M. Breurken et al. / Bioorg. Med. Chem. 19 (2011) 1062–1071
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4.3. Preparation of multivalent CNA35 ligands
2H, J = 7.3 Hz); 6.96–6.87 (m, 3H); 6.20 (br s, 1H); 5.38 (br s, 1H);
4.38 (d, 2H J = 6.2 Hz); 4.13 (t, 1H, J = 7.0 Hz); 3.74 (s, 2H); 2.67
(t, 6H, J = 7.5); 2.20 (t, 6H, J = 7.7 Hz). 13C NMR (CDCl3, 50 MHz):
d 167.7; 147.0; 142.2; 139.9; 136.8; 126.4; 125.7; 123.6; 118.6;
Both dendritic wedges AB2 and AB4 were synthesized as de-
scribed before.33 Native chemical ligation reactions were per-
formed in 20 mM sodium phosphate, 500 mM NaCl, 50 mM
101.9; 66.0; 56.2; 45.7; 44.2; 28.4; 26.3. IR (cmꢁ1):
m
3305, 3080,
MPAA and 2 mM TCEP at a protein concentration of ꢀ450
lM. A
2910, 1786, 1685, 1524, 1488, 1265, 1178, 1097, 957, 760. MAL-
DI-TOF-MS: calcd
993.17.
four and eightfold molar excess CNA35 MESNA thioester was used
for reactions with AB2 and AB4, respectively. The pH was adjusted
to ꢀ6.2 with 1 M sodium phosphate, pH 9.2. After overnight incu-
bation at room temperature, the reaction mixture was buffer ex-
changed to HBS, pH 7.4 to remove MPAA and TCEP by repeated
concentration and dilution using an Amicon Ultra-4 centrifugal
concentrator with a 10 kDa cut-off. The reaction mixtures were
purified using a Superdex 75 size exclusion column (GE Health-
care) connected to an ÅKTA FPLC system (GE Healthcare) using
HBS as a running buffer at a flow rate of 0.8 mL/min.
C
45H30F12N2O9Na+: m/z 993.16; found m/z
4.4.4. tBoc-aminooxy-PEG-N3
tBoc-aminooxy-OSu (500 mg, 2.4 mmol) was dissolved in DCM
(5 mL). To this solution dry DIPEA (1.2 mL, 6.8 mmol) and NH2-
PEG-N3 (800 mg, 2.3 mmol) were added. After stirring for 22 h at
room temperature, the product was purified by a DOWEX MONOS-
PERE 55 Å anion exchange column (in methanol). Evaporation of
the solvent and drying in vacuo resulted in the title compound as
a yellow oil (192 mg, 40%). 1H NMR (CDCl3, 400 MHz): d 7.91 (br
s, 1H); 7.75 (br s, 1H); 4.27 (s, 2H); 3.62–3.31 (m, 28H); 1.42 (s,
9H). 13C NMR (CDCl3, 100 MHz): d 168.7; 156.9; 82.0; 75.4; 70.1;
4.4. Synthesis of the collagen mimic
4.4.1. Fmoc-Gly-(tBu)3 (2)
50.2; 38.5; 27.7. IR (cmꢁ1):
m 2871, 2102, 1660, 1251, 1099, 946,
Fmoc-Gly-OH (894 mg, 3.008 mmol) and DIPEA (1.213 g,
9.385 mmol) were dissolved in DMF (20 mL) before the addition
of aminotriester (1) (1.000 g, 2.406 mmol). The mixture was cooled
to 0 °C and HATU (1.189 g, 3.128 mmol) was subsequently added
as a solid. After stirring for 17 h at room temperature, the solvent
was evaporated under reduced pressure. DCM (100 mL) was added
and the mixture was washed with an aqueous saturated NaHCO3
solution (3 ꢂ 100 mL). After drying on MgSO4 and concentration,
the crude product was purified by flash chromatography (DCM, fol-
lowed by DCM/MeOH; 98:2). The title compound was obtained as a
white solid (1.442 g, 72%). 1H NMR (CDCl3, 200 MHz): d 7.69 (d, 2H,
J = 6.6 Hz); 7.53 (d, 2H, J = 6.6); 7.28 (m, 4H); 6.42 (s, 1H); 5.40 (br
s, 1H); 4.34 (d, 2H, J = 7.0 Hz); 4.16 (t, 1H, J = 6.6 Hz); 3.73 (d, 2H,
J = 4.3 Hz); 2.16 (t, 6H, J = 7.8 Hz); 1.91 (t, 6H, J = 7.4 Hz); 1.37 (s,
27H). 13C NMR (CDCl3, 50 MHz): d 172.7; 169.0; 156.9; 144.4;
141.2; 128.1; 127.6; 125.8; 120.6; 80.1; 66.2; 57.1; 47.1; 43.8;
850, 773. MALDI-TOF-MS: calcd C21H41N5O10H+: m/z 546.27; found
m/z 546.26.
