Full Papers
tra were recorded with a FTIR Nicolet 380 spectrometer in the ATR
mode, and absorption values (n) are in wave numbers [cmÀ1]. Mass
spectra were recorded with an Agilent technology 6520 Accurate
Mass QToF instrument in electrospray ionization (ESI) mode. Mass
data are reported in mass units (m/z). Lipofectamine 2000 and
LysoSensor green DND-189 were obtained from Invitrogen (Cergy
at reflux for 20 h under argon and then cooled down and concen-
trated under reduced pressure at 208C. The crude residue was pu-
rified by flash chromatography over silica gel (CH2Cl2/MeOH 88:12
to 80:20) to yield compound 2 as a waxy solid (130 mg, 20%, two
diastereomers, ammonium chloride). TLC Rf =0.53 (CH2Cl2/MeOH
1
90:10); H NMR (400 MHz, CDCl3): d=5.69–5.59 (m, 2H), 5.35–5.25
(m, 4H), 5.24–5.17 (m, 1H), 5.63–5.61 (brm, 2H), 4.39–4.06 (m, 8H),
Pontoise,
France).
1,2-Dioleoyl-sn-glycero-3-phosphocholine
3
3.71 (t, JH,H =4.7 Hz, 2H), 3.65–3.50 (m, 18H), 3.46 (s, 9H), 3.40 (t,
(DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-ethanolamine (DOPE),
1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1,2-dimyristoyl-
sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene gly-
col)-5000] ammonium salt (DMPE-mPEG5000), 1,2-dioleoyl-sn-gly-
cero-3-phospho-ethanolamine-N-(7-nitro-2–1,3-benzoxadiazol-4-yl
ammonium salt (NBD-PE), and 1,2-dioleoyl-sn-glycero-3-phospho-
ethanolamine-N-(lissamine rhodamine B sulfonyl) (Rh-PE) were
from Avanti Polar Lipids. Cholesterol (chol) was from Sigma–Al-
drich. BHK-21 cells (Syrian hamster kidney cells, CCL-10), Calu-3
cells (epithelial lung adenocarcinoma, HBT-55), A549 cells (human
lung carcinoma, CCL-185), and NCI-H292 cells (human lung muco-
epidermoid carcinoma, CRL-1848) were obtained from ATCC-LGC
(Molsheim, France). pCMV-FLuc expression plasmid (5.5 kbp, BD
Biosciences Clontech, Franklin Lakes, NJ, USA) was used as reporter
gene to monitor DNA transfection activity in vitro.[35] This plasmid
encodes the firefly luciferase gene under the control of a strong
CMV promoter. Plasmid pCMV-GLuc (5.7 kbp, Nanolight Technolo-
gy, Pinetop, AZ, USA) was used as reporter gene to monitor in vivo
DNA transfection activity.[36] This plasmid encodes the Gaussia luci-
ferase gene under the control of the CMV promoter. The A549 cell
line was transformed to allow stable expression of the Photinus
pyralis luciferase gene originating from the pGL3 plasmid (Clon-
tech, Mountain View, CA) to assess siRNA delivery.[37] The pGL3
plasmid also encoded for a gene conferring resistance to the anti-
biotic G418. This antibiotic was thus used to select the transfected
A549-Luc cells. Luciferase-gene-silencing experiments were per-
formed with an RNA duplex (siLuc) of the sense sequence: 5’-CUU
ACG CUG AGU ACU UCG A. Control untargeted RNA duplex (sic)
was of sense sequence: 5’-CGU ACG CGG AAU ACU UCG A. Both
RNAs were from Eurogentec (Angers, France). DNA concentration
refers to phosphate content. Culture media (Dulbecco’s modified
Eagle’s medium, DMEM; Roswell Park Memorial Institute medium,
RPMI 1640), FBS, and supplements were from GIBCO-BRL (Cergy-
Pontoise, France). Lysis and luciferin solutions for monitoring Firefly
luciferase activity were purchased from Promega. Coelenterazine
substrate for monitoring Gaussia luciferase activity was from Nano-
light Technology.
3JH,H =6.9 Hz, 2H), 2.36–2.40 (m, 4H), 2.05–1.90 (m, 8H), 1.65–1.47
3
(m, 2H), 1.36–1.15 (m, 58H), 1.05 (t, JH,H =6.6 Hz, 6H), 0.84 ppm (t,
J=6.7 Hz, 9H); 13C NMR (100 MHz, CDCl3): d=173.4, 173.0, 153.5,
130.2, 129.9, 86.2, 71.7, 70.7, 70.2, 69.4, 68.7, 68.2, 66.8, 66.7, 65.5,
65.4, 62.4, 61.6, 54.7, 34.2, 32.1, 29.9, 29.8, 29.7, 29.6, 29.5, 29.4,
29.3, 29.2, 27.4, 27.3, 26.2, 25.1, 22.9, 14.2 ppm; 31P NMR (162 MHz,
CDCl3): d=À3.2, À3.3 ppm; FTIR (film): n˜ =2922, 2852, 2360, 2341,
1741, 1260 cmÀ1; MS (ESI): m/z calcd for C58H111NO11P(C2H4O)4
1204.89 [MÀCl]+; found: 1204.8.
:
+
Conjugate 3: Intermediate 1-chloroethyl carbonate 10 was pre-
pared from 7 (2.00 g, 5.40 mmol) and chloroethyl chloroformate
(641 mL, 5.94 mmol) by the same procedure as for 9. It was ob-
tained as a colorless oil (2.33 g, 92%) and was used in the next
step without further purification. TLC Rf =0.55 (CH2Cl2/MeOH
90:10); 1H NMR (400 MHz, CDCl3): d=6.4 (q, 3JH,H =5.8 Hz, 1H),
4.34–4.32 (m, 2H), 3.72 (t 3JH,H =4.1 Hz, 2H), 3.63–3.54 (m, 16H),
3
3
3.42 (t, JH,H =6.8 Hz, 2H), 1.81 (d, JH,H =5.8 Hz, 3H), 1.57–1.53 (m,
2H), 1.29–1.23 (m, 18H), 0.86 ppm (t, 3JH,H =6.7 Hz, 3H); 13C NMR
(100 MHz, CDCl3): d=153.1, 84.8, 71.8, 70.9, 70.8, 70.2, 68.9, 68.1,
32.1, 29.8, 29.7, 29.6, 26.3, 25.4, 22.9, 14.3 ppm.
Conjugate 3 was obtained as a waxy solid (290 mg, 37%, four dia-
stereomers, ammonium chloride) from 10 (2.33 g, 4.96 mmol) and
DOPC (488 mg, 0.62 mmol) by the same procedure as for 2. TLC
1
Rf =0.53 (CH2Cl2/MeOH 90:10); H NMR (400 MHz, CDCl3): d=6.41–
6.21 (m, 1H), 5.37–5.24 (m, 4H), 5.24–5.17 (m, 1H), 4.54 (brm, 2H),
4.38–4.05 (m, 8H), 3.83–3.86 (m, 2H), 3.65–3.50 (m, 18H), 3.46 (s,
9H), 3.40 (t, 3JH,H =6.9 Hz, 2H), 2.36–2.23 (m, 4H), 2.05–1.90 (m,
3
8H), 1.65–1.47 (m, 9H), 1.36–1.15 (m, 58H), 0.84 ppm (t, JH,H
=
6.7 Hz, 9H); 13C NMR (100 MHz, CDCl3): d=173.4, 173.0, 153.1, 95.7,
95.3, 71.1, 70.8, 70.7, 70.2, 69.6, 69.5, 69.3, 69.2, 68.8, 68.1, 66.8,
68.1, 66.8, 66.7, 66.6, 66.4, 66.3, 66.2, 65.5, 65.4, 62.5, 62.2, 61.7,
61.6, 61.5, 54.6, 34.3, 34.2, 32.1, 30.4, 29.9, 29.6, 29.4, 29.3, 27.4,
26.3, 25.2, 25.0, 23.7, 22.9, 21.4, 20.9, 14.3 ppm; 31P NMR (162 MHz,
CDCl3): d=À5.3, À5.5, À5.7, À5.9 ppm; FTIR (film): n˜ =2922, 2852,
1743, 1457, 1264, 1108, 1084, 971, 669 cmÀ1; MS (ESI): m/z calcd for
C59H113NO11P(C2H4O)4+: 1218.91 [MÀCl]+; found: 1218.9.
Conjugate 2: Chloromethyl chloroformate (522 mL, 5.94 mmol) was
added to a solution of 7 (2.00 g, 5.40 mmol) and pyridine (547 mL,
6.79 mmol) in freshly distilled CH2Cl2 (40 mL). The reaction mixture
was stirred at rt under argon for 18 h, neutralized with water
(50 mL), and extracted with EtOAc. The organic layer was dried
over Na2SO4 and filtered, and volatiles were removed under re-
duced pressure. Compound 9 was obtained as a colorless oil
(2.26 g, 92%) and was used in the next step without further purifi-
cation. TLC Rf =0.60 (CH2Cl2/MeOH 90:10); 1H NMR (400 MHz,
CDCl3): d=5.68 (s, 2H), 4.33–4.30 (m, 2H), 3.70–3.68 (m, 2H), 3.60–
3.57 (m, 16H), 3.53–3.50 (m, 2H), 1.52–1.50 (m, 2H), 1.26–1.20 (m,
18H), 0.82 ppm (t, 3JH,H =6.6 Hz, 3H); 13C NMR (100 MHz, CDCl3):
d=153.5, 72.3, 71.6, 70.8, 70.7, 70.1, 66.7, 32.0, 29.8, 29.6, 26.2,
22.7, 14.2 ppm; FTIR (film): n˜ =2922, 2854, 2360, 2341, 1766, 1250,
Conjugate 4: Intermediate 1-chloro-2-methylpropyl carbonate 11
(2.58 g, 96%) was prepared from 7 (2.00 g, 5.40 mmol) and 1-
chloro-2-methylpropyl chloroformate (884 mL, 5.94 mmol) by the
same procedure as for 9. It was obtained as a colorless oil (2.58 g,
96%) and was used in the next step without further purification.
TLC Rf 0.50 (CH2Cl2/MeOH 90:10); 1H NMR (400 MHz, CDCl3): d=
3
3
6.16 (d, JH,H =4.5 Hz, 1H), 4.34–4.32 (m, 2H), 3.73 (t, JH,H =4.5 Hz,
2H), 3.64–3.54 (m, 16H), 3.42 (t, 3JH,H =6.8 Hz, 2H), 2.23–2.16 (m,
1H), 1.63–1.48 (m, 2H), 1.29–1.23 (m, 18H), 1.05 (t, 3JH,H =6.6 Hz,
3
6H), 0.86 ppm (t, JH,H =6.7 Hz); 13C NMR (100 MHz, CDCl3): d=92.8,
71.6, 70.7, 70.6 (4C), 70.1, 68.7, 67.8, 35.2, 31.9, 29.6 (4C), 29.5, 29.3,
26.1, 22.7, 17.4, 17.1, 14.1 ppm.
1108 cmÀ1
.
Conjugate 4 was obtained as a waxy solid (174 mg, 20%, four dia-
stereomers, ammonium chloride) from 11 (2.70 g, 5.43 mmol) and
DOPC (533 mg, 0.68 mmol) by the same procedure as for 2. TLC
A solution of compound 9 (1.95 g, 4.28 mmol) in dry CHCl3 (5 mL)
was added to a solution of DOPC (421 mg, 0.54 mmol) in freshly
distilled CHCl3 (10 mL). The resulting reaction mixture was heated
1
Rf =0.53 (CH2Cl2/MeOH 90:10). H NMR (400 MHz, CDCl3): d=6.16–
6.10 (m, 1H), 5.35–5.20 (m, 5H), 4.57 (brm, 2H), 4.47–4.12 (m, 8H),
ChemBioChem 2016, 17, 1 – 14
9
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