Synthesis of novel caspase inhibitors for characterization of the active caspase proteome in vitro and in vivo

Article abstract of DOI:10.1021/jm060385h

Caspases are cysteine proteases that are essential for cytokine maturation and apoptosis. To facilitate the dissection of caspase function in vitro and in vivo, we have synthesized irreversible caspase inhibitors with biotin attached via linker arms of va

Full text of DOI:10.1021/jm060385h

7636
J. Med. Chem. 2006, 49, 7636-7645
Synthesis of Novel Caspase Inhibitors for Characterization of the Active Caspase Proteome in
Vitro and in Vivo
Alexander J. Henzing,† Helen Dodson,† Joel M. Reid,‡ Scott H. Kaufmann,‡,§ Robert L. Baxter,| and William C. Earnshaw*,†
The Wellcome Trust Centre for Cell Biology, ICB, Swann Building, UniVersity of Edinburgh, Mayfield Road, Edinburgh, EH9 3JR, United
Kingdom, Departments of Oncology and Molecular Pharmacology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, and School
of Chemistry, Joseph Black Building, UniVersity of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, United Kingdom
ReceiVed March 31, 2006
Caspases are cysteine proteases that are essential for cytokine maturation and apoptosis. To facilitate the
dissection of caspase function in vitro and in vivo, we have synthesized irreversible caspase inhibitors with
biotin attached via linker arms of various lengths (12a-d) and a 2,4-dinitrophenyl labeled inhibitor (13).
Affinity labeling of apoptotic extracts followed by blotting reveals that these affinity probes detect active
caspases. Using the strong affinity of avidin for biotin, we have isolated affinity-labeled caspase 6 from
apoptotic cytosolic extracts of cells overexpressing procaspase 6 by treatment with 12c, which contains
biotin attached to the Nε-lysine of the inhibitor by a 22.5 Å linker arm, followed by affinity purification on
monomeric avidin-sepharose beads. Compound 13 has proven sufficiently cell permeable to rescue cells
from apoptotic execution. These novel caspase inhibitors should provide powerful probes for the study of
the active caspase proteome during apoptosis both in vitro and in vivo.
Introduction
implicated caspases in apoptotic cleavage, the availability of
the peptide caspase 1 inhibitor tyr-val-ala-asp-chloromethylke-
tone (YVAD-cmk)a allowed demonstration of the critical role
of this class of enzymes in apoptotic events.16 Moreover,
biotinylated derivatives of YVAD-cmk and of benzyloxycar-
bonyl-glu-lys-asp-acyloxymethylketone (Z-EKD-aomk), an in-
hibitor designed with apoptotic effector cleavage sequences in
mind,17 were successfully utilized to identify multiple species
of caspases 3 and 6 as the predominant active caspases in
apoptotic cells17,18 despite the fact that the YVAD and EVD
peptide sequences were subsequently shown to bind caspase 1
more avidly than caspases 3, 6 and 7.19,20 This ability of covalent
caspase inhibitors to derivatize apoptotic caspases despite less
than optimal affinity of their peptide moieties for the enzyme
active sites has been attributed to the high concentrations and
prolonged incubation times often utilized in laboratory experi-
ments.19
Although the previous chloromethylketone (cmk) and acy-
loxymethylketone (aomk) affinity labels have proven useful for
affinity labeling of cell-free extracts prepared from apoptotic
cells, these reagents also had several limitations. First, because
of the short arm linking biotin to the label, they were difficult
to use for affinity purification of caspases. Instead, because the
biotin did not extend far beyond the active site cleft of the
Apoptosis is a highly conserved process by which eukaryotic
cells commit suicide.1,2 This form of programmed cell death
involves a reproducible series of cellular changes that include
cell shrinkage, chromatin condensation, membrane blebbing,
and, in most cases, DNA fragmentation. Studies performed over
the past decade have revealed that many of the changes observed
in apoptotic cells result from the action of a family of cysteine-
dependent aspartate-directed proteases termed caspases.3-5 The
caspase family can be divided into two distinct subfamilies, the
cytokine activators (caspases 1, 4, 5, 11, and 12) and those
involved in apoptosis. Apoptotic caspases can be further
subdivided into initiator caspases (caspases 2, 8, 9, and 10),
which transduce an apoptotic signal into proteolytic activity,
and effector caspases (caspases 3, 6, and 7), which are activated
by initiator caspases and are in turn responsible for the cleavage
of the majority of the substrates leading to the demise of the
cell.3-5
Previous studies have demonstrated that the various caspases
differ in their substrate specificies.4,6 While all cleave on the
carboxyl side of aspartate residues, caspases 3 and 7 prefer acidic
residues in the P4 position of the substrate, whereas caspases 1,
4, 5, 6, and 8-10 prefer aromatic or hydrophobic residues in
this position.7 These substrate specificities have been explained,
at least in part, by differences in the corresponding S4 substrate
binding pockets of the various caspase family members as
crystal structures have become available.8-13
a Abbreviations: Ac, acetyl; Ac-DEVD-pNA, N-R-acetyl-Asp-Glu-Val-
Asp-p-nitroanilide; Ac-IETD-pNA, N-R-acetyl-IIe-Glu-Thr-Asp-p-nitroa-
nilide; Ac-LEHD-pNA, N-R-acetyl-Leu-Glu-His-Asp-p-nitroanilide; Ac-
VEID-pNA, N-R-acetyl-Val-Glu-IIe-Asp-p-nitroanilide; Ac-YVAD-pNA,
N-R-acetyl-Tyr-Val-Ala-Asp-p-nitroanilide; aomk, acyloxymethylketone;
CHAPS, (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate;
DMAP, 4-dimethylaminopyridine; DMF, dimethylformamide; DMPU, 1,3-
dimethyl-3,4,5,6-tetrahydro-2(1H)pyrimidinone; DMSO, dimethylsulfoxide;
DNP, 2,4-dinitrophenyl; DTT, dithiothreitol; EGFP, enhanced green
fluorescent protein; HPLC, high performance liquid chromatography; HRP,
horseradish peroxidase; Ki, inhibition constant; LC, HO2C(CH2)5NH-;
LCLC, HO2C(CH2)5NHCO(CH2)5NH-; MS, mass spectrometry; MOWSE,
molecular weight search; NHS, N-hydroxysuccinimide; NMM, N-methyl-
morpholine; NMR, nuclear magnetic resonance; NOBA, 3-nitrobenzyl
alcohol; PBS, phosphate-buffered saline; pNA, para-nitroanilide; SDS,
sodium dodecyl sulfate; TFA, trifluoroacetic acid; Vmax, maximum velocity;
Z, N-R-benzyloxycarbonyl; Z-EK(label)D-aomk, N-R-benzyloxycarbonyl-
Glu-Lys(label)Asp-acyloxymethylketone.
Earlier progress in understanding the role of caspases in
apoptosis was critically dependent on the availability of caspase
inhibitors and affinity labels. After the demonstration that
apoptotic cleavage of poly(ADP-ribose) polymerase (PARP1)14,15
occurs at the sequence asp-glu-val-aspVgly,16 an observation that
* To whom correspondence should be addressed. Telephone: 0131 650
7101. Fax: 0131 650 8650. E-mail: bill.earnshaw@ed.ac.uk.
† The Wellcome Trust Centre for Cell Biology, University of Edinburgh.
‡ Department of Oncology, Mayo Clinic College of Medicine.
§ Department of Molecular Pharmacology, Mayo Clinic College of
Medicine.
| School of Chemistry, University of Edinburgh.
