Amphiphilic peptoid transporters-synthesis and evaluation

Article abstract of DOI:10.1039/c3ob41139g

Cell-penetrating peptoids are an important class of peptidomimetics, which can replace highly biodegradable cell penetrating peptides for enhanced drug delivery. Typically, they contain positively charged amino side chains which are synthesized via their protected analogues. To avoid the use of amine protecting groups a Click-chemistry based modular synthesis of novel hydrophilic as well as amphiphilic cell penetrating peptoids was developed to generate novel structures for drug delivery in cells.

Full text of DOI:10.1039/c3ob41139g

Organic &
Biomolecular Chemistry
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Amphiphilic peptoid transporters – synthesis and
evaluation†
Sidonie B. L. Vollrath,a Daniel Fürniss,a Ute Schepersb and Stefan Bräse*a,b
Cite this: Org. Biomol. Chem., 2013, 11,
8197
Cell-penetrating peptoids are an important class of peptidomimetics, which can replace highly bio-
degradable cell penetrating peptides for enhanced drug delivery. Typically, they contain positively
charged amino side chains which are synthesized via their protected analogues. To avoid the use
of amine protecting groups a Click-chemistry based modular synthesis of novel hydrophilic as well as
amphiphilic cell penetrating peptoids was developed to generate novel structures for drug delivery
in cells.
Received 1st June 2013,
Accepted 3rd October 2013
DOI: 10.1039/c3ob41139g
www.rsc.org/obc
For more than a decade, cell penetrating peptides (CPPs) have circumvent enzymatic degradation, N-substituted glycine oligo-
been well known for enhancing cellular uptake of a variety of mers (peptoids) are also widely investigated, not only for their
molecules1 ranging from small molecule drugs to larger moi- three-dimensional folding properties but also for biological
eties such as proteins,2 DNA3 or nanoparticles.4 The most pro- applications such as antimicrobials,21 antitumor agents22 and
minent members of this family derive from HIV Tat protein5 molecular transporters.23 Recently, Barron et al. and our group
and penetratin.6 Most of the CPPs are amphiphilic and are showed that amphiphilic peptoids are active cell penetrating
rich in basic amino acids such as lysine and arginine.
agents.24
The synthesis of cell penetrating peptoids (CPPos) mostly
In addition to positively charged side chains, further
potency can be achieved through the introduction of hydro- relies on the submonomer method, which uses repetitive acy-
phobic amino acids. After many years of efforts, the structures lation and substitution steps directly on solid supports.25 The
of polycationic cell-penetrating peptides at atomic level resolu- introduction of functional side chains is usually achieved
tion became most recently available.7–9 These studies have pro- using the respective protected primary amines.
vided valuable structural information to rationalize the CPP
Nowadays, the introduction of functional moieties in a
translocation and may also provide structural insight into the variety of biofunctional molecules is achieved by copper-cata-
design of more efficient alternatives. However, for the more lyzed alkyne azide cycloaddition (CuAAC).26 It has been
hydrophobic peptides, it is not clear which structural para- recently used in the effective design of peptidomimetics, as
meters are important for internalization.10 For example, cat- the resulting triazole is not only an effective mimic of the
ionic amphiphilic peptides have been shown to efficiently amide bond itself but also allows for the design of distinct
target mitochondria.11 Likewise, other side chains and chemi- structural properties.27
cal structures were identified that are specific for the transport
In peptoids, such a functionalization can also be achieved
into a variety of cellular compartments.12 Although well by introducing complex side chains through CuAAC directly
studied, only a few uptake mechanisms have been proposed during solid phase synthesis.28 Inter- and intramolecular coup-
for single CPPs.13
ling of azide and alkyne side chains leads to stable secondary
One major drawback of peptides is their rapid proteolytic structures29 as well as to constrained peptoids.30 This methodo-
degradation in vivo. Therefore, molecules which mimic the cell logy allows for the rapid functionalization with complex
penetrating properties of the CPPs, while showing an building blocks such as nucleobases,31 glycostructures,28b or
enhanced bioavailability, are of broad interest. Besides D-pep- receptor ligands.32
tides or retro-inverso peptides14–18 and β-peptides,19,20 which
Often in the submonomer based peptoid synthesis on solid
supports the incorporation of functionalized side chains is
complicated and results in incomplete conversions and lower
yields. Therefore, effective and easily applicable synthesis strat-
egies for the introduction of functional side chains are
needed.
