Activity Probes for Profiling HDAC Complexes
A R T I C L E S
HATU (84 mg, 0.22 mmol), and i-Pr2EtN (74 µL, 0.45 mmol) in DMF
(0.5 mL) was added to the resin and agitated for 4 h. The resin was
then filtered and washed with DMF (3 × 1 mL), the Fmoc group was
removed (subjected to 20% piperidine in DMF for 30 min), and the
resin was washed again (3 × 1 mL DMF). [For probe 8, a solution of
Fmoc-PABA-OH (79 mg, 0.22 mmol), HATU (84 mg, 0.22 mmol),
and i-Pr2EtN (74 µL, 0.45 mmol) in DMF (0.5 mL) was added to the
resin and agitated for 4 h. The resin was then filtered and washed with
DMF (3 × 1 mL), the Fmoc group was removed, and the resin was
washed successively with DMF, THF, MeOH, THF, and CH2Cl2 (3 ×
1 mL).] A solution of methyl-8-chloro-8-oxooctanoate (51 µL, 0.36
mmol) and i-Pr2EtN (60 µL, 0.36 mmol) in CH2Cl2 (0.5 mL) was added
to the resin and agitated overnight. The acylation was repeated, and
the resin was washed successively with DMF, THF, MeOH, THF, and
CH2Cl2 (3 × 1 mL). The methyl ester analogue of the probe was cleaved
from support by treatment with 95% TFA/5% CH2Cl2 (1 mL), and the
eluant was concentrated by rotary evaporation. DMF (300 µL), THF
(300 µL), and 50% H2NOH in water (300 µL, 5 mmol) were added to
the residue, and the reaction stirred for 48 h. The THF was removed
in Vacuo, and the remainder of the sample was purified using
preparatory C18 reversed-phase HPLC.
0.52 mmol) in DMF (0.5 mL) was added to the resin and agitated
overnight. The resin was then filtered and washed with DMF (3 × 1
mL), the Fmoc group was removed, and the resin was washed again
(3 × 1 mL DMF). A solution of 6-hexynoic acid (36 µL, 0.33 mmol),
HATU (125 mg, 0.33 mmol), and i-Pr2EtN (109 µL, 0.66 mmol) in
DMF (0.5 mL) was added to the resin and agitated for 4 h. The acylation
was repeated overnight, and the resin was then filtered and washed
successively with DMF, THF, MeOH, THF, and CH2Cl2 (3 × 1 mL).
The probes were cleaved from support by treatment with 97.5% TFA/
2.5% H2O, and the eluant was concentrated and then purified using
preparatory C18 reversed-phase HPLC.
Probe 9 (CSIV.102A). The general procedure provided 4.1 mg
1
(15%) of probe 9. tR(analytical): 13.8 min. H NMR (500 MHz) δ
1.60-1.66 (m, 2H), 2.06 (td, J ) 7.2, 2.6, 2H), 2.23 (td, J ) 7.4, 3.3,
2H), 2.79 (t, J ) 2.6, 1H), 2.94-2.98 (m, 1H), 3.19 (dd, J ) 13.7, 5.2,
1H), 4.38 (d, J ) 5.9, 2H), 4.67-4.72 (m, 1H), 7.31 (d, J ) 8.2, 2H),
7.50 (d, J ) 8.2, 2H), 7.61-7.64 (m, 2H), 7.71-7.77 (m, 7H), 8.29
(d, J ) 8.4, 1H), 8.64 (t, J ) 5.9, 1H). 13C NMR (125 MHz) δ 18.10,
25.13, 34.93, 42.732, 54.58, 72.25, 84.95, 100.01, 127.71, 127.76,
128.56, 129.40, 129.41, 130.27, 130.30, 130.32, 130.47, 133.37, 135.95,
138.17, 171.98, 172.39, 196.36. HR-MS m/z calcd for C30H29N3O5 (M
+ H): 512.218. Found: 512.2172.
Probe 8 (CSIV104). The general procedure provided 4.7 mg (37%)
1
of probe 8. tR(analytical): 14.8 min. H NMR (500 MHz) δ 1.33-
Probe 10 (CSIV.102B). The general procedure provided 4.2 mg
1
1.64 (m, 14H), 1.99 (t, J ) 7.0, 2H), 2.20 (m, 2H), 2.37 (t, J ) 7.4,
2H), 2.79 (m, 1H), 2.95 (m, 1H), 4.78 (m, 2H), 7.55-7.84 (m, 13H),
8.16 (br s,1H), 8.52 (br s,1H), 8.70 (br s,1H), 10.15 (s, 1H), 10.38 (s,
1H). HR-MS m/z calcd for C37H42N2O6 (M + H): 639.3177. Found:
639.3159.
