R. Sheng et al. / Bioorg. Med. Chem. 21 (2013) 6366–6377
6375
that, the reaction mixture was poured into distilled water and ex-
tracted with EtOAC, dried with MgSO4 anhydride and concentrated
under reduced pressure, and the crude product was purified by
flash column chromatography (eluent: EtOAC/hexane = 1/4, v/v)
to obtain 3-Bromo-1-propamine-NHBOC (9) as a yellowish oil
(3.1 g, 62.6% yield) .
triazole–CH2–OOC–), 4.44 (t, 2H, C–CH2–triazole), 4.31 (m, 1H,
–OOC–O–CH–), 2.77 (t, 2H, NHþ–CH2–), 2.47 (t, 2H, NHþ–CH2–
3
3
CH2–), 2.26–0.61 (45H, cholesterol).
13C NMR: (CDCl3, 300 Hz) d 154.1, 138.5, 124.8, 121.9, 76.5,
59.7,55.4, 55.2, 54.8, 53.9, 48.7, 45.8, 41.1, 38.9, 38.8, 36.8, 35.5,
34.8, 34.3, 30.5, 26.9, 26.5, 22.9, 22.4, 21.7, 21.6, 19.8, 18.0, 17.5,
11.0
1H NMR (CDCl3, 300 Hz) d 4.31(1H, –CHCON–), 3.34(2H, –CH2–),
3.11(2H, –CH2–), 1.99(2H, –CH2–), 1.27 (9H, Boc).
ESI-MS [M+H+]: 569.4, Calculated: 569.5.
Lipid 2 (Yield:69%).
4.2.2.2. Synthesis of 3-azido-1-propamine-NHBOC (7).
In a
1H NMR: (CDCl3, 300 Hz) d 8.65 (b, NHþ3 ), 8.21 (s, 1H, ArH, tria-
zole), 7.87 (b, NHþ3 ), 5.36 (s, 1H, –C@CH–, cholesterol), 5.16 (s, 2H,
triazole–CH2–OOC–), 4.51(s, 1H, –CH–CONH–), 4.40 (t, 2H, C–CH2–
triazole), 4.33 (m, 1H, –OOC–O–CH–), 3.11(t, 2H, –CONH–CH2–),
2.77 (t, 2H, NHþ–CH2–), 2.47 (t, 2H, NHþ–CH2–CH2–), 2.26–0.62
100 mL flask with 3-Bromo-1-propamine-NHBOC (9, 2.3 g,
0.01 mol) and sodium azide (0.72 g, 0.011 mol) dissolved in 5 mL
DMF the mixture was kept stirring for 8 h at 80 °C. Then the mix-
ture was cooled down and gently poured into 100 mL distilled
water, then extracted with 100 mL EtOAC, the solvent phase was
concentrated under reduced pressure, and crude 3-azido-1-prop-
amine-NHBOC (7) was obtained and utilized directly without fur-
ther purification.
3
3
(45H, cholesterol).
13C NMR: (CDCl3, 300 Hz) d 169.1, 154.1, 139.6, 125.5, 122.8,
77.7, 60.7, 56.5, 55.9, 52.5, 49.8, 47.4, 42.3, 38.9, 38.8, 37.9, 36.7,
36.4, 36.2, 35.6, 31.7, 30.8, 29.9, 28.2, 27.7, 27.6, 27.0, 26.8, 24.1,
23.5, 23.1. 22.8, 21.6, 21.0,19.1, 18.5, 12.1.
4.2.2.3. Synthesis of 3-Bromo-1-propamine-NH-lysine-BOC
ESI-MS [M+H+]: 697.5; Calculated: 697.5.
(10).
In a 100 mL flask with 3-Bromo-1-propamine hydro-
Lipid 3 (Yield:62%).
chloride salt (11, 2.2 g, 0.01 mmol) and Boc-(l-)-lysine (3.5 g,
0.01 mol) were dissolved in 20 mL tetrahydrofuran with 10 mL
pyridine, then EDCꢀHCl/DMAP (3.0 g/0.1 g) was added and the mix-
ture was kept stirring for 24 h under the room temperature. After
that, the mixture was gently poured into 100 mL distilled water
and extracted with 100 mL EtOAC, the organic solvent phase was
washed with distilled water and concentrated under reduced pres-
sure, and purified by flash column chromatography (eluent:
EtOAC/hexane = 1/3, v/v) to get 3-Bromo-1-propamine-NH-lysine
-BOC (10) as a colorless oil (2.6 g, 56.5% yield).
1H NMR: (CDCl3, 300 Hz) d 8.20 (s, 1H, ArH, triazole), 7.89 (b,
NHþ), 5.16 (s, 2H, triazole–CH2–OOC–), 4.48 (m, 1H, –OOC–O–
3
CH–), 4.47 (t, 2H, C–CH2–triazole), 3.56 (s, 3H, -COOCH3), 2.77 (t,
2H, NHþ–CH2–), 2.47 (t, 2H, NHþ–CH2–CH2–), 2.26–0.61 (39H,
3
3
lithocholate).
13C NMR: (CDCl3, 300 Hz) d 174.3, 154.3, 142.0, 125.6, 123.9,
78.3, 60.5, 56.2, 55.9, 52.8, 51.7, 48.3, 47.0, 42.6, 41.5, 38.8, 36.7,
35.7, 35.2, 34.6, 33.1, 32.1, 30.7, 27.3, 27.0, 26.3, 24.2, 23.3, 21.6,
20.6,18.4, 12.1.
