Cell-penetrating peptoids: Introduction of novel cationic side chains

Article abstract of DOI:10.1016/j.ejmech.2014.03.078

During the last decade peptoid-based molecular transporters have been broadly applied. They are highly valued for their easy synthesis and their superior stability against enzymatic degradation. The special structure of peptoids generally allows introducing a variety of different side chains. Yet, the cationic side chains of cell-penetrating peptoids displayed solely lysine- or arginine-like structures. Thus this report is intended to extend the spectrum of cationic peptoid side chains. Herein, we present novel functional groups, like polyamines, aza-crown ethers, or triphenylphosphonium ions that are introduced into peptoids for the first time. In addition, the obtained peptoids were tested for their cell-penetrating properties.2014 Published by Elsevier Masson SAS.

Full text of DOI:10.1016/j.ejmech.2014.03.078

European Journal of Medicinal Chemistry 79 (2014) 231e243
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Cell-penetrating peptoids: Introduction of novel cationic side chains
,
Dominik K. Kölmel a, Anna Hörner a b, Franziska Rönicke c, Martin Nieger d, Ute Schepers c,
,c,
Stefan Bräse a
*
a Karlsruhe Institute of Technology (KIT), Institute of Organic Chemistry, Fritz-Haber-Weg 6, D-76131 Karlsruhe, Germany
b Karlsruhe Institute of Technology (KIT), Light Technology Institute, Engesserstraße 13, D-76131 Karlsruhe, Germany
c Karlsruhe Institute of Technology (KIT), Institute of Toxicology and Genetics, Hermann-von-Helmholtz-Platz 1,
D-76344 Eggenstein-Leopoldshafen, Germany
d University of Helsinki, Laboratory of Inorganic Chemistry, PO Box 55, FIN-00014, Finland
a r t i c l e i n f o
a b s t r a c t
Article history:
During the last decade peptoid-based molecular transporters have been broadly applied. They are highly
valued for their easy synthesis and their superior stability against enzymatic degradation. The special
structure of peptoids generally allows introducing a variety of different side chains. Yet, the cationic side
chains of cell-penetrating peptoids displayed solely lysine- or arginine-like structures. Thus this report is
intended to extend the spectrum of cationic peptoid side chains. Herein, we present novel functional
groups, like polyamines, aza-crown ethers, or triphenylphosphonium ions that are introduced into
peptoids for the first time. In addition, the obtained peptoids were tested for their cell-penetrating
properties.
Received 7 December 2013
Received in revised form
25 March 2014
Accepted 27 March 2014
Available online 28 March 2014
Keywords:
Peptoid
Molecular transporter
Spermine
Ó 2014 Elsevier Masson SAS. All rights reserved.
Cellular uptake
HeLa cell
1. Introduction
transporters can be extremely valuable [3]. In practice, the cellular
uptake of a molecule with low bioavailability can be mediated by its
Although the concept of molecular transporters is known since
the cell-penetrating properties of the HIV-1 Tat protein were
discovered in the late 1980s by Green and Loewenstein [1], and
independently by Frankel and Pabo [2], there is still an ongoing
demand for new transporters. Nowadays, molecular transporters
are defined as substances that possess intrinsic cell-penetrating
properties and can thus assist in transporting other substances
(e.g. drugs or dyes) through the cell membrane. As bioavailability is
covalent conjugation with an appropriate molecular transporter
[3]. By doing this, the molecular uptake of diverse cargoes into
mammalian and even plant cells can be greatly enhanced [4]. To
date, a wide variety of cargoes that greatly differ in size and nature,
like small molecules, oligonucleotides, peptides/proteins, nano-
particles or quantum dots, were successfully delivered by molec-
ular transporters [3]. Furthermore, it is possible to address different
cellular compartments by altering the structure of the molecular
transporter [5]. Therefore, molecular transporters are a promising
delivery system for targeted drug delivery [6].
a
crucial criterion for all bioactive molecules, molecular
Molecular transporters can be built up from numerous different
scaffolds. So far the best characterized transporters are the cell-
penetrating peptides (CPPs) [7]. Among the most frequently used
CPPs are short sequences of the HIV-1 Tat protein [8], derivatives of
the homeoprotein from Antennapedia (e.g. penetratin) [9], the
protein transportan [10] and oligomers of arginine. [11] Besides
Abbreviations: Boc, tert-butoxycarbonyl; CPP, cell-penetrating peptide; CPPo,
cell-penetrating peptoid; CuAAC, copper(I)-catalyzed azide-alkyne cycloaddition;
DIC, N,N0-diisopropylcarbodiimide; DMF, N,N-dimethylformamide; εmax, extinction
coefficient;
FF,
fluorescence
quantum
yield;
Fmoc,
9H-fluoren-9-
ylmethoxycarbonyl; FRET, Förster resonance energy transfer; H, brightness; HeLa,
human cervix carcinoma; HIV, human immunodeficiency virus; HOBt, 1-
those CPPs, peptidomimetics, such as
b-peptides [12] or oligo-
hydroxybenzotriazole; HPLC, high performance liquid chromatography; lmax,abs
,
carbamates [13], have been described that mimic the cell pene-
trating properties but display higher stability against enzymatic
degradation [14]. Despite their structural diversity, they all are
mainly amphiphilic and positively charged under physiological
absorption maximum; lmax,em, emission maximum; Ms, mesyl; PBS, phosphate
buffered saline; RT, room temperature; siRNA, small interfering ribonucleic acid;
SPPS, solid phase peptide synthesis; Tf, triflyl; TFA, trifluoroacetic acid.
* Corresponding author. Karlsruhe Institute of Technology (KIT), Institute of
Organic Chemistry, Fritz-Haber-Weg 6, D-76131 Karlsruhe, Germany.
E-mail address: braese@kit.edu (S. Bräse).
http://dx.doi.org/10.1016/j.ejmech.2014.03.078
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

232
D.K. Kölmel et al. / European Journal of Medicinal Chemistry 79 (2014) 231e243
conditions containing extended regions of lysine- or arginine-like
residues [12,13,15,16].
nucleophilic substitution with the immobilized bromide can take
place. Again, those two steps can be repeated until the desired
peptoid is build up. The advantage of this method is that various
primary amines [35] can be used directly and that there is no need
for a temporary protecting group. Therefore, the submonomer
method is often considered as the more elegant approach.
Although most peptoids are assembled on solid supports, it should
be mentioned that the synthesis can be performed in solution as
well [36].
Besides their application as CPPos [37], peptoids have been
broadly applied [38], e.g. as antimicrobial drugs [39], antifouling
polymers [40], and polymer electrolytes [41], for storing of small
molecules [42], to complex metal ions [43], to stabilize nano-
particles [44,45] and for positron emission tomography [37].
The focus of this work was to extend the spectrum of cationic
peptoid side chains. Since the polycationic character of molecular
transporters plays a crucial role in cellular uptake, changing the
side chain functionalities might lead to novel CPPos with different
uptake properties or even organ specificity in in vivo applications.
Therefore, one goal was to introduce side chains with multiple
amino functions. By doing this, it should be possible to create CPPos
with an increased charge density. Furthermore, we introduced side
chains that are positively charged without the need of protonation.
The respective CPPos were tested for their cellular uptake. In
addition we investigated the influence of the novel side chains on
the intracellular distribution of the CPPos.
Although the cellular uptake mechanism is not yet revealed in
all details, the polycationic character is a very important feature of
molecular transporters. It is assumed that the positive charges
allow the transporters to interact strongly with the negatively
charged structures such as the heparane sulfates and the head
groups of membrane phospholipids on the extracellular side of the
plasma membrane [17,18]. After the adsorption of the transporter
the subsequent translocation through the plasma membrane can
take place. There are many different and partially contrary hy-
potheses for this translocation step, which are controversially dis-
cussed. Currently, it is anticipated that most CPPs are taken up by
endocytosis [19]. But it is very likely that several CPPs utilize other
competing uptake pathways, like direct penetration [19,20].
Furthermore, the actual mechanism may change with the experi-
mental conditions [19]. However, independently from the exact
mechanism, it is quite noteworthy that the plasma membrane is not
significantly damaged during the cellular uptake of such molecular
transporters [19].
