A coumarin-labeled vinyl sulfone as tripeptidomimetic activity-based probe for cysteine cathepsins

Article abstract of DOI:10.1002/cbic.201300806

A coumarin-tetrahydroquinoline hydride 8 was synthesized as a chemical tool for fluorescent labeling. The rigidified tricyclic coumarin structure was chosen for its suitable fluorescence properties. The connection of 8 with a vinyl sulfone building block was accomplished by convergent synthesis thereby leading to the coumarin-based, tripeptidomimetic activity-based probe 10, containing a Gly-Phe-Gly motif. Probe 10 was evaluated as inactivator of the therapeutically relevant human cysteine cathepsins S, L, K, and B: it showed particularly strong inactivation of cathepsin S. The detection of recombinant and native cathepsin S was demonstrated by applying 10 to in-gel fluorescence imaging. Tricycle light: A tripeptidomimetic, vinyl sulfone-type activity-based probe containing a rigid coumarin moiety fluorophore was prepared by convergent synthesis. The probe was evaluated as an inactivator of human cathepsins and, as exemplified with cathepsin S, it proved to be suitable for imaging in SDS-PAGE.

Full text of DOI:10.1002/cbic.201300806

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DOI: 10.1002/cbic.201300806
A Coumarin-Labeled Vinyl Sulfone as Tripeptidomimetic
Activity-Based Probe for Cysteine Cathepsins
Matthias D. Mertens,[a] Janina Schmitz,[a] Martin Horn,[b] Norbert Furtmann,[a, c]
[c]
[a]
ˇ [b]
Jꢀrgen Bajorath, Michael Mares, and Michael Gꢀtschow*
A coumarin-tetrahydroquinoline hydride 8 was synthesized as
a chemical tool for fluorescent labeling. The rigidified tricyclic
coumarin structure was chosen for its suitable fluorescence
properties. The connection of 8 with a vinyl sulfone building
block was accomplished by convergent synthesis thereby lead-
ing to the coumarin-based, tripeptidomimetic activity-based
probe 10, containing a Gly-Phe-Gly motif. Probe 10 was evalu-
ated as inactivator of the therapeutically relevant human cys-
teine cathepsins S, L, K, and B: it showed particularly strong
inactivation of cathepsin S. The detection of recombinant and
native cathepsin S was demonstrated by applying 10 to in-gel
fluorescence imaging.
ABPs as pharmacological tools to better understand its role in
the processes of adaptive immunity.[5,8] Several electrophilic
warheads to interact with the active-site cysteine have been
incorporated into cysteine protease inhibitors. Among these,
peptidyl vinyl sulfones are known to efficiently inactivate their
targets by a Michael-type irreversible reaction.[9] Recent reports
have illustrated the successful development of peptidyl vinyl
sulfones as inactivators of human cathepsins B, L, and K,[10–12]
caspase-3,[13] cathepsin B from Schistosoma mansoni,[14] as well
as falcipain-2 and -3, cruzain, and rhodesain.[10,15] Moreover, an
ABP with a vinyl sulfone substructure was developed for cathe-
psin C,[4] and a panel of vinyl sulfone probes was used in a cys-
teine protease microarray.[16]
Fluorescence labeling is of growing interest in the life scien-
ces. Various classes of fluorophores are synthetically accessible
and commercially available, and these are widely employed for
manifold purposes. In addition to BODIPY and fluorescein de-
rivatives, coumarins constitute a commonly used class of fluo-
rescent label; these are valued for their large Stokes shifts.[17–19]
The coumarins’ small molecular size makes them attractive, in
particular, for incorporation into peptides and for assembling
artificial protease substrates and peptidic protease inhibi-
tors.[20–23] Several derivatization reagents bearing a coumarin
moiety as a fluorophore have been reported.[24] Structure–fluo-
rescence relationships of the coumarin chemotype are well un-
derstood. Among the coumarin derivatives bearing a combina-
tion of an electron-withdrawing group at the 3-position and
an electron-donating substituent at the 7-position, 7-methoxy-
or 7-aminocoumarins produce high fluorescence quantum
yields. The latter possess a typical red shift of absorption and
emission, a favorable feature with regard to background fluo-
rescence. For example, 7-diethylaminocoumarin-3-carboxylic
acid (DEAC) is an established fluorophore,[18,21,22] but its fluores-
cence is quenched in aqueous environment.[22,23] Rigidization
of the amino group in one ring (e.g., tetrahydropyrido-coumar-
ins ATTO390 and ATTO425) or two rings (julolidine-type cou-
marin 343) significantly restores fluorescence in aqueous
media.[17,23,25]
Activity-based probes (ABPs) have been widely used for pro-
tein identification and profiling.[1] Owing to their covalent
mechanism of catalysis, serine and cysteine hydrolases are par-
ticularly well suited to be addressed by ABPs.[2] Most ABPs con-
sist of three main elements: a “warhead” for covalent interac-
tion, a tag (reporter or affinity label) that allows the detection
or isolation of the target, and a linker, which can additionally
control the probe’s selectivity for the protein of interest.