aKarlsruhe Institute of Technology, Institute of Organic Chemistry, Fritz-Haber-Weg
6, 76131 Karlsruhe, Germany. E-mail: braese@kit.edu; Tel: +49 721 608 42902
bKarlsruhe Institute of Technology, Institute of Toxicology and Genetics, Hermann-
von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany
†Electronic supplementary information (ESI) available: Synthetic procedures,
HPLC. See DOI: 10.1039/c3ob41139g
Here, we report a facile synthesis of amphiphilic cell pene-
trating peptoids (CPPos) by a modular approach utilizing
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Fig. 1 Chemical structure of peptoids synthesized for this study via solid phase
peptoid synthesis and CuAAC. Rhod–OH = rhodamine B.
CuAAC conditions to introduce functional side chains without
the use of amine protecting groups. To elucidate the suitability
of this CuAAC reaction for the synthesis of CPPos and especially
amphiphilic peptoids, four different peptoids have been syn-
thesized on Rink amide and 2-chlorotrityl chloride resin
(Fig. 1).
To see whether the CuAAC reaction conditions and the intro-
duction of the triazole ring are interfering with cellular uptake
of the peptoids, cationic peptoids 1 and 2 were synthesized.
In addition hydrophobic side chains were introduced. Peptoids
3 and 4 are two representative examples of amphiphilic pep-
toids consisting of hexamer backbones with cationic hydro-
philic and hydrophobic side chains either separated into
hydrophilic and hydrophobic regions (3) or ordered in an alter-
nating fashion (4). For investigating the cellular uptake by
fluorescence microscopy techniques, all peptoids were coupled
to rhodamine B (Rhod) (Fig. 1).
Scheme 1 Synthesis of linear hydrophilic peptoids 1 and 2.
The hydrophilic peptoids 1 and 2 were synthesized on Rink
amide or 2-chlorotrityl chloride resin as hexamer backbones
with propargylamine as the primary amine (Scheme 1).
Synthesis of the peptoids on both resins differed only in the
coupling of the first N-substituted glycine unit (see ESI† for
experimental details). Afterwards the peptoid was labelled
with rhodamine B in a HOBt and DIC mediated reaction
(Scheme 1) and the completion of the reaction was analysed by
MALDI after cleavage from the resin. Eventually, hexa-
functionalization of the peptoid was achieved by the CuAAC
reaction directly on solid-supports using 3-azidopropylamine,
CuI and DIPEA in THF. Reaction control was achieved by
MALDI after cleavage of a small aliquot. Full conversion to the
desired product was observed after 16 h (Scheme 1). Both pep-
toids were cleaved from the resin by using trifluoroacetic acid
(TFA, 95% in dichloromethane) for Rink amide resin and hexa-
fluoroisopropanol (HFIP, 30% in dichloromethane) for 2-chloro-
trityl chloride resin. The hydrophilic peptoids 1 and 2 were
purified by HPLC and added to HeLa cells to investigate their
cellular uptake.
Scheme 2 Synthesis of amphiphilic peptoid 3.
amphiphilic structure: first, a peptoid trimer was synthesized
with propargylamine as the primary amine building block.
