Tunable Probes with Direct Fluorescence Signals for the Constitutive and Immunoproteasome

Article abstract of DOI:10.1002/anie.201605753

Electrophiles are commonly used for the inhibition of proteases. Notably, inhibitors of the proteasome, a central determinant of cellular survival and a target of several FDA-approved drugs, are mainly characterized by the reactivity of their electrophilic head groups. We aimed to tune the inhibitory strength of peptidic sulfonate esters by varying the leaving groups. Indeed, proteasome inhibition correlated well with the pK_aof the leaving group. The use of fluorophores as leaving groups enabled us to design probes that release a stoichiometric fluorescence signal upon reaction, thereby directly linking proteasome inactivation to the readout. This principle could be applicable to other sulfonyl fluoride based inhibitors and allows the design of sensitive probes for enzymatic studies.

Full text of DOI:10.1002/anie.201605753

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201605753
German Edition: DOI: 10.1002/ange.201605753
Fluorescent Probes
Tunable Probes with Direct Fluorescence Signals for the Constitutive
and Immunoproteasome
+
+
Christian Dubiella ,* Haissi Cui , and Michael Groll*
Abstract: Electrophiles are commonly used for the inhibition
of proteases. Notably, inhibitors of the proteasome, a central
determinant of cellular survival and a target of several FDA-
approved drugs, are mainly characterized by the reactivity of
their electrophilic head groups. We aimed to tune the inhibitory
strength of peptidic sulfonate esters by varying the leaving
groups. Indeed, proteasome inhibition correlated well with the
pK of the leaving group. The use of fluorophores as leaving
a
groups enabled us to design probes that release a stoichiometric
fluorescence signal upon reaction, thereby directly linking
proteasome inactivation to the readout. This principle could be
applicable to other sulfonyl fluoride based inhibitors and
allows the design of sensitive probes for enzymatic studies.
U
p to 2% of the human genome encodes for proteases,
which reflects their indispensable importance for cell func-
[
1]
tion. Although a variety of proteolytic mechanisms have
been discovered for these enzymes, they all follow common
principles of catalysis: 1) direct (aspartic, glutamic, and
metallo-proteases) or 2) indirect (serine, threonine, and
cysteine proteases) activation of a water molecule to facilitate
its nucleophilic attack on a peptide bond. As major intra-
cellular proteolytic complexes, the constitutive 20S protea-
some (cCP; CP = core particle) and immunoproteasome
Figure 1. Carfilzomib (CFZ) and its peptidic sulfonyl fluoride (PSF), as
well as sulfonate ester (PSE) counterparts. IC50 values were determined
À1
against human cCP in fluorogenic (PSEs 1, 2, 3; 10 mgmL cCP) and
À1
luminogenic substrate assays (PSEs 4, 5, 6; 5 mgmL cCP). [a] pK
a
values are given for the conjugate acids of the leaving groups (LGs).
(
iCP) exploit the basicity of an N-terminal threonine (Thr1)
at the active site of their catalytic subunits b1c/i, b2c/i, and
this purpose, we exchanged the fluoride leaving group (LG)
of peptidic sulfonyl fluorides (PSF; Figure 1) with phenolic
LGs to generate sulfonate esters (PSE; Figure 1). This
enables fine-tuning with electron-withdrawing groups on the
[2]
[8]
b5c/i. In contrast to moderately nucleophilic water (pK =
a
[3]
1
5.7), the alkoxide of Thr1 (pK ꢀ 9) readily attacks the
a
carbonyl carbon atom of peptide bonds in protein substrates.
This reactivity is exploited by antineoplastic agents such as
phenol system, which results in differing pK values and thus
a
[
4]
[5]
bortezomib and carfilzomib (CFZ; Figure 1), which cova-
lently bind to Thr1 with C-terminal electrophilic head groups
also called warheads). A systematic evaluation revealed
variable LG ability. Similar studies were conducted using
acyloxy or aryloxy methyl ketones to target cysteine proteases
[
6]
(
and showed a correlation between the LG pK (conjugated
a
[9]
that CP inhibitors with various warheads are mainly charac-
acid) and the inhibition rate. Additionally, the evaluation of
analogues of the CP inhibitor salinosporamide Awith varying
LGs suggested a correlation between LG ability and
[7]
terized by the chemical reactivity of the electrophile.
