Two-step labeling of endogenous enzymatic activities by Diels-Alder ligation

Article abstract of DOI:10.1002/cbic.201000280

A ligation strategy based on the Diels-Alder [4+2] cycloaddition for the two-step activity-based labeling of endogenously expressed enzymes in complex biological samples has been developed. A panel of four diene-derivatized proteasome probes was synthesized, along with a dienophile-functionalized BODIPY(TMR) tag. These probes were applied in a Diels-Alder labeling procedure that enabled us to label active proteasome β-subunits selectively in cellular extracts and in living cells. We were also able to label the activity of cysteine proteases in cell extracts by utilizing a diene-derivatized cathepsin probe. Importantly, the Diels-Alder strategy described here is fully orthogonal with respect to the Staudinger-Bertozzi ligation, as demonstrated by the independent labeling of different proteolytic activities by the two methods in a single experiment.

Full text of DOI:10.1002/cbic.201000280

DOI: 10.1002/cbic.201000280
Two-Step Labeling of Endogenous Enzymatic Activities by
Diels–Alder Ligation
Lianne I. Willems, Martijn Verdoes, Bogdan I. Florea, Gijsbert A. van der Marel, and
Herman S. Overkleeft*[a]
A ligation strategy based on the Diels–Alder [4+2] cycloaddi-
tion for the two-step activity-based labeling of endogenously
expressed enzymes in complex biological samples has been
developed. A panel of four diene-derivatized proteasome
probes was synthesized, along with a dienophile-functionalized
BODIPY(TMR) tag. These probes were applied in a Diels–Alder
labeling procedure that enabled us to label active proteasome
b-subunits selectively in cellular extracts and in living cells. We
were also able to label the activity of cysteine proteases in cell
extracts by utilizing a diene-derivatized cathepsin probe. Im-
portantly, the Diels–Alder strategy described here is fully or-
thogonal with respect to the Staudinger–Bertozzi ligation, as
demonstrated by the independent labeling of different proteo-
lytic activities by the two methods in a single experiment.
Introduction
The introduction of bioorthogonal chemistry has given a great
impetus to chemical biology research. A bioorthogonal reac-
tion can be defined as a chemical transformation between two
functional groups that takes place selectively within complex
biological samples such as cell extracts, tissues, or even living
organisms.[1] By allowing the temporal separation of a molecu-
lar probe and a reporter group or affinity tag, bioorthogonal
chemistry maximizes the potential for selective modification of
a specific biomolecule in a desired experimental setting. The
power of this strategy was initially demonstrated by Bertozzi
and co-workers in the cell-surface labeling of glycoproteins.[2–4]
teasome probe and a biotin-derivatized phosphine reagent for
bioorthogonal ligation, and demonstrated its use in the direct
identification of a proteasome subunit-specific inhibitor.[16,17]
Cravatt and co-workers were the first to report the use of click
chemistry for activity-based profiling of serine hydrolases.[18]
Whereas the examples described above make use of azides
or alkynes as ligation handles and rely either on Staudinger–
Bertozzi ligation or on click chemistry, other small groups also
warrant consideration for use in bioorthogonal ligation reac-
tions. In view of its selectivity and efficiency under mild, aque-
ous conditions, we considered the Diels–Alder [4+2] cycloaddi-
tion as a potential alternative. Several reports have described
the use of Diels–Alder ligation strategies for bioconjugation, in-
cluding the modification of oligonucleotides and DNA
strands,[19–26] surface immobilization of oligonucleotides,[27,28] in-
terior surface modification of viral capsids,[29] microarray devel-
opment,[30–35] conjugation of carbohydrates and proteins,[36,37]
and the surface immobilization and site-specific ligation of
peptides and proteins.[38,39] A few related strategies that use
the inverse-electron-demand Diels–Alder reaction for protein
labeling have recently been reported.[40–42] By using this ap-
proach, live cells could be labeled in vitro by first targeting cell
surface receptors with a dienophile-bearing monoclonal anti-
body, and then by adding a tetrazine conjugated to a fluores-
cent tag in order to label the pretargeted antibody on the cell
surface.[41,42] Here we describe the development of a Diels–
In
a seminal experiment, N-azidoacetylmannosamine was
added to the medium of cultured Jurkat cells, resulting in the
presence of azides on the cell-surface sialic acid residues pres-
ent in N-glycans. These were then selectively modified with a
biotin entity by treatment with a phosphine reagent, a strat-
egy known as the Staudinger–Bertozzi ligation, which allowed
the monitoring of cell surface glycoconjugates. A second im-
portant orthogonal bioconjugation method involves the reac-
tion between an azide and an alkyne—the Huisgen [2+3] cy-
cloaddition or click reaction.[5,6] Nowadays, both copper(I)-cata-
lyzed click reactions[5,6] and copper-free strain-promoted click
strategies[7–11] are used. Bioorthogonal ligation reactions are ap-
plied to study a variety of biological processes, in particular
those involving post-translational modifications.[12–15]
Another area of research that has benefited from bioorthog-
onal chemistry is two-step activity-based protein profiling
(ABPP). The direct introduction of a reporter group into an
active-site-directed chemical probe could affect the cell perme-
ability of the probe, affinity for a target enzyme, and/or selec-
tivity over other enzymes. These problems can be avoided by
introducing the tag in a later stage, after binding of the probe
to a target enzyme. We demonstrated the feasibility of the
Staudinger–Bertozzi ligation for two-step labeling of the pro-
teasome, employing an azide-functionalized activity-based pro-
[a] L. I. Willems, Dr. M. Verdoes, Dr. B. I. Florea, Prof. G. A. van der Marel,
Prof. H. S. Overkleeft
Leiden Institute of Chemistry and
Netherlands Proteomics Centre, Gorlaeus Laboratories
Einsteinweg 55, 2333CC Leiden (The Netherlands)
Fax: (+31)71-5274307
E-mail: h.s.overkleeft@chem.leidenuniv.nl
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201000280.
ChemBioChem 2010, 11, 1769 – 1781
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1769

H. S. Overkleeft et al.
Figure 1. The Diels–Alder ligation in two-step ABPP. Schematic representation of the Diels–Alder-based labeling strategy, entailing: 1) targeting of active pro-
teases by a diene-derivatized probe, 2) ligation of the protease-bound diene with a dienophile-functionalized tag, and 3) visualization of labeled proteases by
SDS-PAGE analysis and fluorescence detection.
Alder ligation procedure for the profiling of endogenous enzy-
matic activities and explore its suitability for labeling of endo-
genously expressed enzymes in complex biological samples.
Our approach is applicable to the labeling of various classes of
enzymes and is orthogonal with respect to the Staudinger–Ber-
tozzi ligation, allowing both strategies to be used in a single
experiment for the independent labeling of different biomole-
cules.
BODIPY moiety in 2 has been reported to be an excellent dien-
ophile for use in Diels–Alder-based ligation procedures on pro-
teins.[38,39] The design of probes 1a–d was based on the pro-
teasome inhibitor epoxomicin,[44,45] extended at its N terminus
with one of four different conjugated dienes.
The probes 1a–d were synthesized as depicted in Scheme 1.
The tripeptide recognition element was obtained by standard
solid-phase peptide chemistry (12) followed by mild acidic
cleavage from the resin (13). The partially protected tripep-
tide 13 was then coupled through an azide-coupling proce-
dure to the leucine-derived epoxyketone warhead amine 17,
which was synthesized essentially as described previously.[45]
After deprotection of the N-terminal amine in 16, treatment
with the N-hydroxysuccinimidyl esters of four different diene-
containing acids (11 a–d) afforded the probes 1a–d.
The dienophile-modified tag 2 was generated by starting
from triethylene glycol (20; Scheme 2). The alcohol moieties
were transformed into the tosylates, which were both substi-
tuted with NaN3 to afford compound 21, after which one of
the azides could be selectively reduced and protected, yielding
compound 23. The free amine 24 resulting from reduction of
the remaining azide was coupled to the activated malei-
mide 19 to give the maleimide-functionalized poly(ethylene
Results and Discussion
Our Diels–Alder-based ABPP strategy, as outlined in Figure 1,
involves the labeling of active enzymes with a diene-derivat-
ized active-site-directed probe followed by reaction with a di-
enophile modified with a fluorescent tag in order to visualize
the labeled enzymes. In the first instance, we evaluated the
validity of this approach for the two-step labeling of catalyti-
cally active subunits of the 20S proteasome, a highly con-
served protease complex that is responsible for degrading the
majority of cellular proteins.[43] For this purpose, a panel of four
diene-functionalized proteasome probes (1a–d) was synthe-
sized along with a dienophile-derivatized BODIPY(TMR)-tag (2).
The maleimide functionality conjugated to the fluorescent
1770
www.chembiochem.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2010, 11, 1769 – 1781

Labeling Endogenous Enzymatic Activities
Scheme 1. Synthesis of the diene-functionalized proteasome probes. Reagents and conditions: a) triethyl orthoacetate, propionic acid, toluene, reflux, 3 h,
94%; b) 1m NaOH/MeOH/1,4-dioxane, RT, 1 h, 68%; c) KCN, DMSO, 608C, 1 h, then RT, overnight (o/n), 94%; d) i: NaOH, 2-methoxyethanol, reflux, 3 h, ii: HCl,
H2O, RT, o/n, 56%; e) i: NaH, THF, RT, 30 min, ii: 2-bromoacetic acid, RT, o/n, 73%; f) HOSu, EDC·HCl, DCE/DMF, RT, o/n, 11 a 71%, 11 b 78%, 11 c 81%, 11 d
80%; g) TFA/DCM (1:99, v/v), RT, 6ꢁ10 min; h) TMS diazomethane, MeOH/toluene, RT, 3.5 h, 64% from MBHA-HMPB resin; i) H2NNH2·H2O, MeOH, reflux, o/n,
76%; j) i: tBuONO, HCl, DMF/EtOAc, ꢀ308C, 1 h, ii: 17, DiPEA, DMF/EtOAc, ꢀ308C to RT, o/n, 89%; k) i: TFA/DCM (1:1, v/v), RT, 15–45 min, ii: R-OSu (11 a–d),
DiPEA, DCE/DMF, RT, 1.5–18 h, 1a 82%, 1b 64%, 1c 39%, 1d 100%.
Scheme 2. Synthesis of the dienophile-functionalized fluorescent tag. Reagents and conditions: a) methyl chloroformate, N-methylmorpholine, EtOAc, 08C,
2 h, 52%; b) i: tosyl chloride, Et3N, DMAP, DCM, RT, 16 h, ii: NaN3, TBAI, DMF, 808C, 16 h, 78%; c) Ph3P, HCl (5%), toluene, 08C, 16 h, 79%; d) Et3N, Boc2O, DCM,
RT, 50 min, 90%; e) i: Ph3P, THF, RT, 5 h, ii: H2O, RT, 2 h, 72% crude); f) 19, saturated aqueous NaHCO3, 08C, 30 min, then RT, 45 min, 31% over 2 steps; g) i:
TFA/DCM (1:1, v/v), RT, 10 min, ii: BODIPY(TMR)-OSu, DiPEA, DCE, RT, o/n, 34%.
glycol) spacer 25. Deprotection and condensation with
BODIPY(TMR)-OSu[46] then afforded the BODIPY(TMR)-malei-
mide 2.
We then set out to establish whether the four diene-contain-
ing probes 1a–d had the ability to inhibit the activity of the
proteasome. This was tested by performing a competition ex-
periment against the fluorescent activity-based proteasome
probe MV151 (Figure 2).[46] EL-4 cell extracts were exposed to
increasing concentrations of the probes 1a–d for 2 hours, fol-
lowed by exposure to MV151 for 1 hour to label residual pro-
teasome activity. After SDS-PAGE analysis, in-gel fluorescence
detection of the labeled proteins revealed that probes 1a–d
inhibited MV151 labeling of the active proteasome b-subunits
ChemBioChem 2010, 11, 1769 – 1781
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
1771