Eventually, functionalization of the newly generated alkynes was
achieved in a Click reaction with 1-azidohexadecane to obtain
The amphiphilic peptoid 3 was synthesized according
to the same but slightly modified procedure to obtain its
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the hydrophobic moiety (Scheme 2). Likewise, full conversion
was observed after 16 h. The synthesis of the hexameric
peptoid was completed using the standard protocol with pro-
pargylamine as a submonomer which again resulted in full
conversions even in the presence of the large hydrophilic side
chains. The peptoid was labelled with rhodamine B and was
then functionalized with 3-azidopropylamine under CuAAC
conditions to finally afford the desired amphiphilic peptoid 3
after cleavage with TFA.
The synthesis of amphiphilic peptoid 4 proved to be rather
challenging. First attempts were made using 1-aminohexa-
decane directly as a submonomer for the peptoid synthesis.
While the synthesis of the peptoid core and also coupling
to rhodamine B could be achieved, the final CuAAC reaction
with 3-azidopropylamine afforded only traces of the desired
product. Probably due to aggregation of the long side chains,
the desired product could not be isolated. Therefore, it was
necessary to use a different approach. It has already been
shown that alkynes can be differentiated by triisopropylsilyl
(TIPS) protecting groups.33 By using this strategy, the peptoid
scaffold was synthesized with propargylamine and TIPS
protected propargylamine respectively and was coupled to
rhodamine B. Functionalization with 1-azidohexadecane was
again performed under the CuAAC conditions. Afterwards,
the TIPS protecting groups were removed with tetrabutyl-
Fig. 2 Cellular uptake of peptoids 1–4 in HeLa cells. 1 × 104 HeLa cells were
treated with 1 µM of hydrophilic peptoids 2 (A) and 1 (B) and amphiphilic pep-
toids 3 (C) and 4 (D) for 24 h at 37 °C. For co-staining of the nuclei and the mito-
chondria the cells were treated with Hoechst 33342 (blue) and 100 nm
Mitotracker Green™ (green) and excited at 364 nm with a UV laser, at 488 nm
ammonium fluoride (TBAF) in THF, which proceeded under with an argon laser and at 561 nm using a DPSS laser. Eventually, the cells were
analysed by fluorescence confocal imaging. The images show the merges of the
full conversion within 16 h. Following CuAAC with 3-azido-
propylamine resulted in the desired peptoid 4. The overall
yields of the peptoids were 0.7 to 3.2% after HPLC purification
respective emission channels of each line by using the PMTs for the emission:
401 nm–464 nm for the detection of the nuclei (blue), 516 nm–552 nm for the
detection of the mitochondria (green), and 600 nm–700 nm for the detection
(purity >90%) (Schemes 1–3). The yields of the crude material
are much higher, but it contains salts and unreacted reagents.
All synthesized peptoids 1–4 were subjected to HPLC purifi-
cation (purity ≥90%) and then tested for their internalization
properties in human cervix carcinoma (HeLa) cells. 104 Cells
were incubated with a 1 µM solution of the respective peptoids
for 24 h at 37 °C. For visualization of the organelle preference,
the cells were co-stained with Mitotracker Green™ for labelling
of the rhodamine B labelled peptoids (red). Scale bar = 10 µm.
mitochondria and with Hoechst 33342 for labelling the
nucleus.
As expected, both hydrophilic peptoids 1 and 2 showed a
fast cellular uptake with an accumulation in the endosomal/
lysosomal compartment and a release to the cytosol after 24 h
(Fig. 2). This result also shows that the triazole rings do not
have a major impact in terms of cellular uptake and intracellular
distribution compared to a peptoid hexamer containing lysine
side chains (Fig. S2†).34 Additionally, the alteration of the C-term-
inal functional group (either carboxylic acid or amide) does
also not influence cellular uptake significantly. Moreover, both
amphiphilic peptoids 3 and 4 showed an increased cellular
uptake after quantification of the cellular fluorescence from
the confocal images using the Leica LASAF software (Fig. 2C
and D) suggesting that a lower overall positive net charge also
allows for effective internalization. Whether this is due to a
mechanistically preferred structural motif has not been deter-
mined so far. Previous studies have shown that the hexameric
peptoid with lysine-like side chains adopts a pseudo-helical
structure in water with an all-cis amide conformation.34
A
helical conformation together with positive charge has been
found to be a prerequisite for some CPPs to penetrate the
membrane.35 Although octaarginine peptides are rapidly taken
up by cells, many of the natural CPPs also have an amphiphilic
Scheme 3 Synthesis of amphiphilic peptoid
4
using TIPS-protected
propargylamine.