Another important class of CPP mimetics is the cell penetrating
peptoids (CPPos). These CPPos are oligomers of N-substituted gly-
cines and thus unnatural regioisomers of the omnipresent peptides
(Fig. 1). Like other peptidomimetics, peptoids are highly stable
against enzymatic degradation [14]. Therefore peptoids are well
suited for biological applications [21]. Unlike peptides, peptoids
have an achiral backbone and typically do not form distinct sec-
ondary structures as the tertiary backbone amides are no longer
able to act as donors for hydrogen bonding. However, it has been
shown that it is possible to stabilize complex secondary or tertiary
structures, like helices [22], sheets [23], threaded loops [24], rib-
bons [25], or turns [26], by introducing suitable side chains. So far,
CPPos always had a high content of lysine- and/or arginine-like side
chains [16,27]. Interestingly, it could be shown that neither the
absence of chirality nor the loss of hydrogen bonding along the
backbone has a negative influence on the cellular uptake [28]. Up to
now, CPPos have been successfully applied to transport many
different cargoes, like siRNA [29], photosensitizers [30], diverse
fluorescence dyes [31], or lanthanide ions [32].
2. Results and discussion
2.1. Synthesis
During this work we focused on the synthesis of hexameric
peptoids, because this length has been approved for CPPos [27,46].
In addition, all peptoids were labeled with rhodamine B at their N-
terminus [27b] to enable visualization via fluorescence microscopy.
The obtained peptoids 1e5 are depicted in Fig. 2.
The first aim was to introduce side chains with multiple amino
functions. Those structures would be very interesting as they
display higher charge density than common CPPos with just one
amino (or guanidino) function per side chain. Unfortunately,
starting with the commercially available primary amine 6 as a
submonomer the coupling yields were too low to allow an efficient
peptoids synthesis via the submonomer approach. Thus, the
respective monomer 10 was built up (Scheme 2). First, primary
amine 6 was treated with ethyl bromoacetate to obtain glycine
ester 8 in nearly quantitative yield. Next, the ester was saponified
and the secondary amine was protected with Fmoc. The resulting
monomer 10 was well suited for the synthesis of a hexameric
peptoid by using the classical monomer approach (see peptoid 1,
Fig. 2).
To further increase the number of amino functions per side
chain, the natural occurring polyamine spermine should be incor-
porated into a peptoid as well. Hence, tris-Boc-protected spermine
7 was synthesized according to a procedure from Geall et al. [47]
But again, primary amine 7 was not suitable for the submonomer
approach. Therefore, monomer 11 was built up by the same
sequence as for the synthesis of monomer 10 (alkylation with ethyl
bromoacetate [48], saponification, and protection of the N-termi-
nus with Fmoc). Monomer 11 was used for the synthesis of a
hexameric peptoid. However, a mixture of the different homolo-
gous peptoids (monomer to hexamer) was obtained with the
hexamer (see peptoid 2, Fig. 2) as the major product, which could
be separated from the other homologues by HPLC. Although the
coupling yields of monomer 11 were only moderate, the monomer
approach was still clearly superior to the submonomer approach.
Peptoids are superior to most other peptidomimetics because
they can be easily assembled on solid supports such as Rink amide
resin. There are two different but convergent synthetic strategies:
the monomer and the submonomer approach (Scheme 1). The
monomer approach is based on the established solid phase peptide
synthesis (SPPS). During this approach Fmoc-protected glycine
derivatives are activated with N,N0-diisopropylcarbodiimide (DIC)
and 1-hydroxybenzotriazole (HOBt) and reacted with the resin
[33]. The coupling step is followed by the deprotection of the N-
terminus. This sequence of coupling glycine derivatives and Fmoc
deprotecting can be repeated iteratively to obtain a peptoid with
the desired length. Finally, the peptoid can be cleaved from the
solid supports with trifluoroacetic acid (TFA).
The second approach for the synthesis of peptoids was originally
introduced by Zuckermann et al. in 1992 [34]. During the first step
of the submonomer approach, the resin is acylated with bromo-
acetic acid. Next, a primary amine is added to the resin and a
Fig. 1. General structure of the natural occurring L-peptides and peptoids.

D.K. Kölmel et al. / European Journal of Medicinal Chemistry 79 (2014) 231e243
233
Scheme 1. General reaction scheme for the solid phase synthesis of peptoids via submonomer [34] or monomer approach. Reagents and conditions: (i) DIC, DMF, RT, 30 min; (ii)
DIC, HOBt, DMF, 60 ꢀC (
-wave), 30 min, double coupling; (iii) RNH2, DMF, RT, 1 h; (iv) piperidine, DMF, RT, 3 ꢁ 5 min.
m
extracellular medium to render the peptoids cationic. To achieve
this, a crown ether moiety was introduced as a side chain. Initially,
N-substituted 1-aza-15-crown-5 derivative 12, which was synthe-
sized according to a procedure of Beer et al., was used as sub-
monomer as such compounds form stable complexes with sodium
as well as potassium ions [49,50]. However, the introduction of this
novel side chain by submonomer method failed. Therefore, we
decided to build up the full length peptoid backbone with subse-
quent introduction of the crown ether moiety via dipolar copper(I)-
catalyzed azide-alkyne cycloaddition (CuAAC) [51]. This strategy of
postsynthetically modifying side chains by CuAAC was shown to be
beneficial for the introduction of various demanding functionalities
into peptoids [52]. By using triflyl azide as diazo transfer reagent
[53], primary amine 12 could be easily converted into the corre-
sponding azide 13 (Scheme 3). To provide a peptoid with terminal
alkynes, propargylamine was used as a submonomer to build up
the respective homohexamer (see experimental section). The
alkyne side chains could be modified with azide 13 via CuAAC.
Finally, the peptoid N-terminus was labeled with rhodamine B to
obtain peptoid 3.
In addition to the previously discussed side chains, different
functionalities bearing intrinsic positive charges were included into
peptoids. To produce peptoids with quaternary ammonium ions
primary amine 14 was exhaustively methylated (Scheme 3).
Eventually, the Boc protecting group of 15 was removed with hy-
driodic acid. The diiodide 16 could be crystallized from methanol/
ethyl acetate. The molecular structure of ammonium ion 16 was
determined by X-ray diffraction (Fig. 3). After diazo transfer with
TfN3 azide 17 was obtained in moderate yield. Most notably the
preparation of azide 17 did not involve purification by column
chromatography as the last two products 16 and 17 can be easily
purified by recrystallization. However, the incorporation of azide 17
into peptoids by CuAAC was not successful (data not shown).
Since quaternary ammonium ions could not be introduced into
peptoids, we tried to incorporate other charged moieties like
phosphonium ions. This would be very interesting, because it is
well known that triphenylphosphonium ions can be used for tar-
geting mitochondria [54]. Those lipophilic cations are able to
overcome the plasma membrane and the mitochondrial membrane
due to the negative transmembrane potential. The triphenylphos-
phonium moieties were integrated into peptoids by CuAAC. Thus,
azide 19 was synthesized according to a procedure of Chen et al.
(Scheme 4) [55]. To reduce electrostatic repulsion between the side
chains during the modification of the peptoid by CuAAC, Boc-
protected aminohexyl residues were used as insulators. This ami-
noalkyl side chain was chosen, as it has repeatedly proven its
effectiveness for cell-penetrating peptoids [16,46]. Hence, the
Fig. 2. Structures of the synthesized hexameric peptoids 1e5. In all cases, the N-ter-
minus was labeled with rhodamine B.
Besides side chains with multiple amino functions, we also tried
other functional groups. The basic idea was to use side chains that
do not rely on pH-dependent protonation to provide the required
positive charges. One attempt was to use alkali metal ions from the
Scheme 2. Syntheses of the peptoid monomers 10 and 11. Reagents and conditions: (i)
ethyl bromoacetate, NEt3, THF, RT, overnight; (ii) 1) LiOH, dioxane/H2O, 0 ꢀC, 3 h; 2)
FmocCl, NaHCO3, RT, overnight.

234
D.K. Kölmel et al. / European Journal of Medicinal Chemistry 79 (2014) 231e243
Scheme 3. Syntheses of azides 13 and 17 by diazo transfer with TfN3. Reagents and conditions: (i) TfN3, CuSO4, K2CO3, MeOH/H2O, RT, overnight; (ii) MeI, NaHCO3, MeOH, RT, 2 d;
(iii) HI(aq), H2O, RT, overnight.
Besides triphenylphosphonium ions, other lipophilic cations
such as triarylmethane dyes were conjugated with peptoids. These
molecules possess a positive charge that is extensively delocalized
and can thus also be applied for targeting mitochondria [56]. To
obtain an azide-substituted derivative of the well-known dye
crystal violet, azide 21 was synthesized from alcohol 20 (Scheme 4).
Finally, azide 21 was reacted with Michler’s ketone to obtain the
intensively colored triarylmethane dye 22 in quantitative yield.
After CuAAC with the same peptoid that was previously used for
the incorporation of azide 19, peptoid 5 (see Fig. 2) could be
obtained.
