Journal of the American Chemical Society
ARTICLE
3.32 (br s, H), 1.26 (t, J = 7.2 Hz, 6 H); 13C NMR (100 MHz, CD3OD)
δ 168.1, 165.4, 164.0, 159.2, 154.7, 149.4, 138.1, 137.0, 133.7, 132.7,
131.9, 131.0, 127.8, 111.7, 110.1, 109.5, 97.3, 71.5, 71.4, 71.3, 70.5, 70.5,
69.5, 67.9, 54.8, 46.0, 40.9, 40.7, 40.3, 12.7; MALDI-TOF-MS calcd for
C31H43N4O10S [M + 1]+ 663.2622, found 663.2451.
Synthesis of 3. The stirred solution of 3,5-bis(trifluoromethyl)-
benzaldehyde (283 μL, 1.72 mmol, 1.3 equiv), 4,40-dimethoxybenzil
(464 mg, 1.72 mmol, 1.3 equiv), 4-(aminomethyl)benzoic acid (200 mg,
4 mmol, 1 equiv), and ammonium acetate (612 mg, 7.94 mmol, 6 equiv)
in acetic acid (6 mL) was heated for 12 h at 100 °C. The reaction mixture
was cooled to room temperature, diluted with ethyl acetate, and washed
with water, saturated NaHCO3, and brine. The organic layer was dried
over MgSO4, filtered, and concentrated under the reduced pressure. The
crude product was purified by flash column chromatography (CH2Cl2/
MeOH 10:1) to give 3 in 48% yield: 1H NMR (CDCl3, 400 MHz) δ 8.07
(s, 2H), 8.00 (d, J = 8.4 Hz, 2H), 7.83 (s, Hz, 1H), 7.52 (dd, J = 6.8, 2.4
Hz, 2H), 7.18 (dd, J = 6.8, 2.0 Hz, 2H), 6.99 (d, J = 8.4, 2H), 6.89 (dd, J =
6.9, 2.0 Hz, 2H), 6.80 (dd, J = 6.8, 2.0 Hz, 2H), 5.16 (s, 2H), 3.82 (s, 3H),
3.77 (s, 3H); 13C NMR (DMSO, 100 MHz) δ 167.0, 159.7, 158.1, 143.3,
141.8, 137.6, 133.0, 132.2, 130.8, 130.6, 130.4, 129.7, 128.4, 127.4,
126.7, 125.8, 124.4, 121.8, 121.7, 114.7, 113.7, 55.1, 55.0, 47.7; MALDI-
TOF-MS calcd for C33H25F6N2O4 [M + 1]+ 627.1713, found 627.1136.
Synthesis of Az-Conjugated Probe 1. To a solution of 2 (3 mg,
4.5 μmol, 1 equiv) in anhydrous DMF (5 mL) were added 3 (2 mg,
10.0 μmol, 2.2 equiv), DIEA (3.9 μL, 22.6 μmol, 5 equiv), and 1-ethyl-3-
(3-dimethylaminopropyl)-carbodiimide (EDC) (1.7 mg, 9.1 μmol,
2 equiv). The mixture was stirred at room temperature for 6 h. The reac-
tion mixture was diluted with ethyl acetate, and washed with water, saturated
NaHCO3, and brine. The organic layer was dried over anhydrous Na2SO4,
filtered, and concentrated under the reduced pressure. The crude residue
was purified by preparative reversed-phase HPLC to give 1: 1H NMR
(400 MHz; CDCl3) δ 8.92 (br s, 1H), 8.66 (s, 1H), 8.38 (s, 1H), 8.16 (d,
J = 7.6 Hz, 1H), 8.06 (s, 2H), 8.03 (d, J = 8.0 Hz, 1H), 7.81 (s, 1H),
7.74 (d, J = 8.4 Hz, 2H), 7.60 (t, J = 5.2 Hz, 1H), 7.50 (d, J = 8.8 Hz,
2H), 7.40 (d, J = 9.2 Hz, 1H), 7.18 (d, J = 8.4 Hz, 2H), 6.96 (d, J = 8.0
Hz, 2H), 6.89 (d, J = 8.8 Hz, 2H), 6.80 (d, J = 8.8 Hz, 2H), 6.64 (dd,
J = 9.2, 2.4 Hz, 1H), 6.48 (dd, J = 8.4, 2.4 Hz, 1H), 5.13 (s, 2H), 4.88
(br s, 2H), 4.21 (t, J = 4.4 Hz, 2H), 3.83 (s, 3H), 3.82 (s, 3H), 3.702
(s, 1H), 3.66ꢀ3.60 (m, 12H), 3.52ꢀ3.42 (m, 9H), 1.23 (t, J = 7.2 Hz,
6H); MALDI-TOF-MS calcd for C64H65F6N6O13S [M + 1]+ 1271.4156,
found 1271.3466.
Figure 2. Az inhibits the ATPase activity of Hsc70 by binding to its
ATPase domain. (A) Chemical structure of Az and an Az-linked resin.
(B) The Az-linked resin was incubated with purified full-length or
truncated His6-tagged Hsc70 (ATPase domain and SBD). Proteins
bound to the resin were visualized by silver staining. (C) The ATP-
agarose was incubated with purified full-length or truncated Hsc70 in the
absence or presence of Az or ATP. Proteins bound to the resin were
visualized by silver staining. (D) Inhibition of the ATPase activity of an
ATPase domain by Az. ATPase activities were measured by malachite
green assay using 200 μM ATP (mean ( SD).