However, this chemical reactivity is difficult to adjust for
the majority of CP inhibitors.
[
10]
potency.
Considering this, we focused on phenols with
In contrast, the recent use of sulfonyl fluorides in iCP and
cCP inhibitors allows alterations in warhead reactivity. For
pK values ranging from 6 to 10 to generate hydrolytically
a
[
8]
[11]
stable peptidic sulfonate esters (PSEs 1–3).
Our probe
design focused on fluorinated LGs that differ in their LG pKa
but have similar steric demands during binding. We therefore
prepared a sulfonyl chloride precursor of l-leucine, which
[
+]
[+]
[
8]
[
*] Dr. C. Dubiella, Dr. H. Cui, Prof. Dr. M. Groll
Center for Integrated Protein Science Munich (CIPSM)
Department of Chemistry, Technische Universitꢀt Mꢁnchen
Lichtenbergstrasse 4, 85748 Garching (Germany)
E-mail: christian.dubiella@mytum.de
was derivatized with phenol (pK = 9.9), 2,4,6-trifluorophenol
a
[12]
(
TFP; pK ꢀ 7.6), or pentafluorophenol (PFP; pK = 5.5).
a
a
In fact, PFP esters are substitutes for acid halides and
succinimidyl esters, which are used in conjugation reactions
michael.groll@tum.de
[
13]
+
due to their water stability and long shelf life. The phenyl
sulfonate ester precursors were fused through HATU-medi-
ated amide coupling to a b5-specific CFZ peptide backbone
prepared by Fmoc-based solid-phase peptide synthesis. This
[
] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under http://dx.doi.org/10.
002/anie.201605753.
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Figure 2. a) Probes optimized for iCP: PSEs 7, 8, and 9. b) In vitro IC50 assays against the chymotrypsin-like (ChTL) b5 activity of human iCP and
cCP after 1 h incubation with various concentrations of PSE 6, 7, 8, and 9 using a luminogenic Suc-LLVY-aminoluciferin substrate assay. The data
were normalized to DMSO-treated controls and are presented as relative activity with standard deviation. c) Time course of the fluorescence
À1
increase when using PSEs 6, 8, and 9 at 1 mm concentration at 308C in the presence of 50 mgmL iCP. It is of note that the reaction between iCP
and the respective PSE does not saturate. The data shown in parts (b) and (c) are depicted as the meanÆSD of triplicates.
convergent synthesis strategy yielded the PSEs 1, 2, and 3
Figure 1). The potency of PSEs 1, 2, and 3 against the
chymotrypsin-like (ChTL) activity of subunit b5c was deter-
similar mechanism is used by fluorescently-quenched activ-
ity-based probes, a variation of activity-based protein profil-
(
[
14]
ing. In this case, a fluorescence-quenching LG is directly
released after protease inactivation, thus retaining the
mined by using a fluorogenic 7-amino-4-methylcoumarin
[15]
(
AMC) substrate assay with purified human cCP. The non-
fluorescent inhibitor bound to the target.
