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tion of the various tagged peptoids in very high yields
and purities. Flow cytometry analysis showed that the
ability to enter cells was fully dependent on the number
of monomer units. Compound 7e (n = 9) proved to be a
quite remarkable carrier for all the cell lines, showing
good penetrability into K562 and L929 cells and
extraordinary cell delivery properties with HeLa cells,
with high entry within 5 min. Additionally, confocal
images supported endocytosis as the main uptake mech-
anism. The NLS-peptoid hybrids allowed specific intra-
cellular trafficking of the fluorescein tag into the nucleus,
thus opening up a potential range of applications for
specific nuclei delivery of these peptide–peptoid hybrids
such as nuclei staining of living cells with non-DNA-
intercalating fluorescent probes. Moreover, the absence
of nuclear targeting capacity evidenced by hybrids
10c,d clearly delineates the importance of the role of
the proline and valine residues in the peptide–protein
interaction between the NLS and the nuclear
transporters.
tion of 3 (5.0 g, 0.0137 mol) in DCM (50 ml). The reac-
tion was monitored by TLC until completion (4 h). The
solvent was removed in vacuo and the crude product
was purified by column chromatography on silica gel
(eluent: EtOAc/Hexane, 1:1) to give 4 as a light yellow
oil (7.2 g, 90%).
1H NMR (300 MHz, CDCl3) two rotamers d 1.13–1.47
(m, 17H), 3.13 and 3.49 (m, 4H), 3.96 and 4.03 (s,
2H), 4.13 and 4.26 (t, J = 6.0 Hz, 1H), 4.40 and 4.53
(d, J = 6.0 Hz, 2H), 5.13 and 5.18 (s, 2H), 7.27–7.44
(m, 9H), 7.53–7.61 (m, 2H), 7.61–7.79 (m, 2H).13C
NMR (75.5 MHz, CDCl3) two rotamers d 26.3, 26.5
and 26.6, 27.9, 28.3 and 28.5, 30.0 and 30.1, 40.5, 47.4,
48.5, 48.8 and 48.9, 49.3, 67.0, 67.4 and 67.6, 79.1,
120.0, 124.9, 125.0, 127.1, 127.7, 128.4, 128.7, 135.4,
135.5, 141.4 and 141.5, 144.1, 155.9 and 156.0, 156.5,
169.6 and 169.7. HRMS (FAB): C35H42N2O6;
[M+H]+: calc 587.3121; found: 587.3113.
4.1.3. Synthesis of [(6-tert-butoxycarbonylamino-hexyl)-
(9H-fluoren-9-ylmethoxycarbomyl)-amino]-acetic
acid
4. Experimental
(1). 10% Pd/C (0.24 g) was added to a solution of 4
(4.0 g, 6.8 mmol) in MeOH (100 ml). The reaction was
stirred under hydrogen atmosphere (1 atm) for 4 h at
room temperature. The reaction mixture was filtered
over Celite and the filtrate concentrated in vacuo. The
product was purified by flash chromatography on silica
gel (eluent: DCM/MeOH, 10:1) to give 1 as a white solid
(2.71 g, 80%).
All chemicals, solvents and biological materials were
purchased from Sigma–Aldrich or Fischer.
4.1. Synthesis of lysine-like peptoid unit 1
4.1.1. Synthesis of benzyl-(6-tert-butoxycarbonylamino-
hexylamino)-acetate (3). Di-tert-butyl carbonate (5.19 g,
0.0298 mol) in DCM (50 ml) was added dropwise to 1,6-
diaminohexane (22.1 g, 0.19 mol) in DCM (100 ml) and
the reaction was stirred overnight. The solvent was
evaporated and the residue was suspended in H2O
(100 ml). The insoluble white solid was removed by fil-
tration and the product was then extracted using
DCM (3 · 100 ml). The combined organic phase was
washed with brine (2 · 50 ml), dried over Mg2SO4 and
concentrated in vacuo to give a pale yellow oil. The
compound was used for the next step without any fur-
ther purification. A solution of benzyl 2-bromacetate
(5.8 g, 0.025 mol) in THF (20 ml) was added dropwise
1H NMR (CDCl3, 300 MHz) two rotamers d 1.3–1.6 (m,
17 H); 3.0 and 3.2 (two br m, 4H); 3.30 and 3.4 (br s,
2H,); 4.10 (m, 1H); 4.45 and 4.51 (two m, 2H); 7.26
(m, 2H) 7.35 (t, J = 7.5 MHz, 2H); 7.53 (d,
J = 7.5 MHz, 2H); 7.72 (d, J = 7.0 MHz, 2H).13C
NMR (CDCl3, 75.5 MHz) d 26.4, 27.6, 28.0, 28.5,
29.9, 40.5, 47.1, 48.5, 51.0, 67.5, 78.9, 119.8, 124.8,
126.9, 127.5, 141.2, 143.9, 156.0, 157.5. HRMS (ES+):
C28H36N2O6; [M+Na]+: calc 519.2466; found: 519.2456.
4.2. Synthesis of labelled peptoids 7a–e, peptide 8 and
peptide–peptoid conjugates 9a–b and 10a–d
to
a stirred solution of N-Boc-1,6-hexanediamine
(5.5 g, 0.025 mol) and triethylamine (5.4 g, 0.075 mol)
in THF (50 ml) over 2 h, then the reaction was stirred
for another 24 h. After filtration of the white solid, the
solution was concentrated in vacuo and the crude prod-
uct was purified by column chromatography using ethyl
acetate as eluent to obtain compound 3 as a pale yellow
oil (5.0 g, 54%, two steps).
The peptoids were assembled on aminomethyl polysty-
rene resin functionalized with a Rink amide linker using
monomer 1.19 Coupling procedure: Three equivalents of
monomer 1, DIC and HOBt in DMF at a concentration
of 0.1 M were mixed with the corresponding resin and
microwave irradiated at 60 ꢁC for 20 min. Deprotection
of the Fmoc group (20% piperidine in DMF) and re-
peated coupling (as above) gave the desired oligomers
of the required length. These were then coupled to an
Fmoc-protected six carbon spacer (Fmoc-aminohexa-
noic acid) using the coupling procedure described above,
deprotected with 20% piperidine in DMF, and conju-
gated with 5(6)-carboxyfluorescein. Finally, the resin
bound peptoids were deprotected and cleaved from the
solid support by treatment with a mixture of TFA/
TIS/H2O (95:2.5:2.5) for 3 h, and precipitated with
diethyl ether as pale yellow solids. The crude materials
were analyzed by MS (ES+) and RP-HPLC (purity as-
sessed via Evaporative Light Scattering detection): 7a,
1H NMR (300 MHz, CDCl3) d 1.23–1.31 (m, 4H), 1.36–
1.50 (m, 13H), 2.56 (t, J = 7.5 Hz, 2H), 3.03–3.10 (m,
2H), 3.42 (s, 2H), 5.14 (s, 2H), 7.31–7.43 (m, 5H).13C
NMR (75.5 MHz, CDCl3) d 26.7, 26.9, 28.5, 30.0,
40.5, 49.5, 51.0, 66.5, 79.0, 128.4, 128.6, 135.7, 156.0,
172.5. HRMS (FAB): C20H32N2O4; [M+H]+: calc:
365.2440; found: 365.2439.
4.1.2. Synthesis of benzyl-[(6-tert-butoxycarbonylamino-
hexyl)-(9H-fluoren-9-ylmethoxycarbomyl)-amino]-acetate
(4). Fmoc-Osu (4.6 g, 0.0137 mol) was added to a solu-