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J. Pan et al. / Tetrahedron 68 (2012) 2943e2949
layer was dried over anhydrous Na2SO4. After concentration, the
residue was purified by silica-gel column chromatography (petro-
leum ether/ethyl acetate) to obtain 13 as colorless oil (1.24 g, yield:
(10 ml) was added dropwise to the reaction. The reaction mixture
was vigorously stirred at room temperature for 2 h. The solution
was then concentrated under vacuum, and the residue was taken
up in ethyl acetate (30 ml) and washed with 1 M HCl (10 ml), 1 M
NaHCO3 (10 ml), and brine (10 ml). The organic layer was dried over
anhydrous Na2SO4. After concentration, the residue was purified
by silica-gel column chromatography (CH2Cl2/MeOH) to obtain
a white waxy solid. Pd/C of 10% (100 mg) was added to a solution of
the obtained white waxy solid in methanol/CH2Cl2 (3:1 v/v 30 ml).
The reaction mixture was stirred under hydrogen atmosphere for
24 h, filtered through membrane filter and, concentrated under
reduced pressure to afford 2a as white foam. (411 mg, yield: 66%).
41%). 1H NMR (CDCl3)
d
: 1.50e1.52 (m, 12H, CHCH3ꢃ4), 2.21e2.68
(m, 12H, succinic acid-CH2CH2ꢃ3), 2.92e3.73 (m, 12H, NCH2ꢃ6),
3.82e3.89 (m, 16H, OCH3ꢃ4þCHCH3ꢃ4), 3.99e4.17 (m, 12H,
NCH2CH2ꢃ6), 5.10 (s, 2H, ph-CH2), 7.06e7.66 (m, 7H, ph-HþNap-
phH), 7.26e7.32 (m, 10H, Nap-phH), 7.59e7.66 (m, 12H, Nap-phH).
ESI MS (m/z): calcd for C87H95N3O21: 1517.65; obsd 1539.42
([MþNaꢁH]þ). Anal. Calcd for C, 68.80; H, 6.31; N, 2.77; O, 22.12.
Found: C, 68.72; H, 6.25; N, 2.73; O, 22.01.
4.2.9. Synthesis of the dendrimer 1a. Compound 12 (315 mg,
0.5 mmol) dissolved in anhydrous THF (25 ml) was cooled at
ꢁ15 ꢀC, then added NMM (10 mmol) and IBCF (10 mmol), stirred
for 10 min, 6a (404 mg, 0.5 mmol) dissolved in anhydrous THF
(10 ml) was added dropwise to the reaction. The reaction mixture
was vigorously stirred at room temperature for 2 h. The solution
was then concentrated under vacuum, and the residue was taken
up in ethyl acetate (30 ml) and washed with 1 M HCl (10 ml), 1 M
NaHCO3 (10 ml), and brine (10 ml). The organic layer was dried over
anhydrous Na2SO4. After concentration, the residue was purified
by silica-gel column chromatography (CH2Cl2/MeOH) to obtain
a white waxy solid. Pd/C of 10% (100 mg) was added to a solution of
the obtained white waxy solid in methanol/CH2Cl2 (3:1 v/v 30 ml).
The reaction mixture was stirred under hydrogen atmosphere for
24 h, filtered through membrane filter, and concentrated under
reduced pressure to afford 1a as white foam (376 mg, yield: 71%).