fluorinated PSE 1 and the tri-fluorinated PSE 2 did not block
ChTL activity (IC > 1 mm). Notably, PSE 3 substantially
Based on the aforementioned finding that a pK range
a
between 4 and 6 is favorable, we utilized methylumbellifer-
ones (MU) as small reporter LGs (PSE 4–6, Figure 1). MUs
are easily tuneable in their pKa values through simple
fluorination and are synthetically accessible through Pech-
mann condensation to yield 6-fluoro-4-methylumbelliferone
5
0
inhibited subunit b5c (IC = 1.12 mm, Figure 1 and Figure S1
5
0
in the Supporting Information), but with at least 40-fold
decreased potency compared to its sulfonyl fluoride counter-
part (IC = 0.028 mm). To investigate the binding mode of
5
0
PSE 3 with CP, we soaked Saccharomyces cerevisiae CP
crystals with the compound for X-ray analysis. Structure
elucidation revealed a mechanism analogous to that of
PSFs: 1) After adduct formation at Thr1 through sulfonyl-
ation, the inhibitor is displaced by intramolecular attack of
the amino function in Thr1 to form an aziridine intermediate
(FMU; pK = 6.4) and 6,8-difluoro-4-methylumbelliferone
a
[16]
(DiFMU; pK = 4.7). Additionally, they feature favorable
a
photophysical properties, including a high quantum yield
[8a]
(F = 0.89), linear fluorescence over an appropriate range of
F
concentrations (Figure S3), and high resistance to photo-
[
16]
bleaching . Moreover, MUs and their derivatives are
relatively small in size, which is favorable in view of the
limited space at the proteasomal active site. Conveniently,
DiFMU bound to sulfonate ester groups (DSE) is non-
fluorescent and is widely used in substrate assays for several
(
2.5 ꢀ resolution, Rfree = 21.9%, PDB ID: 5LAI, Figure S2
and Table ST1). 2) Subsequent nucleophilic aziridine-opening
by the base Lys33 results in irreversible crosslinking of the b5
subunit (2.9 ꢀ resolution, Rfree = 20.1%, PDB ID: 5LAJ,
Figure S2 and Table ST1). The structural data thus confirm
release of the PFP LG as an integral part of the inhibition
mechanism. Since the steric requirements are similar for all of
the phenyl systems used here, the increasing potency suggests
[
17]
enzyme classes.
As a control, we prepared the non-
[
16]
fluorinated PSE 4 (MU; pK = 7.8;
Figure 1). The IC50
a
values against purified human cCP were determined by
using a luminogenic aminoluciferin substrate assay, which is
orthogonal in its readout to the released MU fluorophores. In
line with our previous results, PSE 6 with a DiFMU LG was
over 400-times more active (IC = 0.236 mm, Morrison K =
a direct correlation between the pK value of the leaving
a
groups and the reactivity of the electrophiles.
Since the release of the LG is an integral part of the
inhibitory mechanism, this led us to design PSE probes that
emit a signal upon inhibition. To this end, we employed easily
detectable fluorophores as LGs for the quantification of CP
5
0
I
0.062 mm, K as a function of K = 0.18 mm; Figures S4–S6)
I
Obs
than the monofluorinated PSE 5 (IC > 100 mm) and non-
5
0
fluorinated PSE 4 (IC > 1 mm). No inactivation of b1c
5
0
(
PSE 4–6). In contrast to fluorescent assays, in which peptidic
(caspase-like, IC > 1 mm), b2c (trypsin-like, IC > 1 mm),
5
0
5
0
substrates are constantly cleaved, PSE probes would liberate
a single fluorophore molecule per active site upon inhibition.
As such, these probes could be used for the direct estimation
of proteasome activity without downstream analysis. A
or several other proteases was observed (Figure S7). These
findings demonstrate that the designed PSEs require LGs
with a pK less than 5 for inhibition in the nanomolar range.
a
To rank the DSE head group in comparison with other
2
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[
8a]
Figure 3. a) Proposed mechanism for the reaction of the PSEs with the proteasomal active site. The substituent R indicates the rest of the
1
inhibitor backbone, and P refers to the residue protruding into the S1 specificity pocket. b) Time course of the fluorescence increase with varying
À1
concentrations of cCP (1–100 ngmL cCP) with 0.01% SDS treated with 100 nm PSE 6 for 2 h at 308C and pH 7.5 (excitation: l=360 nm;
emission: l=460 nm). It is of note that the reaction between PSE 6 and cCP does not saturate in the monitored time frame. c) Normalized
À1
fluorescence intensity at the indicated time points [vertical lines in (b)] depicted against the concentration of cCP [mgmL ] displays a linear
2
correlation between cCP activity and fluorescence, irrespective of the chosen time point. r =coefficient of determination. The data in parts (b)
and (c) are shown as the meanÆSD of triplicates.