Synthesis of Cy3-Az. To a stirred solution of N-2-(2-(2-ami-
noethoxy)ethoxy)ethylated apoptozole29,31,32 (27 mg, 36 μmol, 1 equiv)
in DMF were added N-hydroxybenzotriazole (HOBT, 8 mg, 39.6 μmol,
1 equiv), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyl-uronium hexa-
fluorophosphate (HBTU, 15 mg, 39.6 μmol, 1.1 equiv), diisopropy-
lethylamine (DIEA, 13 μL, 72 μmol, 2 equiv), and carboxylic acid-
containing Cy3 (16 mg, 36 μmol, 1 equiv). After being stirred for
8 h at room temperature, the reaction mixture was diluted with ethyl
acetate. The organic layer was washed with brine, dried over anhydrous
Na2SO4, filtered, and concentrated under reduced pressure. The crude
product was purified by flash column chromatography (CH2Cl2/MeOH
10:1) to give Cy3-Az in 24% yield: 1H NMR (400 MHz, CDCl3) δ 8.38
(t, 1H, J = 13.2 Hz), 8.07 (s, 2H), 8.02 (d, 2H, J = 8.0 Hz), 7.78 (s, 1H),
7.49 (d, 2H, J = 8.5 Hz), 7.40ꢀ7.33 (m, 4H), 7.26ꢀ7.20 (m, 4H), 7.17
(d, 2H, J = 8.7 Hz), 7.11 (d, 1H, J = 7.9 Hz), 7.03 (d, 1H, J = 5.5 Hz), 6.99
(d, 2H, J = 8.2 Hz), 6.87 (d, 2H, J = 8.0 Hz), 6.77 (d, 2H, J = 8.8 Hz), 5.11
(s, 2H), 4.13 (t, 2H, J = 7.5 Hz), 3.80 (s, 3H), 3.76 (s, 3H), 3.70ꢀ3.61
(m, 13H), 3.47 (dd, 2H, J = 5.1, 10.7 Hz), 2.54 (t, 2H, J = 6.8 Hz), 1.87 (t,
2H, J = 7.4 Hz), 1.69 (m, 14H); 13C NMR (400 MHz CDCl3): 174.7,
174.0, 173.4, 166.9, 160.2, 158.6, 150.8, 144.3, 142.7, 141.9, 140.6, 140.5,
140.2, 139.0, 134.2, 133.0, 132.4, 132.2, 131.9, 130.7, 129.2, 129.1, 128.7,
128.2, 128.1, 126.8, 125.9, 125.8, 125.6, 122.2, 122.1, 121.8, 114.8, 113.8,
111.1, 110.9, 104.0, 103.5, 70.3, 69.8, 55.4, 55.3, 49.2, 49.1, 48.3, 44.4,
five times. A solution of 1,2-bis(2-aminoethoxy)ethane (40 μL,
0.27 mmol, 3 equiv) and DIEA (94 μL, 0.54 mmol, 6 equiv) in DMF
was added to the resin. After being shaken for 6 h at room temperature,
the resin was washed with 10% DMF in CH2Cl2 five times. A solution of
DIEA (94 μL, 0.54 mmol, 6 equiv) and 3-(chlorosulfonyl)benzoyl
chloride (69 μL, 0.45 mmol 5 equiv) in anhydrous CH2Cl2 was added
to the resin at 0 °C. The mixture was stirred at 0 °C for 15 min and at
room temperature for 3 h. The resin was washed with 10% DMF in
CH2Cl2 five times. A solution of DIEA (94 μL, 0.54 mmol, 6 equiv),
4-dimethylaminopyridine (DMAP, 1.1 mg, 0.009 mmol, 0.1 equiv), and
7-(diethylamino)-N-(2-(2-hydroxyethoxy)ethyl)-2-oxo-2H-chromene-
3-carboxamide (95 mg, 0.27 mmol, 3 equiv) in anhydrous CH2Cl2 was
added to the resin, and the mixture was stirred at room temperature for
12 h. The assembled compound was cleaved from a solid support by
treatment with trifluoroacetic acid (TFA)-triethylsilane (TES) (98:2)
for 1 h. After removal of solvent, the crude residue was purified by
preparative reversed-phase HPLC to give 2: 1H NMR (CDCl3,
400 MHz) δ 9.09 (t, J = 4.0 Hz, 1H), 8.67 (s, 1H), 8.44 (s, 1H), 8.28
(br s, 1H), 8.24 (d, J = 7.6 Hz, 1H), 8.05 (br s, 2H), 8.00 (d, J = 8.0 Hz,
1H), 7.61 (t, J = 8.0 Hz, 1H), 7.47 (d, J = 8.8 Hz, 1H), 6.68 (dd, J = 9.4,
2.4 Hz, 1H), 6.51 (d, J = 2.4 Hz, 1H), 5.43 (br s, 1H), 4.22 (t, J = 4.0 Hz,
2H), 3.82 (t, J = 4.4 Hz, 2H), 3.70ꢀ3.67 (m, 11H), 3.58ꢀ3.46 (m, 9H),
20269
dx.doi.org/10.1021/ja206762p |J. Am. Chem. Soc. 2011, 133, 20267–20276