Incorporation of fluorinated phenylalanine generates highly specific inhibitor of proteasome's chymotrypsin-like sites

Article abstract of DOI:10.1021/jm9015685

Proteasomal processing is conducted by three individual catalytic subunits, namely β11, β2, and β5. Subunit-specific inhibitors are useful tools in dissecting the role of these individual subunits and are leads toward the development of antitumor agents. We here report that the presence of fluorinated phenylalanine derivatives in peptide based proteasome inhibitors has a profound effect on inhibitor potency and selectivity. Specifically, compound 4a emerges as one of the most β5 specific inhibitors known to date.

Full text of DOI:10.1021/jm9015685

J. Med. Chem. 2010, 53, 2319–2323 2319
DOI: 10.1021/jm9015685
Incorporation of Fluorinated Phenylalanine Generates Highly Specific Inhibitor of Proteasome’s
Chymotrypsin-like Sites
Paul P. Geurink,† Nora Liu,† Michiel P. Spaans,† Sondra L. Downey,‡ Adrianus M. C. H. van den Nieuwendijk,†
Gijsbert A. van der Marel,† Alexei F. Kisselev,‡ Bogdan I. Florea,† and Herman S. Overkleeft*,†
†Leiden Institute of Chemistry and Netherlands Proteomics Centre, Gorlaeus Laboratories, Einsteinweg 55, 2333 CC Leiden, The Netherlands,
and ‡Department of Pharmacology and Toxicology, Darthmouth Medical School, 1 Medical Drive, Lebanon, New Hampshire 03756
Received October 23, 2009
Proteasomal processing is conducted by three individual catalytic subunits, namely β1, β2, and β5.
Subunit-specific inhibitors are useful tools in dissecting the role of these individual subunits and are leads
toward the development of antitumor agents. We here report that the presence of fluorinated phenyl-
alanine derivatives in peptide based proteasome inhibitors has a profound effect on inhibitor potency and
selectivity. Specifically, compound 4a emerges as one of the most β5 specific inhibitors known to date.
Introduction
structure analysis by (solid state) 19F NMR spectroscopy or
19F MRI to study, for example, protein aggregation.11
The set of fluorinatedproteasomeinhibitors prepared inthe
context of the here presented studies are depicted in Figure 1.
The majority of all cytosolic and nuclear proteins in
eukaryotic cells are degraded by the ubiquitin-proteasome
pathway. In this system, proteins destined for degradation are
modified with a polyubiquitin chain as a recognition tag for
the 26S proteasome where proteolysis occurs. The 26S protea-
some contains one or two 19S regulatory caps together with
the proteolytically active, cylindrical 20S core. Within the
mammalian constitutive 20S core, three pairs of proteoly-
tically active sites are present displaying different substrate
specificity. Of these, the β1 subunits (caspase-like) cleave after
acidic residues, the β2 subunits (trypsin-like) cleave after basic
residues, and the β5 subunits (chymotrypsin-like) cleave after
bulky, hydrophobic residues.1,2 The peptidyl boronic acid
proteasome inhibitor PS-341 (8)3 is used for the treatment
of multiple myeloma and targets the β5 and β1 subunits. To
study the role of the three individual active subunits, subunit-
specific inhibitors are needed. Inhibitors with moderate to
good selectivity for either one of the subunits have been
developed,4 however there is still room for improvements.