4.4.5. tBoc-aminooxy-PEG-NH2 (5)
tBoc-aminooxy-PEG-N3 (303 mg, 0.579 mmol) was dissolved in
methanol (20 mL) under an inert atmosphere. Pd/C (10% Pd, 60 mg)
in methanol (10 mL) followed by H2. After stirring for 3 h at room
temperature the reaction mixture was filtered through a short plug
of Celite to remove the Pd/C. Evaporation under reduced pressure
yielded the product as a yellow oil (272 mg, 95%) 1H NMR (CDCl3,
400 MHz): d 7.74 (br s, 1H); 4.27 (s, 2H); 3.57–3.41 (m, 28H); 1.41
(s, 9H). 13C NMR (CDCl3, 100 MHz): d 169.3; 157.6; 82.3; 70.6;
41.8; 39.0; 28.3. IR (cmꢁ1):
m 3301, 2871, 1725, 1667, 1548,
1458, 1368, 1350, 1285, 1253, 1110, 951, 851. MALDI-TOF-MS:
calcd C21H43N3O10H+: m/z 498.30; found m/z 498.42.
29.5; 28.2. IR (cmꢁ1):
1314, 1230, 1145, 1100, 1044, 848, 785, 738. MALDITOF-MS: calcd
39H54N2O9Na+: m/z 717.56; found m/z 717.56.
m
3297, 2976, 1721, 1655, 1525, 1366,
4.4.6. Fmoc-Gly-(tBoc-aminooxy)3 (6)
DIPEA (64 mg, 0.515 mmol) and tBoc-aminooxy-PEG-NH2 (5)
(85 mg, 0.170 mol) were dissolved in dry DCM (5 mL). After adding
Fmoc-Gly-(TFP)3 (4) (50 mg, 0.052 mmol) in DCM (2 mL), the mix-
ture was stirred for 5 h at room temperature. The reaction mixture
was diluted with DCM (13 mL), extracted with an aqueous satu-
rated NaHCO3 solution (3 ꢂ 25 mL), filtered and dried overnight
in vacuo. This resulted in the title compound as a yellow oil
(90 mg, 88%). 1H NMR (CDCl3, 400 MHz): d 8.57 (br s, 3H); 7.88
(br s, 3H); 7.68 (d, 2H, J = 7.0 Hz); 7.55 (d, 2H, J = 7.3 Hz); 7.31 (d,
2H, J = 7.0 Hz); 7.25 (d, 2H, J = 7.7 Hz); 6.75 (br s, 3H); 6.19 (br s,
1H); 4.36 (d, 2H, J = 6.6 Hz); 4.27 (s, 6H); 4.16 (t, 1H, J = 6.7 Hz);
3.72 (d, 2H, J = 5.1 Hz); 3.58–3.22 (m, 84H); 2.14 (t, 6H,
J = 7.5 Hz); 1.95 (d, 6H, J = 7.3); 1.40 (s, 27H). 13C NMR (CDCl3,
50 MHz): d 172.1; 168.3; 156.5; 143.3; 140.1; 127.7; 126.5;
124.2; 120.0; 106.8; 81.2; 69.4; 67.7; 56.9; 46.2; 43.9; 38.3;
C
4.4.2. Fmoc-Gly-(OH)3 (3)
Fmoc-Gly-(tBu)3 (2) (1.000 g, 1.504 mmol) was dissolved in for-
mic acid (10 mL) and stirred for 8 h. Evaporation of formic acid un-