ESI-MS [M+H+]: 573.3; Calculated: 573.5.
1H NMR (CDCl3, 300 Hz) d 4.28(1H, –CHCON), 3.42(2H, –CH2–),
3.11(2H, –CH2–), 2.05 (2H, –CH2–), 1.42 (18H, Boc).
Lipid 4 (Yield: 66%).
1H NMR: (CDCl3, 300 Hz) d 8.67 (b, NHþ3 ), 8.20 (s, 1H, ArH, tria-
zole), 7.89 (b, NHþ), 5.15 (s, 2H, triazole–CH2–OOC–), 4.48 (m, 1H, –
3
4.2.2.4. Synthesis of 3-azido-1-propamine-NH-lysine-BOC
(8).
OOC–O–CH–), 4.47 (t, 2H, C–CH2–triazole), 4.17 (m, 1H, –NHþ–CH–
3
In a 100 mL flask with 3-Bromo-1-propamine-NH-ly-
CONH–), 3.56 (s, 3H, –COOCH3), 2.76 (t, 2H, NHþ–CH2–), 2.50(t, 2H,
3
sine-BOC (10, 1.2 g, 0.0025 mol) and sodium azide (0.33 g,
0.005 mol)) dissolved in 10 mL DMF and was kept stirring for 8 h
under 80 °C. Then the mixture was cooled down and gently poured
into 50 mL distilled water, then extracted with 100 mL EtOAC, the
solvent phase was concentrated under reduced pressure, and crude
3-azido-1-propamine-NH-lysine-BOC (8) was obtained and uti-
lized directly without further purification.
NHþ–CH2–CH2–), 2.26–0.61 (39H, lithocholate).
3
13C NMR: (CDCl3, 300 Hz) d 174.3, 169.0, 154.3, 141.7, 125.4,
123.9, 78.1, 60.4, 563, 55.8, 52.6, 52.0, 51.3, 47.5, 42.8, 41.5, 40.4,
38.8, 36.3, 35.6, 35.1, 34.8, 34.6, 32.1, 31.0, 30.9, 29.9, 29.7, 28.0,
26.8, 26.5, 26.2, 24.0, 23.3, 21.6, 20.6, 18.3, 12.0.
ESI-MS [M+H+]: 701.5; Calculated: 701.5.
4.3. NMR and mass spectral measurements
4.2.3. General procedures for the synthesis of sterol-based
cationic lipids (1–4) by CuAAC ‘Click’ method
1H NMR spectra were characterized under room temperature on
In a 100 mL flask added with as-prepared sterol-propynyl build-
ing blocks (5 or 6, 1.0 mmol) and azido-bearing cationic blocks (7
or 8, 1.1 mmol) in 20 mL tetrahydrofuran, and quickly added
CuSO4ꢀ5H2O/Sodium ascorbate (0.5 mmol/1 mmol) which were
firstly dissolved in 5 mL distilled water, the reaction mixture was
kept at 80 °C for 2 h, the yellow solid was removed by filtration
and the filtrate was added with 50 mL 5% EDTA (w/w) aqueous
solution to coordinate excessive Cu(I), then extracted with
100 mL EtOAC. After that, the solvent was removed under reduced
pressure, then the yellow residue was purified by flash column
chromatography (EtOAC/Hexane = 1:1) to get Boc-protected ste-
rol-based cationic lipids, which was reacted with excessive trifluo-
acetic acid (5 mL) at room temperature for 1 h to remove the Boc
protecting group, then the solvent trifluoacetic acid was evapo-
rated, the residue was precipitated with diethyl ether (Et2O) and
dried to get pure sterol-based cationic lipids 1–4 as the final
products.
a Varian VXR-300 Fourier transform NMR spectrometer at
300.0 MHz for proton nuclei, and 13C NMR spectra were measured
on a Bruker Avance NMR spectrometer at 100.0 MHz for the 13C
nuclei, and tetramethylsilane (TMS) was utilized as the internal
chemical shift reference. Mass spectra (ESI-MS) were routinely
measured on a Varian SATURN 2000 instrument.
4.4. Ethidium bromide replacement assay
Ethidium bromide (0.5 lg) and pDNA (Luciferase pDNA, 4.0 lg)
were firstly mixed in 1 mL of 0.01 M PBS buffer solution (pH 7.4),
and kept incubation at room temperature for 2 min. Then, each ste-
rol-based cationic lipids under various +/ꢁ charge ratios of 0.5–20
was separately placed into the pDNA/EB mixture solution, and con-
tinuously kept incubation for 10 min. Subsequently, fluorescence
spectra for the above complex solution were recorded on a Hitachi
F-7000 fluorescence spectrometer with excitation and emission
wavelength at k = 510 and 590 nm, respectively, and the emission
intensities of the initial pDNA/EB mixture solution (I100) and a
pDNA-free EB solution (I0) were taken as the corresponding
Lipid 1 (Yield:79%).
1H NMR: (CDCl3, 300 Hz) d 8.17 (s, 1H, ArH, triazole), 7.89
(b, NHþ3 ), 5.32 (s, 1H, –C@CH–, cholesterol), 5.13 (s, 2H,