10.1021/jm060385h CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/07/2006

Synthesis of NoVel Caspase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26 7637
Scheme 1. Synthesis of Caspase Inhibitors
Reagents and conditions: (a) Ph3PdCHCO2CH2CH3, MeOH, 0 °C; (b) benzylamine, -50 °C, 48 h; (c) H2, Pd/C, EtOH; (d) Z-Lys(Boc) NHS, CH2Cl2,
12 h; (e) H2, Pd/C, EtOH; (f) Z-Glu(Ot-Bu) NHS, CH2Cl2, 12 h; (g) montmorillonite, EtOH/H2O (5:1), 75 °C, 3 h; (h) 2,6-dimethylbenzoylchloride, pyridine,
DMAP, DMPU, 48 h; (i) Dess-Martin periodate, 0 °C to rt, 4 h; (j) 95% TFA/H2O, 12 h; (k) labeling reagent, water, 12 h.
enzymes, the earlier affinity labels were most useful for detection
on blots after polypeptides were denatured. Second, because
biotin was used as a tag, endogenous biotinylated polypeptides
were also detected on blots. Third, the previous affinity labels
penetrated cells poorly and were generally used to label caspases
under cell-free conditions,17,18,21-23 although it has been reported
that caspases in intact cells can sometimes be labeled when cells
are subjected to prolonged incubations with these compounds
prior to introduction of an apoptotic stimulus.24,25
In view of the large number of caspase species that can be
detected when samples of affinity-labeled caspases are separated
by charge and size on two-dimensional polyacrylamide gels,17,18,21
it is clear that further work is required to fully characterize the
post-translational modifications that regulate caspase activation
and activity. Here we describe the synthesis and initial
characterization of several novel irreversible acyloxymethylke-
tone caspase inhibitors labeled with biotin 12a-d or 2,4-
dinitrophenyl (DNP) 13 that overcome some of the limitations
of earlier inhibitors. The construction of affinity probes in which
the biotin was coupled via an extended linker arm enabled the
affinity purification of nondenatured enzyme from cell-free
extracts prepared from apoptotic cells overexpressing caspase
6 fused to enhanced green fluorescent protein (EGFP). The
probe 13 labeled with DNP, rather than biotin, not only
displayed much lower background labeling of polypeptides in
cell-free extracts, but also penetrated cells and efficiently
inhibited apoptosis in situ.
Z-EKD-aomk (11) was synthesized as shown in Scheme 1.
D-Glyceraldehyde acetonide 1 was prepared from D-mannitol.26
Wittig reaction of the chiral acetonide with Ph3PdCHCO2CH2-
CH3 in methanol at 0 °C gave a separable isomeric mixture
(Z/E, 8:1) of the R,â-unsaturated esters 2.27 Reaction of the
mixture of esters with benzylamine in the absence of solvent at
-50 °C for 2 days afforded the (3R)-benzylamino ester 3.
Removal of the Z protecting group by hydrogenation furnished
the amine 4, which was coupled with N-R-benzyloxycarbonyl-
N-ꢀ-butoxycarbonyl-lysine-succinyl ester to give the dipeptide
5. Hydrogenation of 5 afforded amine 6, which was coupled
with N-R-benzyloxycarbonyl-γ-O-tert-butyl-glutamyl-succinyl
ester to give the protected tripeptide analog 7. Selective acid
hydrolysis of the acetonide group of 7 to give the diol 8, without
partial removal of the t-butyl protecting groups, could not be
achieved even under relatively mild solution conditions28 but
was effected in good yield by treatment with Montmorillonite
K10 in aqueous ethanol. Esterification of the primary hydroxyl
of 8 with 2,6-dimethylbenzoyl chloride in pyridine/DMAP/
DMPU at room temperature produced the ester 9. Attempts to
effect oxidation of the sterically crowded secondary alcohol
group of 9 with a variety of mild oxidizing reagents proved
unsuccessful. However, oxidation under Dess-Martin condi-
tions29 afforded the ketone 10 in virtually quantitative yield.
Finally, cleavage of the protecting groups of the glutamyl, lysyl,
and “aspartyl analog” moieties of 10 was achieved with
trifluoroacetic acid (TFA) containing a catalytic amount of water
to give the desired Z-EKD-aomk 11. A series of biotin-labeled
derivatives of 11 (compounds 12a-12d) was then prepared by
reaction of the free ꢀ-amino group of lysine with the N-
hydroxysuccinamides of biotin, iminobiotin, 6-[biotinamido]-
Chemistry
To provide an affinity label that could be readily tagged with
various moieties, the peptidyl acyloxymethylketone inhibitor

7638 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26
Henzing et al.
Table 1. Kinetic Parameters Determined for Human Caspases 1-8
Reacted with LC Biotin-Derivatized Inhibitor 12c
Vmax
Km
Ki
caspase
substrate
(pmol/min)
(µM)
(µM)
1
2
3
4
5
6
7
8
YVAD-pNA
LEHD-pNA
DEVD-pNA
LEHD-pNA
LEHD-pNA
VEID-pNA
DEVD-pNA
IETD-pNA
110
400
110
510
610
230
530
130
100
560
74
1100
2500
220
230
140
0.0063
29
0.84
NT
NT
550
1.2
Figure 1. Epimerization and cyclization of “aspartyl” halo- and
acyloxy-methylketone analogs. R ) H or acyl and X ) halogen or
acyloxy.
hexanoate (LC biotin), and 6-[biotinamido]-6-hexanamidohex-
anoate (LCLC biotin). These have “spacer arms” between the
Nꢀ-lysine of the inhibitor and the avidin-binding ureido ring
nucleus of biotin of ∼13.5, 13.5, 22.5, and 30.0 Å, respectively.
The N-ꢀ-DNP affinity probe 13 was prepared by reaction of 11
with 2,4-dinitrofluorobenzene.
0.54
Results
Synthesis of Novel Caspase Inhibitors. Peptidyl halo- and
acyloxy-methylketone inhibitors of caspases are notoriously
difficult to prepare, and a number of commercially available
preparations of these compounds exhibited variable chemical
composition and biological activity upon testing in our labora-
tory. This variability appears to reflect, at least in part, instability
of the 3-aminoacyl-4-oxopentanoic acid (“aspartyl”) unit sub-
stituted with a good leaving group in the 5-position, which
undergoes a number of reactions, including epimerization and
facile cyclization to the corresponding γ-lactone, during chemi-
cal syntheses (Figure 1). Accordingly, initial attempts to employ
a linear synthetic strategy to the target compound 11 via the
protected acyloxymethylketone 14 proved unsuccessful. While
this intermediate was accessible, albeit in modest yield, by a
classical diazomethylketone route from Z-aspartic acid,30 the
inherent instability of the acyloxymethylketone group gave rise
to significant problems during the subsequent coupling and
deprotection steps in the synthesis. As a result, we elected to
use a new synthetic approach in which the “aspartyl” acyloxym-
ethylketone subunit of the molecule is created only after the
peptide bond-forming steps of the synthesis are complete. Using
this strategy, the tagged inhibitors 12a-d and 13 could all be
prepared in >95% purity.
Figure 2. Michaelis-Menton graphs of caspase 3-catalyzed Ac-
DEVD-pNA cleavage in the absence and presence of peptidyl aomk
inhibitors. (A) Biotinylated inhibitor 12a, (B) DNP-labeled inhibitor
13. Inhibitor concentrations: closed circle, 0 µM; open circle, 0.1 µM;
inverted closed triangle, 1.0 µM; inverted open triangle, 10 µM; closed
square, 100 µM.
Table 2. Inhibition of Caspase 3 by Substituted Peptidyl aomk
Inhibitors
Vmax
Km
Ki
label on N-ꢀ-lysine of 11
biotin, 12a
(pmol/min) (µM) (µM)
140
140
140
150
72
70
52
79
1.4
1.5
0.79
1.1
iminobiotin, 12b
6-[biotinamido]hexanoate (LC-biotin), 12c
6-[biotinamido]-6-hexanamidohexanoate
(LCLC-biotin), 12d
DNP, 13
150
75
1.0
removal of the N-terminal aspartate from the DEVD sequence
diminishes the affinity for caspases 3 and 7 much more
dramatically than for caspases 1 and 4,19 12c had a lower Ki
for caspase 1 than for caspases 3, 7, and 8. Nonetheless,
submicromolar to low micromolar values were calculated for
the Ki for these apoptotic caspases (Table 1 and Figure 2A).