H. S. Overkleeft et al.
Figure 2. Diene-containing epoxomicin derivatives inhibit proteasome activity in vitro. EL-4 cell lysates (10 mg total protein per reaction) were exposed to in-
creasing concentrations of the diene-derivatized probes 1a–d for 2 h at 378C, followed by exposure to MV151 (1 mm) for 1 h at 378C. Labeled proteins were
resolved by SDS-PAGE and detected by fluorescence readout (A) followed by Coomassie staining as a loading control (B). Proteasome b-subunits are designat-
ed on the basis of reported labeling by MV151.[46] DC: Dual Color protein standard.
with similar potencies. Complete inhibition was observed at in-
hibitor concentrations of 0.5–1 mm and above.
Although there is a considerable degree of background fluo-
rescence, three fluorescent bands corresponding to protea-
some b-subunits are clearly visible after labeling with the
diene-derivatized epoxomicin analogues 1d, 1a, and 1b (lanes
1, 5, and 7, respectively). These signals were completely
blocked by addition of an excess of epoxomicin (lanes 2, 6,
and 8), demonstrating that the observed bands indeed reflect
labeling of proteasome active sites. The large differences in la-
beling intensity between the diene-derivatized probes indicate
that the nature of the diene is of crucial importance for the li-
gation efficiency with maleimide 2. The complete absence of
specific labeling in lane 3 suggests that anthracenyl-containing
inhibitor 1c is unable to undergo Diels–Alder ligation with the
maleimide 2 efficiently when bound to the proteasome. The li-
gation step proceeds most efficiently for the noncyclic dienes
in 1d (lane 1) and, especially, 1b (lane 7), which is somewhat
surprising because these dienes are not fixed in the cis config-
uration that is required for Diels–Alder cycloaddition.
We next turned our attention to the two-step Diels–Alder la-
beling procedure. In the first instance, the maleimide-derivat-
ized tag 2 was shown to react with the diene-containing
probes 1a–d in the absence of proteins to give the corre-
sponding Diels–Alder adducts (see the Supporting Informa-
tion). Two-step Diels–Alder ligation experiments were then per-
formed in EL-4 cell extracts. After exposure to inhibitors 1a–d
(1 mm) for 1 hour at 378C, the proteins were denaturated and
cysteine residues were masked with Ellman’s reagent [5,5’-di-
thiobis-(2-nitrobenzoic acid), DTNB]. This is necessary in order
to avoid labeling resulting from Michael addition of (cysteine)
thiol groups to maleimide 2. Remaining thiol-containing re-
agents were removed by chloroform/methanol (c/m) precipita-
tion,[47] after which the proteins were exposed to the BODIPY-
(TMR)-maleimide 2 (25 mm) at 378C overnight at pH 6.0. After
excess maleimide-tag 2 had been washed away in a second
precipitation step, the fluorescently labeled proteins were re-
solved by SDS-PAGE and visualized directly with the aid of a
fluorescence scanner (Figure 3).
In an effort to improve selectivity and to reduce background
labeling, the concentration of the diene-derivatized probe 1b
and the conditions for cysteine masking were varied (Figure 4
and Figure S1 in the Supporting Information, respectively). Pro-
teasome labeling became stronger with increasing concentra-
Figure 4. Effect of diene-functionalized probe concentration on Diels–Alder-
based proteasome labeling. EL-4 cell lysates were exposed to increasing
concentrations of the probe 1b for 1 h at 378C prior to denaturation, mask-
ing of cysteine residues, and c/m precipitation. As a control, lysates were ex-
posed either to 1b in the presence of epoxomicin (100 mm, +ep) or to
MV151 (1 mm). The proteins were then exposed to BODIPY(TMR)-malei-
mide 2 (25 mm) overnight at 378C in Diels–Alder buffer (pH 6.0); 50 mg total
protein per reaction was resolved by SDS-PAGE and labeled proteins were
detected by fluorescence readout.
Figure 3. Labeling of proteasome activity in cell extracts by using the Diels–
Alder ligation. EL-4 cell lysates were exposed to 1a–d (1 mm) in the presence
or in the absence of epoxomicin (100 mm) for 1 h at 378C, prior to denatura-
tion, masking of cysteine residues with DTNB, and c/m precipitation. The
proteins were then exposed to BODIPY(TMR)-maleimide 2 (25 mm) in Diels–
Alder buffer (pH 6.0) overnight at 378C; 50 mg total protein/reaction was re-
solved by SDS-PAGE and labeled proteins were detected by fluorescence
readout (A) followed by Coomassie staining as a loading control (B).
1772
www.chembiochem.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2010, 11, 1769 – 1781

Labeling Endogenous Enzymatic Activities
tions of 1b and reached saturation at around 0.5 mm of the in-
hibitor (Figure 4), which is consistent with the competition
assay shown in Figure 2. The concentration of diene-containing
probe did not affect the amount of background fluorescence,
so we assume that the background fluorescence is not caused
by nonspecific reactions of the inhibitor 1b but rather by the
maleimide-functionalized tag 2, which might react with unpro-
tected cysteine residues.
(lane 6) gives the best results in terms of signal intensity and
background fluorescence. Figure 5B reveals similar increases
both in specific and in nonspecific labeling with increasing re-
action times. The best results were obtained after Diels–Alder
ligation for 4 or 20 hours (lane 5 and lane 6, respectively). At a
reaction time of 20 hours, proteasome labeling reaches levels
comparable to that of MV151 (lane 9), when used at the same
concentration as the diene-derivatized probe. We chose a reac-
tion time of 20 hours and a dienophile concentration of 25 mm
for subsequent Diels–Alder ligation procedures on the protea-
some.
Consistently with this observation, the concentration of cap-
ping reagent (DTNB) proved to be of crucial importance for
the amount of background labeling (Figure S1). The concentra-
tion of the capping reagent needs to be sufficiently high in
order to reduce background fluorescence. A concentration
that is too high, on the other hand, reduces not only back-
ground fluorescence but also specific labeling; this might be
due to the fact that the large amount of reagent is not re-
moved completely by a single c/m precipitation step and can
therefore interfere with the subsequent ligation reaction.
Subsequently, efforts were made to improve the efficiency
of the Diels–Alder ligation by varying the concentration of the
BODIPY(TMR)-maleimide 2 (Figure 5A) and the reaction time of
the ligation step (Figure 5B). From Figure 5A it is apparent
that both background fluorescence and specific labeling are
enhanced at increasing concentrations of maleimide 2. A dien-
ophile concentration between 10 mm (lane 5) and 25 mm
Having shown that the Diels–Alder ligation strategy can suc-
cessfully be applied to the profiling of active proteasomes in
vitro, we also tested the utility of the approach for in situ label-
ing. The diene-functionalized probe 1b was first demonstrated
to be cell-permeable by treatment of cultured EL-4 cells with
the probe for 2 hours, after which the cells were lysed and re-
sidual proteasome activity was labeled with MV151 (Fig-
ure 6A). The probe 1b was able to inhibit the activity of all
proteasome b-subunits in situ, with an inhibitory potency ap-
proximately ten-fold lower in living cells than in cell extracts
(see Figure 2).
Figure 6. In situ labeling of proteasome activity with the diene-derivatized
probe 1b. Living EL-4 cells were exposed for 2 h at 378C either to the indi-
cated concentrations of the probe 1b or to MV151 (1 mm) as a control,
before being harvested and lysed. A) The lysates (10 mg total protein per re-
action) were exposed to MV151 (1 mm) for 1 h at 378C. B) The lysates (20 mg
total protein per reaction) were subjected to denaturation, masking of cys-
teine residues, and c/m precipitation before being exposed to the BODIPY-
(TMR)-maleimide 2 (25 mm) overnight at 378C (pH 6.0). Labeled proteins
were resolved on SDS-PAGE and detected by fluorescence readout.
Figure 5. Effects of dienophile concentration and reaction time on Diels–
Alder ligation efficiency. EL-4 cell lysates were exposed to the probe 1b
(1 mm ) for 1 h at 378C prior to denaturation, masking of cysteine residues,
and c/m precipitation. As a control, the lysates were exposed either to 1b in
the presence of epoxomicin (+ep, 100 mm) or to MV151 (1 mm). The proteins
were then exposed either A) to increasing concentrations of the BODIPY-
TMR-maleimide 2 overnight, or B) to compound 2 (25 mm) for the indicated
time at 378C (pH 6.0); 50 mg total protein/reaction was resolved by SDS-
PAGE and labeled proteins were detected by fluorescence readout.
The activity of the proteasome b-subunits was next labeled
by use of the following Diels–Alder ligation strategy. Viable EL-
4 cells were treated with probe 1b (10 mm) for 2 hours, after
which the cells were harvested and lysed. The cell lysates were
subjected to the Diels–Alder protocol with the maleimide 2 as
before (Figure 6B). In lysates from cells treated with 1b
(lane 2), fluorescent bands can be distinguished that are not
ChemBioChem 2010, 11, 1769 – 1781
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
1773

H. S. Overkleeft et al.
present in lysates from untreated cells (lane 3) and correspond
to the active proteasome b-subunits labeled by MV151
(lane 4). These results reveal specific labeling of proteasome
activity in situ. In comparison with the procedures in which
cell extracts were exposed to the diene-functionalized probe
(Figures 3–5), exposure of cells prior to cell lysis seems to give
a small decrease in background labeling.
The inhibitory potency of the probe 32 was evaluated in a
competition assay against the fluorescent activity-based cys-
teine cathepsin probe N3-BODIPY(TMR)-DCG-04 (Figure 7A).[49]
In order to demonstrate the applicability of the Diels–Alder
strategy in ABPP further, we also employed the procedure for
labeling of cysteine proteases of the papain family. We there-
fore synthesized the diene-functionalized probe 32, derived
from the activity-based cysteine cathepsin probe DCG-04[48]
(Scheme 3). The synthesis of probe 32 involved the generation
of peptidic recognition element 29 and coupling of epoxysuc-
cinate warhead 28 by solid-phase peptide chemistry, cleavage
from the resin and deprotection to give compound 30, and fi-
nally coupling of the lysine e-amine to the diene-functionalized
activated ester 11 b to afford the diene-derivatized DCG-04 an-
alogue 32.
Figure 7. Labeling of cathepsin activity with the aid of the diene-derivatized
DCG-04 analogue 32. RAW cell lysates (50 mg total protein per reaction)
were exposed to the indicated concentrations of the probe 32 for 1 h at
378C. As a control, lysates were exposed either to 32 in the presence of
DCG-04 (+DCG, 25 mm) or to N3-BODIPY(TMR)-DCG-04 (b-DCG, 0.5 mm). The
lysates were next either: A) exposed to N3-BODIPY(TMR)-DCG-04 (0.5 mm) for
1 h at 378C to label residual cathepsin activity, or B) subjected to denatura-
tion, masking of cysteine residues, and Diels–Alder ligation with the indicat-
ed concentrations of the BODIPY(TMR)-maleimide 2 overnight at 378C
(pH 6.0). Labeled proteins were resolved on SDS-PAGE and detected by fluo-
rescence readout.
RAW cell extracts were exposed to increasing concentrations
of compound 32 and then to N3-BODIPY(TMR)-DCG-04 in order
to label residual cathepsin activity. At increasing concentrations
of the diene-derivatized DCG-04 analogue 32 the intensity of
labeling by the fluorescent probe was reduced, demonstrating
that probe 32 is able to inhibit the activity of cysteine cathep-
sins in vitro.
The diene-functionalized cathepsin probe 32 was then used
in a Diels–Alder ligation procedure. Cell extracts were treated
with probe 32 (5 mm) for 1 hour at 378C and subjected to the
Diels–Alder ligation protocol with increasing concentrations of
maleimide tag 2 (Figure 7B). As before, the intensities both of
specific and of background fluorescence were dependent on
the concentration of the dienophile. The labeling profile shows
two bands after treatment with probe 32 (lanes 3–6) that are
not visible in non-treated samples (lane 7). The labeling corre-
sponds to labeling by N3-BODIPY(TMR)-DCG-04 (lane 9) and
was blocked by addition of an excess of the nonfluorescent ca-
thepsin probe DCG-04 (lane 8). This confirms that the two
bands indeed involve labeling of active cathepsins by means
of the two-step Diels–Alder ligation procedure. From the ob-
served molecular weights and a previously reported DCG-04 la-
beling profile in RAW cell extracts,[50] we assume that these
two labeled proteases are cathepsin Z and cathepsin B.
Scheme 3. Synthesis of the diene-functionalized cathepsin probe. Reagents
and conditions: a) i: HBr in AcOH (33%), 08C, 15 min, then RT, o/n, ii: AcCl,
EtOH, reflux, 3.5 h, iii: DBU, Et2O, 08C, 4.5 h, 56%; b) KOH, EtOH, 08C, 3 h,
then RT, 2 h, 86%; c) i: piperidine/NMP (1:4, v/v), RT, 30 min, ii: 28, DiPEA,
BOP, NMP, RT, o/n, iii: TFA/TIS/H2O (95:2.5:2.5, v/v/v), RT, 2 h, 30 67%, 31
33%; d) 11 b, DiPEA, DCE/DMF, RT, o/n, 76%.
Finally, the orthogonality of the two-step Diels–Alder proce-
dure with respect to the Staudinger–Bertozzi ligation was es-
1774
www.chembiochem.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2010, 11, 1769 – 1781

