4. Remove all the organic solvents under reduced pressure at a
rotavapor.
5. Dissolve the residue in DCM (30 mL), dry the solution over
anhydrous MgSO 4 , filter off the solid (see Note 6), and remove
from the filtrate the organic solvent at a rotavapor to obtain the
pure product as a pale brown powder (415 mg, 95% yield).
6. Check the purity of the final compound by ESI-MS and
1 H
NMR. It is possible to compare the collected spectra with the
characterization data of the compound as reported [59].
3.1.4 Synthesis of
25,26,27,28-tetra-nhexyloxycalix[4]arene (5)
1. Stir at room temperature for 24 h in a two-necked roundbottom flask a mixture of aminocalixarene 4 (0.15 g,
0.18 mmol), N α -Boc-N ω -Pbf-L-Arg (0.58 g, 1.09 mmol),
DMAP (0.27 mg, 2.19 mmol), HOBt (0.17 g, 1.24 mmol),
and EDC (0.21 g, 1.09 mmol) in dry DCM (20 mL) (see Notes
9 and 10).
2. Quench the reaction by addition of water, separate with a
separatory funnel the lower organic phase, wash it with water
(2 Â 25 mL) and subsequently with a saturated NaHCO 3
aqueous solution (2 Â 25 mL).
3. Evaporate the organic solvent at the rotavapor to get a crude
material that is treated by flash column chromatography (silica
gel, eluent: gradient from DCM to DCM/MeOH 95:5, v/v)
to isolate the pure product as a white solid (208 mg, 40% yield).
4. Check the purity of the final compound by ESI-MS and
1 H
NMR. It is possible to compare the collected spectra with the
characterization data of the compound as reported [61].
3.1.5 Synthesis of
Arg-amino)-25,26,27,28tetrakis(n-hexyloxy)calix
[4]arene octahydrochloride (1)
1. Stir a solution of calix[4]arene 5 (80 mg, 2.8 Â 10
À2 mmol) in
TFA/triisopropylsilane/H 2 O (5 mL, 95/2.5/2.5, v/v) at
room temperature for 1 h (see Note 11).
2. When the reaction is completed, remove the volatiles under
reduced pressure at the rotavapor.
3. Repeatedly suspend the solid residue in AcOEt (3 Â 5 mL) and
remove the organic solvent under vacuum at the rotavapor (see
Note 12).
4. Wash the crude solid material with distilled diethyl ether
(3 Â 7 mL), each time removing the supernatant liquid after
sample centrifugation.
5. Dissolve the solid in a methanol solution of concentrated HCl
(pH 3–4) and subsequently remove water and the volatiles at
reduced pressure with the rotavapor. Repeat this operation four
times (see Note 13). Compound 1 is obtained as a white solid
(37 mg, 75% yield).
PNA Delivery with Argininocalix[4]arene
131
rotavapor.
5. Dissolve the residue in DCM (30 mL), dry the solution over
anhydrous MgSO 4 , filter off the solid (see Note 6), and remove
from the filtrate the organic solvent at a rotavapor to obtain the
pure product as a pale brown powder (415 mg, 95% yield).
6. Check the purity of the final compound by ESI-MS and
1 H
NMR. It is possible to compare the collected spectra with the
characterization data of the compound as reported [59].
3.1.4 Synthesis of
25,26,27,28-tetra-nhexyloxycalix[4]arene (5)
1. Stir at room temperature for 24 h in a two-necked roundbottom flask a mixture of aminocalixarene 4 (0.15 g,
0.18 mmol), N α -Boc-N ω -Pbf-L-Arg (0.58 g, 1.09 mmol),
DMAP (0.27 mg, 2.19 mmol), HOBt (0.17 g, 1.24 mmol),
and EDC (0.21 g, 1.09 mmol) in dry DCM (20 mL) (see Notes
9 and 10).
2. Quench the reaction by addition of water, separate with a
separatory funnel the lower organic phase, wash it with water
(2 Â 25 mL) and subsequently with a saturated NaHCO 3
aqueous solution (2 Â 25 mL).
3. Evaporate the organic solvent at the rotavapor to get a crude
material that is treated by flash column chromatography (silica
gel, eluent: gradient from DCM to DCM/MeOH 95:5, v/v)
to isolate the pure product as a white solid (208 mg, 40% yield).
4. Check the purity of the final compound by ESI-MS and
1 H
NMR. It is possible to compare the collected spectra with the
characterization data of the compound as reported [61].
3.1.5 Synthesis of
Arg-amino)-25,26,27,28tetrakis(n-hexyloxy)calix
[4]arene octahydrochloride (1)
1. Stir a solution of calix[4]arene 5 (80 mg, 2.8 Â 10
À2 mmol) in
TFA/triisopropylsilane/H 2 O (5 mL, 95/2.5/2.5, v/v) at
room temperature for 1 h (see Note 11).
2. When the reaction is completed, remove the volatiles under
reduced pressure at the rotavapor.
3. Repeatedly suspend the solid residue in AcOEt (3 Â 5 mL) and
remove the organic solvent under vacuum at the rotavapor (see
Note 12).
4. Wash the crude solid material with distilled diethyl ether
(3 Â 7 mL), each time removing the supernatant liquid after
sample centrifugation.
5. Dissolve the solid in a methanol solution of concentrated HCl
(pH 3–4) and subsequently remove water and the volatiles at
reduced pressure with the rotavapor. Repeat this operation four
times (see Note 13). Compound 1 is obtained as a white solid
(37 mg, 75% yield).
PNA Delivery with Argininocalix[4]arene
131