As was the case with covalent inhibitors based on YVAD or
Z-VAD,20 12c was not particularly potent at inhibiting caspase
6, with a calculated Ki of 550 µM. Compounds 12a, 12b, 12d
and 13 were confirmed to have similar potencies by steady-
state kinetics with caspase 3 (Table 2 and Figure 2B). The
similar potencies of these various derivatives are consistent with
previous reports that the P2 side chain of substrate analogues
points away from the caspase active site, permitting extensive
derivatization at this site without altering affinity for the
enzyme.10,33
Novel Inhibitors Detect Active Caspases on Blots. Blotting
with horseradish peroxidase (HRP)-coupled streptavidin or anti-
DNP antibody followed by HRP-coupled secondary antibody
was used to evaluate the ability of the synthesized inhibitors to
affinity label active caspases in extracts from apoptotic Jurkat
cells. In this way, it was shown that all derivatized inhibitors
are able to bind covalently to active caspases in vitro (Figure
3A,B). Pretreatment of extracts with the commercially available
caspase inhibitor DEVD-aomk as a control abolished the
labeling by the inhibitors, thus confirming the detected bands
to be active caspases labeled with the various affinity probes.
Novel Probes Inhibit Caspases 1-8. Synthetic colorimetric
substrates containing a cleavage sequence similar or identical
to the preferred tetrapeptide recognition sequence of each
caspase are commercially available to measure enzyme activity.
Using standard spectrophotometric methods, kinetic parameters
for the cleavage of these substrates catalyzed by caspases 1-8
were determined (Table 1). The Vmax and Km values for the
p-nitroaniline (pNA) substrates are in good agreement with those
reported previously.19,31,32 Assays of caspases 9 and 10 yielded
Km values that were outside the range of substrate concentrations
tested and are not further considered here.
The inhibition constants (Ki) for 12c when added to caspases
1-8 were evaluated using steady-state kinetic methods under
the assumption of a slow, tight binding inhibitor (Table 1).
Compound 12c inhibited all tested caspases (Table 1 and Figure
2A). Consistent with a previous observation showing that

Synthesis of NoVel Caspase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26 7639
Figure 3. Use of new affinity labels for blotting. After preincubation
with diluent or DEVD-aomk, apoptotic cell lysates were reacted with
the DNP-labeled caspase probe 13 (A) or biotinylated probe 12c (B),
subjected to SDS-polyacrylamide gel electrophoresis, transferred to
PVDF membrane, and probed with anti-DNP antibody (A) or HRP-
coupled streptavidin (B).
Figure 4. Affinity purification of caspase 6-EGFP fusion protein from
cell extracts. (A) SDS-PAGE gel stained with colloidal Coomassie blue,
showing the fraction eluted with biotin. (B) Immunoblot characterization
of the biotin-eluted fraction. After SDS-PAGE, proteins were transferred
to a nitrocellulose membrane and reacted with the following: lane 1,
streptavidin-HRP; lane 2, anti-chicken caspase 6; and lane 3, anti-EGFP
antibody. The blot was subjected to a stripping procedure44 between
detection reagents.
Affinity Purification of Native Caspase 6 Using the Novel
Inhibitors. One potential use of inhibitors 12c, 12d, and 13 is
as affinity tags to allow purification from cell extracts of
sufficient quantities of caspases for further studies (e.g., the
analysis of post-translational modifications). This use is il-
lustrated for 12c, which was employed to purify caspase 6-EGFP
from apoptotic DT40 chicken lymphoma cells that were
engineered as previously described34 to overexpress a chicken
procaspase 6-EGFP fusion protein (Figure 4). To avoid the
copurification of endogenous biotinylated proteins, apoptotic
extracts were first passed over an avidin column to remove the
bulk of endogenous biotinylated polypeptides. Extracts were
then reacted with the biotinylated probe 12c, dialyzed to remove
excess probe, and passed over monomeric avidin, which bound
the affinity-labeled caspases. After a series of increasingly
stringent washes, caspases were eluted with D-biotin as a highly
enriched fraction, subjected to sodium dodecyl sulfate-poly-
acrylamide gel electrophoresis (SDS-PAGE) and stained with
Coomassie blue (Figure 4A). This test-of-concept experiment
was performed on cells that overexpressed caspase 6 with the
idea that successful recovery of EGFP-caspase 6, which has a
somewhat low affinity for the inhibitor (Table 1), would indicate
its potential usefulness with other caspases in the future.
The affinity purified caspase fraction contained four major
bands with estimated molecular weights of 18, 28, 42.5, and
44 kDa (Figure 4A). To identify these, a duplicate SDS-
polyacrylamide gel was transferred to a nitrocellulose mem-
brane, probed with HRP-coupled streptavidin, stripped, probed
with rabbit anti-chicken caspase 6 antiserum, stripped again,
and probed with anti-EGFP antiserum (Figure 4B). Band 1
bound HRP-streptavidin very strongly. Bands 1, 3, and 4 were
also recognized by anti-chicken caspase 6, whereas bands 2 and
4 were recognized by anti-EGFP. These results suggested that
band 1 corresponded to the large subunit of caspase 6, band 2
corresponded to EGFP, and bands 3 and 4 corresponded to the
EGFP-caspase 6 fusion species. To verify this identification,
bands were excised, digested with trypsin, and subjected to
matrix-assisted laser desorption/ionization-time-of-flight (MALDI-
TOF) MS. Results of this analysis confirmed the band identi-
fications deduced by immunoblotting (Table 3). Thus, this
experiment demonstrates the utility of this affinity label for the
isolation of caspases from cell extracts under nondenaturing
conditions.
DNP-Derivatized Probe is a Potent Inhibitor of Apoptosis
in Intact Cells. Although Z-VAD(OMe)-fmk and Z-EK-
(biotin)D-aomk 12a have similar modes of action, that is,
reaction with the sulfhydryl group of the active site cysteine,
Z-VAD(OMe)-fmk is sufficiently lipophilic to cross the plasma
membrane and inhibit caspases in situ, whereas the more polar
Z-EK(biotin)D-aomk is not cell-permeable. We hypothesized
that the substitution of an aromatic DNP group for biotin to
yield 13 might increase the lipophilicity of this class of
inhibitors, thus facilitating passage through the plasma mem-
brane and inhibition of apoptosis in intact cells. To address this
possibility, we treated Jurkat cells with etoposide (10 µM) for
5 h in the presence or absence of varying concentrations of 13.
Apoptotic cells were detected by TUNEL labeling and analyzed
by flow cytometry (Figure 5). As a control, the level of apoptosis
inhibition was compared to that achieved with cell permeable
Z-VAD(OMe)-fmk (10 µM). Compound 13 at 1 µM and 10
µM was found to essentially abolish apoptosis in etoposide-
treated Jurkat cells. These data demonstrate that 13 is a cell
permeable inhibitor of caspases and apoptosis in human cells.
Discussion
The human genome encodes at least 11 different caspase gene
products, but many more active caspase species are detected

7640 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26
Henzing et al.
Table 3. Identification of Polypeptides in Biotin-Eluted Fraction by MALDI-TOF MS
a
band
app. Mw
protein ID
GenBank
Mw
Mw total
MOWSEb
cover (%)
1
2
3
18 kDa
28 kDa
38 kDa
caspase 6
GFP
caspase 6
GFP
caspase 6
GFP
AF469049
P42212
AF469049
P42212
AF469049
P42212
18 167
26 887
18 167
26 887
18 167
26 887
4.11E + 04
5.38E + 08
1.79E + 03
1.52E + 07
2.54E + 04
1.52E + 07
46
49
17
45
22
45
45 053
45 053
4
40 kDa
a Apparent masses are based on the SDS preparative gel from which the band was excised. b MOWSE scores were calculated according to the method
of Pappin et al.48 using the MS-Fit tool.