(15%) of probe 10. tR(analytical): 13.8 min. H NMR (400 MHz) δ
1.52-1.60 (m, 2H), 1.99 (td, J ) 7.2, 2.4, 2H), 2.16 (td, J ) 7.3, 2.0,
2H), 2.74 (t, J ) 2.6, 1H), 2.89 (dd, 13.0, 10.2, 1H), 3.13 (m, 13.4,
4.8, 1H), 4.32 (d, J ) 5.9, 2H), 4.60-4.66 (m, 1H), 7.24 (d, J ) 8.1,
2H), 7.43 (d, J ) 8.2, 2H), 7.56 (t, J ) 7.5, 2H), 7.65-7.71 (m, 7H),
8.25 (d, J ) 8.5, 1H), 8.64 (t, J ) 5.7, 1H). 13C NMR (100 MHz) δ
17.25, 24.28, 34.07, 41.87, 53.74, 71.44, 84.10, 100.01, 126.87, 126.88,
128.56, 129.43, 129.49, 129.64, 131.28, 132.54, 135.09, 137.30, 142.53,
143.47, 171.15, 171.54, 195.52. HR-MS m/z calcd for C30H29N3O5 (M
+ H): 512.218. Found: 512.2171.
Probe 17 (CSIV.108B). The general procedure provided 8.2 mg
1
(36%) of probe 17. tR(analytical): 14.2 min. H NMR (500 MHz) δ
1.15-1.24 (m, 4H), 1.40-1.52 (m, 8H), 1.95 (t, J ) 7.4, 2H), 2.10 (t,
J ) 7.4, 2H), 2.19 (td, J ) 6.9, 2.6, 2H), 2.78 (t, J ) 2.7, 1H), 2.92
(dd, J ) 9.4, 4.1, 1H), 3.07-3.15 (m, 3H), 4.58-4.61 (m, 1H), 7.47
(d, J ) 8.2, 2H), 7.62 (t, J ) 7.7, 2H), 7.70-7.76 (m, 5H), 8.04 (t, J
) 5.6, 1H), 8.12 (d, J ) 8.6, 1H), 10.36 (s, 1H). 13C NMR (125 MHz)
δ 18.24, 25.83, 25.95, 26.13, 28.99, 29.11, 29.20, 33.10, 35.99, 38.77,
38.85, 54.42, 72.10, 85.22, 129.40, 130.25, 130.30, 130.41, 133.39,
135.88, 138.16, 144.30, 169.94, 171.63, 172.85, 196.35. HR-MS m/z
calcd for C30H37N3O5 (M + H): 520.2806. Found: 520.2800.
Probe 11 (CSIV.93). The general procedure provided 6.6 mg (24%)
1
of probe 11. tR(analytical): 14.3 min. H NMR (500 MHz) δ 1.62-
1.68 (m, 2H), 2.09 (td, J ) 7.3, 2.6, 2H), 2.25 (td, J ) 7.5, 2.1, 2H),
2.80 (q, J ) 2.6, 1H), 3.04 (dd, J ) 13.4, 9.9, 1H), 3.20-3.23 (dd, J
) 13.5, 4.9, 1H), 4.80-4.85 (m, 1H), 7.54 (d, J ) 8.2, 2H), 7.61 (t, J
) 7.7, 2H), 7.70-7.80 (m, 1H), 8.44 (d, J ) 8.2, 1H), 10.42 (s, 1H).
13C NMR (125 MHz) δ 18.09, 25.12, 34.79, 38.49, 55.40, 72.26, 84.92,
119.51, 128.55, 129.39, 130.28, 130.31, 130.33, 130.43, 133.40, 136.07,
138.11, 142.13, 143.82, 171.34, 172.58, 196.37. HR-MS m/z calcd for
C29H27N3O5 (M + H): 498.2023. Found: 498.2014.