Labeling Endogenous Enzymatic Activities
tablished by performing both procedures in one experiment
for the independent labeling of different proteolytic activities.
HEK cell lysates were first exposed to the azide-derivatized pro-
teasome probe 3, which is selective for the b1 subunit,[51] fol-
lowed by the diene-containing probe 1b to label the b2 and
b5 proteasome subunits. The cell extracts were then treated
with the biotin-functionalized phosphine reagent 4 for Stau-
dinger ligation,[52] prior to denaturation, cysteine masking, and
Diels–Alder reaction with fluorescent maleimide 2. After SDS-
PAGE analysis, the labeled proteins were visualized by fluores-
cence scanning and streptavidin Western blotting (Figure 8).
of endogenously expressed proteases in cellular extracts.[54,55] A
drawback to the procedure described here is the need to mask
cysteine residues prior to dienophile addition, which precludes
in vivo application. A potential solution to this problem would
be the use of a dienophile that is not a Michael acceptor and
should thus be much more selective. Efforts to achieve this
goal are currently being made in our lab. We feel that the
Diels–Alder strategy presented here represents a useful alter-
native to click and Staudinger–Bertozzi ligation approaches. An
attractive aspect of the Diels–Alder approach is that it is fully
orthogonal with respect to the Staudinger ligation and can be
performed in the same sample. This provides the potential to
modify two different biomolecules—two enzymatic activities
or a metabolite and an enzyme, for example—with different
labels in a single experiment and thereby to study their roles
or behavior simultaneously.
Experimental Section
General: All reagents were commercial grade and were used as re-
ceived unless indicated otherwise. Toluene (purum), ethyl acetate
(EtOAc, puriss.), diethyl ether (Et2O), and light petroleum ether
(PetEt, puriss.) were obtained from Riedel–de Haꢂn. Dichloroethane
(DCE), dichloromethane (DCM), dimethyl formamide (DMF), and di-
oxane (Biosolve) were stored over molecular sieves (4 ꢃ). Methanol
(MeOH) and N-methylpyrrolidone (NMP) were obtained from Bio-
solve. Reactions were monitored by TLC analysis (DC-Alufolien,
Merck, Kieselgel60, F254) with detection variously by UV absorp-
tion (254 nm), by spraying with a solution of (NH4)6Mo7O24·4H2O
(25 gLꢀ1) and (NH4)4Ce(SO4)4·2H2O (10 gLꢀ1) in sulfuric acid (10%)
followed by charring at ~1508C, or by spraying with an aqueous
solution of KMnO4 (20%) and K2CO3 (10%). Column chromatogra-
phy was performed on silica gel (Screening Devices, 0.040–
0.063 nm). LC/MS analysis was performed with a LCQ Advantage
Max (Thermo Finnegan) instrument with a Gemini C18 column
(Phenomene x). The applied buffers were H2O (A), MeCN (B), and
Figure 8. Diels–Alder and Staudinger ligations can be performed in the
same sample. HEK cell lysates (50 mg total protein per reaction) were first ex-
posed to the azide-derivatized b1-selective proteasome probe 3 (5 mm) for
1 h at 378C, then to the diene-functionalized probe 1b (5 mm) for 1 h at
378C, and then to the biotin-phosphine 4 (100 mm) for 1 h at 378C. After
protein denaturation, masking of cysteine residues, and c/m precipitation,
Diels–Alder ligation was performed with the BODIPY(TMR)-maleimide 2
(25 mm) overnight at 378C (pH 6.0). The labeled proteins were resolved by
SDS-PAGE and detected by fluorescence readout followed by streptavidin
Western blotting. Proteasome b-subunits are designated on the basis of
known labeling profiles in HEK cell extracts.[53]
When the ligation was performed with maleimide 2 only, fluo-
rescent labeling of the diene-modified active proteasome b-
subunits was observed (upper panel, lane 2). As would be ex-
pected, preincubation with probe 3 completely blocked fluo-
rescent labeling of the b1 subunit because this subunit was
now targeted by azide-derivatized probe 3 (upper panel,
lane 3). Ligation with Staudinger reagent 4 only gave selective
labeling of the b1 subunit (lower panel, lanes 5 and 6).
1
aq. TFA (1.0%, C). H and 13C APT-NMR spectra were recorded with
Jeol JNM-FX-200 (200/50) or Bruker AV400 (400/100 MHz) instru-
ments with a pulsed field gradient accessory. Chemical shifts (d)
are given in ppm relative to tetramethylsilane as internal standard.
Coupling constants are given in Hz. All 13C-APT spectra presented
are proton-decoupled. In-gel fluorescence readout was measured
with a Typhoon Variable Mode Imager (Amersham Biosciences) and
with Cy3/TAMRA settings (excitation wavelength 532 nm, emission
wavelength 580 nm). Western blotting was performed as indicated.
Markers used were Dual Color protein standard (DC) and Biotinylat-
ed protein marker (BM).
Most importantly, the labeling profile obtained by the com-
bination of the two ligation methods in one sample (lane 4) is
very similar to that obtained by the separate methods (lanes 3
and 5). By this combined strategy, b1 activity was selectively la-
beled by Staudinger–Bertozzi ligation (lower panel) whereas at
the same time the b2 and b5 subunits were fluorescently la-
beled by Diels–Alder ligation (upper panel). This experiment
shows that the Diels–Alder ligation is fully compatible with
Staudinger ligation conditions and that there is no cross-reac-
tivity between the ligation reagents and the probes used for
both methods.
Ethyl (E)-hepta-4,6-dienoate (6): Triethyl orthoacetate (2.8 mL,
15 mmol, 5.0 equiv) and propionic acid (one drop) were added to
a solution of penta-1,4-dien-3-ol (5, 0.29 mL, 3.0 mmol) in toluene.
The reaction mixture was stirred under reflux for 3 h, before being
concentrated in vacuo. Purification by column chromatography
(PetEt!5% EtOAc in PetEt) yielded compound 6 as a colorless oil
(0.43 g, 2.8 mmol, 94%). 1H NMR (200 MHz, CDCl3): d=6.44–5.99
(m, 2H), 5.81–5.60 (m, 1H), 5.05 (ddd, J=13.29, 11.48, 1.62 Hz, 2H),
4.14 (q, J=7.14, 7.14, 7.13 Hz, 2H), 2.37 (s, 4H), 1.25 ppm (t, J=
7.14, 7.14 Hz, 3H); 13C NMR (50 MHz, CDCl3): d=172.12, 136.46,
132.18, 131.55, 115.05, 59.72, 33.31, 27.37, 13.75 ppm.
Conclusions
In conclusion, we have developed a Diels–Alder-based ABPP
strategy that has been successfully used to monitor the activity
2-(Anthracen-9-yl)acetonitrile (8): A solution of 9-(chloromethyl)-
anthracene (7, 2.27 g, 10 mmol) in DMSO (15 mL) was heated to
ChemBioChem 2010, 11, 1769 – 1781
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
1775