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structure containing hydrophobic residues, which facilitate the
uptake.35,36 Thus, peptoids 3 and 4 might be good substitutes
for natural CPPs.
4 (a) H. Xia, X. Gao, G. Gu, Z. Liu, Q. Hu, Y. Tu, Q. Song,
L. Yao, Z. Pang, X. Jiang, J. Chen and H. Chen,
Int. J. Pharm., 2012, 436, 840–850; (b) S. Pujals,
N. G. Bastús, E. Pereiro, C. López-Iglesias, V. F. Puntes,
M. J. Kogan and E. Giralt, ChemBioChem, 2009, 10, 1025–
1031.
5 (a) M. Green and P. M. Loewenstein, Cell, 1988, 55, 1179–
1188; (b) A. D. Frankel and C. O. Pabo, Cell, 1988, 55, 1189–
1193.
6 D. Derossi, M. H. Joliot, G. Chassaing and M. Prochiantz,
J. Biol. Chem., 1994, 269, 10444–10450.
7 Y. Su, T. Doherty, A. J. Waring, P. Ruchala and M. Hong,
Biochemistry, 2009, 48, 4587–4595.
Additionally, peptoids 1–4 have been subjected to toxicity tests
for their suitability as molecular transporters (Fig. S3†). With an
LD50 = 91 µM for peptoid 1, LD50 = 86 µM for peptoid 2 and
a slightly higher toxicity for the amphiphilic peptoids with
LD50 = 42 µM for peptoid 3 and LD50 = 38 µM for peptoid 4,
the peptoids show moderate toxicity when compared to the
conventional hexameric peptoid containing alkyl-amino group
side chains with an LD50 > 200 µM37,38 and i.e. the CPP Antp
with an LD50 > 100 µM.39
Initially, it was supposed that the hydrophobic side chains
would influence cellular uptake towards mitochondria, which
was proven for short amphiphilic CPPos.24b However, the
corresponding peptoids 3 and 4 are still accumulating within
8 Y. Su, S. Li and M. Hong, Amino Acids, 2013, 44, 821–833.
9 Y. Su, A. J. Waring, P. Ruchala and M. Hong, Biochemistry,
2010, 49, 6009–6020.
endosomal compartments in HeLa cells. To further under- 10 F. Milletti, Drug Discovery Today, 2012, 17, 850–860.
stand the localization of the peptoids, three dimensional 11 (a) K. L. Horton, K. M. Stewart, S. B. Fonseca, Q. Guo and
images of cells were taken using z-stacks and processed using
Imaris software (see ESI†). These images however verified that
peptoids are localized neither in the nuclei nor in the mito-
S. O. Kelley, Chem. Biol., 2008, 15, 375–382; (b) D. Kalafut,
T. N. Anderson and J. Chmielewski, Bioorg. Med. Chem.
Lett., 2011, 22, 561–563.
chondria but rather stay in the endosomal compartment 12 (a) Y. Niu, G. Bai, H. Wu, R. E. Wang, Q. Qiao, S. Padhee,
before being released into the cytosol.
R. Buzzeo, C. Cao and J. Cai, Mol. Pharmaceutics, 2012, 9,
1529–1534; (b) D. S. Daniels and A. Schepartz, J. Am. Chem.