Figure 5. Compound 13 rescues Jurkat cells from apoptosis induced by etoposide. Jurkat cells were treated with etoposide with or without 13 as
indicated for 5 h. Cells were then stained with TUNEL reagent and analyzed by flow cytometry to assess the level of cell death. A total of 20 000
events were analyzed for each curve.
when apoptotic cell lysates are incubated with affinity probes
such as 12a or biotinylated YVAD-cmk and fractionated by
two-dimensional gel electrophoresis.17,18 In fact, this active
caspase proteome may comprise 30 or more species in certain
cell types.35 In two studies where caspases were labeled, it was
shown that many of the spots in the two-dimensional gels were
derived from caspases 3 and 6, but other spots remained
unidentified.17,18 Many of these different active caspase species
are likely to result from differential processing of the proen-
zymes, phosphorylation,35-37 nitrosylation,38-41 and possibly
other post-translational modifications. To characterize these
modifications, it will be important to be able to purify caspases
away from more abundant cellular polypeptides.
One approach to this is the isolation of caspases tagged at
their C-termini with peptide tags. This has two potential
drawbacks, however. First, unlike affinity labeling, such methods
do not distinguish between active caspases and their procaspase
precursors. Second, there is the possibility that tagging will alter
the post-translational modifications. We have observed, for
example, that tagged caspase 7 no longer exhibits a prominent
post-translational modification that is present in the wild-type
enzyme (N. Korfali; X.W. Meng; W.C.E.; and S.H.K., unpub-
lished). These considerations prompted development of ad-
ditional caspase affinity labeling reagents.
In developing these new agents, we have attempted to address
some of the deficiencies of previous reagents. Several of the
previously available caspase affinity labeling reagents have
proven difficult to use during affinity purification. For example,
caspases affinity-labeled with 12a quantitatively flow through
avidin- and streptavidin-agarose columns under nondenaturating
conditions (F. Durrieu and W.C.E., unpublished observations),
apparently, because the covalently bound inhibitor occupies a
deep pocket shielded by a “flap” of protein that blocks access
to the biotin residue.10,33 In fact, affinity purification of the large
subunits of active caspases was possible with 12a if the labeled
enzymes were first denatured in hot SDS prior to passage over
the affinity column (F. Durrieu and W.C.E., unpublished
observations), suggesting that efficient purification of native
caspases might be possible with a new generation of inhibitor
in which the biotin was linked to the P2 lysine residue by an
extended linker arm.
We were also aware that the repertoire of caspase affinity
labeling reagents was limited, at least in part, by the fact that
synthesis of peptide acyloxymethyl ketone inhibitors is chal-
lenging. The synthetic route described here provides a conve-
nient approach for the preparation of a new generation of
irreversible caspase inhibitors to which a variety of affinity tags
can be attached. This route not only results in a relatively high

Synthesis of NoVel Caspase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26 7641
yield, but also avoids the cyclization reaction between the
aspartate side chain and the acyloxymethylketone moiety that
has plagued previous syntheses. As a result, purity of the
inhibitors prepared by this route is >95%.
might have been activated only following cell homogenization,
when inactive caspases sequestered in various subcellular
compartments would be exposed to active caspases from other
compartments. In Jurkat cells that had been exposed to etopo-
side, striking inhibition of the onset of apoptosis was observed
with 13. Therefore, substitution of the DNP moiety for biotin
(12a-d) appears to enable 13 to cross the plasma membrane,
where it can then inhibit cytosolic caspases and blunt the
apoptotic response. This means that 13 can be used to label
active caspases in situ, thereby avoiding concerns about caspase
activation during cell lysis. Compared to biotinylated VAD-
fmk, which has also recently been utilized for this purpose,24,25
13 has the advantage of not requiring preloading prior to
administration of an apoptotic stimulus. Moreover, 13 lacks a
biotin moiety, thereby diminishing contamination of any pull-
downs by endogenous biotinylated proteins17,25 and, instead,
contains the widely studied hapten DNP, for which im-
munological reagents are commercially available.
In conclusion, the reagents described here comprise a new
generation of small-molecule caspase inhibitors that can be used
for experiments aimed to characterize the active caspase
proteome in apoptotic cells. Identification of the members of
this proteome will be an important step toward understanding
the global regulation of the apoptotic response by protein
kinases, phosphatases, and other enzyme modification systems
in different normal and transformed cell populations, in response
to different stimuli, and when the apoptotic cascade is modulated
by the simultaneous up- or down-regulation of other cytosolic
signaling cascades.
This synthetic route provided a suitable opportunity to ask
whether biotinylated inhibitors with longer aliphatic arms linking
the biotin could be useful for the affinity purification of caspases
under nondenaturing conditions. Accordingly, we synthesized
the acyloxymethylketone inhibitor Z-EKD-aomk, in which the
ꢀ-amino group of the lysine residue was linked to biotin by
spacers of 13.5, 22.5, and 30 Å (12a-d). As predicted from
the fact that the P2 residue extends away from the enzyme active
site, the recognition of these affinity probes by caspases did
not appear to be influenced by the length of the linker arm
(Table 2).
The present inhibitors were based on the Z-EKD peptide
sequence previously shown to allow derivatization of caspases
3 and 6 in apoptotic extracts.17 These new inhibitors displayed
a range of affinity constants (Table 1) similar to that previously
reported for EVD-based inhibitors,19 with high affinity for
caspase 1, intermediate affinity for caspases 3, 7, and 8, and
somewhat lower affinity for caspase 6. These differences reflect
differences in geometry and chemical composition at the S3 and
S4 subsites of the various enzymes.8-12 The present compounds
were not designed to alter this spectrum of affinities, but to
instead test the hypothesis that extended linkers between the
lysine moiety and biotin would allow affinity purification under
nondenaturing conditions and that substitution of the hydro-
phobic DNP moiety would allow penetration of intact cells in
sufficient concentrations to inhibit apoptosis.
Experimental Section
As expected based on our prior unpublished results, when
we synthesized Z-EKD-aomk and linked biotin directly to the
ꢀ-amino group of the lysine, the resulting 12a efficiently labeled
active caspases as detected in immunoblots, but these enzymes
failed to bind avidin under native conditions. In contrast,
caspases bound to inhibitor 12c, in which the biotin was linked
to the ꢀ-amino group of the lysine by a longer linker arm, could
bind to avidin under native conditions. As proof of principle,
we successfully used 12c to purify nondenatured EGFP-caspase
6 from cell-free apoptotic extracts on monomeric avidin. This
protocol allowed us to wash the beads relatively stringently,
but elute the bound enzyme under mild conditions with D-biotin.
Characterization of the purified fraction by both immunoblotting
and MALDI-TOF MS confirmed the presence of active caspase
6 as a major constituent of the fraction. Even though the affinity
of the peptide moiety for caspase 6 was less then optimal (Table
1), prolonged incubation at micromolar concentrations was able
to overcome this obstacle, as had been previously reported for
other covalent caspase inhibitors.19 Accordingly, we expect that
12c will also be useful for purification of caspases for which it
has higher affinity. Thus, this reagent should prove useful in
future studies aimed at characterization of the active caspase
proteome by affinity purification of labeled caspases from
apoptotic cytosolic extracts and identification of the relevant
polypeptides and their post-translational modifications by mass
spectrometric analysis. In addition, because denaturation is not
required, this reagent should be useful for purifying polypeptides
that are associated with active caspases.
Chemistry. Reagents were obtained from the following suppli-
ers: D-mannitol, Ph3PdCHCO2CH2CH3, benzylamine, palladium
on charcoal, montmorillonite K10, 2,6-dimethylbenzoyl chloride,
pyridine, DMAP, DMPU, Dess-Martin periodinane, TFA, and 2,4-
dinitrofluorobenzene from Sigma-Aldrich (Poole, U.K.); N-R-
benzyloxycarbonyl-N-ꢀ-butoxycarbonyl-lysine-succinyl ester and
N-R-benzyloxycarbonyl-γ-O-tert-butyl-glutamyl-succinyl ester from
Bachem AG (Bubendorf, Switzerland); and biotin-NHS, iminobi-
otin-NHS, LC-biotin-NHS and LCLC-biotin-NHS from Pierce
(Rockford, IL). In synthetic procedures, flash column chromatog-
raphy was carried out over silica 60 (230-400 mesh), analytical
thin layer chromatography was carried out on 0.25 mm silica gel
plates (Fisher, Loughborough, U.K.), and organic extracts were
routinely dried over MgSO4, filtered, and concentrated in vacuo.