H. S. Overkleeft et al.
608C, followed by the addition of a solution of KCN (0.98 g,
15 mmol, 1.5 equiv) in H2O (3 mL). The reaction mixture was stirred
at 608C for 1 h and was then allowed to cool to room temperature
and stirred overnight. After addition of H2O (40 mL), the mixture
was filtered and washed with H2O, and the residue was dried in
vacuo to yield compound 8 as yellow crystals (2.04 g, 9.4 mmol,
94%). 1H NMR (200 MHz, CDCL3): d=8.53 (s, 1H), 8.13 (ddd, J1 =
20.68 Hz, J2 =8.43 Hz, J3 =0.42 Hz, 4H), 7.59 (dddd, J1 =14.79 Hz,
J2 =7.67 Hz, J3 =6.63 Hz, J4 =1.08 Hz, 4H), 4.61 ppm (s, 2H);
13C NMR (50 MHz, CDCl3): d=131.21, 129.33, 128.66, 127.07, 125.13,
122.77, 117.65, 77.62, 77.00, 76.35, 15.96 ppm.
3.14–2.90 (m, 4H), 2.84 ppm (s, 4H); 13C NMR (50 MHz, CDCl3): d=
169.04, 167.46, 152.32, 141.50, 109.99, 105.60, 29.09, 25.14,
22.51 ppm.
(E)-Hepta-4,6-dienoyl-OSu (11b): (E)-Hepta-4,6-dienoic acid (10b,
0.17 g, 1.3 mmol) was subjected to the same procedure as de-
scribed above for 11 a, giving the OSu-ester 11 b (0.23 g, 1.0 mmol,
78%). 1H NMR (400 MHz, CDCl3): d=6.30 (td, J=16.90, 10.22,
10.22 Hz, 1H), 6.14 (dd, J=15.11, 10.51 Hz, 1H), 5.76–5.65 (m, 1H),
5.09 (dd, J=51.12, 13.45 Hz, 2H), 2.80 (s, 4H), 2.71 (t, J=7.39,
7.39 Hz, 2H), 2.54–2.47 ppm (m, 2H).
2-(Anthracen-9-yl)acetyl-OSu (11c): 2-(Anthracen-9-yl)acetic acid
(10c, 0.86 g, 3.6 mmol) was subjected to the same procedure as
described above for 11 a, giving the OSu-ester 11 c (0.98 g,
(E)-Hepta-4,6-dienoic acid (10b): Ethyl (E)-hepta-4,6-dienoate (6,
0.43 g, 2.8 mmol) was dissolved in a mixture of 1,4-dioxane/MeOH/
1m NaOH (1:1:1, v/v/v) and the mixture was stirred for 1 h. After
addition of DCM, the mixture was washed with a saturated
NaHCO3 solution (1ꢁ). The aqueous layer was then acidified with
HCl (1m), followed by extraction with DCM (3ꢁ). The combined or-
ganic layers were dried over anhydrous MgSO4, filtered, and con-
centrated in vacuo. The crude product was purified by column
chromatography (PetEt!25% EtOAc in PetEt), yielding hepta-4,6-
dienoic acid (10b, 0.24 g, 1.9 mmol, 68%). 1H NMR (200 MHz,
CDCl3): d=6.43–6.00 (m, 2H), 5.78–5.61 (m, 1H), 5.26–4.87 (m, 2H),
2.56–2.33 ppm (m, 4H); 13C NMR (50 MHz, CDCl3): d=179.26,
136.67, 132.04, 115.74, 33.47, 27.26 ppm.
1
2.9 mmol, 81%). H NMR (200 MHz, CDCl3): d=8.48 (s, 1H), 8.22 (d,
J=9.12 Hz, 2H), 8.03 (d, J=8.4 Hz, 2H), 7.64–7.45 (m, 4H), 4.92 (s,
2H), 2.76 ppm (s, 4H); 13C NMR (50 MHz, CDCl3): d=168.89, 166.58,
131.25, 130.43, 129.13, 128.16, 126.67, 125.01, 123.55, 122.85,
30.30, 25.27 ppm.
2-((2E,4E)-Hexa-2,4-dienyloxy)acetyl-OSu (11d): 2-((2E,4E)-Hexa-
2,4-dienyloxy)acetic acid (10d, 3.42 g, 21.9 mmol) was subjected to
the same procedure as described above for 11 a, giving the OSu-
1
ester 11 d (4.41 g, 17.4 mmol, 80%). H NMR (400 MHz, CDCl3): d=
6.26 (dd, J=15.22, 10.44 Hz, 1H), 6.07 (dd, J=14.09, 11.48 Hz, 1H),
5.81–5.71 (m, 1H), 5.66–5.53 (m, 1H), 4.41 (s, 2H), 4.16 (d, J=
6.60 Hz, 2H), 2.85 (s, 4H), 1.77 ppm (d, J=6.68 Hz, 3H); 13C NMR
(100 MHz, CDCl3): d=168.73, 165.91, 135.31, 131.28, 130.35, 124.34,
72.06, 64.36, 25.52, 18.08 ppm.
2-(Anthracen-9-yl)acetic acid (10c): 2-(Anthracen-9-yl)acetonitrile
(8, 2.04 g, 9.4 mmol) was dissolved in 2-methoxyethanol (15 mL),
and NaOH (0.94 g, 23.5 mmol, 2.5 equiv) was added. The reaction
mixture was heated at reflux for 3 h before addition of H2O
(60 mL). The mixture was then washed with Et2O (2ꢁ) and the
aqueous layer was acidified to pH 2 with HCl (1m), after which pre-
cipitation was allowed to take place overnight. The residue was fil-
tered, washed with H2O, dried in vacuo, and concentrated in the
presence of toluene to give the acid 10c (1.24 g, 5.2 mmol, 56%).
MBHA-HMPB-Thr(tBu)-Ile-Ile-Boc (12): 4-Methylbenzhydrylamine
(MBHA) resin (5.0 g, 6.0 mmol) was coupled to 4-(4-hydroxymethyl-
3-methoxyphenoxy)-butyric acid (HMPB) linker (4.3 g, 18 mmol,
3 equiv) in the presence of BOP (8.0 g, 18 mmol, 3 equiv) and
DiPEA (6.3 mL, 36 mmol, 6 equiv) in NMP. After overnight shaking,
the resin was washed with NMP (3ꢁ) and DCM (3ꢁ). The coupling
was monitored for completion by the Kaiser test. The resulting
MBHA-HMPB resin (~6.0 mmol) was coevaporated with DCE (2ꢁ).
The resin was then condensed with Fmoc-Thr(tBu)-OH (7.2 g,
18 mmol, 3 equiv) in the presence of DIC (3.1 mL, 20 mmol,
3.3 equiv) and DMAP (0.11 g, 0.90 mmol, 15 mol%) in DCM for 2 h.
After the resin had been filtered and washed with DCM, the con-
densation cycle was repeated. The resin was then washed with
NMP (2ꢁ), DCM (2ꢁ), and ether (2ꢁ) and dried in vacuo overnight.
The loading of the resin was determined to be 0.43 mmolgꢀ1 by
spectrophotometric analysis. The obtained resin was washed with
DCM and subjected to two coupling cycles with Fmoc-Ile-OH and
Boc-Ile-OH, respectively, as follows: after deprotection with piperi-
dine in NMP (20%, 20 min), the resin was washed with NMP (2ꢁ)
and DCM (2ꢁ) and coupled to Fmoc-Ile-OH (5.3 g, 15 mmol,
2.5 equiv) or Boc-Ile-OH (3.5 g, 15 mmol, 2.5 equiv) in the presence
of BOP (6.6 g, 15 mmol, 2.5 equiv) and DiPEA (3.1 mL, 18 mmol,
3 equiv) in NMP. The reaction mixture was shaken overnight or for
5 h, respectively, followed by washing with NMP (3ꢁ) and DCM
(3ꢁ). Couplings were monitored for completion by the Kaiser test.
Washing with ether and drying in vacuo overnight yielded the fully
protected resin tripeptide 12.
2-((2E,4E)-Hexa-2,4-dienyloxy)acetic acid (10d): (2E,4E)-Hexa-2,4-
dien-1-ol (9, 3.0 g, 30 mmol) was added under argon to a suspen-
sion of NaH (60% in mineral oil, 2.5 g, 61 mmol, 2 equiv) in freshly
distilled THF. The reaction mixture was stirred for 30 min, followed
by addition of bromoacetic acid (4.2 g, 30 mmol, 1 equiv) and stir-
ring overnight under argon. The reaction was quenched with aque-
ous KOH solution (3m). The aqueous layer was then washed with
Et2O (2ꢁ), acidified with aqueous HCl solution (6m), and extracted
with CHCl3 (3ꢁ). The combined organic layers were dried over an-
hydrous MgSO4, filtered, and concentrated in vacuo. Purification by
column chromatography (DCM+1% AcOH!5% MeOH in DCM+
1% AcOH) yielded the acid 10d (3.4 g, 22 mmol, 73%). 1H NMR
(400 MHz, CDCl3): d=11.58 (s, 1H), 6.20 (dd, J=15.20, 10.45 Hz,
1H), 6.04 (ddd, J=14.73, 10.47, 1.34 Hz, 1H), 5.71 (qd, J=13.57,
6.71, 6.70, 6.70 Hz, 1H), 5.62–5.53 (m, 1H), 4.08 (t, J=3.20, 3.20 Hz,
4H), 1.73 ppm (d, J=7.04 Hz, 3H); 13C NMR (100 MHz, CDCl3): d=
175.58, 134.55, 130.60, 130.27, 124.61, 71.51, 65.80, 17.75 ppm.
3-(2-Furyl)propanoyl-OSu (11a): A solution of 3-(2-furyl)propanoic
acid (10a, 0.98 g, 7.0 mmol) in DCE/DMF was put under argon, and
HOSu (3.2 g, 28 mmol, 4.0 equiv) and EDC·HCl (5.3 g, 28 mmol,
4.0 equiv) were added. The reaction mixture was stirred under
argon overnight, after which EtOAc was added, and the mixture
was washed with aqueous HCl solution (1m, 2ꢁ). The organic layer
was dried over anhydrous MgSO4, filtered, and concentrated in
vacuo and the crude product was purified by column chromatog-
raphy (PetEt!40% EtOAc in PetEt), yielding the OSu-ester 11 a
Boc-Ile-Ile-Thr(tBu)-OH (13): MBHA-HMPB-Thr(tBu)-Ile-Ile-Boc resin
(12, ~6.0 mmol) was subjected to mild acidic cleavage with TFA in
DCM (1%, 10 min, 6ꢁ). The collected fractions were concentrated
in the presence of toluene to yield the crude tripeptide 13, which
was used without further purification. LC/MS analysis: Rf 8.58 min
(linear gradient 10!90% B in 15 min); m/z: 502.3 [M+H]+, 524.5
[M+Na]+, 1025.3 [2M+Na]+, 1521.0 [3M+H2O]+.
1
(1.2 g, 4.9 mmol, 71%). H NMR (200 MHz, CDCl3): d=7.38–7.28 (m,
1H), 6.29 (dd, J=3.18, 1.89 Hz, 1H), 6.11 (dd, J=3.20, 0.80 Hz, 1H),
1776
www.chembiochem.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2010, 11, 1769 – 1781

