Triple bioorthogonal ligation strategy for simultaneous labeling of multiple enzymatic activities

Article abstract of DOI:10.1002/anie.201200923

Three at the same time: A ligation strategy combining tetrazine-norbornene cycloaddition, Staudinger-Bertozzi ligation, and copper(I)-catalyzed click reaction was used to label the three catalytic activities of the proteasome simultaneously in a single experiment. The orthogonality of the three ligation reactions enables selective monitoring of multiple targets at the same time in complex biological samples. Copyright

Full text of DOI:10.1002/anie.201200923

Angewandte
Chemie
DOI: 10.1002/anie.201200923
Activity-Based Profiling
Triple Bioorthogonal Ligation Strategy for Simultaneous Labeling of
Multiple Enzymatic Activities**
Lianne I. Willems, Nan Li, Bogdan I. Florea, Mark Ruben, Gijsbert A. van der Marel, and
Herman S. Overkleeft*
Bioorthogonal chemistry plays an important role in chemical
biology research by creating the means to carry out selective
chemical transformations in complex biological samples. A
ligation reaction classifies as being bioorthogonal when it can
be performed in a biological sample in a chemoselective
manner without any interference with the biological system.
Bioorthogonal reactions have been used in cell-surface
labeling of glycoproteins and studies of biological processes
that involve post-translational modifications.[1] Another area
of research that has benefited from bioorthogonal chemistry
is two-step activity-based protein profiling (ABPP),[2] where it
enables the temporal separation of a reporter group and
a chemical probe that is directed to the active site of an
enzyme (such a chemical probe is also called activity-based
probe, ABP). Two-step ABPP strategies are of particular
interest when the presence of a tag interferes with selectivity,
affinity, cell-permeability, or bioavailability of the probe. A
further advantage of tandem labeling strategies is the option
to use different reporter groups depending on the type of
experiment and the desired method of analysis while using
a single ABP.
Several bioorthogonal ligation strategies have been de-
scribed,[3] and continuing efforts are being made to develop
ligations that are more selective and efficient than existing
methods. At the same time the high complexity of biological
processes often requires the study of multiple targets
simultaneously, thereby creating a need for ligation reactions
that are orthogonal with respect to each other and can thus be
used concurrently in a single experiment. Over the past
decade, a number of tandem ligation strategies has been
described for use in bioconjugation.[4] The first report of
a tandem bioorthogonal ligation in complex biological
samples involved a Staudinger–Bertozzi ligation and Diels–
Alder cycloaddition procedure, which utilizes mutually
orthogonal reagents but suffers from the need to mask free
thiol groups prior to the ligation step to avoid nonspecific
labeling.[5] More recently, it was reported that a copper-free
azide–cyclooctyne cycloaddition can be used concurrently
with an inverse-electron-demand Diels–Alder reaction
between tetrazine and trans-cyclooctene for the simultaneous
labeling of two different receptors on cell surfaces, provided
that the proper reagents are carefully selected so that cross-
reactivity is minimized.[6] Herein, we describe a triple ligation
strategy employing the tetrazine ligation, Staudinger–Ber-
tozzi ligation, and copper(I)-catalyzed Huisgen [2+3] cyclo-
addition (“click” reaction)[7] for the selective and simulta-
neous labeling of three different enzymatic activities in
a single experiment (Scheme 1a).
Several examples of two-step ABPP strategies using click
chemistry[8] and Staudinger–Bertozzi ligation[9] have been
described previously. The tetrazine ligation, however, has thus
far not been used for this purpose. Therefore we set out to
develop a two-step ABPP strategy in which an ABP is
functionalized with norbornene as a ligation handle that can
react with a tetrazine reagent conjugated to a reporter group
to enable detection and analysis of labeled proteins. As
a model system for our studies we selected the 20S protea-
some, containing three catalytically active subunits (b1, b2,
and b5) that can be targeted by either broad-spectrum or
subunit-specific ABPs. We designed two proteasome ABPs
that are functionalized with norbornene as a ligation handle:
ABP 1 is derived from the pan-reactive proteasome inhibitor
epoxomicin,[10] and ABP 2 has a different scaffold based on
a b5-subunit-selective proteasome inhibitor[11] (Scheme 1b).