Among the target enzymes for ABPs, human cysteine cathe-
psins have attracted considerable interest.[3–5] Much of this at-
tention arises from the importance of these proteases in a vari-
ety of (patho)physiological processes and disease states, such
as cancer progression, degradation of the extracellular matrix,
angiogenesis, bone remodeling, prohormone processing,
aneurysm formation, atherosclerosis, and rheumatoid arthri-
tis.[6] Human cathepsin S is the main processing enzyme of the
major histocompatibility complex class II-associated invariant
chain in antigen-presenting cells.[7] Thus, cathepsin S is a target
enzyme for the development of inhibitors as potential thera-
peutics against autoimmune diseases, and for the design of
[a] M. D. Mertens, J. Schmitz, N. Furtmann, Prof. Dr. M. Gꢀtschow
Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn
An der Immenburg 4, 53121 Bonn (Germany)
E-mail: guetschow@uni-bonn.de
Herein, we describe the synthesis of a coumarin-tetrahydro-
quinoline hydride molecule (8, Scheme 1), a new member of
the 3-acceptor-substituted 7-aminocoumarins. Its rigid struc-
ture was expected to allow the desired bathochromic shift and
sufficient fluorescence in aqueous media. The incorporated ni-
trogen can be considered as part of a glycine linker for peptide
bond formation.
ˇ
[b] Dr. M. Horn, Dr. M. Mares
Institute of Organic Chemistry and Biochemistry
Academy of Sciences of the Czech Republic
Flemingovo n. 2, 16610 Prague (Czech Republic)
[c] N. Furtmann, Prof. Dr. J. Bajorath
Bonn–Aachen International Center for Information Technology
University of Bonn
Dahlmannstrasse 2, 53113 Bonn (Germany)
The seven-step synthesis of 8 started from 7-hydroxy-3,4-di-
hydroquinolin-2(1H)-one (1, Scheme 1). After benzylation to
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201300806.
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Scheme 1. Synthesis of coumarin-labeled activity-based probe 10. Reagents and conditions: a) BnBr, K2CO3, DMF, 16 h, 84%; b) BH3·THF, THF, 08C, 99%;
c) BrCH2CO2tBu, K2CO3, DMF, 808C, 16 h, 84%; d) H2, Pd/C, MeOH, 3 h, 93%; e) POCl3, DMF, 08C, 30 min, 908C, 30 min, 92%; f) dimethyl malonate, piperidine,
MeOH, 2 h, 568C, 81%; g) CF3CO2H, CH2Cl2, 2 h, 97%; h) i: MeOH, AcCl, EtOAc, 9, 30 min; ii: 8, HATU, DIPEA, DMF, 2 h, 18% over two steps.
protect the phenolic group, the
lactam moiety of 2 was reduced
with borane to furnish the
1,2,3,4-tetrahydroquinoline deri-
vate 3. Alkylation with tert-butyl
bromoacetate afforded 4, which
was catalytically hydrogenated
to deprotect the phenol. The sal-
icylaldehyde derivative
6 was
obtained by formylation of 5
under Vilsmeier conditions. Sub-
sequent Knoevenagel condensa-
tion with dimethyl malonate
yielded coumarin 7, with two or-
thogonally protected esters. The
tert-butyl ester of 7 was cleaved
Figure 1. Absorption and emission spectra of 10. Left: normalized absorption and emission spectra of 10 (in H2O).
Center: absorption spectra of 10 (10 mm, 1% DMSO) in CH2Cl2 (dotted), MeOH (gray), and H2O (black). Right: emis-
sion spectra of 10 (1 mm, 1% DMSO) in CH2Cl2, MeOH, and H2O.
under acidic conditions to give 8 (overall yield 47%). Building
block 9, which was separately synthesized in five steps accord-
ing to a reported method,[12] was labeled with fluorophore 8.