Purifications were carried out using a Waters 600 HPLC (Milford,
MA) coupled to a Waters 486 detector. A Lichrosorb 100 RP-8
column (7 × 250 mm, 5 µm) was used for preparative HPLC (Jones
Chromatography, Hengoed, U.K.).
Nuclear magnetic resonance (NMR) spectra were recorded on
Varian Gemini 200 (Palo Alto, CA) and Bruker AC 250, 360, or
600 spectrometers (Karlsruhe, Germany). Fast atom bombardment
(FAB) mass spectra were recorded on a Kratos MS 50 TC
(Manchester, U.K.) instrument. HPLC-MS analysis was accom-
plished by electrospray ionization (EI) and analysis on a MicroMass
platform II spectrometer (Manchester, U.K.) after separation on a
Waters Alliance 2795 HPLC using a 10 µm C-18 column (1 ×
150 mm) eluted isocratically with AcCN/H2O/HCOOH (2:98:0.1)
for 2 min, followed by a gradient to 60% AcCN/H2O/HCOOH (97:
3:0.05) over 20 min (flow rate of 1 mL/min).
[(1R)-1-[(4S)-2,2-Dimethyl-1,3-dioxolan-4-yl]-2-carboxyethyl]-
ethylamine (4). (4R)-(2) (400.5 mg, 2.0 mmol), prepared from
D-glyceraldehyde acetonide by the procedure of Matsunaga et al.,27
was stirred with benzylamine (437 µL, 429 mg, 4.0 mmol) at -50
°C for 48 h in the absence of solvent. The reaction mixture was
dissolved in EtOAc (25 mL), washed with water, dried, and
concentrated. Flash column chromatography eluting with EtOAc/
hexane (20:1) afforded N-benzyl-[(1R)-1-[(4S)-2,2-dimethyl-1,3-
Another problem with characterization of the active caspase
proteome is that the previously described affinity-labeling agents
are poorly cell permeable and are generally utilized in cell-free
extracts.17,18,21,23 Because effector caspases are activated both
by themselves and by initiator caspases, this raises the possibility
that some of the active species observed with these reagents

7642 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26
Henzing et al.
dioxolan-4-yl]-2-carboxyethyl]ethylamine (3, 460 mg, 75%) as a
colorless oil. Rf (5% MeOH/CH2Cl2) 0.70; MS (FAB, thioglycerol)
m/z 308.1859 (MH+, C17H26NO4 requires 308.1862). 1H NMR
(CDCl3): 1.24 (t, 3H, CH2CH3), 1.35 (s, 3H, CH3), 1.36 (s, 3H,
CH3), 1.82 (br s, 1H, NH), 2.46 (m, 2H, CH2CO2Et), 3.13 (q, 1H
NCH), 3.81 (t, 2H, OCH2CH3), 4.00 (m, 2H, CH2Ar), 4.12 (m,
3H, CHO + CH2O), 7.30 (m, 5H, Ar). 13C NMR (CDCl3): 13.4
(OCH2CH3), 24.4 (CH3), 25.7 (CH3), 35.4 (CH2CO2Et), 50.7
(NCH), 55.1 (CH2Ar), 59.8 (OCH2CH3), 65.5 (CH2OC), 75.8
(CHOC), 108.5 (-OCO-), 126.3, 127.5, 127.7 and 139.7 (ArC),
171.4 (CO2Et).
(OC(CH3)3), 75.7 (CHOC), 80.9 (CH2Ph), 107.9 (-OCO-), 120.5-
136.0 (ArC), 155.9, 156.0 (2 × CONH), 170.7, 170.9, 171.2, 172.6
(4 × CO2).
N-[N-((R)-N-R-Benzyloxycarbonyl-O-γ-tert-butyl-glutamyl)-
(R)-N-ꢀ-tert-butoxycarbonyl-lysyl]-(1R,2R)-1-(carboxyethylmethyl)-
2,3-dihydroxypropylamine (8). A solution of 7 (100 mg, 0.13
mmol) in EtOH/H2O (5:1, 10 mL) was stirred with Montmorillonite
K10 at 75 °C for 3 h. The suspension was diluted with EtOAc (20
mL), filtered through Celite, washed with H2O, dried, and concen-
trated. Flash column chromatography, eluting with 0-50% acetone/
CH2Cl2, yielded 8 (66 mg, 70%) as a colorless oil. Rf (10% MeOH/
CH2Cl2) 0.55. Anal. Calcd for C35H56N4O12‚2H2O: C, 55.25; H,
7.95; N, 7.36. Found: C, 55.38; H, 7.79; N, 7.30. MS (FAB,
Hydrogenation of 3 (460 mg, 1.50 mmol) over 10% Pd/C in
anhydrous EtOH (25 mL) under atmospheric pressure for 12 h,
removal of the catalyst by filtration, and purification by flash
column chromatography, eluting with 1-10% acetone/CH2Cl2
gradient, afforded 4 (279 mg, 86%) as a colorless oil. Rf (5%
MeOH/CH2Cl2) 0.25. Anal. Calcd for C10H19NO4: C, 55.28; H,
8.81; N, 6.45. Found: C, 54.79; H, 8.94; N, 6.34. MS (FAB,
NOBA) m/z 725.3987 (MH+, C35H57N4O12 requires 725.3974). 13
C
NMR (CDCl3): 13.4 (OCH2CH3), 21.9 (Kγ-CH2), 27.3 (t-Bu-CH3),
27.7 (t-Bu-CH3), 28.6 (2 × CH2, Eâ and Kâ), 30.9 (Eγ-CH2), 33.9
(Kδ-CH2), 35.9 (CH2CO2Et), 39.7 (Kꢀ-CH2), 47.1, 52.8, 53.9 (3
× CHN), 60.6 (OCH2CH3), 62.5 (CH2OH), 66.4 (OC(CH3)3), 72.2
(CHOH), 80.4 (CH2Ph), 127.4, 127.8, 135.6 (ArC), 155.9 (2 ×
CONH), 170.9, 171.8, 172.1, 172.5 (4 × CO2).
1
NOBA) m/z 218.1393 (MH+, C10H20NO4 requires 218.1392). H
NMR (CDCl3): 1.19 (t, 3H, OCH2CH3), 1.27 (s, 3H, CH3), 1.35
(s, 3H, CH3), 2.02 (s, 2H, NH2), 2.33 (m, 2H, CH2CO2Et), 3.13
(m, 1H, NCH), 3.68 (m, 1H, CHO), 3.99 (m, 2H, OCH2CH3), 4.08
(m, 2H, CH2O). 13C NMR (CDCl3): 12.9 (OCH2CH3), 24.3, 25.5
(2 × CH3), 37.6 (CH2CO2Et), 49.2 (CHN), 59.3 (OCH2CH3), 65.0
(CH2OC), 75.6 (CHOC), 108.0 (-OCO-), 170.6 (CO2)
3-[N-((R)-N-R-Benzyloxycarbonyl-glutamyl)-(R)-lysyl]amino-
(3R)-5-(2,6-dimethylbenzoyl)-4-oxopentanoic Acid (Z-EKD-
aomk,11). To a stirring solution of DMAP (22 mg, 0.18 mmol),
DMPU (23 mg, 21 µL, 0.18 mmol), and 2,6-dimethylbenzoyl
chloride (17 mg, 0.10 mmol) in dry pyridine (5 mL) was added 8
(66 mg, 91 µmole), and the solution was stirred under dry Ar at
room temperature for 3 d. The reaction mixture was diluted with
CH2Cl2 (10 mL), washed with H2O, dried, and concentrated.