Furthermore, we chose to create a panel of three tetrazine
reagents functionalized with different tags, being Bodipy-
TMR (3a), BodipyFL (3b), and biotin (3c). Other reagents
used herein for two-step labeling of the proteasome by click
chemistry and Staudinger ligation are shown in Scheme 1c.
The synthesis of all reagents and competition experiments
confirming the ability of the ABPs to target all proteolytically
active proteasome b subunits (1, 4, 5) or only the b5 subunit
(2) in cell extracts and/or in living cells can be found in the
Supporting Information.
The applicability of the tetrazine ligation for two-step
labeling of endogenous proteasome activity was tested by
exposing human embryonic kidney (HEK) cell lysates to
norbornene-functionalized ABP 1 in a concentration that
results in complete proteasome binding followed by ligation
with one of the tetrazine reagents 3a–c for one hour at 378C.
Analysis of labeled proteins by SDS-PAGE using either
fluorescent readout or detection by streptavidin Western
blotting (Figure 1a and Figure S2 in the Supporting Informa-
tion) showed that ligation with all three tetrazine reagents
results in labeling of the three catalytically active proteasome
b subunits in a concentration-dependent manner.[12] In this
[*] L. I. Willems, N. Li, Dr. B. I. Florea, M. Ruben,
Prof. G. A. van der Marel, Prof. H. S. Overkleeft
Leiden Institute of Chemistry and Netherlands Proteomics Centre
Gorlaeus Laboratories
Einsteinweg 55, 2333 CC Leiden (The Netherlands)
E-mail: h.s.overkleeft@chem.leidenuniv.nl
[**] This research was financially supported by the Netherlands
Organisation for Scientific Research (NWO) and the Netherlands
Genomics Initiative (NGI).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201200923.
Angew. Chem. Int. Ed. 2012, 51, 4431 –4434
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4431

.
Angewandte
Communications
Scheme 1. a) Schematic overview of the triple ligation strategy for simultaneous labeling of multiple enzymatic activities involving tetrazine–
norbornene cycloaddition, Staudinger–Bertozzi ligation, and copper(I)-catalyzed click reaction. The triangle, rectangle, and circle depict different
enzyme-reactive probes and the stars correspond to different reporter groups (fluorescent or biotin). b) Structures of norbornene-functionalized
proteasome ABPs 1 and 2 and tetrazines with a fluorescent Bodipy tag (3a, 3b) or biotin (3c). c) Structures of alkyne (4)- and azide (5)-
functionalized proteasome ABPs, azide (6)- or alkyne (7)-derivatized fluorescent click reagents, biotin-phosphine 8 for Staudinger–Bertozzi
ligation, biotinylated dibenzocyclooctyne (DIBO) 9 for strain-promoted click reaction and azide-functionalized ABP 10, selective for the b1 subunit
of the proteasome.
experimental setup the minimal tetrazine concentration that
was needed to detect specific labeling was approximately 5 mm
(Figure S3 in the Supporting Information). The high selectiv-
ity and specificity of the ligation reaction is apparent from the
low degree of background labeling in the absence of the ABP
1 and from the absence of proteasome labeling when
enzymatic activity is inhibited by an excess of epoxomicin
or heat inactivation prior to the labeling procedure (Figure S6
in the Supporting Information). Optimization of the reaction
time and temperature revealed that, at 50 mm of tetrazine,
labeling can be achieved within 30 min and appears to be
complete within 1–2 h (Figure S4 in the Supporting Informa-
tion). The reaction can also be performed at room temper-
ature, but at higher temperature (508C) or a prolonged
reaction time (overnight) nonspecific labeling is increased.
We also tested the compatibility of the ligation reaction with
different pH values (between 4 and 8) and found no marked
effects on reaction efficiency or selectivity.
as low as 1 mm, thus demonstrating that the tetrazine probes
are cell-permeable and that the ligation reaction can be
performed successfully inside living cells. Furthermore, the
ligation proceeded very selectively with even less nonspecific
labeling than in cell lysates (see also Figure S2 in the
Supporting Information). Ligation with tetrazine 3c was less
efficient, presumably owing to a reduced cell permeability
caused by the biotin moiety.