Probe 12 (CSIV.94). The general procedure provided 4.8 mg (18%)
of probe 12. tR(analytical): 14.3 min. 1H NMR (400 MHz) δ 1.28
(quintet, J ) 7.2, 2H), 2.02 (td, J ) 7.2, 2.6, 2H), 2.25 (dd, J ) 7.2,
6.0, 2H), 2.75 (t, J ) 2.6, 1H), 2.96 (dd, J ) 13.4, 10.0, 1H), 3.14 (dd,
J ) 13.8, 5.0, 1H), 4.73-4.78(m, 1H), 7.47-7.73 (m, 13H), 8.40 (d,
J ) 8.2, 1H), 10.38 (s, 1H). 13C NMR (125 MHz) δ 18.10, 25.14,
34.81, 38.49, 55.41, 72.28, 84.94, 119.51, 128.58, 129.40, 130.29,
130.29,130.34, 130.44, 133.41, 136.08, 138.12, 142.16, 143.82, 171.34,
172.58, 196.38. HR-MS m/z calcd for C29H27N3O5 (M + H): 498.2023.
Found: 498.2007.
General Protocol for Preparation of Probes 13-16. A solution
of Fmoc-Amb-OH (31 mg, 0.084 mmol) (for probes 13 and 14) or
Fmoc-PABA-OH (30 mg, 0.08 mmol) (for probes 15 and 16) and i-Pr2-
EtN (46 µL, 0.28 mmol) in dry CH2Cl2 (0.5 mL) was added to a fritted
syringe containing 2-chlorotrityl resin (50 mg, 0.07 mmol). After 4 h,
the resin was filtered, quenched by the addition of i-Pr2EtN/MeOH/
CH2Cl2 (1:2:17), and washed with CH2Cl2 and DMF (3 × 1 mL). The
Fmoc group was removed (subjected to 20% piperidine in DMF for
30 min), and the resin was washed again (3 × 1 mL DMF). A solution
of Fmoc-Bpa-OH (172 mg, 0.350 mmol) (L for probes 13 and 15, D
for probes 14 and 16), HATU (133 mg, 0.350 mmol), and i-Pr2EtN
(116 µL, 0.700 mmol) in DMF (0.5 mL) was added to the resin and
agitated overnight. The resin was then filtered and washed with DMF
Probe 18 (CSIV.108A). The general procedure provided 5.9 mg
1
(26%) of probe 18. tR(analytical): 14.1 min. H NMR (500 MHz) δ
1.16-1.26 (m, 4H), 1.41-1.52 (m, 8H), 1.95 (t, J ) 7.3, 2H), 2.10 (t,
J ) 7.3, 2H), 2.19 (td, J ) 6.8, 2.6, 2H), 2.78 (t, J ) 2.1, 1H), 2.92
(dd, J ) 9.5, 3.9, 1H), 3.05-3.17 (m, 3H), 4.57-4.62 (m, 1H), 7.47
(d, J ) 8.2, 2H), 7.62 (t, J ) 7.7, 2H), 7.70-7.76 (m, 5H), 8.04 (t, J
) 5.7, 1H), 8.12 (d, J ) 8.6, 1H), 10.36 (s, 1H). 13C NMR (125 MHz)
δ 18.22, 25.81, 25.93, 26.10, 28.97, 29.09, 29.18, 33.08, 35.99, 38.75,
38.84, 54.40, 72.10, 85.20, 129.39, 130.24, 130.29, 130.40, 133.38,
135.87, 138.15, 144.30, 169.93, 171.62, 172.85, 196.34. HR-MS m/z
calcd for C30H37N3O5 (M + H): 520.2806. Found: 520.2799.
General Protocol for Preparation of Probes 9-12. Wang resin
(200 mg, 0.220 mmol) was modified to display a resin bound
O-hydroxylamine prepared as previously described.14 A portion of the
resin (60 mg) was added to a fritted syringe and swollen with CH2Cl2
(1 mL) for 1 h. A solution of Fmoc-Amb-OH (97 mg, 0.26 mmol) (for
probes 9 and 10) or Fmoc-PABA-OH (79 mg, 0.22 mmol) (for probes
11 and 12), HATU (99 mg, 0.26 mmol), and i-Pr2EtN (86 µL, 0.52
mmol) in DMF (0.5 mL) was added to the resin and agitated for 4 h.
The resin was then filtered and washed with DMF (3 × 1 mL), the
Fmoc group was removed (subjected to 20% piperidine in DMF for
30 min), and the resin was washed again (3 × 1 mL DMF). A solution
of Fmoc-Bpa-OH (162 mg, 0.33 mmol) (L for probes 9 and 11, D for
probes 10 and 12), HATU (99 mg, 0.26 mmol), and i-Pr2EtN (86 µL,
(14) Floyd, C. D.; Lewis, C. N.; Patel, S. R.; Whittaker, M. Tet. Lett. 1996, 37,
8045-8.
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J. AM. CHEM. SOC. VOL. 130, NO. 7, 2008 2187