The search for subunit selective inhibitors is predominantly
conducted by either screening of natural products,5 rational
design,6 or compound library building.4b,7 We observed that
in these studies the effect of fluorine functionality in protea-
some inhibitors is relatively uncharted.8 In contrast, fluorine
has found wide interest in bioorganic and structural chemistry
over the past decade and has become an important feature in
drug design.9 This is predominantly due to the typical char-
acteristics of fluorine (when bound to carbon) such as its
comparable size to hydrogen, its electron withdrawing ability,
superhydrophobicity of fluorocarbons, and self-association
between fluorinated moieties. In protein structure design, intro-
duction of fluorine can mimic functional groups, alter struc-
tural properties, and thereby (de)stabilize protein structures
or function as recognition motifs.10 In addition, the beneficial
19F nuclear magnetic characteristics have found their use in
a
Compounds 2a and 2b containing pentafluoroPhe (PheF5 )
and 3,5-bis(trifluoromethyl)Phe (Phe(m-CF3)2), respectively,
are based on compound 8 derivative 1 (having a comparable
potency toward the β1 and β5 proteasome subunits with
respect to 8)12 and differ in that the phenylalanine in 8 is
replaced by the corresponding fluorinated analogue. In addi-
tion, incorporation of fluorinated phenylalanines at different
positions in tripeptide epoxyketones2 led to compounds 3-6
in which systematically either one or both of the P2 and P3
positions were altered. We opted for the use of Phe(m-CF3)2)
and PheF5 for the dual reason that these are readily available
and that hydrophobic amino acids (that is the nonfluorinated
analogues) are in principle accepted by all proteasome active
sites. The epoxyketone electrophilic trap was selected based
on the natural product epoxomicin. The epoxyketone war-
head featured by epoxomicin displays a specific and selective
reactivity toward the N-terminal threonine residue that makes
up the proteasome catalytic active sites.2,5 For this reason,
synthetic peptide epoxyketones are now much studied leads in
medicinal chemistry studies in which the proteasome plays a
role.13 The tripeptide epoxyketones 3-6 feature an azide
moiety at the N-terminal end for future modifications (for
instance, coupling to a fluorophore or biotin in either one- or
two-step labeling experiments).14
Results and Discussion
The C-terminally modified oligopeptides were produced fol-
lowing synthesis protocols we reported previously.7 The amino
acids used were either commercially available or prepared
aAbbreviations: Phe(F5), pentafluorophenylalanine; Phe(m-CF3)2,
3,5-bis(trifluoromethyl)phenylalanine, HEK-293, human embryonic
kidney cell lysates; EL-4, mouse lymphoma cell line; DiPEA, N,N-
diisopropylethylamine; DTT, dithiothreitol; Tris, tris(hydroxymethyl)-
aminomethane; amc, 7-amido-4-methylcoumarin; TFA, trifluoroacetic
acid; DMF, N,N-dimethylformamide; DCM, dichloromethane.
*To whom correspondence should be addressed. Phone: þ31 71 5274342.
Fax: þ31 71 5274307. E-mail: h.s.overkleeft@chem.leidenuniv.nl.
r
2010 American Chemical Society
Published on Web 02/04/2010
pubs.acs.org/jmc

2320 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Geurink et al.
Figure 1. Synthesized fluorinated proteasome inhibitors. Indicated are the enzyme pockets (P1, P2, P3).
Table 1. Activity (IC50 in μM) of 2-5 against the Three Active Con-
stitutive 26S Proteasome Subunitsa
compd
β1 nLPnLD
β2 RLR
β5 LLVY
2a
2b
3
0.44
0.16
>15
11
0.031
0.0030
0.0010
0.0020
0.10
>15
>15
>15
>15
>15
1.8
Figure 2. Characterization of the specificity of the fluorinated
dipeptide boronates. Competition assay in HEK-293 lysate. Lysates
were incubated for one hour with compounds 1, 2a, and 2b at the
indicated final concentrations. Residual proteasome activity was
labeled with 0.5 μM 9 for 1 h.
4a
4b
5a
5b
>15
>15
>15
>15
0.20
0.13
a Determined with the indicated subunit specific fluorogenic peptide
substrates. All values are averages of two experiments.
following established procedures.15 See for full experimental
data on the synthesis of the compounds the Supporting
Information (SI). The inhibition potential of compounds 2a
and 2b in comparison with their nonfluorinated analogue
boronic ester 1 (the pinanediol analogue of the clinical
drug 8) was assessed in a competition assay employing human
embryonic kidney (HEK-293) cell lysates in combination
with the fluorescent broad spectrum proteasome probe
MV151 (9).12 Cell lysates were incubated with each of the
three compounds at 0.05, 0.1, and 1 μM final concentrations
prior to treatment with 0.5 μM final concentration of 9. The
samples were denatured, resolved by SDS-PAGE, and the wet
gel slabs were scanned on a fluorescence scanner. The gel
image is shown in Figure 2. Lysates treated with the fluore-
scent probe display three bands that correspond to the three
active subunits (β1, β2, and β5) as depicted in Figure 2 lane 1.
