P. F. van Swieten et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3131–3134
3133
O
(i), (ii)
(iii)
Rink Resin-NHFmoc
FmocHN
NHResin
15: R=Mtt
16: R=biotinyl
NHR
(i), (iv)
(i), (v)
O
O
O
Figure 2. Derivatization of the DCG-04 molecule does not alter tar-
geting of active proteases species in J774 cells. Crude extracts prepared
from J774 cells were labeled for 1 h at 37 °C with different concentra-
tions of compounds 1b, 2a, and 2b. As a control, extracts were pre-
heated for 5 min at 100 °C prior to labeling (lanes 1, 5, and 9). Proteins
were then separated by SDS-PAGE on 12.5% gels and labeled poly-
peptides visualized by streptavidin blotting. Polypeptide species cor-
responding to CTS Z, B, S, and L are indicated based on previous
studies.10
H
N
H
R
N
R
NH
O
NH
O
Resin
O
Resin
S
S
NH
NH
HN
HN
N
N
O
O
H
O
H
17ab: R=NHFmoc
18ab: R=NH2
19ab: R=N3
(i)
(vi)
20ab: R=NH2
Importantly, this concept may be extended toward other
isotopic coded spacers (13C, 15N) and activity-based
probes targeting other proteins.2;3 Current research
efforts are aimed in that direction.
O
O
EtO
(vii), (viii)
(vii), (viii)
OH
O
21
1ab
2ab
Acknowledgements
Scheme 3. Reagents and conditions: (i) 20% piperidine in NMP; (ii)
FmocLys(Mtt)OH, HCTU, DiPEA, NMP; (iii) 1% TFA/CH2Cl2, then
biotin, HCTU, DiPEA, NMP; (iv) 7a or 7b, HCTU, DiPEA, NMP; (v)
14a or 14b, HCTU, DiPEA, NMP; (vi) Me3P, 20% H2O, dioxane; (vii)
Repeated cycles of SPPS: Fmoc cleavage: 20% piperidine in NMP;
amino acid condensation: Fmoc-protected amino acid, HCTU, Di-
PEA, NMP; Fmoc-protected building blocks were used in the fol-
lowing order: FmocTyr(OtBu)OH, FmocLeuOH, 21; (viii) TFA/H2O
95/5.
This work is supported by NIH Grant GM 62502 to
H.L.P., P.F.S. and H.S.O. are financially supported by
the Netherlands Organization for Scientific Research
(NWO). B.M.K. is supported by a Multiple Myeloma
Research Foundation Senior Research Award
(MMRF).
To establish the inhibition profile of the newly synthe-
sized probes, we performed a set of labeling experiments
with cell lysates of the mouse macrophage cell line J774.
Cell lysates were incubated with DCG-04 (1a) as a
control and with the new probes 1b, 2a, and 2b for
60 min at 37 °C. The resulting mixtures were separated
by SDS-PAGE. The separated proteins were transferred
onto a polyvinylidene difluoride (PVDF) membrane,
followed by chemiluminescence induced by horseradish
peroxidase–streptavidin conjugate (Fig. 2). Probes 2ab
label the cysteine proteases CTS B, L, S, and Z in a cell
lysate with the same efficiency as DCG-04, which has
been shown previously to effectively target these proteo-
lytic enzymes.10 This suggests that both sets of isotopic
coded activity-based probes 1ab and 2ab are viable
quantitative functional proteomics tools for the
cathepsin family of cysteine proteases.
References and notes
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In summary, we have presented the efficient synthesis of
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