[
8]
established electrophiles, we compared a set of proteasome
inhibitors in a substrate assay. For this purpose, we evaluated
the reactivity of different warheads (Figure S8) independ-
ently of the peptidic backbone by using inhibitors that feature
the same sequence (Z-LLL). In accordance with our
structural data, the DSE electrophile displayed an irreversible
binding mode and resided between epoxyketone and a-
ketoaldehyde warheads in terms of reactivity.
parable to that of PSF inhibitors. In view of this, we
measured the release of DiFMU by PSE 8 in the presence of
iCP. Surprisingly, altering the backbone resulted in increased
fluorescence signals in comparison to PSE 6 (Figure 2c). To
estimate the reactivity of PSE 6 and 8 aside from proteasome
binding, we evaluated the rates of hydrolysis at different pH
values (Figure S10). Indeed, PSE 8 (P1-phenylalanine) was
more susceptible to hydrolysis than PSE 5 (P1-leucine)
despite having an identical warhead. This unexpected con-
tribution of the P1 sidechain to the reactivity of the probe
points towards an additional possibility for fine-tuning of the
inhibitor. Our hypothesis was verified by synthesizing PSE 9,
which differs from PSE 8 only in its P1 site. Since we propose
that the increased reactivity of PSE 8 originates from the
electron-rich P1 aryl system, we exchanged this position with
a saturated cyclohexyl residue in PSE 9. While this exchange
had no effect on b5i and b5c inhibition (IC50 (b5i) = 0.092 mm,
IC50 (b5c) = 2.46 mm), PSE 9 displayed hydrolysis similar to
PSE 6 in pH-dependent assays (Figure S10). This result
indicates that the reactivity is indeed additionally influenced
by properties of the P1 side chain. Notably, the high sensitivity
given by the release of a single fluorophore upon inhibition
enables detection of these minute changes in affinities, which
are not visible in substrate assays.
[
6]
Following our characterization of the DSE head group, we
utilized b5i-optimized backbones for iCP probes. The iCP is
a potential therapeutic target since it constitutes a key
element in antigen presentation and cytokine production,
[
18]
thereby participating in immune responses.
Since iCP
binding preferences differ from those of cCP, we assessed
state-of-the-art inhibitor backbones (PSEs 7–9, Figure 2a).
The b5i-specific tripeptide of LU-035i (IC50 (b5i) =
[19]
0
.011 mm)
served as a blueprint for PSE 7, while the
PSE 8 tetrapeptide is derived from ONX 0914 (IC (b5i) =
5
0
[
18a]
0
.028 mm).
These backbones were coupled to l-phenyl-
alanine and 3-cyclohexyl-l-alanine DiFMU precursors.
To estimate inhibitor potency, we determined the IC50
values of PSE 7 and 8 against b5i versus b5c for human iCP
and cCP (Figure 2b and Figure S9). Although the backbone
of PSE 7 is identical to that of its potent epoxyketone
equivalent LU-035i, PSE 7 only displays activity above
a concentration of 100 mm. This is in agreement with findings
that PSFs require at least tetrapeptidic backbones for
Based on our proposed mechanism, every released
DiFMU fluorophore correlates with a blocked proteasomal
active site in a single-turnover reaction (Figure 3a). We
investigated whether the fluorescence signal is proportional
to the amount of inhibited CP and thus to the active CP in the
sample. Since iCP-specific tissues are difficult to obtain in
sufficient quantities, we purified human cCP from erythro-
cytes for quantitative assays with PSE 6 as a suitable probe.