The ability of a compound to inhibit the proteasome active
sites is reflected by disappearance of the bands. As apparent
from this image, the fluorinated compounds 2a and 2b are at
least as potent as their nonfluorinated counterpart 1 showing
complete inhibition of the β1 and β5 subunits between 0.1 and
1 μM. In addition, incorporation of fluorinated Phe leaves the
selectivity of β1 and β5 over β2 subunits for this type of
inhibitor unchanged. This result is also apparent from the
activity (IC50) measurements of the inhibitors toward the
different subunits in purified rabbit 26S proteasome as shown
in Table 1. Experiments in murine EL4 cell lysates (mouse
lymphoma cell line) which contain both the immuno- and
constitutive proteasome subunits gave similar results (see SI).
Next, the inhibition properties of the seven epoxyketone
containing compounds were determined in a similar competi-
tion assay against 9 at 1 and 100 μM final concentrations
employing HEK-293 cell lysates (Figure 3) and by measur-
ing inhibition of purified proteasomes for the most active
compounds (Table 1). When comparing nonfluorinated
Figure 3. Characterization of specificity of fluorinated peptide
epoxyketones. Competition assay in HEK-293 lysate. Lysates were
incubated for 1 h with compounds 3, 4, 5, or 6 at the indicated
final concentrations. Residual proteasome activity was labeled with
0.5 μM 9 for 1 h. N.B. compound 3a = 3b = 3.
compound 3 to the fluorinated ones (4-6), it becomes appa-
rent that none of these compounds inhibit the β1 subunit at
concentrations up to 100 μM and that introducing fluorines
(in either position) leads to a decrease in the inhibition of the
β2 subunit and hence to an increase in β5 specificity. When
comparing Table 1 with Figure 3, it appears that the results
from the two independent assays deviate in places. For
example the β2-IC50 value for 4a is <15 μM (Table 1),
whereas the majority of the corresponding band in Figure 3
is gone at 1 μM. Intrinsic differences in both assays, neither of
which deliver ki values are at the basis of these small but
distinct differences. The complementary assays, however,
both show similar trends, which is why we prefer to report
both.16 For instance, in Figure 3, there appears to be almost
no difference in inhibition potential of β5 between the non-
fluorinated compound 3 and either P2 or P3 fluorinated
compounds 4a,b and 5a,b. Although there is a big difference
in IC50 values, all compounds are at least submicromolar
inhibitors.

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2321
Figure 4. Characterization of specificity of compound 4a. Upper
panel: Competition assay in HEK-293 lysate. Lysates were incu-
bated for 1 h with compound 4a at the indicated final concentra-
tions. Residual proteasome activity was labeled with 0.5 μM 9 for
1 h (This image was created from two separate gel images which,
together, show a larger concentration series. The original images are
shown in Figure S-4 in the SI). Lower panel: Remaining subunit
activity after inhibition with compound 4a determined with fluoro-
genic peptides.
Figure 5. Novel probe for the β5 site. Competition assay in
HEK-293 lysate. Lysates were incubated for 1 h with compound 7
at the indicated final concentrations. Residual proteasome activity
was labeled with 0.5 μM 9 for 1 h. Fluorescence readout at (A)
λ
ex 488 nm, λem 520 nm (compound 7) and (B) λex 532 nm, λem 560 nm
(compound 9). Reagents and conditions: (a) Bodipy-FL-alkyne,14
10 mol % CuSO4, 15 mol % sodium ascorbate, toluene/H2O/t-BuOH
1/1/1, 80 °C, yield 87%.
Interestingly, the presence of fluorine substituents at both
P2 and P3 positions (6a,b, Figure 3) has a dramatic effect on
the inhibition. This effect is most pronounced for the Phe-
(m-CF3)2 analogues (b series). In general, the PheF5 com-
pounds (a series) are more active against the β5 subunit than
their hexafluoro-Phe analogues (b series). Experiments in the
lysates of murine EL4 cells gave similar results, but the
difference in inhibition potential between the compounds
was even more pronounced (see SI). Thus, it appears that
introduction of Phe(F5) in the P2 position generates a highly
specific inhibitor of the β5 site. This most potent and β5
selective inhibitor (compound 4a) was further investigated.
