´
S. Wisen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 60–65
65
8. Mosser, D. D.; Morimoto, R. I. Oncogene 2004, 23, 2907.
9. Barral, J. M.; Broadley, S. A.; Schaffar, G.; Hartl, F. U.
Semin. Cell Dev. Biol. 2004, 15, 17.
10. Neckers, L. Curr. Top. Med. Chem. 2006, 6, 1163.
11. Chaudhury, S.; Welch, T. R.; Blagg, B. S. Chem. Med.
Chem. 2006.
added to 55 mL H2O. After 4 h at 60 ꢁC, the mixture was
cooled on ice and the solution acidified by adding
concentrated HCl dropwise. The white precipitate was
filtered, washed with ice-cold water, and dried in air (yield:
ꢀ30%).
33. Biginelli reaction: Five equivalents of 1-ureido-butyric acid
was coupled to the tripeptide in the presence of 3 equiv
DIC and 3 equiv HOBt in 10 mL DMF per 0.25 g resin.
The conditions for the subsequent Biginelli reactions were
as follows: to 0.250 mmol of resin loaded with tripeptide, 4
equiv p-bromobenzaldehyde, 4 equiv ethylacetoacetate,
and 250 lL 4:1 DMF:HCl (conc.) were added. Microwave
conditions: 80 ꢁC for 20 min followed by four washing
steps: 3· 10 mL DMF, 3· 10 mL hexanes, 3· 10 mL
MeOH, 3· 10 mL CH2Cl2. The product was cleaved with
5 mL 1:1 CH2Cl2:TFA for 30 min, followed by washing
the resin with CH2Cl2. The solvent was evaporated under
reduced pressure and the product was purified to over 90%
using reverse phase HPLC (C18; acetonitrile:water;
20–80%, 1% per min). Products were isolated as white
solids. The identities were verified by mass spectrometry
(see Table 1) and 1H NMR: compound 6: [3-((3S)-3-((3S)-
7-amino-3-(4-(4-(4-bromophenyl)-5-(ethoxycarbonyl)-6-methyl-
2-oxo-3,4-dihydropyrimidin-1(2H)-yl)butanamido)hep-
tanamido)-5-phenylpentanamido)propanoic acid] 1H
NMR: d 1.09–1.12 (t, 3H), d 1.25–1.26 (m, 1H), d 1.33–
1.37 (m, 2H), d 1.44–1.48 (m, 2H), d 1.50 (s, 2H), d 1.69 (s,
2H), d 2.04 (s, 2H), d 2.21 (s, 4H), d 2.10–2.29 (m, 2H), d
2.34 (t, 2H), d 2.47 (s, 3H), d 2.54–2.57 (d, 1H), d 2.75 (s,
2H), d 3.21–3.22 (d, 2H), d 3.77 (br s, 1H), d 4.01–4.04 (t,
3H), d 5.13 (d, 1H), d 7.16 (s, 4H), d 7.21–7.25 (t, 2H), d
7.52 (d, 2H), d 7.67 (s, 3H), d 7.78 (d, 1H), d 7.95–7.97 (d,
2H), d 12.22 (br s, 1H). Compound 14: [3-(3-(4-(4-(4-
Bromophenyl)-5-(ethoxycarbonyl)-6-methyl-2-oxo-3,4-dihy-
dropyrimidin-1(2H)-yl)butanamido)-5-phenylpentanami-
do)propanoic acid] 1H NMR: d 1.07–1.13 (t, 3H), d 1.57–1.68
(m, 2H), d 1.68–1.78 (m, 2H), d 2.06 (t, 2H), d 2.17–2.28
(m, 2H), d 2.35 (t, 2H), d 2.47 (s, 3H), d 3.22 (d, 2H),
d 3.76–3.85 (m, 1H), d 4.00–4.04 (q, 3H), d 5.13 (d, 1H), d
7.16–7.20 (m, 5H), d 7.24–7.30 (m, 2H), d 7.50 (d, 1H), d
7.73 (d, 1H), d 7.94 (t, 1H),d 7.99 (d, 1H), d 12.40 (s, 1H).
Compound 15: [3-(3-(4-(4-(4-Bromophenyl)-5-(ethoxycar-
bonyl)-6-methyl-2-oxo-3,4-dihydropyrimidin-1(2H)-yl)
buta-namido)-4-(naphthalen-1-yl)butanamido)propanoic
acid] 1H NMR: d 1.09–1.13 (q, 3H), d 1.43–1.47 (m, 1H), d
1.57 (m, 1H), d 1.87–1.97 (m, 2H), d 2.24 (s, 2H), d 2.31 (s,
1H), d 2.33 (d, 2H), d 2.38–2.40 (t, 2H), d 3.03–3.08 (m,
1H), d 3.19–3.32 (m, 3H), d 3.32–3.34 (t, 2H), d 3.62 (m,
1H), d 3.99–4.05 (t, 2H), d 3.39–4.42 (t, 2H), d 5.11 (s, 1H),
d 7.14 (d, 1H), d 7.18 (d, 1H), d 7.31–7.39 (m, 2H), d 7.47–
7.54 (m, 2H), d 7.55–7.57 (m, 2H), d 7.82–7.85 (t, 1H), d
12. Yaglom, J. A.; Gabai, V. L.; Sherman, M. Y. Cancer Res.
2007, 67, 2373.