der reduced pressure was followed by co-evaporation with toluene
(3 ꢂ 25 mL). After drying in vacuo, the product was obtained
(739 mg, 93%). 1H NMR (DMSO-d6, 400 MHz): d 7.89 (d, 2H,
J = 7.7 Hz); 7.71 (d,2H, J = 7.3 Hz) 7.41 (t, 2H, J = 7.1 Hz); 7.33 (t,
2H, J = 7.3 Hz); 4.28 (d, 2H, J = 6.2 Hz), 3.85 (s, 1H), 3.57 (d, 2H,
J = 5.9 Hz); 2.13 (t, 6H, J = 7.9 Hz); 1.83 (t, 6H, J = 7.7 Hz). 13C
NMR (DMSO-d6, 50 MHz): d 174.5; 168.7; 156.6; 143.9; 141.8;
127.7; 127.1; 125.3; 120.2; 65.5; 56.4; 46.9; 43.5; 29.1; 28.0. IR
(cmꢁ1):
1171, 1136, 1083, 1049, 933, 759, 741. MALDI-TOF-MS: calcd
27H30N2O9Na+: m/z 549.18; found m/z 549.30.
m 3333, 2955, 1731, 1698, 1639, 1533, 1451, 1312, 1258,
37.9; 29.4; 28.7; 27.2. IR (cmꢁ1):
m 3301, 2871, 1721, 1650, 1546,
C
1452, 1368, 1349, 1280, 1251, 1101, 948, 849, 785, 739, 668. MAL-
DI-TOF-MS: calcd C90H153N11O36Na+: m/z 1988.22; found m/z
1988.03.
4.4.3. Fmoc-Gly-(TFP)3 (4)
2,3,5,6-Tetrafluorophenol (90 mg, 0.597 mmol), DMAP (7 mg,
0.054 mmol) and Fmoc-Gly-(OH)3 (3) (95 mg, 0.181 mmol) were
dissolved in dry DCM (2 mL). Subsequently, 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride (114 mg, 0.596 mmol)
in DCM (1 mL) was added. After stirring for 23 h at room temper-
ature, the reaction mixture was purified by flash chromatography
using a gradient of CHCl3 to CHCl3 containing 6% Et2O. Drying of
the product in vacuo resulted in the title compound as a white
foam (89 mg, 51%). 1H NMR (CDCl3, 400 MHz): d 7.66 (d, 2H,
J = 7.3 Hz); 7.49 (d, 2H, J = 7.3 Hz); 7.30 (t, 2H, J = 7.3 Hz); 7.22 (d,
4.4.7. NH2-(tBoc-aminooxy)3 (7)
Fmoc-Gly-(tBoc-aminooxy)3 (6) (420 mg, 0.229 mmol) was dis-
solved in 20% v/v piperidine in DCM (15 mL) and stirred for 2.5 h at
room temperature. The reaction mixture was diluted with DCM
(100 mL), washed with phosphate buffer containing 1 M NaCl pH
5.5 (5 ꢂ 100 mL), dried on MgSO4 and filtered. After evaporation
of the solvent under reduced pressure, the product was obtained
as a yellow oil (385 mg, 95%). 1H NMR (CDCl3, 400 MHz): d 4.33
(s, 6H); 3.76 (s, 2H); 3.69–3.39 (m, 84H); 2.25 (s, 6H); 2.03