Soc., 2007, 129, 14578–14579; (c) D. Kalafut, T. N. Anderson
and J. Chmielewski, Bioorg. Med. Chem. Lett., 2012, 22,
561–563; (d) D. Jha, R. Mishra, S. Gottschalk,
K.-H. Wiesmüller, K. Ugurbil, M. E. Maier and
J. Engelmann, Bioconjugate Chem., 2011, 22, 319–328.
In conclusion, a straightforward modular approach to build
diverse peptoids from an easily accessible alkyne containing
peptoid backbone using the copper mediated cycloaddition is
presented. Using this method, chemically diverse side chains
can be introduced circumventing excessive use of protecting
groups. Two hydrophilic peptoids 1 and 2 as well as two novel
amphiphilic peptoids 3 and 4 have been synthesized. In 13 P. Lundin, H. Johansson, P. Guterstam, T. Holm,
general, it was possible to show that triazole-rings do not influ-
ence cellular uptake and moreover that internalization of
M. Hansen, Ü. Langel and S. E. L. Andaloussi, Bioconjugate
Chem., 2008, 19, 2535–2542.
amphiphilic structures with a reduced density of positive 14 D. S. Youngblood, S. A. Hatlevig, J. N. Hassinger,
charges is also possible. With this method in hand, it should
be possible to design effective peptoid molecular transporters.
P. L. Iversen and H. M. Moulton, Bioconjugate Chem., 2007,
18, 50–60.
15 E. L. Snyder, B. R. Meade, C. C. Saenz and S. F. Dowdy,
PLoS Biol., 2004, 2, E36.
16 V. Parthsarathy, P. L. McClean, C. Holscher, M. Taylor,
C. Tinker, G. Jones, O. Kolosov, E. Salvati, M. Gregori,
M. Masserini and D. Allsop, PLoS One, 2013, 8, e54769.
Acknowledgements
This work was supported by the Landesgraduiertenförderung
Baden-Württemberg (scholarship to S. B. L. V.), and by the 17 X. Zhang, Y. Jin, M. R. Plummer, S. Pooyan, S. Gunaseelan
Carl Zeiss Stiftung (scholarship to D. F.), the Helmholtz and P. J. Sinko, Mol. Pharm., 2009, 6, 836–848.
programme Biointerface and the DFG Excellence Center of 18 D. F. Schorderet, V. Manzi, K. Canola, C. Bonny,
Functional Nanostructures (CFN).
Y. Arsenijevic, F. L. Munier and F. Maurer, Clin. Experiment
Ophthalmol., 2005, 33, 628–635.
19 N. Umezawa, M. A. Gelman, M. C. Haigis, R. T. Raines and
S. H. Gellman, J. Am. Chem. Soc., 2002, 124, 368–369.
20 T. B. Potocky, A. K. Menon and S. H. Gellman, J. Biol.
Chem., 2003, 278, 50188–50194.
Notes and references
1 (a) N. Svensen, J. G. A. Walton and M. Bradley, Trends Phar-
macol. Sci., 2012, 33, 186–192; (b) K. Petrak, Drug Dev. Res., 21 M. L. Huang, S. B. Y. Shin, M. A. Benson, V. J. Torres and
2012, 73, 59–65. K. Kirshenbaum, ChemMedChem, 2012, 7, 114–122.
2 F. Mussbach, M. Franke, A. Zoch, B. Schaefer and 22 D. G. Udugamasooriya, S. P. Dineen, R. A. Brekken and
S. Reissmann, J. Cell. Biochem., 2011, 112, 3824–3833. T. Kodadek, J. Am. Chem. Soc., 2008, 130, 5744–5752.
3 I. Nakase, H. Akita, K. Kogure, A. Gräslund, Ü. Langel, 23 (a) P. A. Wender, D. J. Mitchell, K. Pattabiraman,
H. Harashima and S. Futaki, Acc. Chem. Res., 2012, 45,
1132–1139.