2.2. Photophysical characterization
The obtained peptoids 1e5 were spectroscopically characterized
to investigate the influence of the novel side chains on the incor-
Fig. 3. Molecular structure of diiodide 16 with displacement ellipsoids drawn at 50%
probability level.
porated dye. The compounds were measured in water to facilitate
Scheme 4. Syntheses of azides 19 [54] and 22 by nucleophilic substitution with NaN3. Reagents and conditions: (i) NaN3, EtOH/H2O, reflux, overnight; (ii) 1) MsCl, NEt3, RT, 4 h; 2)
NaN3, DMF, 80 ꢀC, 12 h; (iii) 4,40-bis(dimethylamino)benzophenone, POCl3, toluene, 100 ꢀC, overnight.
obtained peptoid 4 (Fig. 2) comprised two triphenylphosphonium
moieties at the first and fourth side chain, which are separated by
two aminohexyl residues. The side chains at position five and six
were also compromised of aminohexyl residues to diminish the
repulsion between the triphenylphosphonium ions and the posi-
tively charged dye.
optimal comparability with results from biological tests (Table 1).
Besides, peptoid 3 was measured in phosphate buffered saline
(PBS) to enable the formation of complexes between the aza-crown
ethers and sodium ions. In addition, the spectra of rhodamine B
were also recorded for comparison. In every case, the spectral
maxima of the dye were shifted bathochromically (11e15 nm) upon

D.K. Kölmel et al. / European Journal of Medicinal Chemistry 79 (2014) 231e243
235
Table 1
resonance energy transfer (FRET) [58] between the excited dye and
one of the adjacent trityl ions [59].
Spectral properties of rhodamine B-labeled peptoids 1e5, azide 22 and rhodamine B.
If not stated otherwise, all spectra were measured in H2O.
Principally, FRET is a nonradiative process that is based on long-
range dipoleedipole interactions. This energy transfer can always
occur if the absorption band of a suitable acceptor is overlapping
with the emission band of a donor dye and if the distance between
them is not too large (typically 1e10 nm). In the case of peptoid 5
FRET is very likely, as the donor (rhodamine B) and the acceptor
(trityl side chain) are in close proximity. The requirement for
spectral overlap is fulfilled as well, which can be easily seen by
comparing the absorption spectrum of peptoid 5 with the emission
spectrum of undisturbed, peptoid-bound rhodamine B (e.g. peptoid
4, see Fig. 3). Therefore, we concluded that the fluorescence of
peptoid 5 is quenched by a highly efficient intramolecular FRET
between the excited rhodamine B moiety and the nonfluorescent
trityl side chains. Thus, peptoid 5 is only suitable to a limited extent
for fluorescence experiments.
b
Peptoid/Dye
lmax,abs
εmax [Mꢂ1cmꢂ1
]
lmax,em
FF
[nm]a
[nm]c
Rhodamine B
1
2
3 in PBS
4
5
22
554
567
567
568
569
537
589
98,000 ꢃ 3000
75,000 ꢃ 3000
55,000 ꢃ 2000
98,000 ꢃ 8000
53,000 ꢃ 9000
575
587
586
588
587
587
ed
0.23 ꢃ 0.02
0.112 ꢃ 0.013
0.24 ꢃ 0.04
0.21 ꢃ 0.06
0.28 ꢃ 0.03
0.0031 ꢃ 0.0003
ed
90,000 ꢃ 30,000
52,000 ꢃ 1900
a
Absorption maxima have a ꢃ1 nm imprecision.
b
Calculation is based on the molar mass of the trifluoroacetate salts of the
peptoids.
c
Fluorescence maxima are reproducible within a ꢃ2 nm range.
d
Could not be determined.
the conjugation with a peptoid. This slight shift is quite typical for
various dyeepeptoid conjugates [31]. The extinction coefficient
was affected by the peptoid as well. In general, the absorption of the
conjugates decreased. Nevertheless, the extinction coefficients
were still fairly high (5.3e9.8 ꢁ 104 Mꢂ1 cmꢂ1). The quantum yields
of peptoids 2e4 were comparable to the quantum yield of rhoda-
mine B. On the contrary, the quantum yield of peptoid 1 is reduced
by half. This finding suggests that the piperazinyl residues of pep-
toids 1 enable some nonradiative decay ways. Yet, the brightness
2.3. Cell tests
The rhodamine B-labeled peptoids 1e5 were tested for their
cell-penetrating properties by incubating human cervix carcinoma
(HeLa) cells [60] with a 10 mM solution of the respective peptoids.
After incubation for 24 h, the peptoids 1e5 could be detected inside
the cells via live cell fluorescent confocal microscopy (Fig. 5). All
peptoids were found to accumulate at least partially in vesicular,
most likely endosomal structures, which is quite common for many
CPPos [31b,46].
(H ¼ εmax
ꢁ
FF) of the peptoids 1e4 was more than sufficient for
biological tests (H ¼ 0.8e2.1 ꢁ 104 Mꢂ1 cmꢂ1).
Peptoid 2 was the only CPPo accumulating in the cytosol.
Although peptoid 1 had a comparable structure with multiple
amino functions, this transporter was not detected in the cytosol
but exclusively in vesicles. Moreover, we found that the shorter
homologues of peptoid 2 (monomer to tetramer), which were
formed as byproducts during the synthesis and could be separated
by HPLC, accumulated also entirely in vesicles (data not shown).
Thus, we conclude that the cytosol can be targeted if the number of
amino groups in the side chains of a peptoid is large enough. The
fact that peptoid 2 was found in both the cytosolic and the endo-
somal compartment led to the assumption that this transporter is
released from the endosomes after its initial endosomal uptake.
This process is called endosomal escape and there are many ex-
amples for the endosomal release of CPPs [61]. For example, it is
known that the endosomal escape of CPPs can be triggered by
incorporating histidine-rich domains into the peptide. After
endocytosis, the imidazolyl residues will become protonated under
the acidic conditions of the endosome [62]. This will cause osmotic
swelling, which ultimately results in the rupture of the endosome
and the release of its content. It is conceivable that a similar
mechanism is responsible for the cytosolic localization of peptoid 2.
The degree of protonation of peptoid 2 is likely to increase at the
lower pH value of the endosome. As a result peptoid 2 would in-
crease the osmotic pressure of the endosome, which would even-
tually allow endosomal escape.
Unlike the other peptoids, the spectral properties of transporter
5 were heavily influenced by its side chains. Trityl ion 22 exhibited
a strong, broad absorption band in the visible region of the elec-
tromagnetic spectrum by itself but did not fluoresce (see Table 1
and Fig. 4). Incidentally, the absorption maximum of triaryl-
methane dye 22 (589 nm) matched perfectly with the fluorescence
maximum of peptoid-bound rhodamine B (586e588 nm). There-
fore, electronic interactions between the trityl ions and the dye of
peptoid 5 are very likely. The absorption maximum of peptoid 5
was strongly blue-shifted. Furthermore, peptoid 5 showed just a
faint fluorescence. Hence, these trityl side chains have proven to be
efficient quenchers if bound to rhodamine B. Such quenching
properties are known for crystal violet as well [57]. In this case the
quenching could be ascribed to an intramolecular Förster
The aza-crown ether-containing peptoid 3 was shown to accu-
mulate in vesicles as well. Therefore, the uptake mechanism seems
to be comparable to the mechanism of other typical molecular
transporters. Apparently, it does not matter how the positive
charges are generated (either by protonation or by complexation of
alkali metal ions) as long as the transporter is multiply charged
under
physiological
conditions.
Surprisingly,
the
triphenylphosphonium-containing peptoid
4
was also found
exclusively in endosomes, although it is known that such phos-
phonium ions tend to accumulate in mitochondria [54]. Because
molecules that are able to target mitochondria are typically lipo-
philic cations, we think that peptoid 4 is still too polar, mainly due
Fig. 4. Normalized absorption of peptoids 4 (black) and 5 (gray dotted) and azide 22
(gray). The normalized emission spectrum of peptoid 4 (black dashed) is shown as
well. All spectra were recorded in water.

236
D.K. Kölmel et al. / European Journal of Medicinal Chemistry 79 (2014) 231e243
Fig. 5. HeLa cells were incubated with a 10
m
M peptoid solution for 24 h at 37 ꢀC and subsequently subjected to confocal fluorescence microscopy. To determine the intracellular
localization of the peptoids (red), mitochondria were stained with MitoTrackerÒ Green FM (green) and nuclei were stained with Hoechst 33342 stain (blue). (For interpretation of
the references to color in this figure legend, the reader is referred to the web version of this article.)
to the four aminoalkyl residues. Hence it should be possible to
restore the mitochondrial targeting properties by replacing some of
the aminoalkyl groups with more lipophilic residues (e.g. benzyl)
[37]. Expectedly, peptoid 5 showed just a faint fluorescence signal
and only if high laser intensities were applied for the excitation.
Besides its vesicular accumulation, this CPPo was also partially
localized in mitochondria. Consequently, the triarylmethane dye
moieties of peptoid 5 seem to be more lipophilic than the triphe-
nylphosphonium ions of peptoid 4. This is plausible because there
are several triarylmethane dyes that were shown to accumulate
actively in mitochondria, which is again due to the mitochondrial
membrane potential [63]. Nevertheless, triarylmethane dyes are
not particularly suitable for molecular transporters, because of
their strong fluorescence quenching properties.
crown ethers, triphenylphosphonium ions and triarylmethane
dyes. The novel side chains could be introduced into hexameric
peptoids either via monomer approach or via CuAAC. All peptoids
were labeled with rhodamine B to allow visualizing their cellular
uptake. Despite the fact that all peptoids were taken up by HeLa
cells, their intracellular distribution differed in some cases signifi-
cantly. Peptoids 1 and 3e5 aggregated mainly in vesicular, most
likely endosomal structures whereas peptoid 2 was shown to
accumulate in the cytosol. The main feature of peptoid 2 is its
highly polar structure with multiple amino functions. We assume
that those amino functions are required to overcome the endo-
somal entrapment, which was observed for the other less polar
peptoids. In addition, we could show that mitochondria could be
targeted as well by introducing lipophilic cations into peptoid 5.
However, the triarylmethane dye side chains of peptoid 5 had
strong fluorescence quenching properties and thus hamper the
applicability of such CPPos. Instead we recommend using triphe-
nylphosphonium ions, like those of transporter 4, which should be
more suitable to generate mitochondria targeting peptoids. In
conclusion, we think that such cell-penetrating peptoids, which are
3. Conclusion
In summary, we presented a set of novel peptoid side chains that
can be used for the synthesis of molecular transporters. Among
those side chains are piperazinyl and polyamine residues, aza-

D.K. Kölmel et al. / European Journal of Medicinal Chemistry 79 (2014) 231e243
237
capable of targeting specific cell organelles, are very promising, e.g.
for manipulating cells or for drug delivery. Hence we plan to
investigate the influence of different side chains on the intracellular
distribution of peptoid transporters in more detail in the future.
with a C18 Vydac 218TP Series (Grace Davison Discovery Sciences,
5
mm, 22 ꢁ 250 mm). Flow rate: 15 mL/min; solvent A: 0.1% TFA in
water; solvent B: 0.1% TFA in acetonitrile.
4.2. Cell culture techniques for mammalian cells
4. Experimental protocols
All procedures with mammalian cells were carried out under
sterile conditions. 1 ꢁ 104 HeLa (human cervix carcinoma) cells
4.1. General remarks
were plated into each well of an 8-well m-slide from IBIDI (Ibitreat),
Germany, and cultured in 200 mL of Dulbecco’s modified Eagle’s
UV/vis absorption spectra were recorded by using a Varian Cary
300 scan UV/vis spectrophotometer and fluorescence spectra were
recorded by using a Varian Cary Eclipse fluorescence spectrometer.
Closed quartz cuvettes with a 1 cm path length were used in all
experiments. Fluorescence quantum yield measurements were
performed on the previously mentioned fluorometer and UV/vis
instrument. The slit width was 5 nm for both excitation and
emission. Relative quantum efficiencies were obtained by
comparing the absorption values and the areas under the emission
spectrum for the unknown substance with a standard (rhodamine
B in water, FF ¼ 0.23) [64]. The following equation was used to
calculate quantum yields:
medium, high glucose (DMEM, Sigma Taufkirchen), supplemented
with 10% fetal calf serum (FCS, PAA), and 1 U/mL Penicillin/Strep-
tomycin at 37 ꢀC, 5% CO2.
4.3. Treatment of HeLa cells with the rhodamine B-labeled peptoids
The peptoids were dissolved in bidistilled water to yield a 2 mM
stock solution and were further diluted with 10% DMEM to yield the
respective incubation media. The cells cultured as described above
were incubated with the different peptoids at final concentrations
of 10 mM. Cellular uptake of the peptoids was measured by live cell
confocal fluorescence microscopy in DMEM after 24 h as fixation
would alter the intracellular distribution as described for other
polycationic species [65].
Fx
¼
Fs ꢁ ðFx=FsÞ ꢁ ðnx=nsÞ2 ꢁ ðAs=AxÞ
Fs is the reported quantum yield of the standard, F is the integrated
emission spectrum, A is the absorbance at the extinction wave-
length, and n is the refractive index of the solvents used. The
subscript x denotes unknown and s denotes standard. All reactions
were carried out under stirring. Reactions under inert gas were
carried out in flasks equipped with septa under argon (supplied by
using a standard manifold with vacuum and argon lines). Analytical
TLC was performed on MERCK ready-to-use plates with silica gel 60
(F254). Column chromatography: MERCK silica gel 60, 0.04e
0.063 mm. For microwave assisted peptoid synthesis the single
mode CEM Discover microwave was used. IR spectra were recorded
by using Bruker ALPHA-T spectrometer. The samples were
measured as films between KBr plates or by using the attenuated
total reflection (ATR) technique. The transmission intensities are
described as follows: s ¼ strong (11e40%), m ¼ middle (41e70%),
w ¼ weak (71e90%), vw ¼ very weak (91e100%). NMR spectra were
recorded at 25 ꢀC by using Bruker Avance 300 (300 (1H) and
75 MHz (13C)), Bruker AM 400 (400 (1H) and 100 MHz (13C)) and
Bruker DRX 500 (500 (1H) and 125 MHz (13C)) spectrometer. All
4.4. Subcellular localization
For the intracellular localization of the peptoids, the cells were
counterstained with fluorescent markers for different organelles
(Molecular Probes, Germany). For mitochondria labeling the cells
were treated with 125 nM MitoTrackerÒ Green FM for 15 min, ac-
cording to the manufacturer’s manual, and washed three times
with PBS. For the staining of the nuclei, the cells were eventually
treated with Hoechst 33342 dye (2 mg/mL) according to the man-
ufacturer’s instructions. The cells were covered with DMEM and
subjected to live confocal microscopy at 37 ꢀC and 5% CO2
atmosphere.
4.5. Live imaging by confocal microscopy
Simultaneous visualization of the colocalization of the peptoids
and mitochondria and nuclei was achieved by confocal microscopy
using Leica TCS-SP5 II, equipped with a DMI6000 microscope.
MitoTrackerÒ Green FM was excited using the 488 nm line of an
argon ion laser, the nuclei were excited with a UV laser at 364 nm,
and the peptoids were excited at 561 nm using a DPSS laser. The
objective was a HCX PL APO CS 63.0x1.2 Water UV. The exposure
was set to minimize oversaturated pixels in the final images.
Fluorescence emission was measured at 417e468 nm (for Hoechst
33342), 499e552 nm (for MitoTracker Green FM) and 593e696 nm
(for the peptoids) using simultaneous detection. Image acquisition
was conducted at a lateral resolution of 1024 ꢁ 1024 pixels and 8 bit
depth using LAS-AF 2.0.2.4647 acquisition software.
spectra are referenced to tetramethylsilane as standard (
by using the signals of the solvent:
d
¼ 0 ppm)
CDCl3 : 7:26 ppmðCHCl3Þ or 77:0 ppmð13CDCl3Þ
CD3OD : 3:31 ppmðCHD2ODÞ or 49:1 ppmð13CD3ODÞ
Multiplicities of the signals are described as follows: s ¼ singlet,
d ¼ doublet, t ¼ triplet, q ¼ quartet, quin ¼ quintet, m ¼ multiplet,
mc ¼ centered multiplet. Coupling constants (J) are given in Hz.
Multiplicities in the 13C NMR spectra were determined by DEPT
(distortionless enhancement by polarization transfer) measure-
ments. Mass spectra (EI or FAB) were obtained by using a Finnigan
MAT 90 spectrometer. MALDI-TOF mass spectra of the peptoids
were obtained by using a Bruker Biflex IV spectrometer with a
4.6. Chemical synthesis
4.6.1. tert-Butyl 4-(2-((2-ethoxy-2-oxoethyl)amino)ethyl)
piperazine-1-carboxylate (8)
pulsed ultraviolet nitrogen laser, 200
flight mass analyzer with a 125 cm linear flight path. Reversed
phase analytical HPLC was performed using Agilent Series 1100,
equipped with a C18 PerfectSil Target (MZ Analytik, 3e5
4.0 ꢁ 250 mm). Flow rate: 1 mL/min; solvent A: 0.1% TFA in water;
solvent B: 0.1% TFA in acetonitrile. Reversed phase preparative
HPLC was performed using Jasko LC-NetII/ADC Series, equipped
m
J at 337 nm and a time-of-
Ethyl bromoacetate (2.41 mL, 21.8 mmol) was dissolved in THF
(55 mL) and added dropwise over 3 h to a stirred solution of
compound 6 (5.00 g, 21.8 mmol) and NEt3 (9.07 mL, 65.4 mmol) in
THF (85 mL). After stirring overnight at RT, the solvent was
removed under reduced pressure. The residue was dissolved in
water (30 mL) and the product was extracted with CH2Cl2
(4 ꢁ 30 mL). The combined organic layers were washed with water
mm,

238
D.K. Kölmel et al. / European Journal of Medicinal Chemistry 79 (2014) 231e243
(2 ꢁ 30 mL) and brine (30 mL), dried over Na2SO4, and evaporated.
The product was obtained as colorless oil: yield 6.74 g (98%). IR
(KBr): ῦ (cmꢂ1) ¼ 3331 (w), 2977 (m), 2935 (m), 2814 (w), 1738 (m),
1698 (s), 1459 (m), 1421 (m), 1366 (m), 1290 (m), 1245 (m), 1173 (s),
1129 (m), 1027 (m), 1005 (w), 987 (w), 929 (w), 866 (m), 769 (w),
4H, 2 ꢁ CH2), 2.97e3.27 (m, 12H, 6 ꢁ CH2), 3.75 (s, 2H, CH2), 3.34e
3.41 (m,1H, NH), 4.18e4.32 (m, 2H, CH2), 4.44e4.58 (m,1H, CHCH2),
7.28e7.34 (m, 2H, 2 ꢁ CHar), 7.36e7.42 (m, 2H, 2 ꢁ CHar), 7.60e7.67
(m, 2H, 2 ꢁ CHar), 7.76e7.84 (m, 2H, 2 ꢁ CHar), mixture of two
rotamers; 13C NMR was not obtained due to poor signal-to-noise
ratio; HRMS m/z calc for C42H63N4O10: 783.4539; found: 783.4533
[MþH]þ.
574 (vw), 462 (vw); 1H NMR (500 MHz, CDCl3):
d
(ppm) ¼ 1.28 (t,
3J ¼ 7.2 Hz, 3H, CH2CH3), 1.45 (s, 9H, C(CH3)3), 2.42e2.48 (m, 4H,
2 ꢁ CH2), 2.55 (t, 3J ¼ 5.9 Hz, 2H, CH2), 2.76 (t, 3J ¼ 5.9 Hz, 2H, CH2),
3.43e3.49 (m, 6H, 3 ꢁ CH2), 4.19 (q, 3J ¼ 7.2 Hz, 2H, CH2CH3); 13C
4.6.4. 3-(1,4,7,10-Tetraoxa-13-azacyclopentadecan-13-yl)propan-
1-amine (12)
The preparation and properties of compound 12 have been re-
ported in Ref. [48].
NMR (125 MHz, CDCl3):
d
(ppm) ¼ 14.2 (CH3), 28.4 (CH3), 45.5
(CH2), 50.5 (CH2), 52.8 (CH2), 57.5 (CH2), 60.9 (CH2), 79.7 (C), 154.7
(CO), 172.1 (CO); EI MS: m/z (%) ¼ 315 (2) [M]þ, 242 (2) [Me
C3H5O2]þ, 199 (4) [MeC5H10NO2]þ, 143 (9) [MeC8H16N2O2]þ, 84
(99) [MeC11H21NO4]þ, 49 (100) [MeC14H24N3O2]þ; HRMS m/z calc
for C15H29N3O4: 315.2158; found: 315.2156 [M]þ.
4.6.5. General procedure for the preparation of the diazo transfer
reagent TfN3
A solution of triflic anhydride (1.85 equiv.) in CH2Cl2 (7.5 mL)
was added dropwise to a stirred solution of NaN3 (9.65 equiv.) in
water (4.5 mL) at 0 ꢀC. Afterwards, the reaction mixture was stirred
for 2 h at RT. Then, the organic phase was separated and the
aqueous phase was extracted with CH2Cl2 (2 ꢁ 3.75 mL). Finally, the
combined organic layers were washed with brine (4 mL).
4.6.2. Ethyl 9,14-bis(tert-butoxycarbonyl)-2,2-dimethyl-4-oxo-3-
oxa-5,9,14,18-tetraazaicosan-20-oate (9)
The preparation and properties of compound 9 have been re-
ported in Ref. [47].
4.6.3. General procedure for the synthesis of compounds 10 and 11
LiOH (1.5 equiv., 2 M in H2O) was dissolved in water and added
to a solution of secondary amine 8 or 9 (1 equiv., 0.5 M in dioxane)
in dioxane. The mixture was stirred for 5 h at 0 ꢀC. Afterwards,
NaHCO3 (1.5 equiv.) and FmocCl (1.5 equiv.) were added and the
solution was stirred overnight at RT. Next, citric acid (20 mL, 20% in
H2O) was added and the product was extracted with ethyl acetate
(3 ꢁ 40 mL). The combined organic layers were washed with brine
(20 mL) and the solvent was removed under reduced pressure. The
crude product was purified by using column chromatography.
4 . 6 . 5 .1. 13 - ( 3 - A z i d o p r o p y l ) - 1, 4 , 7 ,10 - t e t r a o x a - 13 -
azacyclopentadecane (13). The previously prepared TfN3 solution
was added to a solution of primary amine 12 (800 mg, 2.89 mmol),
K2CO3 (600 mg, 4.34 mmol) and CuSO4$5H2O (7.2 mg, 28.9 mmol) in
MeOH/H2O (2:1, 27 mL). The reaction mixture was stirred overnight
at RT. Afterwards, the organic phase was separated and the aqueous
phase was extracted with CH2Cl2 (3 ꢁ 50 mL). The combined
organic layers were dried over MgSO4 and the solvent was removed
under reduced pressure. The product was obtained as brownish
liquid: yield 615 mg (70%). IR (KBr): ῦ (cmꢂ1) ¼ 3441 (w), 2868 (m),
2096 (s), 1686 (w), 1620 (w), 1561 (vw), 1452 (m), 1356 (m), 1263
(m),1180 (m), 1125 (s), 1032 (m), 978 (w), 849 (w), 638 (w), 614 (w),
4.6.3.1. 2-((((9H-Fluoren-9-yl)methoxy)carbonyl)(2-(4-(tert-butox-
ycarbonyl)piperazin-1-yl)ethyl)amino)acetic acid (10). After purifi-
cation (chromatography with eluent EtOAc / MeOH) the title
compound was obtained as an off-white solid: yield 6.13 g (59%).
Mp 73 ꢀC; IR (ATR): ῦ (cmꢂ1) ¼ 3434 (w), 2974 (vw), 1687 (m), 1595
(w),1477 (w),1449 (w),1420 (w),1364 (w),1243 (w),1149 (w),1047
(w), 1001 (w), 977 (w), 863 (vw), 759 (w), 739 (w), 620 (vw), 540
571 (vw), 517 (vw); 1H NMR (400 MHz, CDCl3):
d
(ppm) ¼ 1.71
(quin, 3J ¼ 6.9 Hz, 2H, CH2), 2.57 (t, 3J ¼ 6.9 Hz, 2H, CH2), 2.72 (t,
3J ¼ 5.9 Hz, 4H, 2 ꢁ CH2), 3.34 (quin, 3J ¼ 6.7 Hz, 2H, CH2), 3.59e3.66
(m,16H, 8 ꢁ CH2); 13C NMR (100 MHz, CDCl3):
(ppm) ¼ 26.8 (CH2),
d
(vw), 426 (vw); 1H NMR (300 MHz, CD3OD):
d
(ppm) ¼ 1.49 (s, 9H,
49.4 (CH2), 53.4 (CH2), 54.7 (CH2), 69.9 (CH2), 70.1 (CH2), 70.4 (CH2),
70.9 (CH2); FAB MS: m/z (%) ¼ 341 (23) [MþK]þ, 325 (8) [MþNa]þ,
303 (100) [MþH]þ, 232 (60) [MeC2H4N3]þ; HRMS m/z calc for
C(CH3)3), 2.19e2.32 (m, 4H, 2 ꢁ CH2), 2.79e2.92 (m, 2H, CH2), 3.06
(t, 3J ¼ 6.4 Hz, 2H, CH2), 3.34e3.68 (m, 4H, 2 ꢁ CH2), 3.73 (s, 2H,
CH2), 4.20e4.24 (m, 1H, CHCH2), 4.28e4.34 (m, 1H, CHCH2), 4.66e
4.74 (m, 1H, CHCH2), 7.26e7.42 (m, 4H, 4 ꢁ CHar), 7.61e7.73 (m, 2H,
C
13H27N4O4: 303.2032; found: 303.2036 [MþH]þ.
