Functionalized triazolopeptoids-a novel class for mitochondrial targeted delivery

Article abstract of DOI:10.1039/c5ob00250h

Here we introduce linear 1,4-triazolopeptoids as a novel class of cell penetrating peptidomimetics suitable as organ targeting molecular transporters of bioactive cargo. Repetitive triazole moieties with up to three residues were assembled on solid supports using copper-catalyzed alkyne-azide cycloadditions (CuAAC) in a submonomer approach. Depending on the lipophilicity of their side chain appendages the 1,4-triazolopeptoids showed either endosomal localization or a strong colocalization with the mitochondria of HeLa cells with moderate toxicity. While the basic triazolopeptoids mainly target the neuromast cells in zebrafish embryos, the lipophilic ones colocalize with either cartilage in the jaws and the blood vessel system. This journal is

Full text of DOI:10.1039/c5ob00250h

Organic &
Biomolecular Chemistry
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Functionalized triazolopeptoids – a novel class for
mitochondrial targeted delivery†
Cite this: Org. Biomol. Chem., 2015,
3, 4226
1
a
b
a
a
Daniela Althuon,‡ Franziska Rönicke, Daniel Fürniss,‡ Jasmin Quan,
b
a,b
b
a,b
Isabelle Wellhöfer, Nicole Jung, Ute Schepers* and Stefan Bräse*
Here we introduce linear 1,4-triazolopeptoids as a novel class of cell penetrating peptidomimetics suitable
as organ targeting molecular transporters of bioactive cargo. Repetitive triazole moieties with up to three
residues were assembled on solid supports using copper-catalyzed alkyne–azide cycloadditions (CuAAC)
in a submonomer approach. Depending on the lipophilicity of their side chain appendages the 1,4-triazo-
lopeptoids showed either endosomal localization or a strong colocalization with the mitochondria of
HeLa cells with moderate toxicity. While the basic triazolopeptoids mainly target the neuromast cells in
zebrafish embryos, the lipophilic ones colocalize with either cartilage in the jaws and the blood vessel
system.
Received 4th February 2015,
Accepted 20th February 2015
DOI: 10.1039/c5ob00250h
www.rsc.org/obc
Among the foldamers, aliphatic β-peptides and cell pene-
Introduction
5
,13,22,34,35
trating peptoids (CPPos)
as well as aromatic amide
2
To date functional peptidomimetics often replace peptides foldamers have already been shown to exhibit cell penetrating
with similar biological activities while displaying an enhanced properties and can be used as transport moieties for targeted
2
3–34
stability towards enzymatic degradation. In addition, they also drug delivery.
However, triazole foldamers, which are
allow for additional spatial arrangements of hydrogen-donor accessible by subsequent click reactions were not tested as
and acceptor moieties. The key feature of most peptidomi- mimetics of CPPs to facilitate drug uptake in vivo and the
metics is the arrangement of moieties that mimic the amido impact of the triazole bioisosters on the cell permeation
1
group. Peptide bond bioisosters, such as hydroxyethylamine characteristics is not well understood. In contrast to the ali-
(Hea), aromatic amides, and tetrazole rings have been used to phatic foldamers, which mostly recapitulate the same struc-
stabilize metabolically labile peptide bonds in peptidomimetic tural features as their peptide counterpart, structural studies
1
,2
drug design. In addition, foldamers containing a triazole of foldamers with triazole containing backbones showed con-
ring peptide bond isoster have recently emerged as a novel siderable differences in the overall structure compared to the
class of peptidomimetics with properties and structural diver- corresponding peptide, which might have an impact on their
3
,4–12
2
sity similar to that of natural peptides.
As such, they not cellular uptake capabilities.
only have a planar geometry like amides but also are enzymati-
cally stable. Poly-1,4-triazoles with three and more triazoles especially the cell penetrating peptoids (CPPos) as molecular
Based on our interest in the use of aliphatic foldamers
1
3
1
4,15
5,13,22,34,35
in a linear backbone containing different spacers (C
2
,
transporters for a variety of cargos,
we were inter-
1
6,17
18–20
C ,
1
C4-sugars
,4-triazoles have been synthesized.
shown to play a pivotal role in materials science, drug of Ke et al., we designed a set of novel triazolopeptoids
and likewise, non-racemic chiral poly- ested to explore linear 1,4-triazolopeptoids as a novel class of
3
1
4,16–20
They have been cell penetrating foldamers. Inspired by the recent publication
2
1
35
development and medicinal chemistry.
(Fig. 1). In contrast to the structure 2 of Ke et al., they are sup-
posed to be less prone to dimerization and aggregation due to
the lack of the amide hydrogen.
The herein presented synthesis of this rather unexplored
3
6–38
a
class of 1,4-triazolopeptoid foldamers,
is based on a
Institute of Organic Chemistry, Karlsruhe Institute of Technology, Fritz-Haber-Weg 6,
modular approach for a straightforward assembly on solid sup-
ports which allows (a) for the incorporation of a huge variety
of functionalized side chains, and (b) for the use of chiral
building blocks. Besides a solid phase synthesis described
by Fujino et al., most of the linear compounds have been
7
6131 Karlsruhe, Germany. E-mail: braese@kit.edu
b
Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Campus
North, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany.
E-mail: ute.schepers@kit.edu
†
Electronic supplementary information (ESI) available: Characterization, toxi-
