EC50 values for 14a, c, and e were similar to those obtained
for 9a, c, and e when tested with PC vesicles and the overlays
of the Hill plots in Figure 4 further suggest that there was little
difference between the activity of the diester homopolymers
and the less hydrophobic random copolymers. When tested
with PC/PS vesicles, there was a slight increase in EC50 values
for the random copolymers compared to the homopolymers,
and there was a noticeable shift in the Hill plots in Figure 5.
However, the shift was about the same for all diester random
copolymers and thus attributed to the lower hydrophobic con-
tent and not due to the p-electronics of the system. Because
no trend was observed, it was concluded that p electronics do
not play a major role in CPPM activity. Alternatively, it is possi-
ble that the assay used herein does not have the fidelity to dis-
tinguish the subtleties of p interactions despite the fact that
Chem. Eur. J. 2015, 21, 3013 – 3019
www.chemeurj.org
3018
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
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Acknowledgements
The work was funded by the NIH (T32 GMO8515) and NSF
(CHE-0910963). The authors would like to thank Dr. Hitesh D.
Thaker, Dr. Jing Jiang, Prof. Ke Zhang, and Dr. Michael Lis for
invaluable scientific discussions and help with half-ester and
monomer purifications. The authors would also like to thank
Ms. Catherine N. Walker, Mr. Ilker Ozay, and Mr. Nicholas Posey
for their feedback on early drafts of this manuscript. Mass
spectral data were obtained at the University of Massachusetts
Mass Spectrometry Facility, which is supported in part by NSF.
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Keywords: peptides · peptidomimetics · pi interactions ·
polymerization · proteins
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Published online on December 23, 2014
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www.chemeurj.org
3019
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim