Molecules 2018, 23, 1540
13 of 18
N-bromosuccinimide (NBS); and the third step was the nucleophilic substitution of bromine of
1
compound
2
with pyridine yielding the target compound D19. H- and 13C-NMR spectra data of
compounds 1, 2, and D19 were identical to those reported in the literature [26].
Didodecyl 2,6-bis-(2-(N,N-dimethylamino)ethyl)-4-phenyl-1,4-dihydropyridine-3,5-dicarboxylate (
3). A mixture
of didodecyl 3,5-bis(dodecyloxycarbonyl)-2,6-dimethyl-4-phenyl-1,4-dihydropyridine (
1, 1.22 g,
2.00 mmol), paraformaldehyde (0.40 g, 0.01 eq, 0.02 mol), dimethylamine hydrochloride (0.98 g,
12.0 mmol), a conc. HCl (0.150 mL) and ethanol (10 mL) was refluxed for 24 h. The solvent was
removed by evaporation in vacuo, after which water (5 mL) was added. The pH of the resulting
mixture was adjusted to approx. 8 with an aqueous solution of sodium carbonate and extracted
with chloroform (6
×
30 mL). The organic layer was washed with brine and dried over MgSO4.
After removal of the solvent, the oily residue was obtained and purified by flash chromatography
(eluent: chloroform:acetone:methanol:Et3N = 9:4:2:0.02) yielding 0.75 g (68%) of light yellow foam. TLC:
Rf: 0.2 (CHCl3/MeOH/Et3N = 1:1:0.01). 1H-NMR (CDCl3):
δ
0.86–0.90 (m, 6H), 1.23–1.34 (m, 36H);
1.55–1.60 (m, 4H), 2.33 (s, 12H), 2.52–2.66 (m, 4H), 2.90–3.09 (m, 4H), 3.98–4.02 (m, 4H), 4.98 (s, 1H),
7.08–7.12 (m, 1H), 7.16–7.20 (m, 2H), 7.26–7.27 (m, 2H), 10.13 (s, 1H) ppm. 13C-NMR (CDCl3):
14.1,
δ
18.4, 22.7, 26.1, 27.8, 28.7, 29.3, 29.4, 29.5, 29.6, 29.7, 31.9, 37.0, 39.4, 44.7, 63.8, 69.2, 102.8, 125.9, 127.7,
127.8, 148.2, 148.3, 167.7 ppm. MS (+ESI) m/z (relative intensity); 725 ([M + H]+, 60). Anal. calcd. for
C45H77N3O4: C, 74.64; H, 10.72; N, 5.80; Found: C, 74.49; H, 10.55; N, 5.91.
N,N0-{[3,5-Bis(dodecyloxycarbonyl)-4-phenyl-1,4-dihydropyridine-2,6-diyl]diethylene}bis N,N-dimethyoctyl
ammonium dibromide (C12-Man-Q). A mixture of compound
3 (290 mg, 0.4 mmol, 1 eq) and
1-bromooctane (232 mg, 1.2 mmol, 3 eq) in nitromethane (3 mL) and DMF (0.5 mL) was refluxed
under argon for 48 h. The solvents were removed in vacuo and the residue was triturated with a small
amount of dry acetone. After cooling the precipitate was filtered off and crystallised from dry acetone
yielding compound C12-Man-Q as a light yellow powder (222 mg, 50%), mp 180–183 ◦C. 1H-NMR
(CDCl3):
δ 0.79–0.84 (m, 12H), 1.21–1.30 (m, 56H), 1.53 (br s, 4H), 1.60–1.87 (m, 8H), 3.22–3.38 (m,
2H)overlap, 3.36 (s, 12H)overlap, 3.48–3.56 (m, 2H), 3.65–3.73 (m, 2H), 3.85–3.97 (m, 4H), 4.24–4.35 (m,
2H), 4.74 (s, 1H), 7.04–7.08 (m, 1H), 7.12–7.15 (m, 2H), 7.20–7.26 (m, 2H)overlap with CDCl3, 10.02 (s,
1H) ppm. 13C-NMR (CDCl3):
δ 14.0, 14.1, 22.5, 22.6, 29.3, 29.6, 29.7, 31.9, 39.2, 51.3, 51.8, 61.6, 64.1, 64.7,
105.4, 126.4, 127.9, 128.0, 143.6, 147.2, 166.9 ppm. MS (+ESI) m/z (relative intensity); 950 ([M-2Br + H]+
10). Anal. calcd. for C61H111Br2N3O4: C, 65.98; H, 10.08; N, 3.78; Found: C, 65.71; H, 10.15; N, 3.72.
3.3. Calculation of Nitrogen to Phosphorus (N/P) Ratio
The N/P ratio is a measure of the ionic balance of the amphiphile/DNA complexes. The positive
charge (N) of amphiphile originates from the nitrogens of 1,4-DHP amphiphiles C12-Man-Q or D19
The negative charge (P) in the plasmid DNA backbone arises from the phosphate group of the
deoxyribose nucleotides. The average molecular weight of the nucleotides is assumed to be 330 g/mol.
.
Example of N/P ratio calculation: Complexation of 1
µg of plasmid DNA with C12-Man-Q at
N/P ratio 1, how much 0.5 mM of C12-Man-Q (VC12ManQneeded ) is needed?
N
P
1 × 10−6
=
= 3 × 10−9M = 3 nmol
= 1 × 3 = 3 µL
330
N = P × 1 = 3 × 1 = 3
VC12ManQ
needed
3.4. Preparation of Amphiphile/pDNA Complexes (Lipoplexes)
The amphiphile/pDNA complexes were prepared using various amounts of cationic lipid and
constant amount of pDNA, ensuring different N/P ratios. Firstly, cationic lipid and pDNA were
diluted separately in various transfection media: (a) medium without any supplements; and (b)