In a next set of experiments, we set out to compare the
efficiency and selectivity of the tetrazine ligation in cell
extracts with three other commonly used ligation methods
(Figure 2 and Figure S5 in the Supporting Information). The
tetrazine ligation procedure showed similar results to Stau-
dinger–Bertozzi ligation using azide-functionalized ABP 5[14]
and biotin-phosphine 8[15] and to both copper(I)-catalyzed
click procedures. A remarkable difference can be seen
between the click reaction using azide-functionalized ABP 5
and alkyne-Bodipy 7,[14] which appears to proceed somewhat
more efficiently but also gives more background fluorescence,
and ligation using alkyne-ABP 4 and azido-Bodipy 6, which
shows much higher selectivity. In the case of strain-promoted
click reaction with DIBO-biotin 9[16] a very high amount of
background labeling was present; thus specific signals were
not detectable. This result confirms our previous finding that
Next, we examined the efficacy of the tetrazine ligation
inside living cells. HEK cell cultures were treated with ABP 1,
washed, and subsequently exposed to tetrazines 3a, 3b, or 3c
for one hour at 378C. After cell lysis the proteins were
analyzed by SDS-PAGE (Figure 1b). In situ tetrazine ligation
with 3a and 3b resulted in efficient labeling of the active
proteasome b subunits, with labeling visible at concentrations
4432
www.angewandte.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2012, 51, 4431 –4434

Angewandte
Chemie
Figure 1. Two-step profiling of proteasome activity by tetrazine ligation
in cell extracts and inside living cells. a) HEK cell extracts were
exposed to ABP 1 (1 mm) for one hour at 378C and then reacted with
the indicated concentrations of tetrazine reagents 3a–c for one hour at
378C. In control experiments tetrazine ligation was performed in the
absence of ABP 1 or after competition by an excess of epoxomicin
(“+ep”). Alternatively, extracts were labeled with fluorescent ABP
MV151[13] (1 mm) as a positive control (“C”). b) HEK cells were
exposed to ABP 1 (10 mm) for three hours at 378C, washed, and then
reacted with the indicated concentrations of tetrazines 3a–c for one
hour, after which the cells were washed and lysed. Proteins were
analyzed by SDS-PAGE and detected by in-gel fluorescent readout (3a,
3b) or streptavidin Western blotting (3c). Annotation of the protea-
some subunits is based on reported labeling profiles.[13]
Figure 2. Two-step profiling of proteasome activity by different ligation
strategies. HEK cell extracts were exposed for one hour at 378C to
proteasome ABP 1 (1 mm) and then reacted with the indicated concentra-
tions of tetrazine 3b or 3c for one hour at 378C. Alternatively, ABP 4 (1 mm)
was used for ligation to Bodipy-azide 6 or ABP 5 (1 mm) for ligation to
Bodipy-alkyne 7, biotin-phosphine 8, and DIBO-biotin 9. Copper-catalyzed
click reactions with 6 and 7 were performed in the presence of CuSO4
(1 mm), TCEP (1 mm), and TBTA (100 mm). Proteins were analyzed by SDS-
PAGE using detection by in-gel fluorescent readout (3b, 6, 7) or streptavidin
Western blotting (3c, 8, 9). TCEP=tris(2-carboxyethyl)phosphine, TBTA=
tris(benzyltriazolylmethyl)amine.
strain-promoted click chemistry is poorly compatible with cell
extracts.[17]
To investigate the orthogonality between tetrazine liga-
tion, Staudinger–Bertozzi ligation, and copper(I)-catalyzed
click reaction, we tested for potential cross-reactivity between
norbornene-, azide-, and alkyne-tagged proteasome ABPs (1,
4, and 5) and azide-, alkyne-, phosphine-, and tetrazine-
functionalized reagents (3b, 6, 7, and 8) in a gel-based assay
(Figure S6 in the Supporting Information). In this assay no
cross-reactivity could be detected, opening up the way to
perform all three ligation reactions simultaneously. However,
we found that tetrazine reagents are not compatible with click
conditions and give a very high degree of background
fluorescence owing to the presence of copper sulfate. As
a result, the tetrazine ligation and copper(I)-catalyzed click
reaction cannot be performed at the same time. Nevertheless
we designed a triple ligation experiment in which HEK cell
extracts were first treated with b5-selective norbornene-
functionalized ABP 2 and b1-selective azide-functionalized
ABP 10[18] and subsequently with alkyne-derivatized probe 4,
which then labels only the available proteasome b2 subunits.