This final step of the convergent synthesis included Boc-depro-
tection of 9 and HATU-mediated amide coupling. The activity-
based probe 10 was designed as a tripeptide whose terminal
nitrogen is part of the tricyclic fluorophore and whose carbox-
ylic group is replaced by the vinyl unit. If there is a preference,
10 would be expected to favor cathepsin S rather than the
other cathepsins included in this study, considering the struc-
ture of a related vinyl sulfone inhibitor.[12]
cence of 10 was similarly intense in each solvent; no quench-
ing was observed in methanol or water (in contrast to DEAC
derivatives).[22,23] The logD7.4 value, a reliable predictor of cell
permeability, was determined by an HPLC-based procedure.[26]
The logD7.4 value of 1.93 indicates the ability of the fluoro-
phore-labeled probe 10 to cross membranes and penetrate
into cells.
The inhibitory potency of 10 against four human cysteine
cathepsins (S, L, K, and B) was evaluated by activity assays with
chromogenic or fluorogenic peptide substrates. Time-depen-
dent inactivation of the cathepsins was monitored over 60 min
(Figure 2). The probe was able to inactivate all tested cathep-
sins, with cathepsin S being most efficiently affected (second-
The activity-based probe 10 was characterized for its spec-
troscopic properties in three solvents of different polarity
(Figure 1). The spectra show slight bathochromic shifts in polar
solvents for both absorption maxima (413 nm in CH2Cl2,
420 nm in MeOH, 424 nm in H2O) and emission maxima
(456 nm in CH2Cl2, 466 nm in MeOH, 470 nm in H2O), thus re-
sulting in Stokes shifts of 43–46 nm. Noteworthy, the fluores-
order rate constant 49000mÀ1 À1; Table 1).
s
AutoDock 4.2 (Scripps Research Institute, La Jolla, CA) was
used to predict the binding mode of 10 at the active site of
human cathepsin S (Figure 3). The covalent sulfur–carbon
bond was manually constructed involving the active-site cys-
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tematic exploration of the conformational space available to
covalently linked 10 at the active site revealed that the tripep-
tide backbone is oriented in a substrate-like manner, thus
forming multiple hydrogen bond interactions with Gly69 and
Asn163 (see Figure S1 in the Supporting Information).
An overlay of the putative binding mode of 10 with the
crystal structure revealed a similar orientation (Figure S2).[27]
The S2 subsite of cathepsin S (shaped by residues Met71,
Gly137, Gly165, Phe70, Val162, and Phe211)[27] provides a hydro-
phobic environment that well accommodates the side chain of
phenylalanine at the P2 position of 10. The coumarin-tetrahy-
droquinoline moiety is directed towards the S3 pocket but ori-
entated away from the enzyme, and is thus solvent exposed.
However, its position might be stabilized by hydrogen bond
interactions between its two carbonyl groups and the side
chain of Arg141 and the backbone NH of Val162 (Figure S1).
The S1’ subsite is occupied by the phenyl sulfone group of the
inhibitor, thus enabling formation of p–p interactions with the
side chains of His164 and Trp186. The SO2 group is potentially
in hydrogen bonding distance from Gln19 and the indole ni-
trogen of Trp186.
Figure 2. Inhibition of human cathepsin S by 10. Cathepsin S-catalyzed hy-
drolysis of Z-Phe-Arg-AMC (40 mm) in the presence of 10 (top to bottom:
0 nm, 5 nm, 10 nm, 15 nm, 20 nm, and 25 nm). Inset: plot of kobs against con-
centration of 10.
The feasibility of 10 for direct in-gel fluorescence detection
of human cysteine cathepsins was demonstrated by using
cathepsin S and a common imaging tool. Cathepsin S (500 ng)
was treated with 5 mm 10 for 30 min and subjected to SDS-
PAGE; a strong fluorescent band at 25 kDa was clearly detect-
ed (Figure 4). The binding mode of 10 was confirmed by a
Table 1. Inhibition of human cysteine cathepsins by 10.
Cat S
Cat L
Cat K
Cat B
[a]
k
obs/[I] [mÀ1 sÀ1
]
22820Æ1100
2450Æ160
61.7Æ8.6 128Æ12
[b]
kinac/Ki [mÀ1 sÀ1
]
49000Æ1900 16900Æ1100 884Æ123
186Æ17
[a] Data kobs/[I]ÆSEM are from duplicate measurements with five different
inhibitor concentrations. The kobs values were obtained by nonlinear
regression of the progress curves. The kobs/[I] values were obtained by
linear regression from plots of kobs against [I]. [b] The kinac/Ki values were
calculated by using the equation kinac/Ki =(1+[S]/Km)ꢂkobs/[I].
