K.M. Tewari et al.
Bioorganic Chemistry 109 (2021) 104667
OH, Fmoc-Arg(Pmc)-OH, or Fmoc-Ala-OH), 3 eq of PyBOP as coupling
agent, 6 eq of DIEA as base and DMF (6.0 mL) as solvent. The peptide
resin was washed thoroughly with DMF (2 × 5.0 mL), DCM (2 × 5.0 mL),
MeOH (5.0 mL) and Et2O (2 × 5.0 mL), and dried in vacuo to give 8 (1.3
g) of peptide resin. The final loading of the peptide was found to be
0.745 mmol/g by Fmoc loading test.
4.4.9. Dend-(Tris-ALA)3-LARLLT-OH. 10TFA (13)
4.4.9.1. Click coupling AT RT. A solution of peptide-targeted core unit
11 (5 mg, 3.55 µmol) in DMSO (100 µL) was treated with 4 (18 mg, 21
µmol) and copper(I) trifluoromethanesulfonate benzene complex (11
mg, 21 µmol), and the reaction mixture was stirred for 36 h and moni-
tored by analytical HPLC (Gradient 1) which showed the disappearance
of the starting material and appearance of new species at 9.94 min. The
reaction mixture was diluted with 0.1% aq. TFA and the product was
isolated by semi-preparative HPLC (Gradient 2) and freeze-dried from
H2O to give Boc-protected peptide-targeted dendrimer 12 (9.5 mg,
69%).
4.4.6. H-Leu-Ala-Arg-Leu-Leu-Thr-OH. 2TFA (9)
Peptide resin 8 (100 mg, mmol/g) was placed in a 5 mL vial and was
treated with TFA/TIS/H2O (2 mL, 95:2.5:2.5 v/v/v) for 3 h at RT. The
resin beads were filtered off, washed with TFA, and the combined fil-
trates were collected into a Falcon tube containing Et2O (5.0 mL) to
precipitate the peptide. The resulting precipitate was collected by
centrifugation and was washed repeatedly with Et2O to remove excess
TFA. The precipitated material was dissolved in 0.1% aq. TFA, filtered
using a 0.2 µm syringe filter and the resulting solution was directly
purified by semi-preparative HPLC (Gradient 1). The purified peptide
was then freeze-dried from H2O to give 9 as a white solid (55 mg, 85%);
Analytical HPLC (Gradient 1) Rt = 5.27 min; 1H NMR (500 MHz,
CD3OD) δ 0.93–1.04 (m, 18H), 1.18 (d, J = 6.0, 3H), 1.41 (d, J = 7.0,
3H), 1.63–1.79 (m, 12H), 1.85–1.92 (m, 1H), 3.21 (t, J = 7.0, 2H),
3.90–3.92 (m, 1H), 4.32–4.50 (m, 6H); 13C NMR (125 MHz, CD3OD) δ
17.99, 20.55, 21.86, 21.99, 22.05, 23.15, 23.47, 25.35, 25.81, 25.88,
25.96, 30.19, 41.70, 41.72, 41.97, 42.03, 50.53, 52.83, 53.18, 53.40,
53.95, 58.97, 68.44, 158.65, 170.57, 173.36, 173.47, 174.33, 174.89;
[Found (ESI+) 686.4605 [M+H]+, C31H60N9O8 requires 686.4559].
4.4.9.2. Under microwave irradiation. A solution of peptide-targeted
core unit 11 (5 mg, 3.55 µmol) in DMSO (100 µL) was treated with 4
(18 mg, 21 µmol) and copper(I) trifluoromethanesulfonate benzene
complex (11 mg, 21 µmol), and the reaction was carried out in a mi-
crowave (MW) reaction vial (Biotage Initiator Microwave reactor)
which was irradiated for 10 min (at constant power, 10–15 W; temp =
70 ◦C). The reaction was allowed to reach RT and the vial was removed
from the MW cavity. The reaction mixture was analysed by HPLC
(Gradient 1) which showed complete disappearance of the starting
material 11 and formation of a new species at 9.94 min. The product was
isolated by semi-preparative HPLC and freeze-dried from H2O to give
the Boc-protected peptide-targeted dendrimer 12 (10.3 mg, 75%).
Analytical HPLC (Gradient 1) Rt = 9.94 min; [Found (ESI+) 1915.9682
[M+2H]2+, C173H278N30O66 requires 1915.9654]. The resulting product
12 was dissolved in dry DCM (3 mL) and the reaction mixture cooled to
0 ◦C. TFA (3 mL) was added dropwise and the reaction mixture stirred
for 30 min at RT and monitored by analytical HPLC (Gradient 1) which
showed complete disappearance of 12 and appearance of new species at
5.18 min. The solvent was evaporated under reduced pressure and the
residue was co-evaporated with Et2O (x 3) to remove excess TFA. This
material was freeze dried from H2O to give final product 13 as a white
4.4.7. Azido PEG gallic acid spacer-H-Leu-Ala-Arg(Pmc)-Leu-Leu-Thr
(tBu)-2-Chlorotrityl resin (10)
Peptide resin 8 (115 mg, 65.5 µmol) was swollen in DCM (3.0 mL) for
10 min and then in DMF (3 × 3.0 mL) for 5 min. A solution of 5 (84.0 mg,
131 µM) in DMF (400 µL) was treated with HATU (49.0 mg, 131 µmol)
followed by DIEA (45.0 µL, 262 µmol). After 3 min of preactivation, the
mixture was added to the peptide resin. The resin was shaken for 24 h,
then the solvent was removed and the resin beads were washed with
DMF (3 × 2.0 mL), DCM (3 × 2.0 mL), MeOH (2.0 mL) and DCM (2 ×
2.0 mL). Successful attachment of 10 was confirmed by a negative Kaiser
test.