The inhibitory potential at much lower concentrations (1 nM
to 1 μM) is shown in Figure 4 (competition assay against 9).
Already, at 5 nM, inhibition of β5 is apparent, and between
100 and 250 nM, all β5 subunits are saturated while β1 and β2
are unaffected or even upregulated (a phenomenon which is
not fully understood but has been observed by others as
well).17 Having an IC50 value of 2 nM for the β5 subunit
against >15 μM for β1 and β2, this compound is one of the
most β5 selective inhibitors known to date. For instance, this
compound compares well with NC005, a β5 selective inhibitor
we recently found.6 Comparison of nonfluorinated com-
pound 3 with 4a reveals that both compounds are equally
active toward the β5 subunit. Enhanced selectivity for β5
arises by the dramatic drop in activity for the β2 subunit when
fluorine is introduced, as in 4a.
For the direct labeling of β5, a new fluorescent probe was
made by reacting compound 4a with Bodipy-FL-alkyne18 in a
Cu(I) mediated Huisgen 1,3-dipolar cycloaddition, giving
green fluorescent probe 7 (Figure 5). The potential of this
probe to label the β5 subunit was explored in a competition
assay against 9 as explained before. The wet gel slab was
scanned on a fluorescence scanner at two different wave-
lengths, allowing visualization of one of the two fluorescent
dyes at a time. Figure 5A shows the read-out at 520 nm
visualizing the appearance of one band: labeling of the β5
subunit by compound 7. The labeling is already visible at a
concentration of 1 nM, and the subunit appears to be
saturated (no more increase in the bands intensity) between
50 and 100 nM. At this point, only a faint β2 band is visible.19
The fluorescence read-out at 560 nm in Figure 5B (displaying
labeling with9 of remaining activities) reveals thatthe β5 band
disappears while leaving the remaining subunit-bands intact.
Conclusions
In summary, the effect of incorporation of fluorinated Phe
in proteasome inhibitors was studied. We found that substitu-
tion of nonfluorinated Phe in compound 8 analogue 1 with
fluorinated versions does not affect its selectivity for the
different active subunits, whereas the potency is slightly
increased for the Phe(m-CF3)2 version. In addition, we found
that the effect of incorporation of fluorinated Phe in peptide
epoxyketone proteasome inhibitors depends on the site of
substitution. Fluorination of both the P2 and P3 sites de-
creases potency dramatically, however, fluorinated Phe at the
P2 position hardly affects the potency but instead yields much
more β5 selective inhibitors. Comparison of the results ob-
tained with the boronic esters with those from the epoxyke-
tone studies invites the tentative conclusion that β2 is most
sensitive toward fluorine substituents. Compound 1 is ineffec-
tive toward β2 and introduction of fluorines into this sequence
has no apparent effect. When comparing epoxyketone 3 with
4-6, however, the major difference is that the latter, fluorinated
analogues leave β2 largely intact. Further studies, for instance,
making use of different fluorine amino acids, are needed to
substantiate this finding. Finally, compound 4a was identified
as one of the most β5 selective inhibitors known to date and was
converted to a β5 selective fluorescent probe (7), which can be
used to label and visualize the β5 subunit selectively.
Experimental Section
All compounds tested are >95% pure on the basis of LC-MS.
LC-MS analysis was performed on a Jasco HPLC system with a
Phenomenex Gemini 3 μm C18 50 mm ꢀ 4.60 mm column
(detection simultaneously at 214 and 254 nm), coupled to a PE
Sciex API 165 mass spectrometer with ESI.

2322 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Geurink et al.
Compounds 2a and 2b were synthesized via the same method
described in literature for compound 1.12
(3) Adams, J.; Behnke, M.; Chen, S. W.; Cruickshank, A. A.; Dick, L.
R.; Grenier, L.; Klunder, J. M.; Ma, Y. T.; Plamondon, L.; Stein,
R. L. Potent and selective inhibitors of the proteasome: Dipeptidyl
boronic acids. Bioorg. Med. Chem. Lett. 1998, 8, 333–338. For the
structure of compound 8, see SI .