Column chromatography, eluting with a 0-20% acetone/CH2Cl2
gradient, gave N-[N-((R)-N-R-Benzyloxycarbonyl-O-γ-tert-butyl-
glutamyl)-(R)-N-ꢀ-tert-butoxycarbonyl-lysyl]-(1R,2R)-1-(carboxy-
ethylmethyl)-2-hydroxy-3-(2,6-dimethylbenzoyl)propylamine (9, 56
mg, 72%) as a colorless oil. Rf (10% MeOH/CH2Cl2) 0.65. MS
(FAB, thioglycerol) m/z 857.4547 (MH+, C44H65N4O13 requires
857.4548). 13C NMR (CDCl3): 13.9 (OCH2CH3), 19.2 (2 ×
ArCH3), 22.0 (Kγ-CH2), 27.3 (t-Bu), 27.7 (t-Bu), 28.7 (2 × CH2,
Eâ and Kâ), 30.5 (Eγ-CH2), 31.1 (Kδ-CH2), 34.1 (CH2CO2Et),
39.9 (Kꢀ-CH2), 51.6, 52.6, 54.3 (3 × CHN), 60.6 (OCH2CH3),
66.3(CH2OCOAr), 66.5 (OC(CH3)3), 75.8 (CHOH), 80.6 (CH2Ph),
127.0-135.6 (ArC), 155.9, 156.0 (2 × CONH), 168.4, 171.3, 171.4,
172.4 (4 × CO2). Dess-Martin periodinane (0.92 mL, 138 mg,
0.325 mmol) in CH2Cl2 was added dropwise to a stirred solution
of 9 (56 mg, 65 µmole) in dry CH2Cl2 under Ar, and the reaction
was allowed to stir overnight. The reaction mixture was diluted
with CH2Cl2 (10 mL), washed with H2O, dried, and concentrated.
Column chromatography, eluting with a 0-10% acetone/CH2Cl2
gradient, gave N-[N-((R)-N-R-Benzyloxycarbonyl-O-γ-tert-butyl-
glutamyl)-(R)-N-ꢀ-tert-butoxycarbonyl-lysyl]-(1R,2R)-1-(carboxy-
ethylmethyl)-3-(2,6-dimethylbenzoyl)-2-oxopropylamine (10, 44
mg, 80%) as a colorless glass. Rf (10% MeOH/CH2Cl2) 0.65. MS
(FAB, thioglycerol) m/z 855.4392 (MH+, C44H67N4O13 requires
855.4393). 13C NMR (CDCl3): 13.9 (OCH2CH3), 19.2 (2 ×
ArCH3), 22.0 (Kγ-CH2), 27.3 (t-Bu), 27.7 (t-Bu), 28.7 (2 × CH2,
Eâ and Kâ), 30.5 (Eγ-CH2), 31.1 (Kδ-CH2), 34.1 (CH2CO2Et),
39.9 (Kꢀ-CH2), 51.6, 52.6, 54.3 (3 × CHN), 60.6 (OCH2CH3), 66.3
(OC(CH3)3), 66.5 (OC(CH3)3), 75.8 (COCH2O), 80.6 (CH2Ph),
127.0-135.6 (ArC), 155.9, 156.0 (2 × CONH), 168.4, 171.3, 171.4,
172.4 (4 × CO2), 199.9 (CO). TFA/water (95:5, 300 µL) was added
dropwise to a stirred solution of 10 (5.0 mg, 5.8 µmole) in CH2Cl2
(2 mL) at 0 °C, and the mixture was allowed to come to room
temperature. After 2 h, the reaction mixture was filtered and
concentrated in vacuo, affording essentially homogeneous 11 (3.1
mg, 80%) as a colorless residue. Rf (10% MeOH/CH2Cl2) 0.05.
HPLC-MS Rt (MH+) 24.14 min (694). MS (FAB, NOBA) m/z
694.3189 (MHNa+, C33H43N4O11Na requires 694.2826). 13C NMR
(CDCl3): 17.3 (2 × ArCH3), 21.2 (Kγ-CH2), 25.4 (2 × CH2, Eâ
& Kâ), 26.9 (Eγ-CH2), 31.1 (Kδ-CH2), 32.9 (CH2CO2), 37.9 (Kꢀ-
CH2), 51.6, 52.6, 54.3 (3 × CHN), 78.9 (COCH2O), 80.6 (CH2-
Ph), 126.1-135.4 (ArC), 156.1, 160.2 (2 × CONH), 168.5, 169.6,
172.0, 174.2 (4 × CO2), 199.5 (CO)
N-[N-((R)-N-R-Benzyloxycarbonyl-O-γ-tert-butyl-glutamyl)-
(R)-N-ꢀ-tert-butoxycarbonyl-lysyl]-[(1R)-1-[(4S)-2,2-dimethyl-
1,3-dioxolan-4-yl]-2-carboxyethyl]ethylamine (7). A solution of
4 (279 mg, 1.28 mmol) in 2 mL dry CH2Cl2 was added to a solution
of (R)-N-R-benzyloxycarbonyl-N-ꢀ-tert-butoxycarbonyl-lysine NHS
ester (673 mg, 1.41 mmol) in dry CH2Cl2 (10 mL), and the mixture
was stirred overnight at room temperature. The reaction mixture
was diluted with CH2Cl2 (10 mL), washed with H2O (10 mL), dried,
and concentrated. Column chromatography using 0-10% acetone
in CH2Cl2 as eluent afforded N-[(R)-N-R-Benzyloxycarbonyl-N-ꢀ-
tert-butoxycarbonyl-lysyl]-[(1R)-1-[(4S)-2,2-dimethyl-1,3-dioxolan-
4-yl]-2-carboxyethyl]ethylamine (5, 632 mg, 85%) as a colorless
oil. Rf (5% MeOH/CH2Cl2) 0.45. MS (FAB, NOBA) m/z 580.3234
(MH+, C29H46N3O9 requires 580.3235). 13C NMR (CDCl3): 13.3
(OCH2CH3), 21.7 (Kγ-CH2), 24.0 (Kâ-CH2), 25.4 (2 × CH3), 27.7
(t-Bu-CH3), 28.9 (Kδ-CH2), 36.1 (CH2CO2Et), 35.9 (Kꢀ-CH2), 45.9,
54.5 (2 × CHN), 60.1 (OCH2CH3), 65.2 (CH2OC), 66.2 (C(CH2)3),
75.5 (CHOC), 78.2 (CH2Ph), 108.6 (-OCO-), 127.4, 127.8, 135.7
(ArC), 155.6 (CONH), 170.3, 171.4 (2 × CO2). Hydrogenation of
5 (630 mg, 1.09 mmol) with H2 over 10% Pd/C in anhydrous EtOH
(25 mL) under atmospheric pressure for 3-4 h gave the crude amine
after filtration and concentration. Column chromatography eluting
with a 1-10% acetone/CH2Cl2 gradient gave N-[(R)-N-ꢀ-tert-
Butoxycarbonyl-lysyl]-[(1R)-1-[(4S)-2,2-dimethyl-1,3-dioxolan-4-
yl]-2-carboxyethyl]ethylamine (6, 446 mg, 92%) as a colorless oil.