13. Smith, D. F.; Whitesell, L.; Katsanis, E. Pharmacol. Rev.
1998, 50, 493.
14. Lee, A. S. Cancer Res. 2007, 67, 3496.
15. Workman, P.; Powers, M. V. Nat. Chem. Biol. 2007, 3,
455.
16. Workman, P.; Burrows, F.; Neckers, L.; Rosen, N. Ann.
N.Y. Acad. Sci. 2007.
17. Pick, E.; Kluger, Y.; Giltnane, J. M.; Moeder, C.; Camp,
R. L.; Rimm, D. L.; Kluger, H. M. Cancer Res. 2007, 67,
2932.
18. Neckers, L. Handb. Exp. Pharmacol. 2006, 259.
19. Brodsky, J. L. Biochem. Pharmacol. 1999, 57, 877.
20. Otaka, M.; Yamamoto, S.; Ogasawara, K.; Takaoka, Y.;
Noguchi, S.; Miyazaki, T.; Nakai, A.; Odashima, M.;
Matsuhashi, T.; Watanabe, S.; Itoh, H. Biochem. Biophys.
Res. Commun. 2007, 353, 399.
21. Liebscher, M.; Jahreis, G.; Lucke, C.; Grabley, S.; Raina,
S.; Schiene-Fischer, C. J. Biol. Chem. 2007, 282, 4437.
22. Fewell, S. W.; Day, B. W.; Brodsky, J. L. J. Biol. Chem.
2001, 276, 910.
23. Fewell, S. W.; Smith, C. M.; Lyon, M. A.; Dumitrescu, T.
P.; Wipf, P.; Day, B. W.; Brodsky, J. L. J. Biol. Chem.
2004, 279, 51131.
24. Rodina, A.; Vilenchik, M.; Moulick, K.; Aguirre, J.; Kim,
J.; Chiang, A.; Litz, J.; Clement, C. C.; Kang, Y.; She, Y.;
Wu, N.; Felts, S.; Wipf, P.; Massague, J.; Jiang, X.;
Brodsky, J. L.; Krystal, G. W.; Chiosis, G. Nat. Chem.
Biol. 2007, 3, 498.
´
25. Evans, C. G.; Wisen, S.; Gestwicki, J. E. J. Biol. Chem.
2006, 281, 33182.
26. Peptide synthesis: To 1 equiv Wang resin, 2 equiv of the
Fmoc-b-amino acid, 3 equiv DIC, and 3 equiv HOBt were
added. The reaction was performed in DMF (5 mL DMF
per 0.25 mmol Wang resin), and the mixture was irradi-
ated with microwaves at 70 ꢁC for 20 min. The resin was
washed with 4· 10 mL DMF, followed by incubation with
5 mL of 20% acetic anhydride for 20 min in order to block
uncoupled sites before Fmoc deprotection. The resin was
washed (4· 10 mL DMF) followed by deprotection of the
N-terminus with 20% piperidine in DMF. The coupling
efficiency was estimated by transferring approximately 1–
2 mg resin into 3 mL of 20% piperidine in DMF in two
quartz cuvettes. After 2–3 min of stirring, the absorbance
at 290 nm was determined, using 20% piperidine in DMF
as a reference. The average coupling efficiency was 80%.
27. Murray, J. K.; Farooqi, B.; Sadowsky, J. D.; Scalf, M.;
Freund, W. A.; Smith, L. M.; Chen, J.; Gellman, S. H. J.
Am. Chem. Soc. 2005, 127, 13271.
7.96 (s, 1H), d 8.00 (s, 1H), d 8.31 (d, 1H).
34. Bukau, B.; Horwich, A. L. Cell 1998, 92, 351.
´
35. Chang, L.; Bertelsen, E. B.; Wisen, S.; Larsen, E. M.;
Zuiderweg, E. R.; Gestwicki, J. E. Anal. Biochem. 2007.
28. Murray, J. K.; Gellman, S. H. J. Comb. Chem. 2006, 8, 58.
29. Wipf, P.; Cunningham, M. Tetrahedron Lett. 1995, 36, 7819.
30. Dallinger, D.; Kappe, C. O. Nat. Protoc. 2007, 2, 1713.
31. Kappe, C. O.; Stadler, A. Methods Enzymol. 2003, 369, 197.
32. Synthesis of 1-ureido-butyric acid: Reaction at 60 ꢁC:
25 mmol d-amino butyric acid and 16.7 g KOCN were
36. The malachite green-based ATPase assay was performed
as described in Ref. 35, except that the reactions were
performed without GrpE and the incubation time was 4 h.
37. Feder, M. E.; Hofmann, G. E. Annu. Rev. Physiol. 1999,
61, 243.
38. Rensing, S. A.; Maier, U. G. J. Mol. Evol. 1994, 39, 80.