General Procedure for the Synthesis of Compounds 3-6.
Hydrazine hydrate (20 equiv) was added to N3Phe-Phe-
PheOMe (1 equiv) in MeOH (20 mL/mmol) and refluxed until
TLC analysis revealed complete consumption of the starting
material (usually after 3 h). Toluene was added, and the mixture
was concentrated under reduced pressure followed by coeva-
poration with toluene (2ꢀ). The acylhydrazide (1 equiv) was
dissolved in a 10/1 (v/v) mixture of dichloromethane/N,N-
dimethylformamide (DCM/DMF) (10 mL/mmol) and cooled
to -35 °C under argon. To this were added tert-butylnitrite
(1.1 equiv) and HCl (2.8 equiv as a 4 M solution in 1,4-dioxane)
and the mixture was stirred for 3 h at -35 °C. Next, a mixture of
the deprotected amine-epoxyketone warhead (1.1 equiv, as a
trifluoroacetic acid (TFA) salt) and N,N-diisopropylethylamine
(DiPEA) (5 equiv) in DMF (1 mL) were added. The reaction was
slowly warmed to room temperature and stirred for another 12 h
before being diluted with DCM and extracted with 1 M HCl
(2ꢀ), saturated NaHCO3 (2ꢀ) and brine. After drying (MgSO4)
and concentrating, the obtained crude product was purified by
column chromatography, applying a 1% f 15% MeOH/DCM
eluent system.
(4) Examples of selective inhibitors for (a) β1: Van Swieten, P. F.;
ꢀ
Samuel, E.; Hernandez, R. O.; van den Nieuwendijk, A. M. C. H.;
Leeuwenburgh, M. A.; van der Marel, G. A.; Kessler, B. M.;
Overkleeft, H. S.; Kisselev, A. F. A cell-permeable inhibitor and
activity-based probe for the caspase-like activity of the proteasome.
Bioorg. Med. Chem. Lett. 2007, 17, 3402-3405; (b) β2: Nazif, T.;
Bogyo, M. Global analysis of proteasomal substrate specificity using
positional-scanning libraries of covalent inhibitors. Proc. Natl. Acad.
Sci. U.S.A. 2001, 98, 2967-2972; (c) β5: Elofsson, M.; Splittgerber,
U.; Myung, J.; Mohan, R.; Crews, C. M. Towards subunit-specific
proteasome inhibitors: synthesis and evaluation of peptide R0,β0-epoxy-
ketones. Chem. Biol. 1999, 6, 811-822.
(5) Kim, K. B.; Myung, J.; Sin, N.; Crews, C. M. Proteasome inhibi-
tion by the natural products epoxomicin and dihydroeponemycin:
insights into specificity and potency. Bioorg. Med. Chem. Lett.
1999, 9, 3335–3340.
(6) Britton, M.; Lucas, M. M.; Downey, S. L.; Screen, M.; Pletnev, A.
A.; Verdoes, M.; Tokhunts, R. A.; Amir, O.; Goddard, A. L.;
Pelphrey, P. M.; Wright, D. L.; Overkleeft, H. S.; Kisselev, A. F.
Selective inhibitor of proteasome’s caspase-like sites sensitizes cells
to specific inhibition of chymotrypsin-like sites. Chem. Biol. 2009,
16, 1278–1289.
Competition Experiments. Whole cell lysates of HEK-293T
were made by sonication in 3 volumes of lysis buffer containing
50 mM Tris pH 7.5, 1 mM dithiothreitol (DTT), 5 mM MgCl2,
250 mM sucrose, and 2 mM ATP. Protein concentration was
determined by the Bradford assay. Cell lysates (13.5 μg total
protein) were exposed to the inhibitors for 1 h prior to incuba-
tion with 9 (0.5 μM) for 1 h at 37 °C. Reaction mixtures were
boiled with Laemmli’s buffer containing β-mercaptoethanol for
3 min before being resolved on 12.5% SDS-PAGE. In-gel detec-
tion of residual proteasome activity was performed in the wet gel
slabs directly on a Typhoon variable mode imager (Amersham
Biosciences) using the Cy3/Tamra settings (λex 532, λem 560 nm).