The fluorescence emission was determined at physiological
[
8]
sufficient stabilization at the active site. Comparison of
the DiFMU sulfonate ester electrophile with other warheads
indicates that it reacts rather slowly with Thr1 (Figure S8). In
contrast, PSE 8 exhibited inhibition of b5i in the low nano-
molar range (IC = 0.075 mm) and moderate inhibition of b5c
5
0
(
IC = 2.14 mm), which represents a 25-fold selectivity com-
50
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À1
relevant concentrations (ca. 190 nm =^ 140 mgmL in living
cells), while the concentration of PSE 6 was kept constant
[
20]
(
100 nm: Figure 3b, or 1 mm: Figure S11). We monitored
the fluorescence over the course of 2 h and found that the
signal intensified linearly with increasing amounts of cCP
(
Figure 3b). A quantitative estimation of cCP concentrations
[1] B. Turk, Nat. Rev. Drug Discovery 2006, 5, 785 – 799.
[
[
[
2] E. M. Huber, W. Heinemeyer, X. Li, C. S. Arendt, M. Hoch-
strasser, M. Groll, Nat. Commun. 2016, 7, 10900.
3] A. F. Kisselev, Z. Songyang, A. L. Goldberg, J. Biol. Chem. 2000,
was possible with PSE 6 below its IC50 value (100 nm PSE;
Figure 3c), where background fluorescence resulting from
probe hydrolysis is negligible (Figure S12). Discernible signal
resulting from the action of the inhibitor was found up to
5
2
75, 14831 – 14837.
4] P. G. Richardson, T. Hideshima, K. C. Anderson, Cancer Control
003, 10, 361 – 369.
À1
mgmL cCP, despite the lack of any signal amplification.
2
This corresponds to an absolute protein amount of 340 fmol
in a sample volume of 50 mL.
To verify that the fluorescence is not caused by unspecific
reactions of PSE 6 with nucleophilic side chains in the
protein, we used varying amounts of bovine serum albumin
[5] S. D. Demo, C. J. Kirk, M. A. Aujay, T. J. Buchholz, M. Dajee,
M. N. Ho, J. Jiang, G. J. Laidig, E. R. Lewis, F. Parlati et al.,
Cancer Res. 2007, 67, 6383 – 6391.
[
6] M. L. Stein, H. Cui, P. Beck, C. Dubiella, C. Voss, A. Krꢁger, M.
Groll, B. Schmidt, M. Groll, Angew. Chem. Int. Ed. 2014, 53,
1679 – 1683; Angew. Chem. 2014, 126, 1705 – 1709.
(
BSA) as a negative control. The fluorescence in the presence
[
[
7] M. Groll, R. Huber, L. Moroder, J. Pept. Sci. 2009, 15, 58 – 66.
8] a) C. Dubiella, H. Cui, M. Gersch, A. J. Brouwer, S. A. Sieber,
A. Krꢁger, R. M. J. Liskamp, M. Groll, Angew. Chem. Int. Ed.
2014, 53, 11969 – 11973; Angew. Chem. 2014, 126, 12163 – 12167;
b) A. J. Brouwer, A. Jonker, P. Werkhoven, E. Kuo, N. Li, N.
Gallastegui, J. Kemmink, B. I. Florea, M. Groll, H. S. Overkleeft
et al., J. Med. Chem. 2012, 55, 10995 – 11003.
of BSA was comparable to background hydrolysis (Fig-
ure S13). In order to confirm that PSE 6 derived fluorescence
is caused by CP activity, we inactivated the latter with 50 mm
of different, well-characterized proteasome inhibitors prior to
the measurement (Figure S14). We were able to show that
pre-incubated CP samples exhibited strongly reduced fluo-
rescence (Figure S15). This confirms that fluorophore release
from PSE 6 results directly from the inhibition mechanism
and reflects the active concentration of CP in the sample.
However, the applicability of PSEs as probes in cells or cell
lysates is so far limited. This is in part due to side reactions
occurring at physiological concentrations of other cellular
components, for example glutathione (Figure S15).
[
9] a) R. A. Smith, L. J. Copp, P. J. Coles, H. W. Pauls, V. J.
Robinson, R. W. Spencer, S. B. Heard, A. Krantz, J. Am.