Figure 4. Imaging human cathepsin S with the fluorescent probe 10. Left:
purified recombinant cathepsin S (500 ng) was labeled with 10 (5 mm). In the
control experiment, the enzyme was treated with E64 (5 mm) prior to 10
(5 mm). Right: cathepsin S (250 to 0.5 ng) was treated with 10 (5 mm). Label-
ing reactions were performed for 1 h at pH 6.5. The mixtures were resolved
by SDS-PAGE and visualized with a Typhoon 9410 fluorescence imager.
competition experiment with the active-site-directed broad-
spectrum cysteine protease inhibitor E64 to protect cathe-
psin S from inactivation by 10. First, cathepsin S (500 ng) was
treated with E64 (5 mm), then incubated with 10 (5 mm). No
fluorescence at 25 kDa was visible, as the active-site cysteine
of cathepsin S was blocked by covalently bound E64 (Figure 4,
left). These findings confirmed the covalent interaction be-
tween the active-site cysteine and inactivator 10, and suggest
that the surface nucleophiles of the enzyme were not affected
by this probe.
Figure 3. Predicted binding mode of compound 10 (dark gray) within the
active site of human cathepsin S. AutoDock 4.2 was used for covalent ligand
docking. Active-site residues are depicted in light gray; the surface is ren-
dered as transparent.
teine and the prochiral electrophilic b-carbon of the inactiva-
tor. The preceding noncovalent complex gives rise to nucleo-
philic attack of the Cys25 thiolate from the si face, thereby
leading to the S-configuration of the resulting chiral center (in
accordance with the X-ray crystal structure of another vinyl sul-
fone inactivator in complex with human cathepsin S).[27] Sys-
Next, the sensitivity of the probe 10 was tested. The amount
of cathepsin S in the labeling reaction was decreased to 0.5 ng.
In each lane, a clear band was observed thus indicating the
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probe’s suitability for sub-nanogram detection of human cath-
epsin S (Figure 4, right).
was carried out at mild acidic pH as well as neutral pH (at
which cathepsin S is active and stable, in contrast to other cys-
teine cathepsins).[28] It can therefore be concluded that the
probe is suitable for selectively detecting human cathepsin S
in complex protein mixtures.
For studying its selectivity, 10 was applied to label cathe-
psin S in the presence of a complex proteome. Lysate from
HEK293 cells was prepared, spiked with cathepsin S, and treat-
ed with 10. As controls, cathepsin S and HEK293 lysate were
separately incubated with 10. The incubation mixtures were
subjected to SDS-PAGE and visualized by protein staining and
fluorescence imaging (Figure 5). This analysis revealed selective
In summary, we have introduced a straightforward synthesis
of building block 8 with a tailored tricyclic structure account-
ing for suitable fluorescent properties. This coumarin-tetrahy-
droquinoline hydride is considered to be of great value for ver-
satile applications in the design of fluorescently tagged mole-
cules. One implementation was realized in this study by incor-
porating 8 in the tripeptidic activity-based probe 10, the first
coumarin-based ABP. The Gly-Phe-Gly motif was particularly
favorable for recognition and inactivation of the therapeutical-
ly relevant cysteine protease cathepsin S. A plausible binding
mode of 10 in the active site of cathepsin S is proposed. Final-
ly, we successfully employed 10 for direct in-gel fluorescence
imaging of recombinant and native cathepsin S with high sen-
sitivity and specificity. In future studies, the activity-based
probe 10 will be examined for its feasibility in the determina-
tion of active cathepsin S by HPLC in combination with a fluo-
rescence detector.
Acknowledgements
The authors thank Erik Gilberg for assistance. J.S. is supported by
the Gender Equality Center of the Bonn-Rhein-Sieg University of
Applied Sciences, and N.F. by a fellowship from the Jꢀrgen Man-
chot Foundation, Dꢀsseldorf, Germany. M.H. and M.M. are sup-
ported by the Czech Grant Agency (P302/11/1481) and institu-
tional project RVO 61388963.
Figure 5. Imaging of human cathepsin S with the fluorescent probe 10 in
the presence of cell lysate. Purified recombinant cathepsin S (250 ng) was
mixed with HEK293 cell lysate (25 mg) and treated with 10 (5 mm) for 1 h at
pH 6.5. The reaction mixture was resolved by SDS-PAGE and visualized by
fluorescence in a Typhoon 9410 imager (right) followed by protein staining
with Coomassie Brilliant Blue (left).
Keywords: activity-based probes · cathepsins · coumarins ·
fluorescent probes · inhibitors · peptidyl vinyl sulfones
labeling of the target cathepsin S in the presence of a large
excess of lysate proteins, without detectable nonspecific inter-
actions of 10 (Figure 5, lanes 2 and 5).
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Published online on March 19, 2014
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