E. T. Pelkey, L. Steinman and J. B. Rothbard, Proc. Natl.
Acad. Sci. U. S. A., 2000, 97, 13003–13008; (b) T. Schröder,
8200 | Org. Biomol. Chem., 2013, 11, 8197–8201
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
N. Niemeier, S. Afonin, A. S. Ulich, H. F. Krug and S. Bräse, 29 J. M. Holub, H. Jang and K. Kirshenbaum, Org. Lett., 2007,
J. Med. Chem., 2008, 51, 376–379; (c) K. Eggenberger, 9, 3275–3278.
E. Birtalan, T. Schröder, S. Bräse and P. Nick, ChemBio- 30 S. B. L. Vollrath, S. Bräse and K. Kirshenbaum, Chem. Sci.,
Chem, 2009, 10, 2504–2512; (d) B. Rudat, E. Birtalan, 2012, 3, 2726–2731.
S. B. L. Vollrath, D. Fritz, D. K. Kölmel, M. Nieger, 31 H. Jang, A. Fafarman, J. M. Holub and K. Kirshenbaum,
U. Schepers, K. Müllen, H.-J. Eisler, U. Lemmer and Org. Lett., 2005, 7, 1951–1954.
S. Bräse, Eur. J. Med. Chem., 2011, 46, 4457–4465; 32 P. M. Levine, K. Imberg, M. J. Garabedian and
(e) T. Schröder, K. Schmitz, N. Niemeier, T. S. Balaban, K. Kirshenbaum, J. Am. Chem. Soc., 2012, 134, 6912–6915.
H. F. Krug, U. Schepers and S. Bräse, Bioconjugate Chem., 33 I. E. Valverde, A. F. Delmas and V. Aucagne, Tetrahedron,
2007, 18, 342–354. 2009, 65, 7597–7602.
24 (a) W. Huang, J. Seo, J. S. Lina and A. E. Barron, Mol. 34 U. Sternberg, E. Birtalan, I. Jakovkin, B. Luy, U. Schepers,
BioSyst., 2012, 8, 2626–2628; (b) D. K. Kölmel, D. Fürniss,
S. Susanto, A. Lauer, C. Grabher, S. Bräse and U. Schepers,
Pharmaceuticals, 2012, 5, 1265–1281.
S. Bräse and C. Muhle-Goll, Org. Biomol. Chem., 2013, 11,
640–647.
35 J. S. Bahnsen, H. Franzyk, A. Sandberg-Schaal and
H. M. Nielsen, Biochim. Biophys. Acta, 2013, 1828, 223–232.
36 I. D. Alves, N. Goasdoue, I. Correia, S. Aubry, C. Galanth,
S. Sagan, S. Lavielle and G. Chassaing, Biochim. Biophys.
Acta, 2008, 1780, 948–959.
25 R. N. Zuckermann, J. M. Kerr, S. B. H. Kent and
W. H. Moos, J. Am. Chem. Soc., 1992, 114, 10646–10647.
26 (a) V. V. Rostovtsev, L. G. Green, V. V. Fokin and
K. B. Sharpless, Angew. Chem., Int. Ed., 2002, 41, 2596–
2599; (b) C. W. Tornøe, C. Christensen and M. Meldal, 37 T. Schröder, K. Schmitz, N. Niemeier, T. S. Balaban,
J. Org. Chem., 2002, 67, 3057–3064.
27 J. M. Holub and K. Kirshenbaum, Chem. Soc. Rev., 2010,
39, 1325–1337.
H. F. Krug, U. Schepers and S. Bräse, Bioconjugate Chem.,
2007, 18, 342–354.
38 T. Schröder, N. Niemeier, S. Afonin, A. S. Ulrich, H. F. Krug
and S. Bräse, J. Med. Chem., 2008, 51, 376–379.
28 (a) J. M. Holub, H. Jang and K. Kirshenbaum, Org. Biomol.
Chem., 2006, 4, 1497–1502; (b) D. Fürniss, T. Mack, 39 A. Hansen, I. Schafer, D. Knappe, P. Seibel and
F. Hahn, S. B. Vollrath, K. Koroniak, U. Schepers and
S. Bräse, Beilstein J. Org. Chem., 2013, 9, 56–63.
R. Hoffmann, Antimicrob. Agents Chemother., 2012, 56,
5194–5201.
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