Labeling Endogenous Enzymatic Activities
Boc-Ile-Ile-Thr(tBu)-methyl ester (14): TMS diazomethane (2m in
hexane, 24 mL, 48 mmol, 8 equiv) was added in four equal portions
over 1.5 h to a solution of crude Boc-Ile-Ile-Thr(tBu)-OH (13, ~3.0 g,
~6.0 mmol) in MeOH/toluene (1:1, v/v, 25 mL). The reaction mixture
was then stirred for 2 h, before being concentrated in the presence
of toluene. Purification by column chromatography (DCM!0.5%
MeOH in DCM) yielded the partially purified product 14 (pure frac-
tion 0.55 g, 1.1 mmol) as a colorless foam. The impure fraction
(2.1 g, ~4.1 mmol) was dissolved in MeOH/toluene (1:1, v/v) and
TMS diazomethane (2m in diethyl ether, 4.1 mL, 8.2 mmol, 2 equiv)
was added. After 30 s, the reaction mixture was concentrated in
vacuo. Purification by column chromatography (DCM!0.5%
MeOH in DCM) yielded a second batch of the title compound 14
(dd, J=93.10, 5.02 Hz, 2H), 1.97–1.53 (m, 5H), 1.52 (s, 3H), 1.44 (s,
9H), 1.28 (s, 9H), 1.06 (d, J=6.44 Hz, 3H), 1.37–1.02 (m, 4H), 0.99–
0.82 ppm (m, 18H); 13C NMR (50 MHz, CDCl3): d=208.01, 171.74,
171.13, 169.52, 155.91, 79.24, 75.27, 66.48, 59.11, 59.00, 57.29,
56.77, 52.35, 50.62, 39.64, 37.27, 37.06, 28.21, 27.96, 25.30, 24.90,
24.60, 23.26, 21.20, 16.62, 16.38, 15.35, 15.20, 11.23, 11.04 ppm; LC/
MS analysis: Rf 11.44 min (linear gradient 10!90% B in 15 min);
m/z: 599.13 [MꢀtBu+H]+, 655.3 [M+H]+, 677.3 [M+Na]+, 1331.4
[2M+Na]+.
3-(2-Furyl)propanoyl-Ile-Ile-Thr(tBu)-leucinyl-(R)-2-methyloxirane
(1a): The tetrapeptide epoxyketone 16 (0.16 g, 0.25 mmol) was
treated with TFA/DCM (1:1, v/v) for 15 min, before being concen-
trated in the presence of toluene. The TFA salt of the deprotected
compound was dissolved in DCE/DMF and neutralized with DiPEA
(0.17 mL, 1.0 mmol, 4.0 equiv), followed by addition of a solution
of the OSu-ester 11 a (0.18 g, 0.75 mmol, 3.0 equiv) in DCE/DMF.
The reaction mixture was stirred under argon for 1.5 h. DCM was
then added, and the mixture was washed with H2O (1ꢁ). The
aqueous layer was extracted with EtOAc (1ꢁ), the organic layers
were combined, and MeOH was added until the solution became
clear. The organics were dried over anhydrous MgSO4, filtered, and
concentrated in vacuo. Purification by column chromatography
(DCM!4% MeOH in DCM) afforded the title compound 1a
1
(total yield 2.0 g, 3.8 mmol, 64% from MBHA-HMPB resin). H NMR
(400 MHz, CDCL3): d=6.51 (d, J=8.12 Hz, 1H), 6.43 (d, J=9.25 Hz,
2H), 4.48 (dd, J=9.09, 1.61 Hz, 1H), 4.40 (dd, J=8.56, 6.49 Hz, 1H),
4.24 (dq, J=6.21, 6.16, 6.16, 1.59 Hz, 1H), 3.70 (s, 3H), 3.98–3.88
(m, 1H), 2.07–1.97 (m, 2H), 1.92–1.81 (m, 4H), 1.44 (s, 9H), 1.14 (d,
J=6.28 Hz, 3H), 1.11 (s, 9H), 0.99–0.86 ppm (m, 12H); 13C NMR
(50 MHz, CDCl3): d=171.05, 170.86, 67.21, 57.83, 57.50, 57.46,
52.14, 37.86, 37.23, 37.22, 37.20, 37.03, 28.32, 28.29, 24.88, 24.79,
24.76, 20.92, 15.53, 15.08, 11.41, 11.31 ppm; LC/MS analysis: Rf
9.68 min (linear gradient 10!90% B in 15 min); m/z: 538.5
[M+Na]+, 1053.5 [2M+Na]+.
1
(0.13 g, 0.20 mmol, 82%) as a white solid. H NMR (200 MHz, CDCl3/
MeOD): d=8.34 (d, J=9.35 Hz, 1H), 7.87 (d, J=7.38 Hz, 1H), 7.79
(d, J=7.88 Hz, 1H), 7.26 (s, 1H), 7.15 (d, J=8.80 Hz, 1H), 6.24 (dd,
J=3.12, 1.88 Hz, 1H), 6.00 (dd, J=3.18, 0.71 Hz, 1H), 4.80–4.50 (m,
3H), 4.28–4.01 (m, 2H), 3.29 (d, J=4.77 Hz, 1H), 3.03–2.90 (m, 2H),
2.88 (d, J=4.86 Hz, 1H), 2.75–2.46 (m, 3H), 1.87–1.54 (m, 7H), 1.52
(s, 3H), 1.49–1.34 (m, 1H), 1.10 (d, J=6.20 Hz, 3H), 0.93–0.71 ppm
(m, 18H); LC/MS analysis: Rf 8.50 min (linear gradient 10!90% B
in 15 min); m/z: 621.3 [M+H]+, 1241.3 [2M+H]+; HRMS: calcd. for
[C32H52N4O8Na]+ 643.36774; found: 643.36751.
Boc-Ile-Ile-Thr(tBu)-hydrazide (15): Hydrazine monohydrate
(11.1 mL, 228 mmol, 60 equiv) was added to a solution of the fully
protected tripeptide methyl ester 14 (2.0 g, 3.8 mmol) in MeOH
and the reaction mixture was heated at reflux overnight, before
being concentrated in the presence of toluene. The white precipi-
tate was filtered and washed with MeOH to give the hydrazide 15
(0.88 g, 1.7 mmol, 45%). The filtrate was concentrated in the pres-
ence of toluene and recrystallized from toluene/MeOH to yield a
second batch of the product 15 (0.61 g, 1.2 mmol, 31%). 1H NMR
(400 MHz, CDCL3): d=8.50 (s, 1H), 7.79–7.71 (m, 1H), 7.54–7.45 (m,
2H), 6.03–5.95 (m, 2H), 4.47–4.35 (m, 1H), 4.28 (d, J=7.27 Hz, 1H),
4.08 (d, J=1.20 Hz, 1H), 3.94 (d, J=6.40 Hz, 1H), 1.97–1.72 (m, 4H),
1.63–1.49 (m, 2H), 1.45 (s, 9H), 1.21 (s, 9H), 1.08 (d, J=6.28 Hz,
3H), 0.92 ppm (dd, J=14.72, 7.62 Hz, 12H); 13C NMR (50 MHz,
CDCl3): d=172.90, 171.27, 169.84, 79.50, 74.62, 66.18, 58.82, 58.74,
57.90, 57.84, 57.74, 56.79, 36.38, 36.14, 27.64, 27.51, 24.32, 24.24,
18.04, 14.96, 14.85, 14.75, 10.36 ppm; LC/MS analysis: Rf 6.39 min
(linear gradient 10!90% B in 15 min); m/z: 516.1 [M+H]+, 460.1
[MꢀtBu+H]+.
(E)-Hepta-4,6-dienoyl-Ile-Ile-Thr(tBu)-leucinyl-(R)-2-methyloxirane
(1b): The same procedure as described above (for 1a) was used
with the OSu-ester 11 b (0.13 g, 0.60 mmol, 3.0 equiv) to yield the
title compound 1b (78 mg, 0.13 mmol, 64%). 1H NMR (200 MHz,
CDCl3): d=8.27 (d, J=8.08 Hz, 1H), 7.85 (d, J=7.86 Hz, 1H), 7.63
(d, J=6.91 Hz, 1H), 7.01 (d, J=8.92 Hz, 1H), 6.37–6.00 (m, 2H), 5.66
(td, J=14.52, 6.19, 6.19 Hz, 1H), 5.03 (ddd, J=13.25, 11.12, 1.58 Hz,
2H), 4.79–4.49 (m, 3H), 4.28–4.08 (m, 2H), 3.10 (dd, J=80.88,
4.92 Hz, 2H), 2.86–2.82 (m, 1H), 2.52–2.28 (m, 4H), 1.85–1.56 (m,
6H), 1.53 (s, 3H), 1.48–1.33 (m, 2H), 1.11 (d, J=6.45 Hz, 3H), 0.96–
0.76 ppm (m, 18H); LC/MS analysis: Rf 8.94 min (linear gradient
10!90% B in 15 min); m/z: 607.3 [M+H]+, 1213.3 [2M+H]+,
1819.1 [3M+H]+; HRMS: calcd. for [C32H54N4O7Na]+ 629.38847;
found: 629.38831.
Boc-Ile-Ile-Thr(tBu)-leucinyl-(R)-2-methyloxirane (16): A solution
of Boc-Ile-Ile-Thr(tBu)-hydrazide (15, 0.52 g, 1.0 mmol) in DMF/
EtOAc (1:1, v/v) was cooled under argon to a temperature of
ꢀ308C [N2 (l)+DCE]. After addition of HCl (4m in dioxane, 0.70 mL,
2.8 mmol, 2.8 equiv) and tBuONO (0.13 mL, 1.1 mmol, 1.1 equiv),
the reaction mixture was stirred under argon for 1 h. A solution of
leucinyl-(R)-2-methyloxirane TFA salt (17,[45] 1.1 mmol, 1.1 equiv)
and DiPEA (0.19 mL, 1.1 mmol, 1.1 equiv) in DMF was then added
to the acyl azide reaction mixture at ꢀ308C. After addition of more
DiPEA (0.66 mL, 3.8 mmol, 3.8 equiv), the reaction mixture was
stirred overnight under argon (ꢀ308C!RT). The mixture was then
extracted with EtOAc and washed with H2O (3ꢁ), and the com-
bined organic layers were dried over anhydrous MgSO4, filtered,
and concentrated in vacuo. Purification by column chromatogra-
phy (PetEt!25% EtOAc in PetEt) afforded the fully protected ep-
oxyketone 16 as white crystals (058 g, 0.89 mmol, 89%). 1H NMR
(200 MHz, CDCl3): d=7.64 (d, J=7.06 Hz, 1H), 6.85 (d, J=5.84 Hz,
1H), 6.46 (d, J=8.57 Hz, 1H), 5.07 (d, J=8.31 Hz, 1H), 4.54–4.38 (m,
1H), 4.37–4.25 (m, 2H), 4.20–4.06 (m, 1H), 3.99–3.87 (m, 1H), 3.13
2-(Anthracen-9-yl)acetyl-Ile-Ile-Thr(tBu)-leucinyl-(R)-2-methyloxir-
ane (1c): The same procedure as described above (for 1a) with
the OSu-ester 11 c (0.25 g, 0.75 mmol, 3.0 equiv) afforded the title
compound 1c (70 mg, 0.098 mmol, 39%). 1H NMR (200 MHz,
CDCl3/MeOD): d=8.455 (s, 1H), 8.18 (d, J=8.8 Hz, 2H), 8.03 (d, J=
7.7 Hz, 2H), 7.61–7.41 (m, 5H), 6.74 (d, J=8.0 Hz, 1H), 4.65 (s, 2H),
4.61–4.48 (m, 1H), 4.29–4.01 (m, 4H), 3.09 (dd, J=79.9, 4.94 Hz,
2H), 1.80–1.24 (m, 8H), 1.50 (s, 3H), 1.00–0.64 ppm (m, 22H); LC/
MS analysis: Rf 10.17 min (linear gradient 10!90% B in 15 min);
m/z: 717.3 [M+H]+, 1455.4 [2M+Na]+; HRMS: calcd. for
[C41H56N4O7Na]+ 739.40412; found: 739.40443.
2-(Hexa-2,4-dienyloxy)acetamido-Ile-Ile-Thr-leucinyl-2-methylox-
irane (1d): The same procedure as described above (for 1a) with
the OSu-ester 11 d (0.21 g, 0.81 mmol, 3.2 equiv) afforded the title
ChemBioChem 2010, 11, 1769 – 1781
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
1777