city, spectra for compounds 14 to 18. See DOI: 10.1039/c5ob00250h
These authors contributed equally.
1
6,17,25,39
‡
obtained by solution phase syntheses so far.
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As known from peptoid chemistry, the synthesis on solid
supports not only allows for the coupling of fully functiona-
2
2
lized monomers but also for the assembly of submonomers.
We therefore devised two different strategies in order to syn-
thesize the 1,4-triazolopeptoids 1 (Scheme 1).
In the first submonomer-based approach, a stepwise
addition of azidoacetic acids 3 and functionalized propargylic
amines 4 were used to synthesize the immobilized 1,4-triazolo-
peptoids on solid supports.
In the second approach, a so-called monomer method
adapted from well-established peptide and peptoid syntheses
with the incorporation of building blocks of type 5 was used.
We came to the conclusion that the stepwise approach is more
promising. For the CuAAC reaction, we used copper(I) iodide
in N,N-diisopropylethylamine (DIPEA) in THF furnishing the
product 14c in overall 32% yield (Scheme 2).
Fig. 1 Generic structure of 1,4-triazolopeptoids synthesized in this
report 1 and by Ke et al. 2.
3
5
In order to test different 1,4-triazolopeptoids for their
ability to act as cell penetrating moieties for the delivery of bio-
active molecules, we synthesized a small library containing
fluorophore-labelled 1,4-triazolopeptoids with different side
chains with (14c, 17a–c, 18a–c) and without (15a–c, 16a–c) an
N-methoxyethyl glycine spacer (Table 1, Fig. 2). As the syn-
thesis of 14c resulted in significantly higher yields compared
Table 1 Yields of purified triazolopeptoids, see ESI and Fig. 2
Peptoid
Yield [%]
32
Peptoid
Yield [%]
6.3
14c
15a
15b
15c
16a
16b
16c
17b
17c
18c
a
a
7.2–29
1.6–9.5
9.4–1.9
1.4–4.6
a
1.6
1.8
7.0
a
2.0
a
Result of several runs and/or variants (see ESI).
Scheme 1 Strategies to form polymer-bound triazolopeptoids 6.
Scheme 2 Solid–phase synthesis of 1,4-triazolopeptoid 14c.
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The solid phase synthesis was either conducted with pro-
tected propargyl amines (4-Fmoc) or free amines (4-H). All 1,4-
triazolopeptoids were labelled with rhodamine
fluorophore.
B as a
Biological application
To investigate, whether the 1,4-triazolopeptoids are capable to
act as peptidomimetics of CPPs, we tested the trimeric 1,4-tri-
azolopeptoids 15c (n = 3), 16c (n = 3), 17c (n = 3) and 18c
(
1
n = 3) for their uptake in living cells and zebrafish embryos.
× 10 human cervix carcinoma cells (HeLa cells) were treated
4
with 10 µM of the 1,4-triazolopeptoids, respectively.
Fig. 2 Small library of 1,4-triazolopeptoids bearing different side chains
(
see Table 1).
to the reactions without spacer-unit, the spacer was used with
the intention to increase the yields of the other 1,4-triazolo-
peptoids bearing different side chains. However, following
syntheses did not confirm that the presence of the spacer
improves yields of all derivatives suggesting that the charges of
the side chains have considerable effect on the synthesis.
Fig. 4 96 hpf zebrafish were incubated with a 5 µM triazolopeptoids in
E3-medium solution for 3 h at 28 °C in the dark. Control embryos were
only treated with E3-medium. For subsequent live imaging, embryos
were washed (3 × E3-medium), anesthetized (0.02% tricaine) and posi-
tioned in an 8 well µ-slide from IBIDI (Ibitreat). Confocal fluorescence
microscopy was performed on a Leica SP5-TCS (DMI6000) inverse
microscope using a HC PL FLUOTAR 10.0 × 0.30 DRY objective. 1st
column: RhodB-triazolopeptoid, emission bandwidth: 593–696 nm; 2nd
column: brightfield; 3rd column: merge of column 1 and 2. Scale bar =
600 µm.
4
Fig. 3 1 × 10 HeLa cells were incubated with a 10 µM of 15c, 16c, 17c,
and 18c in DMEM at 37 °C for 24 h and subsequently subjected to fluor-
escent confocal microscopy using a Leica SP5-TCS (DMI6000) inverse
microscope. Objective: HCX PL APO CS 63.0 × 1.20 WATER UV. 1st
column: Hoechst 33342, emission bandwidth: 417–468 nm; 2nd
column: MitoTracker® Green, emission bandwidth: 499–522 nm; 3rd
column: RhodB-triazolopeptoid, emission bandwidth: 593–696 nm; 4th
column: merge of columns 1 to 3. Scale bar = 20 µm.
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To determine their route of cellular uptake and their intra- in solution but also the structure–activity relationship for drug
cellular localization, the cells were counterstained with a mito- delivery purposes.
chondrial marker (MitoTracker®Green) and the nuclear stain
Hoechst 33342. While the 1,4-triazolopeptoids 16c and 18c
comprising the lysine side chain accumulate in endosomal
vesicles, 1,4-triazolopeptoids 15c and 17c, bearing the more
Acknowledgements
lipophilic chlorobenzyl moiety show a strong accumulation in We acknowledge the Carl–Zeiss–Stiftung (D.F.) for financial
the mitochondria and a less pronounced colocalization with support. The work was further supported by the Helmholtz
the endosomes (Fig. 3). This mitochondrial localization programme BioInterfaces in Technology and Medicine (BIFTM)
suggested a more amphiphilic nature of 15c and 17c and (U.S., F.R., I.W., N.J. and S.B.) and the GRK 2039 of the DFG
therefore a direct transport through the plasmamembrane via (F.R., U.S, S.B).
inverted micelles driven by the membrane potential, while 16c
and 18c are taken up by classical endocytosis (Fig. 3).
All 1,4-triazolopeptoids showed moderate to low toxicity
with LD50 values between 10 µM (15c) and 40 µM (17c) for the
Notes and references
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