Rf (10% MeOH/CH2Cl2) 0.55. MS (FAB, NOBA) m/z 446.2866
(MH+, C21H40N3O7 requires 446.2866). 13C NMR (CDCl3): 13.3
(OCH2CH3), 21.7 (Kγ-CH2), 24.0 (Kâ-CH2), 25.4 (2 × CH3), 27.6
(t-Bu-CH3), 28.8 (Kδ-CH2), 36.1 (CH2CO2Et), 39.2 (Kꢀ-CH2), 45.9
(CHN), 53.9 (CHN), 59.8 (OCH2CH3), 65.2 (CH2OC), 75.4
(CHOC), 77.8 (CH2Ph), 108.5 (-OCO-), 155.4 (CONH), 170.2,
173.3 (2 × CO2). To a solution of N-R-benzyloxycarbonyl-γ-O-
tert-butyl-glutamyl NHS ester (478 g, 1.1 mmol) in dry CH2Cl2
(10 mL) was added 6 (440 mg, 0.99 mmol), and the mixture was
stirred overnight under dry N2 at room temperature. The reaction
mixture was diluted with CH2Cl2 (10 mL), washed with H2O, dried,
and concentrated. Column chromatography using a 0-20% acetone/
CH2Cl2 gradient afforded 7 (620 mg, 81%) as a colorless amorphous
solid (mp 62-63 °C). Rf (10% MeOH/CH2Cl2) 0.60. Anal. Calcd
for C38H60N4O12: C, 59.67; H, 7.91; N, 7.09. Found: C, 59.28; H,
7.93; N, 7.32. MS (FAB, NOBA) m/z 765.4286 (MH+, C38H61N4O12
requires 765.4285). 13C NMR (CDCl3): 13.9 (OCH2CH3), 22.3 (Kγ-
CH2), 24,2, 25.8 (2 × CH3), 27.3 (t-Bu-CH3), 27.9 (t-Bu-CH3),
28.3, 29.1, 29.2 (3 × CH2), 36.5 (CH2CO2Et), 39.9 (Kꢀ-CH2), 48.2,
53.1, 54.6 (3 × CHN), 60.6 (OCH2CH3), 65.9 (CH2OC), 66.9

Synthesis of NoVel Caspase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26 7643
N-Biotinylated Z-EKD-aomk Derivatives (12a-d). These
compounds were prepared on a 4-5 mmolar scale in 70-80%
yields by treatment of 11 with the N-hydroxysuccinamides of biotin,
iminobiotin, LC biotin, and LCLC biotin. Typically, to a stirred
solution of 11 (3.1 mg, 4.6 µmole) in dry DMF (500 µL) was added
biotin N-hydroxysuccinamide (1.0 mg, 5.1 µmole). After 3 h, the
reaction mixture was diluted with CH2Cl2 (20 mL) and washed
with H2O to remove unreacted reagent and byproducts. Concentra-
tion in vacuo afforded 12a (2.9 mg, 75%) as a colorless glass that
was further purified by HPLC on a RP-8 silica column using
MeOH/H2O/TFA (66:33:0.1) as eluent.
(ArC), 156.1, 160.2 (2 × CONH), 170.6, 171.1, 171.3, 173.6 (4 ×
CO2), 199.5 (ketone-CO).
Spectrophotometric Assays of Caspase Inhibition. Recombi-
nant human caspases 1-10 were purchased from Biomol (Plymouth
Meeting, PA). Peptidyl caspase substrates conjugated to p-nitroa-
niline (pNA), including Ac-YVAD-pNA, Ac-LEHD-pNA, Ac-
DEVD-pNA, Ac-VEID-pNA, and Ac-IETD-pNA, from Biomol
were confirmed to be >95% pure by HPLC, as were the peptidyl
inhibitors prepared in this study. Assays were conducted in 96-
well microtiter plates and contained 150 µL of assay buffer [10
mM Tris-HCl, 1 mM DTT, 10% (v/v) glycerol, 0.1% (w/v)
CHAPS, pH 7.5], 10 µL of enzyme in assay buffer (final
concentration 1-100 nM), 20 µL of substrate solution (dissolved
in DMSO and diluted in assay buffer to 3.9-1000 µM), and 20
µL of inhibitor solution (dissolved in DMSO and diluted in assay
buffer to 1-1000 µM). Assays were conducted in triplicate with
the assay buffer, substrate, and inhibitor preincubated in the
microtiter plate at 37 °C. Reactions were started by the addition of
enzyme warmed to 37 °C. Production of pNA was measured by
following the absorbance at 405 nm minus that at 650 nm using an
ELx808 Ultra microplate reader from Bio-Tek Instruments, Inc.
(Winooski, VT).
Progress curves of product formation versus time were fitted
using the program KinetiCalc for Windows (Bio-Tek Instruments,
Winooski, VT) to measure the initial velocity for reactions over
the range of 3.9-1000 µM substrate and 0.1-100 µM inhibitor.
Michaelis-Menton curves of the initial velocity data were fitted
by nonlinear least-squares regression analysis using SigmaPlot,
version 9.01, with the Enzyme Kinetics Module (Systat Software,
Inc., Richmond, CA). The substituted Z-EKD-aomk caspase inhibi-
tors were modeled as tight binding inhibitors.42
Tissue Culture, Cytosolic Extract Preparation, and Affinity
Labeling. The human Jurkat T cell lymphoma cell line was cultured
in RPMI 1640 supplemented with 10% fetal bovine serum. The
chicken lymphoma B-cell line DT40 expressing chicken procaspase
6 fused to EGFP34 was cultured as previously described.43 To induce
apoptosis, cells were treated with 10 µM etoposide for 5 hours.
Cytosolic extracts were prepared from untreated and apoptotic cells
according to the procedure described by Ruchaud et al.34 Aliquots
of apoptotic cytosol were incubated for 30 min at 37 °C with 2
µM inhibitor. At the completion of the incubation, samples were
diluted with 0.5 volume of 3× SDS sample buffer, heated to 95
°C for 5 min, subjected to SDS-PAGE on 16% (w/v) acrylamide
gels, transferred to nitrocellulose or polyvinylidene fluoride mem-
branes (Amersham Biosciences, Uppsala, Sweden), probed with
HRP-coupled streptavidin (Sigma, St. Louis, MO). Alternatively,
blots were probed with antibodies as described below.
Immunoblotting. Samples containing 30 µg of total cellular
protein were subjected to electrophoresis for 75 min at 200 V on
SDS polyacrylamide minigels containing 16% (w/v) acrylamide and
electrophoretically transferred to nitrocellulose or polyvinylidene
fluoride membranes (Amersham Biosciences) at ambient temper-
ature for 90 min at 40 mA per minigel. Immunoblotting followed
by enhanced chemiluminescent detection was performed as de-
scribed34 using mouse monoclonal anti-DNP from Molecular Probes
(Leiden, NL), rabbit anti-EGFP antiserum from Ilan Davis (Univ.
of Edinburgh), or rabbit polyclonal anti-chicken caspase 6 (R549)
raised against the large subunit of chicken caspase 634 as primary
antibodies and HRP-conjugated anti-mouse IgG or anti-rabbit IgG
(Amersham Biosciences) as secondary antibody.
Compound 12a: 13C NMR (CD3CN-H2O): 19.8 (2 × ArCH3),
23.6 (Kγ-CH2), 26.3 (biotin γ-CH2), 27.6 (Kâ-CH2), 28.9 (biotin
â-CH2), 29.2 (biotin δ-CH2), 30.2 (Eâ-CH2), 31.0 (Kδ-CH2), 31.3
(CH2CO2), 35.4 (Eγ-CH2), 36.4 (Kꢀ-CH2), 39.7 (biotin R-CH2),
40.8 (biotin C-5), 53.9, 54.5, 55.5 (3 × CHN), 56.2 (biotin C-2),
61.1 (biotin C-3), 62.3 (COCH2O), 62.9 (biotin C-4), 67.5
(COCH2O), 68.0 (CH2Ph), 128.6, 129.1, 129.5, 130.9, 133.5, 136.3,
137.5 (12 × ArC), 158.0 (CO), 165.5 (biotin ureido-C), 170.3,
172.2, 172.3, 174.0, 176.0, 176.8 (6 × CO), 202.1 (ketone-CO).
Compound 12b: 13C NMR (CD3CN-H2O): 20.3 (2 × ArCH3),
23.7 (Kγ-CH2), 26.7 (biotin γ-CH2), 28.4 (Kâ-CH2), 29.1 (biotin
â-CH2), 29.2 (biotin δ-CH2), 30.7 (Eâ-CH2), 31.9 (Kδ-CH2), 32.6
(CH2CO2), 35.9 (Eγ-CH2), 36.9 (Kꢀ-CH2), 40.0 (biotin R-CH2),
40.7 (biotin C-5), 54.2, 54.4, 56.1 (3 × CHN), 56.6 (biotin C-2),
62.7 (biotin C-3), 64.0 (COCH2O), 64.8 (biotin C-4), 68.1
(COCH2O), 68.5 (CH2Ph), 129.1, 129.5, 129.9, 131.3, 134.2, 136.8,
138.0 (12 × ArC), 156.1, 158.0, 158.6, 170.3, 172.2, 172.3, 174.0,
176.1 (CO and CdN), 202.1 (ketone CO).