H. S. Overkleeft et al.
1
compound 1d (0.16 g, 0.25 mmol, quant.) as a white solid. H NMR
3.50 (m, 2H), 3.45–3.24 (m, 6H), 1.45 ppm (s, 9H); 13C NMR
(50 MHz, CDCl3): d=154.91, 77.46, 69.36, 69.12, 68.96, 49.50, 39.28,
27.27 ppm; LC/MS analysis: Rf 7.00 min (linear gradient 10!90% B
in 15 min); m/z: 274.8 [M+H]+.
(400 MHz, CDCl3): d=8.43 (d, J=8.99 Hz, 1H), 7.94 (d, J=7.41 Hz,
1H), 7.45 (d, J=9.08 Hz, 1H), 7.30 (s, 1H), 6.19 (dd, J=15.16,
10.43 Hz, 1H), 6.05 (dd, J=14.02, 11.42 Hz, 1H), 5.79–5.66 (m, 1H),
5.63–5.54 (m, 1H), 4.86–4.69 (m, 1H), 4.55 (td, J=9.77, 5.65,
5.65 Hz, 3H), 4.18–3.99 (m, 1H), 3.97 (s, 2H), 3.89 (d, J=15.42 Hz,
2H), 3.12 (dd, J=182.56, 4.90 Hz, 2H), 1.76 (d, J=6.73 Hz, 2H),
1.71–1.58 (m, 2H), 1.52 (s, 3H), 1.43–1.17 (m, 2H), 1.11 (d, J=
6.29 Hz, 3H), 1.08–0.94 (m, 2H), 0.90 (d, J=6.47 Hz, 6H), 0.87–
0.75 ppm (m, 12H); 13C NMR (50 MHz, CDCl3): d=208.20, 171.81,
171.21, 170.53, 169.80, 134.45, 130.80, 130.40, 125.02, 71.78, 68.65,
67.12, 59.17, 57.52, 56.86, 56.69, 52.35, 50.73, 39.13, 38.01, 37.92,
25.13, 25.05, 24.91, 23.21, 21.09, 17.98, 17.26, 16.74, 15.22, 15.20,
11.33, 11.25 ppm; HRMS: calcd. for [C33H56N4O8Na]+ 659.39904;
found: 659.39896.
tert-Butyl 2-(2-(2-(2,5-dioxopyrrol-1-yl)ethoxy)ethoxy)ethylcarba-
mate (25): The Boc-protected azide 23 (1.0 g, 3.6 mmol) was dis-
solved in THF and triphenylphosphine (1.2 g, 4.4 mmol, 1.2 equiv)
was added. The reaction mixture was stirred for 5 h, after which a
few drops of H2O were added. After the mixture had been stirred
for an additional 2 h, toluene was added, and the mixture was ex-
tracted with HCl solution (1m, 4ꢁ). The combined aqueous layers
were made basic with saturated aqueous NaHCO3 solution and
KOH solution (3m) and were extracted with DCM (3ꢁ) and EtOAc
(3ꢁ). The combined organics were dried over anhydrous MgSO4,
filtered, and concentrated in vacuo to give the crude partially pro-
tected amine 24 (0.65 g, 2.6 mmol, 72%); LC/MS analysis: Rf
3.86 min (linear gradient 10!90% B in 15 min); m/z: 249.0
[M+H]+, 496.9 [2M+H]+.
Methyl maleimido-N-carboxylate (19): A solution of maleimide
(18, 0.99 g, 10 mmol) and N-methylmorpholine (1.1 mL, 10 mmol,
1.0 equiv) in EtOAc was cooled to 08C. Methyl chloroformate
(0.80 mL, 10 mmol, 1.0 equiv) was added and the reaction mixture
was stirred at 08C for 2 h. The mixture was then washed with satu-
rated aqueous NaHCO3 solution (3ꢁ) and the aqueous layer was
extracted with EtOAc (1ꢁ). The combined organics were dried
over anhydrous MgSO4, filtered, and concentrated in vacuo to yield
the crude product 19 (0.80 g, 5.2 mmol, 52%), which was used
without further purification. 1H NMR (200 MHz, [D6]acetone): d=
7.05 (s, 2H), 3.89 ppm (s, 3H); 13C NMR (50 MHz, [D6]acetone): d=
166.93, 148.80, 136.27, 54.12 ppm; LC/MS analysis: Rf 1.29 min
(linear gradient 10!90% B in 15 min); m/z: 156.0 [M+H]+.
The crude compound 24 was then dissolved in saturated aqueous
NaHCO3 solution and cooled to 08C, and the activated malei-
mide 19 (0.52 g crude, ~3.4 mmol, 1.3 equiv) was added. The reac-
tion mixture was stirred at 08C for 30 min and then at room tem-
perature for 45 min. The mixture was extracted with chloroform
(3ꢁ) and the combined organics were dried over anhydrous
MgSO4, filtered, and concentrated in vacuo. Purification by column
chromatography (PetEt!70% EtOAc in PetEt) yielded the (Boc)-
amine-PEG-maleimide 25 (0.36 g, 1.1 mmol, 31% over two steps).
1H NMR (200 MHz, CDCl3): d=6.72 (s, 2H), 5.04 (s, 1H), 3.85–3.41
(m, 10H), 3.39–3.20 (m, 2H), 1.45 ppm (s, 9H); 13C NMR (50 MHz,
CDCl3): d=170.37, 155.66, 133.86, 78.70, 69.81, 69.51, 67.42, 40.00,
36.67, 28.06 ppm; LC/MS analysis: Rf 6.37 min (linear gradient 10!
90% B in 15 min); m/z: 328.9 [M+H]+, 351.1 [M+Na]+.
1,2-Bis(2-azidoethoxy)ethane (21): Triethyleneglycol (20, 0.30 g,
2.0 mmol) was dissolved in DCM and put under argon, after which
tosyl chloride (1.14 g, 6 mmol, 3 equiv), Et3N (0.83 mL, 6.0 mmol,
3.0 equiv), and N,N-dimethyl-4-aminopyridine (12 mg, 0.1 mmol,
5 mol%) were added. After 16 h, the reaction mixture was washed
with H2O and the organic layer was dried over MgSO4, filtered, and
concentrated in vacuo. NaN3 (0.26 g, 4.0 mmol, 2.0 equiv) and tet-
rabutylammonium iodide (37 mg, 0.1 mmol, 5 mol%) were added
to a solution of the resulting yellow oil in DMF. The reaction mix-
ture was stirred for 16 h at 808C before being washed with saturat-
ed aqueous NaHCO3. The organic layer was dried over MgSO4, fil-
tered, and concentrated to a yellow oil. Purification by column
chromatography (toluene!15% EtOAc in toluene) afforded the
bis-azide 21 as a colorless oil (0.31 g, 1.6 mmol, 78%). 1H NMR
(200 MHz, CDCl3): d=3.68 (m, 8H), 3.39 ppm (t, J=5.1 Hz, 4H);
13C NMR (50 MHz, CDCl3): d=70.3, 69.8, 50.3 ppm.
(N)-Bodipy(TMR)-2-(2-(2-(2,5-dioxopyrrol-1-yl)ethoxy)ethoxy)-
ethylamine (2): The Boc-protected compound 25 (65 mg,
0.20 mmol) was treated with TFA/DCM (1:1, v/v) for 10 min before
being concentrated in the presence of toluene. The TFA salt of the
deprotected compound was dissolved in DCE and neutralized with
DiPEA (0.20 mL, 1.2 mmol, 6.0 equiv), followed by addition of
BODIPY(TMR)-OSu[46] (99 mg, 0.20 mmol, 1.0 equiv). The reaction
mixture was stirred under argon overnight. DCE was added, and
the mixture was washed with H2O (1ꢁ). The aqueous layer was ex-
tracted with EtOAc (2ꢁ), the organic layers were combined, and
MeOH was added until the solution became clear. The organics
were dried over anhydrous MgSO4, filtered, and concentrated in
vacuo. Purification by column chromatography (toluene!30%
acetone in toluene) afforded the title compound 2 (41 mg,
2-(2-(2-Azidoethoxy)ethoxy)ethanamine (22): An aqueous HCl so-
lution (5%, 10 mL) was added to a cooled solution (08C) of 1,2-
bis(2-azidoethoxy)ethane (21, 2.0 g, 10 mmol) in toluene (10 mL),
after which triphenylphosphine (2.5 g, 9.5 mmol, 0.95 equiv) was
added. The reaction mixture was allowed to warm to RT and stirred
for 16 h, after which the aqueous layer was separated and concen-
trated in vacuo to yield the crude monoazide 22 (1.67 g, 7.9 mmol,
79%).
1
0.068 mmol, 34%) as a purple solid. H NMR (400 MHz, CDCl3): d=
7.89–7.83 (m, 2H), 7.07 (s, 1H), 6.99–6.95 (m, 2H), 6.94 (d, J=
4.07 Hz, 1H), 6.61 (s, 2H), 6.53 (d, J=4.04 Hz, 1H), 6.35 (t, J=4.93,
4.93 Hz, 1H), 3.85 (s, 3H), 3.68 (t, J=5.49, 5.49 Hz, 2H), 3.58 (t, J=
5.39, 5.39 Hz, 2H), 3.52–3.41 (m, 6H), 3.41–3.35 (m, 2H), 2.77 (t, J=
7.44, 7.44 Hz, 2H), 2.53 (s, 3H), 2.36 (t, J=7.45, 7.45 Hz, 2H),
2.21 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=171.82, 170.72,
160.28, 159.69, 139.88, 134.80, 134.07, 130.77, 130.62, 127.67,
125.45, 122.63, 118.16, 113.66, 70.07, 69.76, 69.74, 67.74, 55.21,
39.17, 37.09, 36.16, 20.05, 13.12, 9.54 ppm; LC/MS analysis: Rf
8.55 min (linear gradient 10!90% B in 15 min); m/z: 589.4 [MꢀF]+,
1216.8 [2M+H]+.
tert-Butyl 2-(2-(2-azidoethoxy)ethoxy)ethylcarbamate (23): Tri-
ethylamine (1.5 mL, 11 mmol, 2.0 equiv) and Boc2O (1.3 g,
5.9 mmol, 1.1 equiv) were added to a solution of crude 2-[2-(2-azi-
doethoxy)ethoxy]ethanamine HCl salt (22, 1.5 g, 5.4 mmol) in DCM.
The reaction mixture was stirred for 50 min before being concen-
trated in vacuo. Purification of the crude product by column chro-
matography (DCM!3% MeOH in DCM) gave the Boc-protected
compound 23 (pure yield 1.3 g, 4.9 mmol, 90%) as a colorless oil.
1H NMR (200 MHz, CDCl3): d=5.07 (s, 1H), 3.73–3.59 (m, 6H), 3.58–
Diethyl (2S,3S)-oxirane-2,3-dicarboxylate (27): (ꢀ)-Diethyl d-tar-
trate (26, 29 g, 0.14 mol) was cooled to 08C, after which a solution
of HBr in acetic acid (33%, 120 mL) was added dropwise over
1778
www.chembiochem.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2010, 11, 1769 – 1781

Labeling Endogenous Enzymatic Activities
45 min. After complete addition, the reaction mixture was stirred
at 08C for 15 min and then at RT overnight. Next, the mixture was
poured onto crushed ice/H2O (300 mL) and extracted with Et2O
(3ꢁ). The combined organics were washed with H2O (3ꢁ), dried
over anhydrous MgSO4, filtered, and concentrated in vacuo. Re-
maining solvents were concentrated in the presence of toluene.
The crude oil was dissolved in EtOH, and acetyl chloride (5.1 mL,
0.07 mol, 0.5 equiv) was added. The reaction mixture was stirred
under reflux for 3.5 h before being concentrated at a temperature
of 308C. The remaining yellowish oil was dissolved in Et2O
(175 mL), cooled to 08C, and put under argon. A solution of DBU
(21 mL, 0.14 mol, 1.0 equiv) in Et2O (90 mL) was added dropwise
over 100 min. The reaction mixture was then stirred at 08C for 1 h,
more DBU (2.1 mL, 14 mmol, 0.1 equiv) was added, and the reac-
tion mixture was stirred for an additional 2 h, followed by addition
of H2O. The mixture was washed with KHSO4 solution (1m) and
H2O and the organic layer was dried over anhydrous MgSO4, fil-
tered, and concentrated in vacuo. Purification by column chroma-
tography (PEtEt!15% EtOAc in PEtEt) yielded the title com-
pound 27 (15 g, 79 mmol, 56% over 3 steps). 1H NMR (400 MHz,
CDCl3): d=4.32–4.22 (m, 4H), 3.66 (s, 2H), 1.32 ppm (t, J=7.15,
7.15 Hz, 6H); 13C NMR (100 MHz, CDCl3): d=166.69, 62.14, 51.96,
13.96 ppm.
for 5 h and to Fmoc-Leu-OH (0.80 g, 2.3 mmol, 2.5 equiv) over-
night, respectively. Couplings were monitored for completion by
the Kaiser test. Washing with ether and drying in vacuo overnight
yielded the resin-bound compound 29.
(2S,3S)-3-(Ethoxycarbonyl)oxirane-2-carboxyl-Leu-Tyr-Ahx-
Lys·TFA (30): The resin-bound compound 29 (~0.90 mmol) was de-
protected in piperidine/NMP (1:4, v/v) for 30 min. The resin was
washed with NMP (2ꢁ) and DCM (3ꢁ) before being subjected to a
condensation cycle with the free acid 32 (0.36 g, 2.3 mmol,
2.5 equiv) in the presence of BOP (1.0 g, 2.3 mmol, 2.5 equiv) and
DiPEA (0.45 mL, 2.7 mmol, 3.0 equiv) in NMP; the reaction mixture
was shaken overnight, followed by washing with NMP (3ꢁ) and
DCM (3ꢁ). The condensation cycle was repeated, after which the
Kaiser test indicated complete coupling. Cleavage from the resin
was then accomplished by treatment with TFA/TIS/H2O (95:2.5:2.5,
v/v/v) for 2 h. After filtration and concentration in vacuo in the
presence of toluene, the residue was recrystallized first from ace-
tone/MeOH/EtOAc and then from MeOH/Et2O, yielding a 2:1 mix-
ture of the fully deprotected ester 30 and the free acid 31 (total
1
yield 0.73 g, 0.93 mmol, quant.) according to NMR analysis. H NMR
(400 MHz, MeOD): d=6.98 (d, J=7.87 Hz, 2H), 6.66 (d, J=7.76 Hz,
2H), 4.44 (t, J=7.23, 7.23 Hz, 1H), 4.37 (dd, J=7.59, 5.82 Hz, 1H),
4.30 (dd, J=7.96, 5.15 Hz, 1H), 4.26–4.16 (m, 1H), 3.79–3.61 (m,
1H), 3.61–3.46 (m, 1H), 3.17–3.07 (m, 2H), 3.06–2.98 (m, 2H), 2.98–
2.91 (m, 2H), 2.91–2.73 (m, 2H), 2.20 (t, J=6.87, 6.87 Hz, 2H), 1.66–
1.63 (m, 2H), 1.59–1.31 (m, 9H), 1.26 (t, J=6.99, 6.99 Hz, 3H), 1.23–
1.13 (m, 2H), 0.88 (d, J=5.77 Hz, 3H), 0.84 ppm (d, J=5.75 Hz,
3H); LC/MS analysis: Rf 4.97 min (linear gradient 10!90% B in
15 min); m/z: 677.4 [M+H]+, 1353.2 [2M+H]+.
(2S,3S)-3-(Ethoxycarbonyl)oxirane-2-carboxylic acid (28): A solu-
tion of compound 27 (14 g, 76 mmol) in absolute EtOH (200 mL)
was cooled to 08C and a solution of KOH (5.0 g, 76 mmol,
1.0 equiv) in absolute EtOH (100 mL) was added dropwise over
20 min. The reaction mixture was next stirred at 08C for 3 h and
then at RT for 2 h, before being concentrated in vacuo. H2O
(200 mL) was added to the residue, and washing was performed
with DCM (1ꢁ 30 mL). The aqueous layer was acidified with con-
centrated HCl (7.0 mL), NaCl (60 g) was added, and the mixture
was extracted with EtOAc (4ꢁ 200 mL). The combined organics
were dried over anhydrous MgSO4, filtered, and concentrated in
vacuo to give the title compound 28 (11 g, 66 mmol, 86%).
1H NMR (400 MHz, CDCl3): d=6.24 (brs, 1H), 4.33–4.20 (m, 2H),
3.74–3.61 (m, 2H), 1.32 ppm (t, J=7.10, 7.10 Hz, 3H); 13C NMR
(100 MHz, CDCl3): d=168.54, 166.92, 61.95, 51.49, 51.42,
13.38 ppm.
(2S,3S)-3-(Ethoxycarbonyl)oxirane-2-carboxyl-Leu-Tyr-Ahx-((N)-
(E)-hepta-4,6-dienoyl)Lys-H2N (32):
A solution of 30 (0.31 g,
0.39 mmol crude, non-hydrolyzed/hydrolyzed 2:1) in DCE/DMF
under argon was made basic (pH 8.5) with DiPEA (0.13 mL,
0.78 mmol, 2.0 equiv), after which a solution of the OSu-ester 11 b
(0.23 g, 1.0 mmol, 2.6 equiv) in DCE/DMF was added. After the re-
action mixture had been stirred overnight under argon, it was con-
centrated in vacuo. The portion of the residue that was soluble in
MeOH/acetone was purified by column chromatography (CHCl3!
10% MeOH in CHCl3), yielding the diene-modified title com-
pound 32 (0.16 g, 0.20 mmol, 76% from non-hydrolyzed starting
MBHA-Rink amide-Lys(Boc)-Ahx-Tyr(tBu)-Leu(Fmoc) (29): 4-Meth-
1
compound). H NMR (400 MHz, [D6]DMSO): d=8.54 (d, J=8.19 Hz,
ylbenzhydrylamine-functionalized
(MBHA-functionalized)
Rink
amide resin (3.2 g, 0.64 mmolgꢀ1, 2.1 mmol) was washed with
DCM and deprotected in piperidine/NMP (1:4, v/v) for 20 min. After
washing with NMP (2ꢁ) and DCM (2ꢁ), the resin was coupled to
Fmoc-Lys(Boc)-OH (2.4 g, 5.2 mmol, 2.5 equiv) in the presence of
BOP (2.3 g, 5.2 mmol, 2.5 equiv) and DiPEA (1.1 mL, 6.2 mmol,
3.0 equiv) in NMP; the reaction mixture was shaken overnight, fol-
lowed by washing with NMP (2ꢁ) and DCM (2ꢁ). The remaining
free amines were capped with acetic anhydride (0.98 mL, 10 mmol,
5.0 equiv) in the presence of DiPEA (3.6 mL, 21 mmol, 10 equiv) in
DCM for 30 min. The resin was then washed with ether and dried
in vacuo overnight, and the loading of the resin was determined
by spectrophotometric analysis to be 0.30 mmolgꢀ1 (3.7 g,
1.1 mmol, 53%). The obtained resin (3.0 g, 0.90 mmol) was subject-
ed to three cycles of Fmoc solid-phase synthesis as follows: after
deprotection in piperidine/NMP (1:4, v/v) for 30 min, the resin was
washed with NMP (2ꢁ) and DCM (3ꢁ) and coupled to e-Ahx-Fmoc
(0.80 g, 2.3 mmol, 2.5 equiv) in the presence of BOP (1.0 g,
2.3 mmol, 2.5 equiv) and DiPEA (0.45 mL, 2.7 mmol, 3.0 equiv) in
NMP for 3 days, followed by washing with NMP (3ꢁ) and DCM
(3ꢁ). The second and third cycles were performed in the same
way, with coupling to Fmoc-Tyr(tBu)-OH (1.0 g, 2.3 mmol, 2.5 equiv)
1H), 8.11 (d, J=8.24 Hz, 1H), 7.82–7.74 (m, 2H), 7.30 (s, 1H), 6.96
(d, J=8.40 Hz, 2H), 6.61 (d, J=8.32 Hz, 2H), 6.28 (td, J=17.03,
10.26, 10.26 Hz, 1H), 6.04 (dd, J=15.16, 10.53 Hz, 1H), 5.74–5.64
(m, 1H), 5.02 (dd, J=50.10, 13.54 Hz, 2H), 4.37–4.28 (m, 2H), 4.23–
4.08 (m, 3H), 3.73–3.69 (m, 1H), 3.62–3.57 (m, 1H), 3.07–2.88 (m,
4H), 2.82 (dd, J=13.69, 5.57 Hz, 1H), 2.67 (dd, J=13.64, 8.85 Hz,
1H), 2.26 (dd, J=14.23, 7.06 Hz, 2H), 2.17–2.05 (m, 4H), 1.59 (td,
J=9.47, 6.89, 6.89 Hz, 1H), 1.54–1.26 (m, 12H), 1.23 (t, J=7.10,
7.10 Hz, 3H), 1.20–1.12 (m, 2H), 0.83 ppm (dd, J=14.79, 6.46 Hz,
6H).
In vitro competition assay versus MV151: EL-4 cells were cultured
on Dulbecco’s modified Eagle’s medium supplemented with fetal
calf serum (10%), penicillin (10 unitsmLꢀ1), and streptomycin
(10 mgmLꢀ1) in a CO2 (5%) humidified incubator at 378C. Cells
were harvested, washed with PBS (2ꢁ), and lysed in digitonin lysis
buffer [Tris (pH 7.5, 50 mm), sucrose (250 mm), MgCl2 (5 mm), di-
thiothreitol (DTT) (1 mm), ATP (2 mm), digitonin (0.025%)] for
30 min on ice, followed by sonication. After centrifugation at
16100g for 15 min at 48C, the supernatants containing the cyto-
solic fraction were collected and the protein concentration was de-
termined by Bradford assay. The lysates (10 mg total protein per ex-
ChemBioChem 2010, 11, 1769 – 1781
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
1779