IC50 Determinations. Purified 26S proteasome (∼10 ng/mL)
was incubated with various concentrations of inhibitors at 37 °C
for 30 min in the assay buffer (50 mM Tris-HCl, pH 7.5, 40 mM
KCl, 2 mM EDTA, 1 mM DTT, 100 μM ATP, 50 μg/mL BSA).
In the meantime, 100 μM solution of the fluorogenic peptide
substrates (Suc-LLVY-7-amido-4-methyl-coumarin (amc) for
the β5 site, Ac-nLPnLD-amc for the β1 site, and Ac-RLR-amc
or Ac-RQR-amc for the β2 site) in the assay buffer were pre-
incubated at 37 °C. Immediately after the end of this incubation,
an aliquot of the inhibitor-treated proteasome was mixed with
the substrate, and fluorescence of released amc was measured
continuously for 30 min at 37 °C. The rate of reaction was
determined from the slope of the reaction progress curves.
Mock-treated proteasomes served as control. Residual activity
in inhibitor treated samples were plotted against concentration
of inhibitors, and IC50 values were determined from these plots.
(7) Verdoes, M.; Florea, B. I.; van der Linden, W. A.; Renou, D.; van
den Nieuwendijk, A. M. C. H.; van der Marel, G. A.; Overkleeft,
H. S. Mixing of peptides and electrophilic traps gives rise to potent,
broad-spectrum proteasome inhibitors. Org. Biomol. Chem. 2007,
5, 1416–1426.
ꢀ
(8) Formicola, L.; Marechal, X.; Basse, N.; Bouvier-Durand, M.;
Bonnet-Delpon, D.; Milcent, T.; Reboud-Ravaux, M.; Ongeri, S.
Novel fluorinated pseudopeptides as proteasome inhibitors.
Bioorg. Med. Chem. Lett. 2009, 19, 83–86.
(9) (a) Berkowitz, D. B.; Karukurichi, K. R.; de la Salud-Bea, R.;
Nelson, D. L.; McCune, C. D. Use of fluorinated functionality in
enzyme inhibitor development: mechanistic and analytical advan-
tages. J. Fluor. Chem. 2008, 129, 731–742. (b) Molteni, M.; Pesenti,
C.; Sani, M.; Volonterio, A.; Zanda, M. Fluorinated peptidomimetics:
synthesis, conformational and biological features. J. Fluor. Chem.
2004, 125, 1735–1743. (c) Zanda, M. Trifluoromethyl group: an
effective xenobiotic function for peptide backbone modification.
New. J. Chem. 2004, 28, 1401–1411.
(10) For examples see: (a) Zheng, H.; Comeforo, K.; Gao, J. Expanding
the fluorous arsenal: tetrafluorinated phenylalanines for protein
design. J. Am. Chem. Soc. 2009, 131, 18–19. (b) Holmgren, S. K.;
Taylor, K. M.; Bretscher, L. E.; Raines, R. T. Code for collagen's
€
stability deciphered. Nature 1998, 392, 666–667. (c) Jackel, C.;
Salwiczek, M.; Koksch, B. Fluorine in a native protein environ-
ment;how the spatial demand and polarity of fluoroalkyl groups affect
protein folding. Angew. Chem., Int. Ed. 2006, 45, 4198–4203.
(11) (a) Flaherty, D. P.; Walsh, S. M.; Kiyota, T.; Dong, Y.; Ikezu, T.;
Vennerstrom, J. L. Polyfluorinated bis-styrylbenzene β-amyloid
plaque binding ligands. J. Med. Chem. 2007, 50, 4986–4992.
(b) Wadhwani, P.; B€urck, J.; Strandberg, E.; Mink, C.; Afonin, S.;
Ulrich, A. S. Using a strerically restrictive amino acid as a 19F NMR
label to monitor and to control peptide aggregation in membranes.
J. Am. Chem. Soc. 2008, 130, 16515–16517.
(12) Verdoes, M.; Florea, B. I.; Menendez-Benito, V.; Maynard, C. J.;
Witte, M. D.; van der Linden, W. A.; van den Nieuwendijk, A. M.
