146
4. Dialyze against isotonic Tris buffer at pH = 7.4 (0.150 M NaCl,
0.010 M Tris).
5. Place a small stir bar in the dialysis bag and vigorously stir during the exchange of the solutions (600 rpm).
6. Remove excess of insoluble Pacl in the buffer by centrifugation
at 112 × g at 20 °C for 10 min.
7. Separate the supernatant very carefully from the solid (Pacl).
8. Repeat the centrifugation three times (see Note 20).
4 Notes
1. N,N-dimethylformamide is further purified by short-path
fractional distillation under high vacuum in custom-made
apparatus. Use always the middle fraction.
2. 1,6-Diaminohexane is a highly hygroscopic compound, leave
it to dry over a sodium mirror for 24 h, then dilute with purified DMF, subdivide into ampoules, and store under high
vacuum (HV) at room temperature.
3. Benzene is purified by stirring over concentrated sulfuric acid
for a week. It is subsequently washed with an aqueous solution
of NaOH and then with water many times until it becomes
neutral and finally dry with CaCl 2 . The dry material is then
transferred into a round-bottom flask containing fresh finely
grounded CaH 2 and a magnetic stirring bar, and is attached to
the vacuum line and degassed. Leave the flask for reaction of
CaH 2 with moisture overnight, degas again, and distill in a
calibrated cylinder containing n-BuLi and styrene.
4. Triethylamine is dried over calcium hydride for 1 day and then
distilled and stored in the vacuum line over sodium. The
appropriate quantity needed is freshly distilled in a vacuum
line prior to use.
5. For in vitro and in vivo experiments use the injectable forms of
Dox (adriamycin 2 mg
.
mL
−1
) and Pacl (taxol 6 mg
.
mL
−1
).
6. Since the benzyl groups are deuterated, the composition of
the protected copolypeptides cannot be obtained by
1
H NMR
spectroscopy, but is verified using UV spectroscopy in DMF,
since the PBLG-d7 block absorbs at 267 nm. Anisole is used
as a scavenger of the tert-butyl cations. It is well established
that Boc and benzyl groups can be orthogonally deprotected:
the former under acidic and the latter under basic conditions.
Confirm the complete removal of the Boc groups by
1
H NMR
spectroscopy.
7. The Boc groups are selectively deprotected.
8. Use a custom-made 100 mL glass apparatus (equipped with a
filter), which has been previously flame dried several times.
Hermis Iatrou et al.
4. Dialyze against isotonic Tris buffer at pH = 7.4 (0.150 M NaCl,
0.010 M Tris).
5. Place a small stir bar in the dialysis bag and vigorously stir during the exchange of the solutions (600 rpm).
6. Remove excess of insoluble Pacl in the buffer by centrifugation
at 112 × g at 20 °C for 10 min.
7. Separate the supernatant very carefully from the solid (Pacl).
8. Repeat the centrifugation three times (see Note 20).
4 Notes
1. N,N-dimethylformamide is further purified by short-path
fractional distillation under high vacuum in custom-made
apparatus. Use always the middle fraction.
2. 1,6-Diaminohexane is a highly hygroscopic compound, leave
it to dry over a sodium mirror for 24 h, then dilute with purified DMF, subdivide into ampoules, and store under high
vacuum (HV) at room temperature.
3. Benzene is purified by stirring over concentrated sulfuric acid
for a week. It is subsequently washed with an aqueous solution
of NaOH and then with water many times until it becomes
neutral and finally dry with CaCl 2 . The dry material is then
transferred into a round-bottom flask containing fresh finely
grounded CaH 2 and a magnetic stirring bar, and is attached to
the vacuum line and degassed. Leave the flask for reaction of
CaH 2 with moisture overnight, degas again, and distill in a
calibrated cylinder containing n-BuLi and styrene.
4. Triethylamine is dried over calcium hydride for 1 day and then
distilled and stored in the vacuum line over sodium. The
appropriate quantity needed is freshly distilled in a vacuum
line prior to use.
5. For in vitro and in vivo experiments use the injectable forms of
Dox (adriamycin 2 mg
.
mL
−1
) and Pacl (taxol 6 mg
.
mL
−1
).
6. Since the benzyl groups are deuterated, the composition of
the protected copolypeptides cannot be obtained by
1
H NMR
spectroscopy, but is verified using UV spectroscopy in DMF,
since the PBLG-d7 block absorbs at 267 nm. Anisole is used
as a scavenger of the tert-butyl cations. It is well established
that Boc and benzyl groups can be orthogonally deprotected:
the former under acidic and the latter under basic conditions.
Confirm the complete removal of the Boc groups by
1
H NMR
spectroscopy.
7. The Boc groups are selectively deprotected.
8. Use a custom-made 100 mL glass apparatus (equipped with a
filter), which has been previously flame dried several times.
Hermis Iatrou et al.