2 ꢁ CHar), 7.77e7.82 (m, 2H, 2 ꢁ CHar), mixture of two rotamers; 13
C
4.6.5.2. 1-(3-(Azidomethyl)phenyl)-N,N,N-trimethylmethanaminium
iodide (17). The previously prepared TfN3 solution was added to a
solution of primary amine 16 (1.00 g, 2.30 mmol), K2CO3 (954 mg,
NMR (75 MHz, CD3OD):
d
(ppm) ¼ 28.7 (CH3), 45.6 (CH2), 48.6 (CH),
52.5 (CH2), 53.4 (CH2), 53.7 (CH2), 56.5 (CH2), 69.5 (CH2), 81.7 (C),
121.1 (CHar), 125.7 (CHar), 128.4 (CHar), 128.8 (CHar), 142.8 (Car),
145.5 (Car), 156.0 (CO), 158.2 (CO), 177.6 (CO), mixture of two
rotamers; FAB MS: m/z (%) ¼ 532 (12) [MþNa]þ, 510 (8) [MþH]þ,
408 (9) [MeC5H9O2]þ, 199 (14) [MeC18H16NO4]þ, 179 (100) [Me
6.90 mmol) and CuSO4$5H2O (5.7 mg, 23.0 mmol) in MeOH/H2O
(2:1, 27 mL). The reaction mixture was stirred overnight at RT. Af-
terwards, the organic phase was separated and the aqueous phase
was extracted with CH2Cl2 (2 ꢁ 50 mL). The combined organic
layers were washed with a saturated solution of NaI in H2O (20 mL)
and the solvent was removed under reduced pressure. The crude
product was taken up in a small amount of EtOH and precipitated
by the addition of Et2O. The precipitate was thoroughly washed
with Et2O and dried in vacuum. The product was obtained as an off-
C
C
14H24N3O6]þ, 143 (55) [MeC21H22N2O4]þ, 99 (26) [Me
23H24NO6]þ; HRMS m/z calc for C28H36N3O6: 510.2604; found:
510.2600 [MþH]þ.
4.6.3.2. 18-(((9H-Fluoren-9-yl)methoxy)carbonyl)-9,14-bis(tert-
butoxycarbonyl)-2,2-dimethyl-4-oxo-3-oxa-5,9,14,18-tetraazaicosan-
20-oic acid (11). After purification (chromatography with eluent
cyclohexane/EtOAc 3:1 / MeOH) the title compound was obtained
as a white solid: yield 3.73 g (64%). Mp 70 ꢀC; IR (ATR): ῦ
(cmꢂ1) ¼ 3367 (vw), 2973 (w), 2930 (w), 1676 (m), 1604 (w), 1478
(w), 1449 (w), 1417 (w), 1364 (w), 1245 (w), 1158 (m), 1072 (w), 999
(w), 866 (w), 759 (w), 740 (w), 670 (vw), 621 (vw), 543 (vw), 493
(vw), 459 (vw), 425 (vw); 1H NMR (500 MHz, CD3OD):
white solid: yield 309 mg (40%). Mp 127 ꢀC; IR (ATR):
ῦ
(cmꢂ1) ¼ 2999 (w), 2080 (m), 1477 (m), 1445 (w), 1368 (w), 1230
(m),1161 (w), 1005 (w), 973 (w), 915 (w), 884 (w), 865 (m), 806 (m),
777 (w), 746 (m), 714 (m), 684 (w), 569 (w), 555 (w), 443 (w), 411
(vw); 1H NMR (300 MHz, CDCl3):
d
(ppm) ¼ 3.43 (s, 9H, 3 ꢁ CH3),
4.41 (s, 2H, CH2), 5.11 (s, 2H, CH2), 7.45e7.51 (m, 2H, 2 ꢁ CHar), 7.66
(s, 1H, CHar), 7.71 (d, 3J ¼ 6.5 Hz, 1H, CHar); 13C NMR (75 MHz,
CDCl3):
d
(ppm) ¼ 53.0 (CH3), 53.9 (CH2), 68.2 (CH2), 127.8 (Car),
d
(ppm) ¼ 1.39e1.55 (m, 31H, 2 ꢁ CH2, 3 ꢁ C(CH3)3), 1.61e1.85 (m,
129.9 (CHar), 130.7 (CHar), 132.7 (CHar), 133.0 (CHar), 136.8 (Car); FAB

D.K. Kölmel et al. / European Journal of Medicinal Chemistry 79 (2014) 231e243
239
MS: m/z (%) ¼ 205 (100) [M]þ; HRMS m/z calc for C11H17N4:
bis(dimethylamino)benzophenone (2.74 g, 10.2 mmol) in toluene
(3.8 mL). The reaction mixture was heated overnight at 100 ꢀC.
After cooling to RT, CH2Cl2 (400 mL) was added and the organic
phase was washed with water (20 mL). The solvent was removed
under reduced pressure. After purification (chromatography with
eluent CH2Cl2 / MeOH) the title compound was obtained as a
205.1448; found: 205.1450 [M]þ.
4.6.6. tert-Butyl 3-(aminomethyl)benzylcarbamate (14)
The preparation and properties of compound 9 have been re-
ported in Ref. [66].
bronze solid: yield 4.70
g ῦ
(>99%). Mp 75 ꢀC; IR (KBr):
4.6.7. 1-(3-(((tert-Butoxycarbonyl)amino)methyl)phenyl)-N,N,N-
trimethylmethanaminium iodide (15)
(cmꢂ1) ¼ 2853 (w), 2090 (m), 1573 (m), 1476 (w), 1331 (m), 1291
(m), 1160 (m), 938 (m), 909 (m), 822 (w), 757 (w), 717 (w), 663 (w),
619 (w), 557 (w), 506 (w), 418 (w); 1H NMR (400 MHz, CD3OD):
To a solution of primary amine 14 (14.4 g, 61.0 mmol) and
NaHCO3 (51.2 g, 610 mmol) in MeOH (875 mL) was added MeI
(38.1 mL, 610 mmol). The resulting mixture was stirred for 2 d at RT.
Afterwards, the solution was filtrated and the solvent was removed
under reduced pressure. The product was obtained as white solid
and was used without further purification. Mp 126 ꢀC; IR (ATR): ῦ
(cmꢂ1) ¼ 3350 (vw), 3006 (w), 2997 (w), 1689 (m), 1517 (w), 1475
(w),1446 (w),1393 (w),1365 (w),1339 (w),1248 (w),1164 (m),1121
(w), 1045 (w), 1021 (w), 998 (w), 971 (w), 955 (w), 915 (w), 879 (w),
803 (w), 745 (w), 704 (w), 639 (vw), 571 (vw), 442 (w); 1H NMR
d
(ppm) ¼ 3.24e3.27 (m, 15H, 5 ꢁ CH3), 3.64 (t, 3J ¼ 5.4 Hz, 2H, CH2),
3.82 (t, 3J ¼ 5.4 Hz, 2H, CH2), 6.93e7.04 (m, 6H, 6 ꢁ CHar), 7.26e7.38
(m, 6H, 6 ꢁ CHar); 13C NMR (100 MHz, CD3OD):
d
(ppm) ¼ 39.5
(CH3), 40.6 (CH3), 50.3 (CH2), 52.4 (CH2), 113.6 (CHar), 113.7 (CHar),
127.9 (Car), 128.4 (Car), 140.6 (CHar), 141.0 (CHar), 156.4 (Car), 157.5
(Car), 179.8 (Car); FAB MS: m/z (%) ¼ 427 (100) [M]þ, 413 (58)
[MꢂN]þ; HRMS m/z calc for C26H31N6: 427.2610; found: 427.2608
[M]þ.
