130
1 mg/mL fluorescamine in acetonitrile, 15 μL of 0.1 M borate
buffer at pH 8.0, and 145 μL of Milli-Q water. After 5 min,
measure the fluorescence using excitation at 405 nm and emission at 485 nm.
Perform the synthesis using high-vacuum techniques [2]. The
purity of the NCA monomers is crucial for their successful living
polymerization utilizing ROP through primary amine difunctional
initiator and confirms by FTIR along with
1
H NMR spectroscopy
analysis [3].
1. Add N
α
,N
ε
-Di-(tert-butoxycarbonyl)-l-lysine into a flask,
place it on the vacuum line, and pump overnight.
2. Distill purified ethyl acetate, followed by argon insertion, in
order to reach atmospheric pressure and by the addition of
triphosgene. Leave the mixture to react for 10 min.
3. Dilute triethylamine in dry ethyl acetate, add it dropwise, and
immerse the solution in an ice-water bath for 6 h.
4. Filter the precipitate, in order to remove the HCl salt of triethylamine, and immerse the clear solution in an ice bath.
5. Extract the NCA repeatedly with Milli-Q water, until neutral
pH of the aqueous phase is achieved.
6. Recrystallize the purified NCA three times under high vacuum
in a custom-made apparatus, with ethyl acetate/hexane
(1/5 v/v) pair at −20 °C. The yield is 60%.
1. Add in a 500 mL round-bottom flask 20 g (40.2 mmol) of
Boc-His(Trt)-OH and dry overnight under high vacuum.
2. Distill 150 mL of THF in the flask, giving a clear yellowish
solution.
3. In an ice bath place the reaction flask, filled with argon.
4. Dilute 3.25 mL (44.2 mmol) of thionyl chloride in 20 mL of
THF and add dropwise in a period of 10 min.
5. After 2 h, pour the solution in 2 L of cold (Et) 2 O with precipitation of Trt-His-NCA.HCl as the major product.
6. Finally, filter the solid (glass sintered filter 3) and then transfer
it to a round-bottom flask of 500 mL. Drying in HV gives
17.2 g.
7. Recrystallize the above solid mixture, containing the HCl salt,
free anhydride, and the initial substrate by distilling 300 mL of
ethyl acetate under HV.
8. Remove the round-bottom flask of 500 mL, containing the
suspension, from HV and place it into a water bath, at 45 °C
for 1 h, resulting in dissolution.
3.2 Synthesis
of Monomers
3.2.1 Synthesis
of ε-tert-Butoxycarbonyl-lLysine N-Carboxy
Anhydride (Ν
ε
BOC-l-LYS
NCA)
3.2.2 Synthesis
of N
im
-trityl-l-Histidine-NCarboxy Anhydride
(Trt-HIS-NCA) [4]
Hermis Iatrou et al.
1 mg/mL fluorescamine in acetonitrile, 15 μL of 0.1 M borate
buffer at pH 8.0, and 145 μL of Milli-Q water. After 5 min,
measure the fluorescence using excitation at 405 nm and emission at 485 nm.
Perform the synthesis using high-vacuum techniques [2]. The
purity of the NCA monomers is crucial for their successful living
polymerization utilizing ROP through primary amine difunctional
initiator and confirms by FTIR along with
1
H NMR spectroscopy
analysis [3].
1. Add N
α
,N
ε
-Di-(tert-butoxycarbonyl)-l-lysine into a flask,
place it on the vacuum line, and pump overnight.
2. Distill purified ethyl acetate, followed by argon insertion, in
order to reach atmospheric pressure and by the addition of
triphosgene. Leave the mixture to react for 10 min.
3. Dilute triethylamine in dry ethyl acetate, add it dropwise, and
immerse the solution in an ice-water bath for 6 h.
4. Filter the precipitate, in order to remove the HCl salt of triethylamine, and immerse the clear solution in an ice bath.
5. Extract the NCA repeatedly with Milli-Q water, until neutral
pH of the aqueous phase is achieved.
6. Recrystallize the purified NCA three times under high vacuum
in a custom-made apparatus, with ethyl acetate/hexane
(1/5 v/v) pair at −20 °C. The yield is 60%.
1. Add in a 500 mL round-bottom flask 20 g (40.2 mmol) of
Boc-His(Trt)-OH and dry overnight under high vacuum.
2. Distill 150 mL of THF in the flask, giving a clear yellowish
solution.
3. In an ice bath place the reaction flask, filled with argon.
4. Dilute 3.25 mL (44.2 mmol) of thionyl chloride in 20 mL of
THF and add dropwise in a period of 10 min.
5. After 2 h, pour the solution in 2 L of cold (Et) 2 O with precipitation of Trt-His-NCA.HCl as the major product.
6. Finally, filter the solid (glass sintered filter 3) and then transfer
it to a round-bottom flask of 500 mL. Drying in HV gives
17.2 g.
7. Recrystallize the above solid mixture, containing the HCl salt,
free anhydride, and the initial substrate by distilling 300 mL of
ethyl acetate under HV.
8. Remove the round-bottom flask of 500 mL, containing the
suspension, from HV and place it into a water bath, at 45 °C
for 1 h, resulting in dissolution.
3.2 Synthesis
of Monomers
3.2.1 Synthesis
of ε-tert-Butoxycarbonyl-lLysine N-Carboxy
Anhydride (Ν
ε
BOC-l-LYS
NCA)
3.2.2 Synthesis
of N
im
-trityl-l-Histidine-NCarboxy Anhydride
(Trt-HIS-NCA) [4]
Hermis Iatrou et al.