1H NMR (DMSO-d6)
d
: 1.40e1.42 (m, 12H, CHCH3ꢃ4), 2.26e2.75 (m,
32H, succinic acid CH2ꢃ6þAsp- -CH2ꢃ4þNCH2CH2CONHꢃ6),
b
3.17e3.66 (m, 24H, NCH2CH2Oꢃ6þNCH2CH2CONHꢃ6), 3.83 (s, 12H,
OCH3ꢃ4), 3.93e4.12 (m, 22H, NCH2CH2Oꢃ6þCHCH3ꢃ4þGly-
CH2ꢃ3), 4.49e4.55 (m, 4H, Asp- -CHꢃ4), 7.11e7.33 (m, 12H, Nap-
a
phH), 7.64e7.75 (m, 12H, Nap-phH), 7.85 (br s, 1H, CONH), 7.95 (br s,
1H, CONH), 8.08 (br s, 1H, CONH), 8.26 (br s, 2H, CONHꢃ2), 8.39 (br
s, 2H, CONHꢃ2), 12.55 (br s, 8H, Asp-COOHꢃ8). ESI TOF MS (m/z):
calcd for C120H145N13O45: 2487.95; obsd 2487.85 ([M]ꢁ). Anal. Calcd
for C, 57.89; H, 5.87; N, 7.31; O, 28.92. Found: C, 57.75; H, 5.74; N,
7.26; O, 28.84.
4.2.12. Synthesis of the dendrimer 2b. The same procedure as de-
scribed above for preparation of 2a. Dendrimer 2b: white foam
(401 mg, yield: 63%). 1H NMR (DMSO-d6)
d: 1.41e1.42 (m, 12H,
CHCH3ꢃ4), 1.77e1.97 (m, 8H, Glu-
b
-CH2ꢃ4), 2.27 (br s, 8H, Glu-
1H NMR (DMSO-d6)
d
: 1.41e1.44 (m, 6H, CHCH3ꢃ2), 2.31e2.70 (m,
g
CH2ꢃ4), 2.36e2.51 (m, 24H, succinic acid CH2ꢃ6þNCH2CH2
12H, succinic acid CH2ꢃ2þAsp-
b
-CH2ꢃ2þNCH2CH2CONHꢃ2),
CONHꢃ6), 3.24e3.67 (m, 24H, NCH2CH2Oꢃ6þNCH2CH2CONHꢃ6),
3.84 (s, 12H, OCH3ꢃ4), 3.94e4.12 (m, 22H, NCH2CH2Oꢃ6þ
3.19e3.45 (m, 8H, NCH2CH2Oꢃ2þNCH2CH2CONHꢃ2), 3.86 (s, 6H,
OCH3ꢃ2), 3.94e4.06 (m, 8H, NCH2CH2Oꢃ2þCHCH3ꢃ2þGly-
CHCH3ꢃ4þGly-CH2ꢃ3), 4.18e4.20 (m, 4H, Glu- -CHꢃ4), 7.11e7.33
a
CH2ꢃ1), 4.50e4.52 (m, 2H, Asp-
a-CHꢃ2), 7.12e7.33 (m, 4H, Nap-
(m, 12H, Nap-phH), 7.65e7.75 (m, 12H, Nap-phH), 7.86 (br s, 1H,
CONH), 7.96 (br s, 1H, CONH), 8.09 (br s, 1H, CONH), 8.18 (br s, 2H,
CONHꢃ2), 8.31 (br s, 2H, CONHꢃ2),12.40 (br s, 8H, Asp-COOHꢃ8). ESI
TOF MS (m/z): calcd for C124H153N13O45: 2544.01; obsd2544.92 ([M]ꢁ).
Anal. Calcd for C, 58.51; H, 6.06; N, 7.15; O, 28.28. Found: C, 58.43; H,
6.00; N, 7.06; O, 28.15.
phH), 7.64e7.78 (m, 6H, Nap-phH), 7.80 (br s, 1H, CONH), 8.24 (br s,
1H, CONH), 8.37 (br s, 1H, CONH), 12.57 (br s, 4H, Asp-COOHꢃ4). ESI
MS (m/z): calcd for C52H61N5O19: 1059.4; obsd 1059.52 ([M]ꢁ). Anal.
Calcd for C, 58.92; H, 5.80; N, 6.61; O, 28.68. Found: C, 58.83; H,
5.69; N, 6.49; O, 28.60.