(500 MHz, CD3OD):
3 ꢁ CH3), 4.29 (s, 2H, CH2), 4.57 (s, 2H, CH2), 7.49 (mc, 4H, 4 ꢁ CHar);
13C NMR (125 MHz, CD3OD):
(CH3), 53.2 (CH3), 53.3 (CH3), 70.4 (CH2), 80.4 (C), 129.2 (Car), 130.5
(CHar), 130.7 (CHar), 132.7 (CHar), 142.5 (Car), 158.6 (C); FAB MS: m/z
(%) ¼ 279 (100) [M]þ, 223 (17) [MꢂC4H8]þ, 178 (2) [MeC5H9O2]þ,
164 (19) [MeC7H17N]þ; HRMS m/z calc for C16H27N2O2: 279.2073;
found: 279.2069 [M]þ.
d
(ppm) ¼ 1.45 (s, 9H, 3 ꢁ CH3), 3.13 (s, 9H,
4.6.12. General procedure for the synthesis of the peptoids 1 and 2
Fmoc-protected Rink amide resin (0.64 mmol/g, 50 mg) was
swollen in DMF (1 mL) for 1 h. Multiple washing steps using DMF
were performed between each step as described below. Fmoc
deprotection was completed by adding piperidine (20% in DMF,
1 mL, 3 ꢁ 5 min). Following the respective peptoid monomer was
coupled to the resin. To achieve this, the monomer 10 or 11
d
(ppm) ¼ 28.8 (CH3), 44.7 (CH2), 53.2
(96.0
mmol, 3 equiv.), diisopropylcarbodiimide (15.0
mL, 96.0
mmol)
and 1-hydroxybenzotriazole hydrate (14.7 mg, 96.0
mmol) were
4.6.8. 1-(3-(Ammoniomethyl)phenyl)-N,N,N-
trimethylmethanaminium diiodide (16)
dissolved in DMF biograde (1 mL) and added to the resin. The re-
action vessel was subjected to microwave irradiation to keep the
constant temperature at 60 ꢀC (max. 20 W) for 30 min while being
stirred. The reaction solution was filtered and the resin was treated
a second time with freshly prepared reaction solution under the
same conditions as described above (double coupling). Afterwards,
the resin was thoroughly washed with DMF (5 ꢁ 3 mL). This pro-
cedure of Fmoc deprotection and monomer coupling was repeated
six times in total, so that a resin bound hexamer was obtained. Then
another Fmoc deprotection step was carried out under the previ-
ously described conditions. Subsequently, a solution of rhodamine
To a solution of Boc-protected amine 15 (24.8 g, 61.0 mmol) in
H2O (525 mL) was added hydriodic acid (57%, 40.3 mL, 305 mmol).
Afterwards, the mixture was stirred overnight at RT. The solution
was filtrated and the aqueous phase was washed with CH2Cl2
(4 ꢁ 200 mL). The solvent was removed under reduced pressure
and the residue was taken up in MeOH (450 mL). Following, the
product was precipitated by the addition of EtOAc (900 mL) and
washed with EtOAc (300 mL) and MeOH (30 mL). After drying in
vacuum, the product was obtained as slightly yellow crystals: yield
18.5 g (70% over two steps). Mp 236 ꢀC; IR (ATR): ῦ (cmꢂ1) ¼ 3000
(w), 1736 (vw), 1566 (w), 1475 (w), 1441 (w), 1397 (w), 1377 (w),
1365 (w), 1282 (vw), 1234 (vw), 1173 (w), 1124 (vw), 1100 (w), 1070
(w), 1000 (w), 969 (w), 925 (w), 917 (w), 876 (w), 860 (w), 808 (w),
744 (w), 706 (w), 635 (vw), 574 (vw), 483 (vw), 445 (w), 418 (vw);
B
96.0
96.0
(46.0 mg, 96.0
m
mol), diisopropylcarbodiimide (15.0
mol) and 1-hydroxybenzotriazole hydrate (14.7 mg,
mol) in DMF biograde (1 mL) was added to the washed resin.
mL,
m
m
A double coupling was performed under the same conditions
(microwave irradiation: 60 ꢀC for 30 min) as described for the
coupling of the monomer. Afterwards, the resin was thoroughly
washed with DMF until the washing solution remained colorless.
For the final cleavage the resin was incubated overnight with TFA
(95% in CH2Cl2, 2 mL) at RT. Following, the resin was washed with
MeOH until the washing solution remained colorless. The crude
product was lyophilized and purified by HPLC.
1H NMR (500 MHz, CD3OD):
d
(ppm) ¼ 3.19 (s, 9H, 3 ꢁ CH3), 4.26 (s,
2H, CH2), 4.68 (s, 2H, CH2), 7.62e7.65 (m, 1H, CHar), 7.68e7.70 (m,
2H, 2 ꢁ CHar), 7.78 (s, 1H, CHar); 13C NMR (125 MHz, CD3OD):
d
(ppm) ¼ 43.9 (CH2), 55.5 (CH3), 55.5 (CH3), 55.6 (CH3), 69.9 (CH2),
130.1 (Car), 131.3 (CHar), 132.6 (CHar), 134.9 (CHar), 134.9 (CHar),
135.7 (Car); FAB MS: m/z (%) ¼ 179 (100) [M]þ, 120 (18) [Me
C3H9N]þ; HRMS m/z calc for C11H19N2: 179.1548; found: 179.1550
[M]þ.
4.6.12.1. Peptoid 1. After purification the title compound was ob-
tained as red solid: yield 15.9 mg (17% over 15 steps). Analytical
HPLC (5e95% acetonitrile þ 0.1% TFA in 30 min, tret ¼ 9.9 min)
purity: 98%; MALDI-TOF MS: m/z ¼ 1458 [MþH]þ.
4.6.9. (4-Azidobutyl)triphenylphosphonium bromide (19)
The preparation and properties of compound 19 have been re-
ported in Ref. [54].
4.6.12.2. Peptoid 2. After purification the title compound was ob-
tained as red solid: yield 1.77 mg (1.4% over 15 steps). Analytical
HPLC (5e95% acetonitrile þ 0.1% TFA in 30 min, tret ¼ 8.9 min)
purity: 98%; MALDI-TOF MS: m/z ¼ 1896 [MþH]þ.
4.6.10. N-(2-Azidoethyl)-N-methylaniline (21)
The preparation and properties of compound 21 have been re-
ported in Ref. [67].
4.6.11. (4-((2-Azidoethyl)methyl)amino)phenyl)bis(4-
4.6.13. Peptoid 3
(dimethylamino)phenyl)methylium chloride (22)
Fmoc-protected Rink amide resin (0.64 mmol/g, 100 mg) was
swollen in DMF (2 mL) for 1 h. Multiple washing steps using DMF
(5 ꢁ 3 mL) were performed between each step as described below.
Phosphoryl chloride (1.90 mL, 20.8 mmol) was added to a stirred
solution of aniline 21 (2.51 g, 14.2 mmol) and 4,40-

240
D.K. Kölmel et al. / European Journal of Medicinal Chemistry 79 (2014) 231e243
Fmoc deprotection was completed by adding piperidine (20% in
methoxy)carbonyl)(6-((tert-butoxycarbonyl)amino)hexyl)amino)
acetic acid was repeated once more to obtain a resin bound hex-
amer. Then another Fmoc deprotection step was carried out under
the previously described conditions. Subsequently, a solution of
DMF, 2 mL, 3 ꢁ 5 min). Afterwards a solution of bromoacetic acid
(89.0 mg, 640
mmol) and diisopropylcarbodiimide (99.7 mL,
640
reaction mixture was shaken for 30 min at RT. Next, a solution of
propargylamine (34.0 L, 531 mol) in 530 L DMF biograde was
mmol) in 640 mL DMF biograde was added to the resin. The
rhodamine
(29.9 L, 192
192 mol) in DMF biograde (2 mL) was added to the washed resin. A
B
(92.0 mg, 192
mmol), diisopropylcarbodiimide
m
m
m
m
mmol) and 1-hydroxybenzotriazole hydrate (29.4 mg,
added and the mixture was shaken for 1 h at RT. This procedure of
acylation with bromoacetic acid and amination with propargyl-
amine was repeated six times in total, so that a resin bound hex-
amer was obtained. The side chains were functionalized by adding
m
double coupling was performed under the same conditions (mi-
crowave irradiation: 60 ꢀC for 30 min) as described for the coupling
of the monomer. Afterwards, the resin was thoroughly washed with
DMF until the washing solution remained colorless. Finally, the
respective azide was introduced. A solution of azide 19 or 22
a solution of azide 13 (406 mg, 1.36 mmol), 2,6-lutidine (358
3.07 mmol), and tetrakis(acetonitrile)copper(I) hexa-
fluorophosphate (143 mg, 384 mol) in THF (1.9 mL) to the washed
mL,
m
(384
m
mol, 6 equiv.), 2,6-lutidine (59.7
mL, 512
mmol), and tetraki-
resin. After shaking for 2.5 d at RT, the resin was thoroughly washed
with DMF. Subsequently, a solution of rhodamine B (92.0 mg,
s(acetonitrile)copper(I) hexafluorophosphate (47.7 mg, 128
m
mol)
in THF was added to the resin and shaken at RT. Afterwards, the
resin was thoroughly washed with DMF. For the final cleavage the
resin was incubated with TFA (95% in CH2Cl2) at RT. Following, the
resin was washed with MeOH until the washing solution remained
colorless. The crude product was lyophilized and purified by HPLC.
