Development of an isotope-coded activity-based probe for the quantitative profiling of cysteine proteases

Article abstract of DOI:10.1016/j.bmcl.2004.04.046

Quantification studies of complex protein mixtures have been restricted mainly to whole cell extracts. Here we describe the synthesis of two sets of isotope-coded activity-based probes that allow quantitative functional proteomics experiments on the cathe

Full text of DOI:10.1016/j.bmcl.2004.04.046

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3131–3134
Development of an isotope-coded activity-based probe for
the quantitative profiling of cysteine proteasesq
Paul F. van Swieten,a Rene Maehr,b Adrianus M. C. H. van den Nieuwendijk,a
Benedikt M. Kessler,b Michael Reich,c,d Chung-Sing Wong,a Hubert Kalbacher,c
Michiel A. Leeuwenburgh,a Christoph Driessen,d Gijsbert A. van der Marel,a
Hidde L. Ploeghb and Herman S. Overkleefta,*
aGorlaeus Laboratories, Leiden University, Einsteinweg 55, 2300 RA Leiden, The Netherlands
bDepartment of Pathology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston MA 02115, USA
cMedical and Natural Sciences Research Centre, University of Tu€bingen, D-72074 T€ubingen, Germany
dDepartment of Medicine II, University of T€ubingen, D-72074 T€ubingen, Germany
Received 25 February 2004; revised 8 April 2004; accepted 8 April 2004
Abstract—Quantification studies of complex protein mixtures have been restricted mainly to whole cell extracts. Here we describe
the synthesis of two sets of isotope-coded activity-based probes that allow quantitative functional proteomics experiments on the
cathepsins.
Ó 2004 Elsevier Ltd. All rights reserved.
Proteomics research aims at the study of the expression
levels and functioning of (subsets of) the proteins pres-
ent in a biological sample. Proteomics research presents
a number of challenges to the researchers. The protein
content in a cell is dynamic and cannot be amplified
easily. Further, there is a difference of several orders of
magnitude between the most and the least abundant
protein. Traditionally, 2D-gel electrophoresis is used to
separate mixtures of proteins, allowing identification of
the proteins, originally using Edman degradation.
Nowadays, protein sequencing is normally performed
using mass spectrometry techniques, as introduced by
Watanabe and co-workers in their groundbreaking
report.1
affinity––or fluorescence tag.2–5 For instance, broad
spectrum, irreversible protease inhibitors have been used
in the profiling of serine proteases,6 cysteine proteases,7
and the catalytically active subunits of the protea-
some.8;9 A relevant example of a chemical proteomics
probe is represented by 1a (DCG-04), developed by
Bogyo and co-workers as an irreversible cysteine pro-
tease inhibitor, and applied by us to monitor the pro-
teolytic activity of maturing phagosomes in live antigen-
presenting cells.10;11 Compound 1a consists of three
functionalities: (1) an electrophilic epoxysuccinate, that
alkylates the active site cysteine residue, (2) a short
peptide sequence that allows recognition of the probe by
the cathepsin family of cysteine proteases, and (3) bio-
tin, for the detection and isolation of the modified
proteins (Fig. 1). The biotin is connected to the peptide
epoxysuccinate by an aminohexanoic acid residue.
In chemical proteomics approaches, a complex biologi-
cal mixture of proteins is simplified before analysis
by labeling a specific set of related proteins with an
We reasoned that incorporation of an isotopic encoded
entity, in analogy to the isotope-coded affinity tag
(ICAT) reagent developed by Aebersold and co-workers
for the quantitative analysis of cysteine containing
proteins,12;13 would allow for both quantitative and
functional assessment of the cathepsin family of cysteine
proteases from complex biological samples.14 The ICAT
strategy is based on the presence (heavy) or absence
Keywords: Cysteine proteases; Active site-directed probe; Isotopic
labeling; Suicide inhibitor; Synthesis; Chemical proteomics.
q Supplementary data associated with this article can be found, in the
online version, at doi:10.1016/j.bmcl.2004.04.046
* Corresponding author. Tel.: +31-71-527-4342; fax: +31-71-527-4307;
e-mail: h.s.overkleeft@chem.leidenuniv.nl
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.04.046