Chem. Soc. 1988, 110, 4429 – 4431; b) A. Krantz, L. J. Copp,
P. J. Coles, R. A. Smith, S. B. Heard, Biochemistry 1991, 30,
4678 – 4687; c) B. R. Ullman, T. Aja, T. L. Deckwerth, J. L. Diaz,
J. Herrmann, V. J. Kalish, D. S. Karanewsky, S. P. Meduna, K.
Nalley, E. D. Robinson et al., Bioorg. Med. Chem. Lett. 2003, 13,
3623 – 3626.
[
10] a) R. R. Manam, K. A. McArthur, T.-H. Chao, J. Weiss, J. A. Ali,
V. J. Palombella, M. Groll, G. K. Lloyd, M. A. Palladino, S. T. C.
Neuteboom et al., J. Med. Chem. 2008, 51, 6711 – 6724; b) M.
Groll, K. A. McArthur, V. R. Macherla, R. R. Manam, B. C.
Potts, J. Med. Chem. 2009, 52, 5420 – 5428.
In summary, we have described a new class of inhibitors
for the constitutive proteasome and immunoproteasome, the
efficiency of which can be fine-tuned through altering the
properties of the LG. By using fluorescent probes as LGs, we
were able to generate tools that allow direct detection of
relative proteasomal activity and concentration in solution
through a simple readout. The lack of linkers, spacers, or the
need for downstream reactions for signal amplification avoids
possible side reactions, thus rendering the system precise and
sensitive. Our findings suggest that DiFMU sulfonate esters
as electrophiles show a similar range of reactivity to the
sulfonyl fluoride warhead. Therefore, this strategy for the
design of fluorogenic probes as research tools could in
principle be applied to a range of sulfonyl fluoride based
inhibitors, which are widely used against a diverse set of
[
[
[
11] S. C. Miller, J. Org. Chem. 2010, 75, 4632 – 4635.
12] W. A. Sheppard, J. Am. Chem. Soc. 1970, 92, 5419 – 5422.
13] a) J. Bornholdt, K. Wilhemsen, J. Felding, J. Langgaard, Tetra-
hedron 2009, 65, 9280 – 9284; b) S. Caddick, J. D. Wilden, D. B.
Judd, J. Am. Chem. Soc. 2004, 126, 1024 – 1025.
[14] a) A. E. Speers, G. C. Adam, B. F. Cravatt, J. Am. Chem. Soc.
2003, 125, 4686 – 4687; b) G. de Bruin, B. T. Xin, M. Kraus, M.
van der Stelt, G. A. van der Marel, A. F. Kisselev, C. Driessen,
B. I. Florea, H. S. Overkleeft, Angew. Chem. Int. Ed. 2016, 55,
4199 – 4203; Angew. Chem. 2016, 128, 4271 – 4275; c) N. Li, C.-L.
Kuo, G. Paniagua, H. van den Elst, M. Verdoes, L. I. Willems, W.
van der Linden, M. Ruben, E. van Genderen, J. Gubbens et al.,
Nat. Protoc. 2013, 8, 1155 – 1168; d) C. Gu, I. Kolodziejek, J.
Misas-Villamil, T. Shindo, T. Colby, M. Verdoes, K. H. Richau, J.
Schmidt, H. S. Overkleeft, R. A. L. Van Der Hoorn, Plant J.
[
21]
targets.
2010, 62, 160 – 170.
[
15] a) G. Blum, S. R. Mullins, K. Keren, M. Fonovi cˇ , C. Jedeszko,
Acknowledgements
M. J. Rice, B. F. Sloane, M. Bogyo, Nat. Chem. Biol. 2005, 1, 203 –
2
09; b) G. Blum, G. von Degenfeld, M. J. Merchant, H. M. Blau,
This work was funded by SFB 749/A10. We thank Richard
Feicht and Dr. Sabine Schneider for experimental support
and our students Regina Baur, Josef Braun, Theresa Rauh &
Michael Heilmann. We thank the staff of PXI of the Paul
Scherrer Institute, Swiss Light Source (Villigen, Switzerland)
for help with data collection.
