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C8ec) at a flow rate of 4 mLminꢀ1. Fractions containing only the
desired a-aminoxy peptide were collected and lyophilized from
the HPLC solvents, yielding the purified a-aminoxy peptides
(>95% purity in all cases, for details see Table 2 and the Support-
ing Information).
identified a right-handed 28-helical conformation with precisely
two residues per turn and a helical pitch of 5.8 ꢂ. This 28-helix
can mimic the spatial arrangement of the peptide side chains
in a-helices and 310-helices. By 2D ROESY experiments, MD sim-
ulations, and CD spectroscopy we identified a 28-helix as the
predominant conformation in organic solvents. However, in
aqueous solutions, we observed a pH-dependent equilibrium
between the 28-helix and another conformation. As CD spec-
troscopy in the presence of liposomes revealed that long-
chained a-aminoxy peptides have an increased propensity to
take up a 28-helical conformation in a membrane environment,
we conclude that this conformation is responsible for the
membranolytic activity of the decameric a-aminoxy peptides
observed in our mode of action studies.
Biological evaluation
Cell lines and cell culture: The human esophagus cell line
Kyse510 and human embryonic kidney cells HEK293 were obtained
from the German Collection of Microorganism and Cell Cultures
(DSMZ, Braunschweig, Germany). The human ovarian carcinoma
cell line A2780 was obtained from the European Collection of Cell
Cultures (ECACC, Salisbury, United Kingdom). The cisplatin resistant
CisR cell lines were generated by exposing the parental cell lines
to weekly cycles of cisplatin at an inhibitory concentration of 50%
according to Gosepath et al. and Eckstein et al.[18,19] The cell lines
were grown at 378C under humidified air supplemented with 5%
CO2 in RPMI 1640 (A2780, Kyse510) or DMEM (HEK293) media con-
taining 10% fetal calf serum, penicillin (120 IUmLꢀ1), and strepto-
mycin (120 mgmLꢀ1).
In summary, the improved access to a-aminoxy peptide hex-
amers and decamers allowed for a thorough analysis of their
anticancer activity, conformational fold, and modes of action of
this novel class of bioactive compounds. These findings should
improve the understanding of a-aminoxy peptides as folda-
mers and enable the structure-based design of peptide ana-
logs that mimic helical structures such as a-helices and 310-
helices in the future.
MTT cell viability assay: MTT assays were performed as previously
described.[20] MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli-
um bromide) was purchased from Applichem (Darmstadt, Germa-
ny). In brief, A2780, Kyse510, and HEK293 cell lines were seeded in
96-well plates. After 24 h, the cells were exposed to increased con-
centrations of the test compounds. Incubation was ended after ad-
ditional 72 h, and the cell survival was determined by adding the
MTT solution (5 mgmLꢀ1 in PBS buffer). The formazan precipitate
was dissolved in DMSO and the absorbance was measured at 544
and 690 nm in a FLUOstar microplate reader (BMG LabTech, Offen-
burg, Germany).
Experimental Section
Chemistry
Materials and general methods, synthetic protocols for the prepa-
ration of the dimeric building blocks, and the compound character-
ization data for all novel compounds are given in the Supporting
Information.
Analysis of the cell membrane integrity and apoptosis: To mea-
sure the membrane integrity, Kyse510 cells were grown in 6-well
plates and treated with the indicated concentrations of the com-
pounds for 24 h. Treatment with 0.2% Triton X-100 for 20 min
served as a positive control. After harvesting and washing with
PBS, the cells were stained with 20 mgmLꢀ1 propidium iodide (PI,
Santa Cruz Biotechnology, Heidelberg, Germany) dissolved in 0.9%
NaCl for 15 min in the dark at 378C. Then, the cells were analyzed
by flow cytometry (Partec, Mꢀnster, Germany). To determine apop-
tosis, Kyse510 cells were treated in 24-well plates with the indicat-
ed concentrations of the compounds for 24 h. 5% DMSO was used
as a positive control. After incubation, the cells were lysed over-
night in hypotonic staining buffer (0.1% Triton X-100, 0.1%
sodium citrate in sterile filtrated water) containing 100 mgmLꢀ1 PI
and analyzed by flow cytometry.
Solid-phase synthesis of a-aminoxy oligopeptides 1a–i: The
manual peptide synthesis was conducted in fritted PE syringes on
a 0.2 mmol scale. After the resin swelling for 60 min in DMF, the
Fmoc deprotection of the Rink Amide PEG resin (loading
0.56 mmolgꢀ1) was accomplished with 20% piperidine in DMF (2ꢃ
15 min, 2 mL/100 mg of resin) before the resin was sequentially
washed with DMF, CH2Cl2, and DMF (3ꢃ2 mL/100 mg resin, agita-
tion for 15 s and then drained). A solution of the free acid[9] of the
respective phthaloyl-protected a-aminoxy dipeptide (0.6 mmol,
3.0 equiv), BOP (265.4 mg, 0.6 mmol, 3.0 equiv), and HOBt
(81.1 mg, 0.6 mmol, 3.0 equiv) in DMF (5 mL) was agitated for
1 min, NEM (101 mL, 0.8 mmol, 4.0 equiv) was added, and this solu-
tion was then added to the resin. The amide coupling was per-
formed for 24 h at room temperature. Afterwards, the resin was se-
quentially washed with DMF, CH2Cl2 and DMF. Then, the phthaloyl
group was removed by treatment with 5% hydrazine monohydrate
in methanol for 15 min (2ꢃ) and the resin was sequentially washed
with DMF, MeOH, CH2Cl2, and DMF. After the assembly of the a-
aminoxy hexamer or decamer by iterative cycles of the phthaloyl
deprotection, the amide coupling reaction, and several washing
cycles (final washing with CH2Cl2), the crude product was cleaved
from the support with TFA/TES (98:2, v/v, 5 mL) for 1.5 h. The fil-
trate was concentrated in a stream of nitrogen to a volume of
smaller than 1 mL. The crude product was precipitated with cold
diethyl ether, centrifuged, and the diethyl ether was discarded.
This procedure was repeated twice to obtain the crude a-aminoxy
peptide. For the semipreparative purification, the crude a-aminoxy
peptides were redissolved in acetonitrile and purified on a Macher-
ey-Nagel Nucleosil C8 RP-HPLC column (VP 250/10 Nucleosil 100–5
Data analysis: Concentration–effect curves were obtained using
Prism 4.0 from GraphPad (San Diego, CA, USA) by fitting the
pooled data to the four-parameter logistic equation. Flow cytome-
try data were analyzed using FloMax 2.82 (Partec, Mꢀnster, Germa-
ny).
Crystal structure analysis
Long rod shaped crystals of the hexamer 1b with an average size
of 15ꢃ15ꢃ600 mm3 were obtained after one day from acetonitrile/
water (60:40). After overlaying the crystal containing the mother
liquid with mineral oil for cryoprotection, the crystals were harvest-
ed with litho loops and flash frozen in liquid nitrogen. A dataset of
the hexamer 1b was collected at 100 K at the European Synchro-
tron Radiation Facility (ESRF, Grenoble, France) on beamline
ID30A-3. According to the instrumentation at the beamline, the
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Chem. Eur. J. 2016, 22, 1 – 13
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ÝÝ These are not the final page numbers!