C. H.; Hofmann, T.; Berkers, C. R.; van Leeuwen, F. W. B.;
Groothuis, T. A.; Leeuwenburgh, M. A.; Ovaa, H.; Neefjes, J. J.;
Filippov, D. V.; van der Marel, G. A.; Dantuma, N. P.; Overkleeft,
H. S. A fluorescent broad-spectrum proteasome inhibitor for
labeling proteasomes in vitro and in vivo. Chem. Biol. 2006, 13,
1217–1226. For the structure of compound 9, see SI .
Acknowledgment. This work was supported by The
Netherlands Organization for Scientific Research (NWO),
The Netherlands Genomics Centre Initiative (NGI) and The
NCI (grant RO1CA124634). Wethank Hans van denElst and
Nico Meeuwenoord for HPLC and LC-MS assistance.
(13) (a) Muchamuel, T.; Basler, M.; Aujay, M. A.; Suzuki, E.; Kalim, K.
W.; Lauer, C.; Sylvain, C.; Ring, E. R.; Shields, J.; Jiang, J.;
Shwonek, P.; Parlati, F.; Demo, S. D.; Bennett, M. K.; Kirk,
C. J.; Groettrup, M. A selective inhibitor of the immuno-
proteasome subunit LMP7 blocks cytokine production and
attenuates progression of experimental arthritis. Nat. Med. 2009,
15, 781–788. (b) Demo, S. D.; Kirk, C. J.; Aujay, M. A.; Buchholz, T. J.;
Dajee, M.; Ho, M. N.; Jiang, J.; Laidig, G. J.; Lewis, E. R.; Parlati, F.;
Shenk, K. D.; Smyth, M. S.; Sun, C. M.; Vallone, M. K.; Woo, T. M.;
Molineaux, C. J.; Bennett, M. K. Antitumor activity of PR-171, a novel
irreversible inhibitor of the proteasome. Cancer Res. 2007, 67, 6383–
6391.
Supporting Information Available: Complete synthetic details
and characterization of all compounds and additional biological
experiments. This material is available free of charge via the
Internet at http://pubs.acs.org.
References
(1) Borissenko, L.; Groll, M. 20S Proteasome and its inhibitors:
crystallographic knowledge for drug development. Chem. Rev.
2007, 107, 687–717.
(2) Kisselev, A. F.; Goldberg, A. L. Proteasome inhibitors: from
(14) Van Swieten, P. F.; Leeuwenburgh, M. A.; Kessler, B. M.;
Overkleeft, H. S. Bioorthogonal organic chemistry in living cells:
research tools to drug candidates. Chem. Biol. 2001, 8, 739–758.

Brief Article
novel strategies for labeling biomolecules. Org. Biomol. Chem.
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2323
A. L. The caspase-like sites of proteasomes, their substrate specifi-
city, new inhibitors and substrates, and allosteric interactions with
the trypsin-like sites. J. Biol. Chem. 2003, 278, 35869–35877.
(18) Verdoes, M.; Hillaert, U.; Florea, B. I.; Sae-Heng, M.; Risseeuw,
M. D. P.; Filippov, D. V.; van der Marel, G. A.; Overkleeft, H. S.
Acetylene functionalized BODIPY dyes and their application in
the synthesis of activity based proteasome probes. Bioorg. Med.
Chem. Lett. 2007, 17, 6169–6171.
2005, 3, 20–27.
(15) Park, H. G.; Jeong, B. S.; Yoo, M. S.; Lee, J. H.; Park, M. K.; Lee,
Y. J.; Kim, M. J.; Jew, S. S. Highly enantioselective and practical
cinchona-derived phase-transfer catalysis for the synthesis of
R-amino acids. Angew. Chem., Int. Ed. 2002, 41, 3036–3038.
(16) The fluorescence intensity of the bands in Figure 3 was quantified
to get an impression of the residual proteasome subunit activity.
These values are shown in Table 2 in the SI.
(19) The IC50 values of compound 7 for each active subunit were
determined in the same manner as for the other compounds:
β1>10 μM, β2>10 μM, β5 0.30 μM.
(17) Kisselev, A. F.; Garcia-Calvo, M.; Overkleeft, H. S.; Peterson, E.;
Pennington, M. W.; Ploegh, H. L.; Thornberry, N. A.; Goldberg,