H. S. Overkleeft et al.
periment) were exposed to the indicated concentrations of 1a, 1b,
1c, or 1d (1 mL 10ꢁ solution in DMSO) for 2 h at 378C in a total re-
action volume of 10 mL (buffer/DMSO 9:1, v/v), prior to incubation
with MV151 (1 mm) for 1 h at 378C in a total reaction volume of
11 mL (buffer/DMSO 9:2, v/v). The reaction mixtures were heated to
558C for 20 min with 4ꢁ Laemmli’s sample buffer (4 mL) containing
2-mercaptoethanol and resolved by SDS-PAGE (12.5%). In-gel visu-
alization of the fluorescent labeling was performed directly in the
wet gel slabs with use of Cy3/Tamra settings.
digitonin (0.013%)] for 30 min on ice followed by sonication. After
centrifugation at 16100g for 15 min at 48C, the supernatants con-
taining the cytosolic fraction were collected and the protein con-
centration was determined by Bradford assay. The lysates (50 mg
total protein per experiment) were exposed to the indicated con-
centrations of compound 32 (1 mL 10ꢁ solution in DMSO) for 1 h
at 378C in a total reaction volume of 10 mL (buffer/DMSO 9:1, v/v),
prior to incubation with N3-BODIPY(TMR)-DCG-04 (0.5 mm) for 1 h
at 378C in a total reaction volume of 11 mL (buffer/DMSO 9:2, v/v).
The reaction mixtures were boiled for 3 min with 4ꢁ Laemmli’s
sample buffer (4 mL) containing 2-mercaptoethanol and resolved
by SDS-PAGE (12.5%). In-gel visualization of the fluorescent label-
ing was performed directly in the wet gel slabs with use of Cy3/
Tamra settings.
General procedure for Diels–Alder-based proteasome labeling in
vitro: EL-4 cell lysates (50 mg total protein per experiment) were
exposed to 1a, 1b, 1c, or 1d (1 mm, 1 mL, 10 mm DMSO) for 2 h at
378C in the presence or in the absence of epoxomicin (100 mm,
1 mL, 1 mm in DMSO) in a total reaction volume of 11 mL (buffer/
DMSO 9:2, v/v). The proteins were then denatured for 15 min at
room temperature in urea (8m) and treated with DTT (5 mm) for
30 min at 558C, after which cysteine residues were capped with
DTNB (50 mm) for 3.5 h at RT. Next, the mixtures were subjected to
c/m precipitation and the proteins were taken up in Diels–Alder
buffer [NaH2PO4 (5 mm), NaCl (20 mm), MgCl2 (0.2 mm), pH 6.0]
containing urea (2m), followed by exposure to the indicated con-
centrations of the BODIPY(TMR)-maleimide 2 (1.7 mL 10ꢁ solution
in DMSO) overnight at 378C in a total reaction volume of 17 mL
(buffer/DMSO 15:2, v/v). Where indicated, reaction times were re-
duced to 1, 2 or 4 h. After c/m precipitation, the proteins were
taken up in Laemmli’s sample buffer (10 mL) containing 2-mercap-
toethanol, heated to 558C for 15 min, and resolved by SDS-PAGE
(12.5%). In-gel visualization of the fluorescent labeling was per-
formed directly in the wet gel slabs with use of Cy3/Tamra set-
tings.
General procedure for Diels–Alder-based cathepsin labeling in
vitro: RAW cell lysates (50 mg total protein per experiment) were
exposed to compound 32 (5 mm, 0.5 mL 100 mm DMSO) for 1 h at
378C in the presence or in the absence of DCG-04 (25 mm, 2.5 mL
100 mm in DMSO) in a total reaction volume of 12 mL (buffer/DMSO
9:3, v/v). The proteins were then subjected to denaturation, cys-
teine capping, c/m precipitation, exposure to the BODIPY(TMR)-
maleimide 2 overnight at 378C, and again c/m precipitation (see
proteasome labeling procedure). The labeled proteins were taken
up in Laemmli’s sample buffer (10 mL) containing 2-mercaptoetha-
nol, heated to 558C for 15 min, and resolved by SDS-PAGE (12.5%).
In-gel visualization of the fluorescent labeling was performed di-
rectly in the wet gel slabs with use of Cy3/Tamra settings.
Combined Diels–Alder and Staudinger ligation for labeling of
proteasome b-subunits in vitro: EL-4 cell lysates (50 mg total pro-
tein per experiment) were exposed to compound 3 (5 mm, 1 mL
50 mm DMSO) for 1 h at 378C in a total reaction volume of 10 mL
(buffer/DMSO 9:1, v/v), followed by exposure to compound 1b
(5 mm, 1 mL 50 mm DMSO) for 1 h at 378C. The biotin-phosphine 4
(100 mm) was then added (1.2 mL 1 mm in DMF) and the lysates
were again incubated for 1 h at 378C. The proteins were then de-
natured for 15 min at room temperature in urea (8m) and treated
with DTT (5 mm) for 30 min at 558C, after which cysteine residues
were capped with DTNB (50 mm) for 3.5 h at RT. The mixtures were
next subjected to c/m precipitation and the proteins were taken
up in Diels–Alder buffer [NaH2PO4 (5 mm), NaCl (20 mm), MgCl2
(0.2 mm), Cu(NO3)2 (10 mm), pH 6.0] containing urea (2m), followed
by exposure to the indicated concentrations of the BODIPY(TMR)-
maleimide 2 (1.7 mL 10ꢁ solution in DMSO) overnight at 378C in a
total reaction volume of 17 mL (buffer/DMSO 15:2, v/v). Following
c/m precipitation, the proteins were taken up in Laemmli’s sample
buffer (10 mL) containing 2-mercaptoethanol, heated to 558C for
15 min, and resolved by SDS-PAGE (12.5%). In-gel visualization of
the fluorescent labeling was performed directly in the wet gel
slabs with use of Cy3/Tamra settings, which was followed by West-
ern blotting. Blots were blocked with BSA (1%) in TBS-Tween 20
(0.1% Tween 20) for 60 min at RT, hybridized for 40 min with strep-
tavidin/HRP (1:10000) in blocking buffer, washed, and visualized
with the aid of an ECL+ Western Blotting detection kit (Amersham
Biosciences).
Diels–Alder-based proteasome labeling in situ: EL-4 cells were
cultured on Dulbecco’s modified Eagle’s medium supplemented
with fetal calf serum (10%), penicillin (10 unitsmLꢀ1), and strepto-
mycin (10 mgmLꢀ1) in a CO2 (5%) humidified incubator at 378C.
Some 1ꢁ 106 cells per experiment were grown overnight in
medium (5.5 mL), before being exposed to increasing concentra-
tions of 1b for 2 h at 378C in a total volume of 6 mL (medium con-
taining 0.25% DMSO). As a control, cells were exposed to MV151
(1 mm). The cells were then harvested, washed with PBS (2ꢁ) and
lysed in digitonin lysis buffer [Tris (pH 7.5, 50 mm), sucrose
(250 mm), MgCl2 (5 mm), DTT (1 mm), ATP (2 mm), digitonin
(0.025%)] for 30 min on ice followed by sonication. After centrifu-
gation at 16100g for 15 min at 48C, the supernatants containing
the cytosolic fraction were collected and the protein concentration
was determined by Bradford assay. In case of a competition experi-
ment, the lysates were exposed to MV151 and visualized as de-
scribed above. For Diels–Alder ligation, the lysates from cells treat-
ed with compound 1b (10 mm, 20 mg total protein per experiment)
were denatured, capped, and subjected to c/m precipitation as de-
scribed above, after which the proteins were taken up in Diels–
Alder buffer [15 mL; NaH2PO4 (5 mm), NaCl (20 mm), MgCl2
(0.2 mm), pH 6.0] containing urea (2m) and exposed to the indicat-
ed concentrations of the BODIPY(TMR)-maleimide 2 (1.7 mL 10ꢁ so-
lution in DMSO) overnight at 378C. After c/m precipitation, the
proteins were taken up in Laemmli’s sample buffer (10 mL) contain-
ing 2-mercaptoethanol, heated to 558C for 15 min, and resolved
by SDS-PAGE (12.5%). In-gel visualization of the fluorescent label-
ing was performed directly in the wet gel slabs with use of Cy3/
Tamra settings.
Acknowledgements
Financial support from the Netherlands Organisation for Scientif-
ic Research (NWO) and the Netherlands Genomics Initiative (NGI)
is gratefully acknowledged.
In vitro competition assay versus N3-BODIPY(TMR)-DCG-04: RAW
cell lysates were prepared by incubation of harvested RAW cells in
MES lysis buffer [MES (pH 5.5, 50 mm), NaCl (50 mm), DTT (5 mm),
1780
www.chembiochem.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2010, 11, 1769 – 1781