1
solid (9.1 mg, 95%); Analytical HPLC (Gradient 1) Rt = 5.18 min; H
NMR (500 MHz, CD3OD) δ 0.92–0.94 (m, 6H), 0.97–1.03 (m, 12H),
1.19–1.23 (m, 3H), 1.38–1.43 (m, 3H), 1.66–1.85 (m, 12H), 1.92–1.95
(m, 1H), 2.61 (t, J = 7.5, 6H), 2.68–2.73 (m, 18H), 2.87–2.91 (m, 18H),
2.94 (t, J = 7.0, 6H), 3.21 (t, J = 7.0, 2H), 3.64–3.70 (m, 12H),
3.74–3.79 (m, 2H), 3.84–3.87 (m, 4H), 3.88–3.92 (m, 6H), 4.08 (s, 18H),
4.18–4.21 (m, 2H), 4.22–4.25 (m, 4H), 4.28–4.31 (m, 1H), 4.33–4.37
(m, 2H), 4.38–4.45 (m, 20H), 4.46–4.52 (m, 1H), 4.54–4.59 (m, 7H),
7.29–7.33 (m, 2H), 7.79–7.83 (m, 3H); 13C NMR (125 MHz, CD3OD) δ
17.50, 20.50, 21.78, 22.23, 23.38, 23.56, 23.60, 25.75, 25.91, 26.09,
28.27, 35.31, 36.46, 41.34, 41.61, 41.94, 48.14, 51.25, 51.32, 53.36,
53.61, 54.50, 54.85, 59.10, 63.29, 68.57, 69.97, 70.44, 70.73, 71.28,
71.43, 71.57, 71.65, 73.63, 108.08, 124.32, 147.59, 153.66, 158.58,
173.47, 173.55, 174.94, 175.08, 203.26; [Found (ESI+) 977.4865
[M+3H]3+, C128H204N30O48 requires 977.4888].
4.4.8. Azido PEG gallic acid spacer-H-Leu-Ala-Arg-Leu-Leu-Thr-OH. TFA
(11)
Peptide resin 10 (50 mg, 37.7 µmol) was placed in a 3 mL vial and
was treated with TFA/TIS/H2O (1 mL, 95:2.5:2.5 v/v/v) for 3 h at RT.
The resin beads were filtered off, washed with TFA, and the combined
filtrates were collected into a Falcon tube containing Et2O (5.0 mL) to
precipitate the peptide. The resulting precipitate was collected by
centrifugation and was washed repeatedly with Et2O. The precipitated
material was dissolved in 0.1% aq. TFA, filtered using a 0.2 µm syringe
filter and the resulting solution was purified by semi-preparative HPLC
(Gradient 2). The purified peptide was then freeze-dried from H2O to
give 11 as the TFA salt (29 mg, 54%); Analytical HPLC (Gradient 1) Rt =
8.44 min; 1H NMR (500 MHz, CD3OD) δ 0.90–1.02 (m, 18H), 1.18 (d, J
= 6.5, 3H), 1.38 (d, J = 7.0, 3H), 1.63–1.79 (m, 12H), 1.87–1.94 (m,
1H), 3.16–3.19 (m, 2H), 3.34–3.38 (m, 6H), 3.63–3.69 (m, 12H),
3.71–3.75 (m, 6H), 3.81–3.85 (m, 2H), 3.87–3.92 (m, 4H), 4.21–4.36
(m, 9H), 4.38–4.42 (m, 2H), 4.45–4.51 (m, 2H), 7.25 (s, 2H); 13C NMR
(125 MHz, CD3OD) δ 17.44, 20.49, 21.76, 21.82, 22.21, 23.34, 23.54,
23.57, 25.77, 25.92, 26.00, 26.11, 29.89, 41.38, 41.63, 41.70, 41.94,
51.26, 51.79, 53.32, 53.48, 54.27, 54.82, 59.04, 68.58, 70.25, 70.94,
71.15, 71.62, 71.65, 71.89, 73.65, 108.37, 153.84, 173.29, 173.90,
174.85; [Found (ESI+) 677.3840 [M+2Na]2+, C56H96N18O18Na2 re-
quires 677.3467].
4.5. Cell lines and cultivation
Cell culturing was carried out in 10% FCS DMEM (high glucose
Dulbecco’s modified Eagle’s medium) with 30 mM NaHCO3, 2 mM L-
glutamine, and 50 IU/mL of each of penicillin/ streptomycin. The FCS
◦
stock was heat-inactivated at 56 C for 45 min before use. Cells were
passaged once every 5 days and seeded at a density of 8 × 103 cells per
well in media (200 µL), and grown for 24 h prior to the treatment with
compounds for 4 or 24 h, depending on the experimental requirement.
4.6. Time course fluorescence studies
Cells were seeded into clear 96-well cell culture micro-plates
(CELLSTAR®, Greiner Bio-one, United Kingdom) at a density of 8 ×
103 cells per well for 24 h. The culture medium was removed and each
7