4.2.10. Synthesis of the dendrimer 1b. The same procedure as de-
scribed above for preparation of 1a. Dendrimer 1b: white foam
4.3. In vitro HAP binding assay
(408 mg, yield: 75%). 1H NMR (DMSO-d6)
d: 1.42e1.44 (m, 6H,
The dendrimers 1a,b, 2a,b were dissolved in PBS (pH¼7.4), re-
spectively, with various precise concentrations and the adsorption
amounts were determined by an UV spectrophotometer at 273 nm
to obtain the AeC linear regression equation. Then, in tubes, 0.05 M
1a,b, 2a,b were dissolved in 10 ml PBS (pH¼7.4), respectively
(C0¼5 mmol/ml), and incubated with HAP 20 mg in a shaker bath at
37 ꢀC. Each tube was cultured for 1 h and 24 h. After the prescribed
time, tubes were centrifuged for 3 min at 5000 rpm. The adsorption
amounts were determined by UV at 273 nm and C was obtained by
the special equation. The bound percentage was calculated by
(C0ꢁC)/C0ꢃ100% and the result was presented in Fig. 1.
CHCH3ꢃ2), 1.71e1.96 (m, 4H, Glu-
b
-CH2ꢃ2), 2.26 (br s, 4H, Glu-
g
CH2ꢃ2), 2.31e2.47 (m, 8H, succinic acid CH2ꢃ2þNCH2CH2CONHꢃ2),
3.19e3.47 (m, 8H, NCH2CH2Oꢃ2þNCH2CH2CONHꢃ2), 3.88 (s, 6H,
OCH3ꢃ2), 3.93e4.06 (m, 8H, NCH2CH2Oꢃ2þCHCH3ꢃ2þGly-CH2ꢃ1),
4.17e4.22 (m, 2H, Glu-
a
-CHꢃ2), 7.12e7.34 (m, 4H, Nap-phH),
7.67e7.81 (m, 6H, Nap-phH), 7.81 (br s, 1H, CONH), 8.16 (br s, 1H,
CONH), 8.28 (br s, 1H, CONH), 12.43 (br s, 4H, Asp-COOHꢃ4). ESI MS
(m/z): calcd for C54H65N5O19: 1087.43; obsd 1087.89 ([M]ꢁ). Anal.
Calcd for C, 59.61; H, 6.02; N, 6.44; O, 27.94. Found: C, 59.49; H, 5.93; N,
6.32; O, 27.88.
4.2.11. Synthesis of the dendrimer 2a. To the solution of trifluoro-
acetic acid TFA/anhydrous CH2Cl2 (1:1, v/v), 7a (475 mg,
0.25 mmol) was added at room temperature. When the protecting
groups were completely removed, the solvent was rotary evapo-
rated to obtain 8a (yield: 99%). The mixture of 13 (379 mg,
0.25 mmol) and 10% Pd/C (100 mg) in methanol/CH2Cl2 (5:1 v/v
30 ml) was stirred at the room temperature under hydrogen at-
mosphere. After 24 h the mixture passed through a membrane
filter to remove the catalyst, then evaporated under reduced
pressure to afford 14 (yield: 99%).
4.4. Solubility assessment
The solubility of the Janus dendrimers was determined at
pH¼7.4 in PBS. The method used for sample preparation was sim-
ilar to each system,38 i.e., AeC linear regression equation was ob-
tained in 4.3. Excess compounds 1a,b, 2a,b were added to 1 ml of
the PBS (pH¼7.4) solution, respectively, to ensure the compound
solution reaching saturation. The solutions were mechanically
shaken for 10 min at 37 ꢀC and centrifuged at 10,000 rpm for
10 min. Supernatant of 200 ml was pipetted and diluted by PBS
Without further purification, 14 dissolved in anhydrous THF
(25 ml) was cooled at ꢁ15 ꢀC, then added NMM (0.25 mmol) and
IBCF (0.25 mmol), stirred for 10 min, 8a dissolved in anhydrous THF
(pH¼7.4) as proper multiple to make the concentration of the di-
luted solution in the range of the obtained AeC linear regression
equation. The adsorption amounts were determined by UV at