Biomacromolecules
Article
TBAB (4.86 g, 0.0151 mol, 0.1 eq) was added, and the mixture was
cooled down to 10 °C and epichlorohydrin (70 mL, 0.755 mol, 5 eq)
was added slowly to the mixture. The mixture was stirred for 48 h at
room temperature, diluted with diethylether, and washed with
saturated NaHCO3, NaCl, and water. The organic phase was dried
over MgSO4. After concentration under vacuum, the product was
obtained as a colorless liquid and was stored over dried molecular
sieve before polymerization (yield 82%).
as viscous clear oil (yield 90%). (GPC THF-Mn : 23.5 kDa, PDI:
1.13). 1H NMR (500 MHz, Methanol-d4): δ 4.26 (m, 2H), 4.07 (dd, J
= 8.2, 6.4 Hz, 2H), 3.87−3.44 (br, 6H), 1.40 (s, 3H), 1.35 (s, 3H).
13C NMR (500 MHz, Methanol-d4) δ 110.45 (CH acetal), 76.12 (CH
backbone, CH acetal), 72.76 (CH2), 71.11 (CH2), 67.86 (CH2
backbone, CH2 acetal), 27.42 (CH3 acetal), 25.98 (CH3 acetal).
Polymerization of First-Generation Glycerol Dendrons with
Epoxide. In a flame-dried 50 mL Schlenk flask, N(Oct)4Br (50.4 mg,
0.092 mmol, 0.017 eq) was dried by melting and flushed with argon.
After cooling down, the salt was dissolved in 12 mL of dry toluene.
Thereafter, the monomer (2 mL, 5.3 mmol, 1 eq) was added to afford
50 repeating units per chain, and the mixture was cooled down to 0
°C. Then (1.23 mL of 1.1 M stock solution), i-Bu3Al was added all at
once under brisk stirring. The reaction was allowed to run over night
and was quenched by the addition of 1 mL of water. Then, the
mixture was dried with MgSO4, filtered, and concentrated under
vacuum. The mixture was precipitated in cold diethylether and
dialyzed in acetone with a 2 kDa MWCO membrane for further
purification. The product was obtained as viscous clear oil (yield
1H NMR (500 MHz, Methanol-d4): δ 4.24 (m, 1H), 4.10−3.97 (m,
1H), 3.80 (dt, J = 11.7, 2.3 Hz, 1H), 3.73−3.65 (m, 1H), 3.53 (m,
1H), 3.16−3.07 (m, 1H), 2.75 (m, 1H), 2.57 (dd, J = 5.1, 2.7 Hz,
1H), 1.36 (s, 3H), 1.31 (s, 3H). 13C NMR (500 MHz, Methanol-d4):
δ 109.23 (quaternary C acetal), 76.16 (CH acetal), 74.85 (CH2
acetal), 66.28 (CH2),51.24 (CH epoxide), 43.31 (CH2 epoxide),
25.82 (CH3 acetal), 24.46 (CH3 acetal). ESI-MS: [M + Na]+: 211.09
(theoretical), 211.09 (observed).
Modification of First-Generation Glycerol Dendrons with
Epoxide 10. The synthesis was carried out similar to isopropylidene
glyceryl glycidyl ether (IGG) synthesis. G1-dendrons (10.5 g, 0.032
mol, 1 eq) were dissolved in a mixture of 17 mL of toluene and 17 mL
of 50% NaOH. TBAB (2.11 g, 0.006 mol, 0.2 eq) was added, and the
mixture was cooled down to 10 °C. Epichlorohydrin (13 mL, 0.165
mol, 7 eq) was added dropwise, and the mixture was left to stir for 48
h. The reaction was diluted with diethylether and was extracted three
times with saturated NaHCO3, NaCl, and water. The organic phase
was separated and dried with MgSO4. The product was purified with
flash chromatography (85% cyclohexan, 15% ethylacetate) (yield
70%). The product was dried over activated molecular sieve before
93%). (GPC THF-Mn : 19.34 kDa, PDI: 1.21). 1H NMR (500 MHz,
Methanol-d4): δ 4.25 (m, 2 H), 4.05 (m, 2H), 3.81−3.71 (m, 3H),
3.65−3.45 (m, 9H), 1.38 (m, 6H), 1.33 (m, 6H). 13C NMR (500
MHz, Methanol-d4): δ 109.13 (quaternary C acetal), 78.46(tertiary
CH, G1), 74.79 (tertiary CH, acetal), 71.16 (CH2 acetal), 66.56 (CH2
arms), 26.17 (CH3 acetal), 24.75 (CH3 acetal).
Deprotection of Acetal-Protected PGs 3, 7, 11. The respective
polymers were dissolved in ethanol (1 g in 10 mL), and then HCl
37% (3% of the ethanol) was added to the solution. The mixture was
left to stir overnight and dialyzed in water (MWCO: 2 kDa) for 2
days to afford the desired polymers in quantitative yields.
Sulfation of the PGs. All polymers were sulfated according to an
already published protocol.29 The completely dry corresponding
polymer was dissolved in dry dimethylformamide (10 mL for 1 g).
The mixture was then heated up to 60 °C, and a respective amount of
SO3/pyridine (1.5 eq of −OH groups) was added to it. The reaction
was allowed to run overnight. Thereafter, the pH was brought to 9 by
the addition of 1 M NaOH to the solution. Then, the polymer was
dialyzed against saturated solution of NaCl for 2 days and 2 days in
water. After drying in high vacuum, the crude product was obtained as
a white solid powder. The degree of functionalization was determined
via elemental analysis.