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P. F. van Swieten et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3131–3134
Recognition Element
FmocHN
FmocHN
O
4, CuCl, TMEDA,
FmocHN
O2, THF, 45oC,
OBn
3
5
O
O
O
H
N
36%
O
H
N
EtO
D2, EtOAc,
Pd/C, 84%
N
H
N
H
NH2
NH
O
O
O
Reactive Group
OBn
OR
S
O
HN
HO
O
4
DCl/D2O, dioxane
N
H
6: R=Bn,
7: R=H,
= CD2
= CD2
O
O
O
6h, 90oC, 85%
1a: =CH2
1b: =CD2
Affinity Tag
Scheme 1.
OH
oxide and dioxane to give isotopically coded Fmoc-
AhxOH-d8 (7).
O
O
H
H
N
H
N
O
N
EtO
N
H
O
O
NH2
NH
The syntheses of N3Aoh-d0 (14a) and N3Aoh-d8 (14b)
were accomplished as follows (Scheme 2). Upon trityl-
ation of deuterated 8, monotrityl ether 9 was obtained
in 23% and deuterated butanediol was recovered easily
(68%). Tosylation of the primary alcohol followed by
the replacement of the tosylate by azide gave protected
azidoalcohol 11. Detritylation (TFA/TES in CH2Cl2)
afforded azidoalcohol 12b. Both known 4-azido-1-
butanol21 12a and deuterated 12b were alkylated under
phase transfer conditions with tert-butyl bromoacetate
to furnish the corresponding tert-butyl ester 13a and
13b. Subsequent acidolysis of the tert-butyl esters
employing 50% v/v TFA/CH2Cl2 yielded, respectively,
azido acid 14a in 49% over two steps and 14b in an
overall yield of 38% over six steps.
O
O
S
HN
O
2a: =CH2
2b: =CD2
N
H
O
Figure 1. Target structures.
(light) of eight deuterium atoms in a biotin-containing
cysteine-reactive probe. After labeling a denatured
protein sample from two different sources with either the
light or heavy ICAT probe, trypsinolysis of the com-
bined fragments and enrichment in the biotinylated
fragments, both the relative quantity and sequence
identity of the proteins from which the biotinylated
peptides originated are determined by automated mul-
tistage mass spectrometry.15
The incorporation of spacers 7b and 14ab into the
respective cysteine protease inhibitors 1b and 2ab is
shown in Scheme 3. Immobilization of biocytin on a
Rink linker was accomplished as described earlier.22
Standard solid phase peptide synthesis (SPPS) with the
sequential addition of a spacer (7, 14a, and 14b,
respectively), FmocTyr(OtBu)OH, FmocLeuOH, and
ethyl (2S,3S)oxirane-2,3-dicarboxylate,23 acidic cleavage
from the resin and purification by repeated precipitation
afforded target compounds 1b, and 2a and 2b in 34%,
29%, and 40%, respectively, in 80–90% purity as judged
by LCMS. A small portion of each product was purified
to homogeneity by HPLC.
Here we describe the synthesis of a new azide-protected
amino acid spacer, 7-azido-3-oxaheptanoic acid
(N3AohOH, 14a) and two d8-enriched amino acids, 2,2,-
3,3,4,4,5,5-octadeutero-6-(Fmoc-amino)hexanoic acid
(FmocAhxOH-d8, 7) and 4,4,5,5,6,6,7,7-octadeutero-7-
azido-3-oxaheptanoic acid (N3AohOH-d8, 14b). Their
use in the synthesis of two sets of DCG-04-based iso-
topic encoded activity-based probes: compound 1b (in
combination with DCG-04 1a) and compounds 2a and
2b is demonstrated. We also show here that neither
incorporation of an isotopic label (compounds 1b and
2b, Fig. 1) nor substitution of the aminohexanoic acid
moiety by an 7-amino-3-oxa-heptanoic acid residue
(compounds 2ab) affects the broad spectrum affinity for
cysteine proteases of the cathepsin family.
TrCl, TEA, DMAP
TrO
HO
OR
OH
8: =CD2
23% conv,
91% yield
TosCl, TEA
9: R=H
95%
10: R=Ts
NaN3, DMF,
50oC, quant
The synthesis of FmocAhx-d8 7 (Scheme 1) starts with a
copper(I) catalyzed oxidative Glaser coupling16 of
Fmoc-protected propargylamine17 (3) and benzyl pro-
piolate18 (4) to furnish diyne 5. Upon reduction of diyne
5 with deuterium gas and palladium on carbon, the
benzyl ester remained intact, yielding deuterated 6.
Contrary to the results of reduction of several analogs of
5,19 no isotopic scrambling had taken place. Finally, the
benzyl ester was hydrolyzed20 using DCl in deuterium
tBu bromoacetate,
O
Bu4NBr, NaOH, Tol
O
RO
13a 61%, 13b 61%
RO
N3
N3
TFA, TES,
11: R=Tr
12b: R=H
12a: =CD2, R=H
13ab: R=tBu
14ab: R=H
50% TFA/DCM
14a 81%
DCM, 83%
14b 87%
Scheme 2.

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.
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