Next the lysates were exposed to tetrazine-BodipyTMR 3a
together with biotin-phosphine 8 for one hour, excess
reagents was removed by buffer exchange, and click ligation
was performed for one hour with azido-BodipyFL 6, after
which the proteins were analyzed by SDS-PAGE (Figure 3
and Figure S7 in the Supporting Information). Using this
tandem labeling strategy we were able to selectively label the
three catalytic activities of the proteasome by the three
different ligation reactions in a single sample.
In conclusion, we have developed a two-step ABPP
strategy based on the tetrazine ligation and have shown that
this method can be used to label endogenous proteasome
activity with high selectivity in cell extracts and inside living
cells. We have demonstrated that the reagents used for the
tetrazine ligation are orthogonal to those used in Staudinger–
Bertozzi ligation and copper(I)-catalyzed click reactions and
have used these three ligation reactions for the simultaneous
labeling of multiple enzymatic activities in a single experi-
ment. Although tetrazines are not directly compatible with
copper-catalyzed click chemistry, this problem can easily be
overcome by a simple washing step between the two ligation
reactions. The triple ligation strategy will not only be useful
Angew. Chem. Int. Ed. 2012, 51, 4431 –4434
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
4433

.
Angewandte
Communications
Boltje, V. V. Popik, G.-J. Boons, J. Am. Chem. Soc. 2011, 133,
949; f) E. M. Sletten, C. R. Bertozzi, J. Am. Chem. Soc. 2011,
133, 17570; g) D. M. Beal, V. E. Albrow, G. Burslem, L. Hitchen,
C. Fernandes, C. Lapthorn, L. R. Roberts, M. D. Selby, L. H.
Jones, Org. Biomol. Chem. 2012, 10, 548.
[5] L. I. Willems, M. Verdoes, B. I. Florea, G. A. Van der Marel,
H. S. Overkleeft, ChemBioChem 2010, 11, 1769.
[6] M. R. Karver, R. Weissleder, S. A. Hilderbrand, Angew. Chem.
2012, 124, 944; Angew. Chem. Int. Ed. 2012, 51, 920.
[7] a) C. W. Tornøe, C. Christensen, M. Meldal, J. Org. Chem. 2002,
67, 3057; b) V. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B.
Sharpless, Angew. Chem. 2002, 114, 2708; Angew. Chem. Int. Ed.
2002, 41, 2596.
Figure 3. Triple ligation strategy using tetrazine ligation, Staudinger–
Bertozzi ligation, and copper(I)-catalyzed click reaction in a single
sample. HEK cell extracts were exposed to b5-selective ABP 2 (1 mm)
and b1-selective azide-functionalized ABP 10 (5 mm) for one hour at
378C and then to ABP 4 (1 mm) for one hour at 378C. Next the lysates
were reacted with tetrazine 3a (25 mm) and phosphine 8 (100 mm) for
one hour at 378C, followed by buffer exchange to click buffer (50 mm
Tris pH 7.5, 1 mm CuSO4, 1 mm TCEP, 100 mm TBTA) and reaction
with azide 6 (25 mm) for one hour at 378C. Proteins were then
analyzed by SDS-PAGE and detected by in-gel fluorescent readout (Cy2
(6) and Cy3 (3a) settings) and streptavidin Western blotting (8).
[8] A. E. Speers, G. C. Adam, B. F. Cravatt, J. Am. Chem. Soc. 2003,
125, 4686.
[9] H. Ovaa, P. F. Van Swieten, B. M. Kessler, M. A. Leeuwenburgh,
E. Fiebiger, 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; Angew. Chem. Int. Ed. 2003, 42, 3626.
[10] M. Hanada, K. Sugawara, K. Kaneta, S. Toda, Y. Nishiyama, K.
Tomita, H. Yamamoto, M. Konishi, T. Oki, J. Antibiot. 1992, 45,
1746.
[11] M. Verdoes, L. I. Willems, W. A. Van der Linden, B. A. Duiven-
voorden, G. A. Van der Marel, B. I. Florea, A. F. Kisselev, H. S.