Used cell lines and cell cultivation conditions: For the different cell-
based assays and tests used in this publication, cells were routinely
maintained at 37 °C and 5% CO2 and passaged every 3−4 days after
reaching 70 to 90% confluency or before being used for a test or assay.
A549 human lung carcinoma cells (DSMZ ACC 107) and Vero E6
African green monkey kidney epithelial cells (ATCC CRL-1586) were
cultured in Dulbecco’s Modified Eagle Medium (DMEM), while
16HBE14o human bronchial epithelial cells (Millipore SCC150) were
cultivated in minimum essential media all supplemented with 10%
fetal bovine serum, penicillin/streptomycin, and glutamine (all from
Gibco BRL, Eggenstein, Germany). Cells were routinely tested for the
absence of mycoplasma by a published PCR test protocol using the
primer pair MGSO-(TGCACCATCTGTCACTCTGTTAACCTC-
3′) and GPO-3 (5′- GGGAGCAAACAGGATTAGATACCCT-3′).38
Cell Viability Assay CCK-8. To analyze the cytotoxicity and
determine the half-maximal cytotoxic concentration (CC50) of
compounds, the cell viability assay Cell Counting Kit 8 (CCK-8)
from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany) was
used according to the manufacturer’s instructions. In short, A549,
Vero E6 16HBE14o cells were cultivated as described above and
seeded 24 h prior to the compound treatment in all inner wells of a
96-well plate (4.000 cells/well); in all outer wells, the same cell
culture medium as used for the respective seeded cell line without
cells was added. After an overnight incubation, compounds were
added in serial dilutions in triplicates to the wells containing cells and
additionally in one outer well without cells for subsequent background
subtraction. Sodium dodecyl sulfate (1%) and the nontreated cells
served as a control. The 96-well plates were incubated for another day
1
polymerization. H NMR (500 MHz, Methanol-d4): δ 4.25 (m, 2H),
4.05 (dd, J = 8.3, 6.5 Hz, 2H), 3.91 (dd, J = 11.8, 2.7 Hz, 1H), 3.76−
3.66 (m, 3H), 3.64−3.46 (m, 9H), 3.16−3.12 (m, 1H), 2.76 (dd, J =
5.1, 4.2 Hz, 1H), 2.61 (m, 1H), 1.38 (t, J = 0.7 Hz, 6H), 1.33 (d, J =
1.0 Hz, 6H). 13C NMR (500 MHz, Methanol-d4): δ 110.39
(quaternary C acetal), 79.60 (tertiary CH, G1), 76.06 (tertiary CH,
acetal), 73.34 (CH2), 72.40 (CH2 acetal), 67.51(CH2 arms), 52.02
(CH epoxide), 44.72 (CH2 epoxide), 27.08 (CH3 acetal), 25.69 (CH3
acetal). ESI-MS: [M + Na]+: 399.43 (theoretical), 399.20 (observed),
[M + K]+: 415.54 (theoretical), 415.18 (observed).
All the polymerizations were done according to a reported protocol
for monomer-activated ring-opening polymerization.36
Polymerization of Ethoxyethyl Glycidyl Ether. In a flame-
dried Schlenck flask, Oct4NBr (172.78 mg, 0.310 mmol, 0.0047 eq)
was dried under high vacuum and dissolved in 60 mL of dry toluene.
Afterward, ethoxyethyl glycidyl ether (10 mL, 65.6 mmol, 1 eq) was
added under an argon atmosphere to the solution to afford 200
repeating units per chain. The mixture was cooled down in an ice bath
to 0 °C, and i-Bu3Al (1.4 mL, 1.55 mmol, 0.023 eq) was added all at
once under an argon atmosphere and brisk stirring. The reaction was
left to proceed overnight, which was quenched by the addition of 1
mL of ethanol. The crude product was dissolved in cold Et2O to
precipitate the excess i-Bu3Al. The product was dialyzed in acetone
(MWCO: 2 kDa) for further purification. After drying, the product
was obtained as 8.38 g colorless viscous oil (50%). (GPC THF-Mn :
32.4 kDa, PDI: 1.07). 1H NMR (500 MHz, Acetone-d6): δ 4.81−4.60
(m, 1H), 3.83−3.32 (m, 7H, monomer unit), 1.33−1.22 (m, 3H),
1.14 (t, J = 7.1 Hz, 3H). 13C NMR (500 MHz, Acetone-d6): δ 102.69
(tertiary CH acetal), 79.12 (CH backbone), 70.24 (CH2), 65.19
(CH2 backbone), 62.37 (CH2 acetal), 19.55 (CH3 acetal), 15.03
(CH3 acetal).
Polymerization of 1,2-IGG (DenPG0.5). In a flame-dried Schlenk
flask, N(Oct)4Br (50.2 mg, 0.089 eq, 0.09 mmol) was dried by
melting and flushed with argon. After cooling down, the salt was
solved in 11 mL of dry toluene. Thereafter, the monomer (1.8 mL,
11.3 mmol, 1 eq) was added to afford 100 repeating units per chain
and the mixture was cooled down to 0 °C. Then, i-Bu3Al (0.4 mL of
1.1 M stock solution) was added all at once under brisk stirring. The
reaction was allowed to run overnight and was quenched by the
addition of 1 mL of ethanol and concentrated under vacuum. The
activator was precipitated in cold Et2O and dialyzed in acetone
(MWCO: 2 kDa) for further purification. The product was obtained
1547
Biomacromolecules 2021, 22, 1545−1554