132
2. Distill 250 mL of highly dry THF; remove the flask from the
vacuum line and brink to atmospheric pressure by the careful
addition of dry argon. The flask is equipped with a condenser
and an inlet for Ar streaming.
3. Add under stirring 29.6 mL (183 mmol, 1.6 eq.) of purified
(+) (−) limonene and allow the suspension to warm under vigorous stirring at 50–55 °C. At this time, add 13.6 g (46 mmol,
1.2/3 eq.) of triphosgene. Allow the reaction mixture to stir
at this temperature until a clear dark orange solution is formed
(after 1–2 h).
4. Stir the clear solution for another 1 h under Ar flow and transfer the solution by filtration to the crystallization apparatus.
Attach the apparatus to the vacuum line and pump out the
solvent to dryness. Then, distill a small amount of highly dry
THF (~20 mL) capable to dissolve the solid monomer and a
clear solution is formed.
5. Slowly distill, under vigorous stirring, n-hexane (~500 mL) in
order for the NCA to precipitate in the form of a fine powder.
Keep the apparatus at −20 °C overnight.
6. Next day, filter off the orange supernatant and dry the solid in
vacuo for 1 h.
7. Perform three additional crystallizations and finally dissolve
the white solid product in dry THF and cannula transfer it in
a sealed flask. Attach the flask to the vacuum line, remove the
solvent, and dry overnight the final product.
8. Finally, move the flask to the glove box and weigh solid yielding 16.6 g (95 mmol) of Leu-NCA (83%).
1. Add an α,ω-hexamethylenediamine solution in DMF, in a
solution of ε-tert-butoxycarbonyl-l-lysine N-carboxy anhydride in DMF (Lys-NCA) (see Note 7).
2. After completion of polymerization (middle block), dissolve in
DMF appropriate amounts of N
im
-trityl-protected l-histidine
N-carboxy anhydride (HIS-NCA) and γ-benzyl-l-glutamate
N-carboxy anhydride (BLG-NCA) (molar ratio 1:1) and add
to the reaction flask. Allow the polymerization to continue for
6 days (two hydrophobic blocks).
3. Add then Lys-NCA, followed by precipitation, in diethyl ether
after the completion of the polymerization (see Note 8).
4. Mix the deprotected pentablock with Milli-Q water and dialyze (membrane molecular weight cutoff 3500 Da) against
dilute HCl solution (pH = 3) for 2 days, dilute NaOH solution (pH = 8) for 2 days, and DI water for 2 days (for each step
change the water every 12 h).
3.3 Synthetic
Approach of
Polypeptides PLys-b(PHIS-co-PBLG (or
PLEU))-b-PLys-b(PHIS-co-PBLG (or
PLEU))-b-PLys (See
Fig. 2)
Hermis Iatrou et al.
2. Distill 250 mL of highly dry THF; remove the flask from the
vacuum line and brink to atmospheric pressure by the careful
addition of dry argon. The flask is equipped with a condenser
and an inlet for Ar streaming.
3. Add under stirring 29.6 mL (183 mmol, 1.6 eq.) of purified
(+) (−) limonene and allow the suspension to warm under vigorous stirring at 50–55 °C. At this time, add 13.6 g (46 mmol,
1.2/3 eq.) of triphosgene. Allow the reaction mixture to stir
at this temperature until a clear dark orange solution is formed
(after 1–2 h).
4. Stir the clear solution for another 1 h under Ar flow and transfer the solution by filtration to the crystallization apparatus.
Attach the apparatus to the vacuum line and pump out the
solvent to dryness. Then, distill a small amount of highly dry
THF (~20 mL) capable to dissolve the solid monomer and a
clear solution is formed.
5. Slowly distill, under vigorous stirring, n-hexane (~500 mL) in
order for the NCA to precipitate in the form of a fine powder.
Keep the apparatus at −20 °C overnight.
6. Next day, filter off the orange supernatant and dry the solid in
vacuo for 1 h.
7. Perform three additional crystallizations and finally dissolve
the white solid product in dry THF and cannula transfer it in
a sealed flask. Attach the flask to the vacuum line, remove the
solvent, and dry overnight the final product.
8. Finally, move the flask to the glove box and weigh solid yielding 16.6 g (95 mmol) of Leu-NCA (83%).
1. Add an α,ω-hexamethylenediamine solution in DMF, in a
solution of ε-tert-butoxycarbonyl-l-lysine N-carboxy anhydride in DMF (Lys-NCA) (see Note 7).
2. After completion of polymerization (middle block), dissolve in
DMF appropriate amounts of N
im
-trityl-protected l-histidine
N-carboxy anhydride (HIS-NCA) and γ-benzyl-l-glutamate
N-carboxy anhydride (BLG-NCA) (molar ratio 1:1) and add
to the reaction flask. Allow the polymerization to continue for
6 days (two hydrophobic blocks).
3. Add then Lys-NCA, followed by precipitation, in diethyl ether
after the completion of the polymerization (see Note 8).
4. Mix the deprotected pentablock with Milli-Q water and dialyze (membrane molecular weight cutoff 3500 Da) against
dilute HCl solution (pH = 3) for 2 days, dilute NaOH solution (pH = 8) for 2 days, and DI water for 2 days (for each step
change the water every 12 h).
3.3 Synthetic
Approach of
Polypeptides PLys-b(PHIS-co-PBLG (or
PLEU))-b-PLys-b(PHIS-co-PBLG (or
PLEU))-b-PLys (See
Fig. 2)
Hermis Iatrou et al.