M. Bogyo, Nat. Chem. Biol. 2007, 3, 668 – 677; c) L. E. Edging-
ton, A. B. Berger, G. Blum, V. E. Albrow, M. G. Paulick, N.
Lineberry, M. Bogyo, Nat. Med. 2009, 15, 967 – 973; d) S. Serim,
U. Haedke, S. H. L. Verhelst, ChemMedChem 2012, 7, 1146 –
1
159; e) M. Verdoes, S. H. L. Verhelst, Biochim. Biophys. Acta
Proteins Proteomics 2016, 1864, 130 – 142; f) M. Hu, L. Li, H.
Wu, Y. Su, P. Y. Yang, M. Uttamchandani, Q. H. Xu, S. Q. Yao, J.
Am. Chem. Soc. 2011, 133, 12009 – 12020.
Keywords: fluorescent probes · inhibitors · probe design ·
proteasome · structure–activity relationships
[
16] W. C. Sun, K. R. Gee, R. P. Haugland, Bioorg. Med. Chem. Lett.
1998, 8, 3107 – 3110.
4
www.angewandte.org
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
These are not the final page numbers!

Angewandte
Communications
Chemie
[
17] a) C. C. Fjeld, J. M. Denu, J. Biol. Chem. 1999, 274, 20336 –
0343; b) V. Ahmed, M. Ispahany, S. Ruttgaizer, G. Guillemette,
H. Cui, J. D. Hegemann, M. A. Marahiel, A. Krꢁger, M. Groll,
ChemMedChem 2015, 10, 1969 – 1973.
2
S. D. Taylor, Anal. Biochem. 2005, 340, 80 – 88; c) Y. Ge, E. G.
Antoulinakis, K. R. Gee, I. Johnson, Anal. Biochem. 2007, 362,
[21] a) D. A. Shannon, C. Gu, C. J. McLaughlin, M. Kaiser, R. A. L.
van der Hoorn, E. Weerapana, ChemBioChem 2012, 13, 2327 –
2330; b) J. C. Powers, J. L. Asgian, O. D. Ekici, K. E. James,
Chem. Rev. 2002, 102, 4639 – 4750; c) A. Narayanan, L. H. Jones,
Chem. Sci. 2015, 6, 2650 – 2659.
63 – 68.
[
18] a) T. Muchamuel, M. Basler, M. A. Aujay, E. Suzuki, K. W.
Kalim, C. Lauer, C. Sylvain, E. R. Ring, J. Shields, J. Jiang et al.,
Nat. Med. 2009, 15, 781 – 787; b) M. Groettrup, C. J. Kirk, M.
Basler, Nat. Rev. Immunol. 2010, 10, 73 – 78.
[
19] G. De Bruin, E. M. Huber, B.-T. Xin, E. J. van Rooden, K. Al-
Ayed, K.-B. Kim, A. F. Kisselev, C. Driessen, M. Van Der Stelt,
G. A. van der Marel et al., J. Med. Chem. 2014, 57, 6197 – 6209.
20] a) S. Asano, Y. Fukuda, F. Beck, A. Aufderheide, F. Fçrster, R.
Danev, W. Baumeister, Science 2015, 347, 439 – 442; b) P. Beck,
Received: June 14, 2016
Revised: July 20, 2016
Published online: && &&, &&&&
[
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
Fluorescent Probes
A tunable light: The potency of peptidic
sulfonate esters as proteasome inhibitors
C. Dubiella,* H. Cui,
M. Groll*
can be fine-tuned by altering the pK of
a
&&&& — &&&&
the leaving group. The introduction of
a fluorescent dye as a leaving group
allows direct monitoring of proteasomal
activity and can be used for quantifica-
tion. This concept might inspire future
work on other hydrolases.
Tunable Probes with Direct Fluorescence
Signals for the Constitutive and
Immunoproteasome
6
www.angewandte.org
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
These are not the final page numbers!