Compound 12c: 13C NMR (CD3CN-H2O): 19.3 (2 × ArCH3),
23.0 (Kγ-CH2), 25.6, 25.8 (2 × linker CH2), 26.3 (biotin γ-CH2),
27.6 (Kâ-CH2), 28.3 (biotin â-CH2), 28.6 (biotin δ-CH2), 28.8
(linker CH2), 29.7 (Eâ-CH2), 30.8 (Kδ-CH2), 31.6 (CH2CO2), 34.9
(Eγ-CH2), 36.0 (linker CH2CO), 36.1 (Kꢀ-CH2), 39.3 (linker CH2N),
39.9 (biotin R-CH2), 40.3 (biotin C-5), 53.3, 53.9, 55.3 (3 × CHN),
55.8 (biotin C-2), 60.5 (biotin C-3), 61.8 (COCH2O), 62.2 (biotin
C-4), 67.1 (COCH2O), 67.5 (CH2Ph), 128.1, 128.5, 130.0, 130.3,
133.0, 135.7, 137.0, 139.0 (12 × ArC), 157.5 (CO), 164.8 (biotin
ureido-C), 169.7, 171.5, 171.6, 173.6, 173.7, 175.3, 175.4 (7 ×
CO), 201.8 (ketone-CO).
Compound 12d: 13C NMR (CD3CN-H2O): 19.3 (2 × ArCH3),
23.0 (Kγ-CH2), 25.6, 25.8 (4 × linker CH2), 26.3 (biotin γ-CH2),
27.5 (Kâ-CH2), 28.3 (biotin â-CH2), 28.7 (biotin δ-CH2), 29.8 (2
× linker CH2), 29.6 (Eâ-CH2), 31.0 (Kδ-CH2), 31.4 (CH2CO2),
34.9 (Eγ-CH2), 36.0 (2 × linker CH2CO), 36.2 (Kꢀ-CH2), 39.1,
39.3 (2 × linker CH2N), 39.9 (biotin R-CH2), 40.3 (biotin C-5),
53.3, 53.9, 55.3 (3 × CHN), 55.8 (biotin C-2), 60.5 (biotin C-3),
61.8 (COCH2O), 62.2 (biotin C-4), 67.1 (COCH2O), 67.5 (CH2-
Ph), 128.1, 128.5, 130.0, 130.3, 133.0, 135.8, 137.0, 139.0 (12 ×
ArC), 157.6 (CO), 164.9 (biotin ureido-C), 169.7, 171.6, 171.7,
173.6, 173.7, 175.4, 175.5 (7 × CO), 201.8 (ketone-CO).
HPLC-MS (Rt, MH+): 12a (18.50 min, 897), 12b (18.34 min,
896), 12c (18.28 min, 1010), 12d (18.11 min, 1123). Because exact
mass determinations could not easily be obtained for these
compounds directly, they were derivatized as their ethyl esters by
treatment with 3% HCl in EtOH for 1 h at 10 °C. MS (FAB,
thioglycerol) of the monoethyl esters of 12a-d gave exact masses
within a deviation of 0.8 ppm of the calculated MH+ values (see
Supporting Information).
N-(2,4-Dinitrophenyl) Z-EKD-aomk (13). To a stirred solution
of 11 (3.1 mg, 4.6 µmole) in dry DMF (500 µL) were added NMM
(50 µg, 5 µmol) and 2,4-dinitrofluorobenzene (950 µg, 5.1 µmol).
After 12 h, the reaction mixture was diluted with CH2Cl2 (20 mL),
washed with H2O, dried, and concentrated to afford 13 (2.9 mg,
75%) as a colorless glass. Rf (10% MeOH/CH2Cl2) 0.55. HPLC-
MS Rt (MH+) 20.41 min (837). MS (FAB, NOBA) m/z MH+
837.3028 (MH+, C39H45N6O15 requires 837.2943). 13C NMR
(CDCl3): 19.1 (2 × ArCH3), 21.5 (Kγ-CH2), 22.4 (Kâ-CH2), 27.4
(Eâ-CH2), 29.0 (Kδ-CH2), 30.8 (Εγ-CH2), 35.8 (CH2CO2), 42.5
(Kꢀ-CH2), 50.5, 51.6, 52.7 (3 × CHN), 78.9 (COCH2O), 79.2 (CH2-
Ph), 113.4 (CNO2), 123.7 (CNO2), 126.1-135.4 (15 × ArC), 147.7
Affinity Purification of Derivatized Caspase 6. Avidin-
sepharose, monomeric avidin-sepharose beads, and slide-a-lyze
dialysis slides were purchased from Pierce (Rockford, IL). Endog-
enous biotinylated proteins were removed from apoptotic cytosolic
extracts of DT40 cells expressing EGFP/caspase 634 by incubation
with avidin-sepharose beads for 30 min at 4 °C. Unbound
polypeptides were eluted with calcium- and magnesium-free
Dulbecco’s phosphate buffered saline (PBS) and labeled with Z-EK-
(LC-biotin)D-aomk (12c, 2 µM) by incubation for 30 min at 37
°C. Excess inhibitor was removed by dialysis against PBS overnight
at 4 °C. After the labeled fraction was incubated with monomeric

7644 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26
Henzing et al.
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avidin-sepharose beads for 30 min at ambient temperature, the
column was washed (three washes of 3× bed volume) with PBS.
The nonspecifically bound polypeptides were removed with in-
creasingly stringent washes (four times 3× bed volume of 0.5 M
NaCl, four times 3× bed volume of 1.0 M NaCl). Bound
polypeptides were then eluted with 2 mM biotin in PBS (four times
3× bed volume). Eluted fractions were concentrated using 5 K spin
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Each fraction was loaded onto two SDS-polyacrylamide gels
containing 16% (w/v) acrylamide. One gel was stained with
colloidal Coomassie blue (Genomic Solutions, Huntington, U.K.),
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MALDI-TOF Mass Spectrometry. Polypeptide identification
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Coomassie blue-stained gels, in-gel digestion with trypsin was
performed as previously described,46 followed by sample preparation
using miniaturized sample concentration/desalting techniques.47 For
the mass spectrometric analysis, a PerSeptive Biosystems Voyager
DESTR MALDI-TOF mass spectrometer (Applied Biosystems,
Foster City, CA) was used. Peptide ion signals were assigned with
a mass error less than 50 ppm. Lists of tryptic peptide masses were
used to search protein sequence databases using the MS-Fit tool
(http://prospector.ucsf.edu/ucsfhtml4.0u/msfit.htm). For positive
identification, a molecular weight search (MOWSE) score48 of 104
and at least 20% coverage of the protein by the peptide fragments
was required.
Flow Cytometry. Jurkat cells were treated with 10 µM etoposide
(Calbiochem, San Diego, CA) plus 10 µM Z-VAD(OMe)-fmk
(Calbiochem) or Z-EK(DNP)D-aomk (13) at 0.1, 1 or 10 µM for
5 h, harvested, washed in PBS, fixed in 4% (w/v) paraformaldehyde
in PBS, permeabilized in 0.1% (w/v) Triton X-100 containing 0.1%
(w/v) sodium citrate in PBS for 2 min on ice, and labeled using
the TUNEL (terminal deoxyribonucleotidyl transferase nick end
labeling) reaction according to the manufacturer’s recommendations
(In situ cell death detection kit, TMR red, Roche, Indianapolis, IN).
The cells were then analyzed on a FACSCalibur flow cytometer
(Becton Dickinson, San Jose, CA) using Becton Dickinson CellQuest
software.
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Acknowledgment. We are grateful for the financial support
from the NIH (CA69008 to S.H.K. and W.C.E.) and the
Wellcome Trust. A.J.H. was supported by a Wellcome Prize
Studentship and Wellcome Prize Fellowship. W.C.E. is a
Principal Research Fellow of the Wellcome Trust.
(21) Martins, L. M.; Mesner, P. W.; Kottke, T. J.; Basi, G. S.; Sinha, S.;
Tung, J. S.; Svingen, P. A.; Madden, B. J.; Takahashi, A.; McCor-
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Supporting Information Available: Further documentation of
the characterization of target compounds 12a-d and 13. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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