Labeling Endogenous Enzymatic Activities
[32] W. S. Dillmore, M. N. Yousaf, M. Mrksich, Langmuir 2004, 20, 7223–7231.
[33] W. S. Yeo, M. N. Yousaf, M. Mrksich, J. Am. Chem. Soc. 2003, 125, 14994–
14995.
Keywords: activity-based protein profiling · bioorthogonal
chemistry · cycloaddition · hydrolases · proteomics
[34] M. N. Yousaf, B. T. Houseman, M. Mrksich, Proc. Natl. Acad. Sci. USA
2001, 98, 5992–5996.
[1] W. P. Heal, E. W. Tate, Org. Biomol. Chem. 2010, 8, 731–738.
[2] L. K. Mahal, K. J. Yarema, C. R. Bertozzi, Science 1997, 276, 1125–1128.
[3] E. Saxon, C. R. Bertozzi, Science 2000, 287, 2007–2010.
[4] J. A. Prescher, D. H. Dube, C. R. Bertozzi, Nature 2004, 430, 873–877.
[5] V. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless, Angew. Chem.
2002, 114, 2708–2711; Angew. Chem. Int. Ed. 2002, 41, 2596–2599.
[6] Q. Wang, T. R. Chan, R. Hilgraf, V. V. Fokin, K. B. Sharpless, M. G. Finn, J.
Am. Chem. Soc. 2003, 125, 3192–3193.
[7] N. J. Agard, J. A. Prescher, C. R. Bertozzi, J. Am. Chem. Soc. 2004, 126,
15046–15047.
[8] J. M. Baskin, J. A. Prescher, S. T. Laughlin, N. J. Agard, P. V. Chang, I. A.
Miller, A. Lo, J. A. Codelli, C. R. Bertozzi, Proc. Natl. Acad. Sci. USA 2007,
104, 16793–16797.
[35] M. N. Yousaf, M. Mrksich, J. Am. Chem. Soc. 1999, 121, 4286–4287.
[36] V. Pozsgay, N. E. Vieira, A. Yergey, Org. Lett. 2002, 4, 3191–3194.
[37] A. Berkin, B. Coxon, V. Pozsgay, Chem. Eur. J. 2002, 8, 4424–4433.
[38] A. D. De Araffljo, J. M. Palomo, J. Cramer, M. Kçhn, H. Schrçder, R.
Wacker, C. Niemeyer, K. Alexandrov, H. Waldmann, Angew. Chem. 2005,
117, 300–302; Angew. Chem. Int. Ed. 2005, 44, 296–301.
[39] A. D. De Araffljo, J. M. Palomo, J. Cramer, O. Seitz, K. Alexandrov, H. Wald-
mann, Chem. Eur. J. 2006, 12, 6095–6109.
[40] M. L. Blackman, M. Royzen, J. M. Fox, J. Am. Chem. Soc. 2008, 130,
13518–13519.
[41] N. K. Devaraj, R. Weissleder, S. A. Hilderbrand, Bioconjugate Chem. 2008,
19, 2297–2299.
[42] N. K. Devaraj, R. Upadhyay, J. B. Haun, S. A. Hilderbrand, R. Weissleder,
Angew. Chem. 2009, 121, 7147–7150; Angew. Chem. Int. Ed. 2009, 48,
7013–7016.
[43] M. Verdoes, B. I. Florea, G. A. Van der Marel, H. S. Overkleeft, Eur. J. Org.
Chem. 2009, 3301–3313.
[44] M. Hanada, K. Sugawara, K. Kaneta, S. Toda, Y. Nishiyama, K. Tomita, H.
Yamamoto, M. Konishi, T. Oki, J. Antibiot. 1992, 45, 1746–1752.
[45] N. Sin, K. B. Kim, M. Elofsson, L. Meng, H. Auth, B. H. B. Kwok, C. M.
Crews, Bioorg. Med. Chem. Lett. 1999, 9, 2283–2288.
[46] M. Verdoes, B. I. Florea, V. Menendez-Benito, C. J. Maynard, M. D. Witte,
W. A. Van der Linden, A. M. C. H. Van den Nieuwendijk, T. Hofmann, C. R.
Berkers, F. W. Van Leeuwen, T. A. Groothuis, M. A. Leeuwenburgh, H.
Ovaa, J. J. Neefjes, D. V. Filippov, G. A. Van der Marel, N. P. Dantuma,
H. S. Overkleeft, Chem. Biol. 2006, 13, 1217–1226.
[47] D. Wessel, U. I. Flügge, Anal. Biochem. 1984, 138, 141–143.
[48] D. Greenbaum, K. F. Medzihradszky, A. Burlingame, M. Bogyo, Chem.
Biol. 2000, 7, 569–581.
[49] U. Hillaert, M. Verdoes, B. I. Florea, A. Saragliadis, K. L. Habets, J. Kuiper,
S. Van Calenbergh, F. Ossendorp, G. A. Van der Marel, C. Driessen, H. S.
Overkleeft, Angew. Chem. 2009, 121, 1657–1660; Angew. Chem. Int. Ed.
2009, 48, 1629–1632.
[50] A-M. Lennon-DumØnil, A. H. Bakker, R. Maehr, E. Fiebiger, H. S. Over-
kleeft, M. Rosemblatt, H. L. Ploegh, C. Lagaudri›re-Gesbert, J. Exp. Med.
2002, 196, 529–540.
[9] X. Ning, J. Guo, M. A. Wolfert, G. J. Boons, Angew. Chem. 2008, 120,
2285–2287; Angew. Chem. Int. Ed. 2008, 47, 2253–2255.
[10] M. F. Debets, S. S. Van Berkel, S. Schoffelen, F. P. J. T. Rutjes, J. C. M. Van
Hest, F. L. Van Delft, Chem. Commun. 2010, 46, 97–99.
[11] A. A. Poloukhtine, N. E. Mbua, M. A. Wolfert, G. J. Boons, V. V. Popik, J.
Am. Chem. Soc. 2009, 131, 15769–15776.
[12] D. J. Vocadlo, H. C. Hang, E. J. Kim, J. A. Hanover, C. R. Bertozzi, Proc.
Natl. Acad. Sci. USA 2003, 100, 9116–9121.
[13] Y. Kho, S. C. Kim, C. Jiang, D. Barma, S. W. Kwon, J. Cheng, J. Jaunbergs,
C. Weinbaum, F. Tamanoi, J. Falck, Y. Zhao, Proc. Natl. Acad. Sci. USA
2004, 101, 12479–12484.
[14] H. C. Hang, E. J. Geutjes, G. Grotenbreg, A. M. Pollington, M. J. Bijlmak-
ers, H. L. Ploegh, J. Am. Chem. Soc. 2007, 129, 2744–2745.
[15] H. Ban, J. Gavrilyuk, C. F. Barbas III, J. Am. Chem. Soc. 2010, 132, 1523–
1525.
[16] H. Ovaa, P. F. Van Swieten, B. M. Kessler, M. A. Leeuwenburgh, E. Fiebig-
er, A. M. C. H. Van den Nieuwendijk, P. J. Galardy, G. A. Van der Marel,
H. L. Ploegh, H. S. Overkleeft, Angew. Chem. 2003, 115, 3754–3757;
Angew. Chem. Int. Ed. 2003, 42, 3626–3629.
[17] P. F. Van Swieten, E. Samuel, R. O. Hernꢄndez, A. M. C. H. Van den Nieu-
wendijk, M. A. Leeuwenburgh, G. A. Van der Marel, B. M. Kessler, H. S.
Overkleeft, A. F. Kisselev, Bioorg. Med. Chem. Lett. 2007, 17, 3402–3405.
[18] A. E. Speers, G. C. Adam, B. F. Cravatt, J. Am. Chem. Soc. 2003, 125,
4686–4687.
[51] M. Britton, M. M. Lucas, S. L. Downey, M. Screen, A. A. Pletnev, M. Ver-
does, R. A. Tokhunts, O. Amir, A. L. Goddard, P. M. Pelphrey, D. L. Wright,
H. S. Overkleeft, A. F. Kisselev, Chem. Biol. 2009, 16, 1278–1289.
[52] M. Verdoes, B. I. Florea, U. Hillaert, L. I. Willems, W. A. Van der Linden, M.
Sae-Heng, D. V. Filippov, A. F. Kisselev, G. A. Van der Marel, H. S. Over-
kleeft, ChemBioChem 2008, 9, 1735–1738.
[19] B. Seelig, A. Jꢅschke, Tetrahedron Lett. 1997, 38, 7729–7732.
[20] K. W. Hill, J. Taunton-Rigby, J. D. Carter, E. Kropp, K. Vagle, W. Pieken,
D. P. C. McGee, G. M. Husar, M. Leuck, D. J. Anziano, D. P. Sebesta, J. Org.
Chem. 2001, 66, 5352–5358.
[21] D. Graham, A. Grondin, C. McHugh, L. Fruk, W. E. Smith, Tetrahedron
Lett. 2002, 43, 4785–4788.
[53] B. M. Kessler, D. Tortorella, M. Altun, A. F. Kisselev, E. Fiebinger, B. G. Hek-
king, H. L. Ploegh, H. S. Overkleeft, Chem. Biol. 2001, 8, 913–929.
[54] During the preparation of this manuscript two related studies on Diels–
Alder ligation for in situ and in vivo imaging of small molecules ap-
peared; see N. K. Devaraj, S. Hilderbrand, R. Upadhyay, R. Mazitschek, R.
Weissleder, Angew. Chem. Int. Ed. 2010, 49, 2869–2872; Angew. Chem.
2010, 122, 2931–2934. See also ref. [55].
[55] R. Rossin, P. R. Verkerk, S. M. Van den Bosch, R. C. M. Vulders, I. Verel, J.
Lub, M. S. Robillard, Angew. Chem. Int. Ed. 2010, 49, 3375–3378; Angew.
Chem. 2010, 122, 3447–3450.
[22] L. Fruk, A. Grondin, W. E. Smith, D. Graham, Chem. Commun. 2002,
2100–2101.
[23] R. Tona, R. Hꢅner, Chem. Commun. 2004, 1908–1909.
[24] R. Tona, R. Hꢅner, Bioconjugate Chem. 2005, 16, 837–842.
[25] V. Marchꢄn, S. Ortega, D. Pulido, E. Pedroso, A. Grandas, Nucleic Acids
Res. 2006, 34, e24.
[26] V. Borsenberger, S. Howorka, Nucleic Acids Res. 2009, 37, 1477–1485.
[27] G. M. Husar, D. J. Anziano, M. Leuck, D. P. Sebesta, Nucleosides Nucleo-
tides Nucleic Acids 2001, 20, 559–566.
[28] H. A. Latham-Timmons, A. Wolter, J. S. Roach, R. Giare, M. Leuck, Nucleo-
sides Nucleotides Nucleic Acids 2003, 22, 1495–1497.
[29] J. M. Hooker, E. W. Kovacs, M. B. Francis, J. Am. Chem. Soc. 2004, 126,
3718–3719.
[30] B. T. Houseman, M. Mrksich, Chem. Biol. 2002, 9, 443–454.
[31] B. T. Houseman, J. H. Huh, S. J. Kron, M. Mrksich, Nat. Biotechnol. 2002,
20, 270–274.
Received: May 11, 2010
Published online on July 7, 2010
ChemBioChem 2010, 11, 1769 – 1781
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
1781