192
m
mol), diisopropylcarbodiimide (29.9
mL, 192
mmol) and 1-
hydroxybenzotriazole hydrate (29.4 mg, 192
mmol) in DMF bio-
grade (2 mL) was added to the washed resin. The reaction vessel
was subjected to microwave irradiation to keep the constant tem-
perature at 60 ꢀC (max. 20 W) for 30 min while being stirred. The
reaction solution was filtered and the resin was treated a second
time with freshly prepared reaction solution under the same con-
ditions as described above (double coupling). Afterwards, the resin
was thoroughly washed with DMF until the washing solution
remained colorless. For the final cleavage the resin was incubated
for 3 ꢁ 30 min with TFA (95% in CH2Cl2, 3 mL) at RT. Following, the
resin was washed with MeOH until the washing solution remained
colorless. The crude product was lyophilized and purified by HPLC.
After purification the title compound was obtained as red solid:
yield 3.49 mg (1.5% over 16 steps). Analytical HPLC (5e95%
acetonitrile þ 0.1% TFA in 30 min, tret ¼ 14.2 min) purity: 89%;
MALDI-TOF MS: m/z ¼ 2826 [M]þ.
4.6.15.1. Peptoid 4. Azide 19 was dissolved in 1.9 mL THF and
shaken for 1.5 d at RT. The cleavage was performed with 3 mL TFA
(95% in CH2Cl2, 3 ꢁ 30 min). After purification the title compound
was obtained as red solid: yield 2.36 mg (1.3% over 16 steps).
Analytical HPLC (5e95% acetonitrile þ 0.1% TFA in 30 min,
tret ¼ 12.3 min) purity: 85%; MALDI-TOF MS: m/z ¼ 1976 [MþH]þ.
4.6.15.2. Peptoid 5. Azide 22 was dissolved in 3.8 mL THF and
shaken for 1.5 d at RT. Afterwards, the reaction solution was filtered
and the resin was treated a second time with freshly prepared re-
action solution. The mixture was shaken again for 2 d at RT. The
cleavage was performed with 3 mL TFA (95% in CH2Cl2, 30 min).
After purification the title compound was obtained as violet solid:
yield 0.69 mg (0.4% over 16 steps). Analytical HPLC (5e95%
acetonitrile þ 0.1% TFA in 30 min, tret ¼ 13.6 min) purity: 98%;
MALDI-TOF MS: m/z ¼ 2111 [MþH]þ.
4.6.14. 2-((((9H-Fluoren-9-yl)methoxy)carbonyl)(6-((tert-
butoxycarbonyl)amino)hexyl)amino)acetic acid
The preparation and properties of the title compound have been
reported in Ref. [46].
4.6.15. General procedure for the synthesis of the peptoids 4 and 5
Fmoc-protected Rink amide resin (0.64 mmol/g, 100 mg) was
swollen in DMF (2 mL) for 1 h. Multiple washing steps using DMF
(5 ꢁ 3 mL) were performed between each step as described below.
Fmoc deprotection was completed by adding piperidine (20% in
DMF, 2 mL, 3 ꢁ 5 min). The first monomer was introduced by the
submonomer method. A solution of bromoacetic acid (89.0 mg,
4.7. Crystal structure determination of 16
The single-crystal X-ray diffraction study was carried out on a
Bruker-Nonius Kappa-CCD diffractometer at 123(2) K using MoK
a
ꢀ
radiation (
l
¼ 0.71073 A). Direct Methods (SHELXS-97) [68] were
used for structure solution and refinement was carried out using
SHELXKL-97 [68] (full-matrix least-squares on F2). Non hydrogen
atoms were refined anisotropically, hydrogen atoms were localized
by difference electron density determination and refined using a
riding model (H(N) free). A semi-empirical absorption correction
was applied.
640
640
m
mol) and diisopropylcarbodiimide (99.5
mL, 640 mmol) in
mL DMF biograde was added to the resin. The reaction mixture
was shaken for 30 min at RT. Next, a solution of propargylamine
(34.0 L, 531 mol) in 530 L DMF biograde was added and the
m
m
m
mixture was shaken for 1 h at RT. Following, the peptoid monomer
2-((((9H-fluoren-9-yl)methoxy)carbonyl)(6-((tert-butox-
ycarbonyl)-amino)hexyl)amino)acetic acid was introduced. To
16: colorless crystals, C11H20N22þ e 2 Iꢂ, M ¼ 434.09, crystal size
0.30 ꢁ 0.12 ꢁ 0.06 mm, monoclinic, space group P21/n (No. 14),
ꢀ
ꢀ
ꢀ
achieve this, the monomer (95.5 mg, 192
m
mol), diisopropylcarbo-
mol) and 1-hydroxybenzotriazole hydrate
mol) were dissolved in DMF biograde (2 mL) and
a
b
¼
5.3325(ꢀ4) A,
b
¼
17.0104(17) A,
c
¼
16.4987(11) A,
3
ꢀ
diimide (29.9
(29.4 mg, 192
m
m
L, 192
m
¼ 96.915(7) , V ¼ 1485.7(2) A , Z ¼ 4,
r
(calc) ¼ 1.941 Mg mꢂ3
,
F(000) ¼ 824,
m
¼ 4.210 mmꢂ1, 11882 reflections (2qmax ¼ 55ꢀ),
added to the resin. The reaction vessel was subjected to microwave
irradiation to keep the constant temperature at 60 ꢀC (max. 20 W)
for 30 min while being stirred. The reaction solution was filtered
and the resin was treated a second time with freshly prepared re-
action solution under the same conditions as described above
(double coupling). Next, the Fmoc protecting group was removed as
described above. The same monomer was introduced once more
under equal conditions (double coupling with microwave irradia-
tion). The sequence of submonomer coupling with proparglyamine
and two monomer coupling steps with 2-((((9H-fluoren-9-yl)
3338 unique [Rint ¼ 0.038], 145 parameters, 9 restraints, R1 (for
2957 I > 2
s
(I)) ¼ 0.024, wR2 (all data) ¼ 0.055, S ¼ 1.04, largest diff.
ꢀꢂ3
peak and hole 0.822 and ꢂ0.745 e A
.
Crystallographic data (excluding structure factors) for the
structure reported in this work have been deposited with the
Cambridge Crystallographic Data Centre as supplementary publi-
cation no. CCDC 969830 (16). Copies of the data can be obtained
free of charge on application to The Director, CCDC, 12 Union Road,
Cambridge CB2 1EZ, UK (Fax: int.codeþ(1223)336-033; e-mail:
deposit@ccdc.cam.ac.uk).

D.K. Kölmel et al. / European Journal of Medicinal Chemistry 79 (2014) 231e243
a TAT-derived peptide and a
241
b
-peptide after endocytic uptake into HeLa cells,
Acknowledgments
Journal of Biological Chemistry 278 (2003) 50188e50194.
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This work was supported by a Feasibility Studies of Young Sci-
entists (FYS) of the KIT (fellowship to D.K.K.), the Fonds der
Chemischen Industrie (fellowship to D.K.K.), the Karlsruhe School
of Optics & Photonics (fellowship to D.K.K. and A.H.) the Carl-Zeiss
foundation (fellowship to A.H.) and the Helmholtz program Bio-
interface (F.R., U.S., and S.B.) The authors gratefully acknowledge
the help of Karolin Niessner for her assistance on preparative HPLC
purification of the compounds.
D. Seebach, On the biodegradation of
b-peptides, ChemBioChem 3 (2002)
424e432;
c) J. Farrera-Sinfreu, E. Giralt, M. Royo, F. Albericio, Cell-penetrating proline-
rich peptidomimetics, Methods in Molecular Biology 386 (2007) 241e267;
d) J.-K. Bang, Y.H. Nan, E.K. Lee, S.Y. Shin, A novel Trp-rich model antimicrobial
peptoid with increased protease stability, Bulletin of the Korean Chemical
Society 31 (2010) 2509e2513.
Appendix A. Supplementary data
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