Overkleeft, Org. Biomol. Chem. 2010, 8, 2719.
[12] It should be noted that proteasome subunits b1 and b5, which
have close to identical molecular weights, sometimes overlay on
an SDS-PAGE gel, especially when visualized by Western
blotting. However, by tuning the conditions of the gel (e.g.
running time and composition) they can be separated well.
[13] 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 Nieu-
wendijk, 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.
[14] M. Verdoes, U. Hillaert, B. I. Florea, M. Sae-Heng, M. D. P.
Risseeuw, D. V. Filippov, G. A. Van der Marel, H. S. Overkleeft,
Bioorg. Med. Chem. Lett. 2007, 17, 6169.
[15] 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. Overkleeft, ChemBioChem 2008, 9,
1735.
for application in ABPP but also for the study of other
biological processes such as post-translational modifications,
allowing the selective monitoring of multiple targets at the
same time. Moreover, the possibility to perform both
tetrazine and Staudinger–Bertozzi ligation inside living
cells[19,9] and in living animals[20] should also enable tandem
labeling procedures in vivo.
Received: February 2, 2012
Published online: March 21, 2012
Keywords: activity-based profiling · bioorthogonal reactions ·
.
click chemistry · cycloadditions · tetrazines
[1] a) E. M. Sletten, C. R. Bertozzi, Acc. Chem. Res. 2011, 44, 666;
b) N. K. Devaraj, R. Weissleder, Acc. Chem. Res. 2011, 44, 816;
c) J. T. Ngo, D. A. Tirell, Acc. Chem. Res. 2011, 44, 677; d) Z.
Hao, S. Hong, X, Chen, P. R. Chen, Acc. Chem. Res. 2011, 44,
742; e) W. P. Heal, E. W. Tate, Org. Biomol. Chem. 2010, 8, 731,
and references therein.
[16] X. Ning, J. Guo, M. A. Wolfert, G.-J. Boons, Angew. Chem. 2008,
120, 2285; Angew. Chem. Int. Ed. 2008, 47, 2253.
[17] W. A. Van der Linden, N. Li, S. Hoogendoorn, M. Ruben, M.
Verdoes, J. Guo, G. J. Boons, G. A. Van der Marel, B. I. Florea,
H. S. Overkleeft, Bioorg. Med. Chem. 2012, 20, 662.
[18] M. Britton, M. M. Lucas, S. L. Downey, M. Screen, A. A.
Pletnev, M. Verdoes, R. Tokhunts, O. Amir, A. L. Goddard,
P. M. Pelphrey, D. L. Wright, H. S. Overkleeft, A. F. Kisselev,
Chem. Biol. 2009, 16, 1278.
[19] N. K. Devaraj, S. Hilderbrand, R. Upadhyay, R. Mazitschek, R.
Weissleder, Angew. Chem. 2010, 122, 2931; Angew. Chem. Int.
Ed. 2010, 49, 2869.
[20] a) J. A. Prescher, D. H. Dube, C. R. Bertozzi, Nature 2004, 430,
873; b) R. Rossin, P. Renart Verkerk, S. M. Van den Bosch,
R. C. M. Vulders, I. Verel, J. Lub, M. S. Robillard, Angew. Chem.
2010, 122, 3447; Angew. Chem. Int. Ed. 2010, 49, 3375.
[21] See for a study which was carried out in a similar spirit and that
details the introduction of two or three individual noncanonical
amino acids in a ribosomally synthesized protein: S. Lepthien, L.
Merkel, N. Budisa, Angew. Chem. 2010, 122, 5576; Angew.
Chem. Int. Ed. 2010, 49, 5446.
[2] L. I. Willems, W. A. Van der Linden, N. Li, K.-Y. Li, N. Liu, S.
Hoogendoorn, G. A. Van der Marel, B. I. Florea, H. S. Over-
kleeft, Acc. Chem. Res. 2011, 44, 718, and references therein.
[3] a) E. Saxon, C. R. Bertozzi, Science 2000, 287, 2007; b) Q. Wang,
T. R. Chan, R. Hilgraf, V. V. Fokin, K. B. Sharpless, M. G. Finn, J.
Am. Chem. Soc. 2003, 125, 3192; c) N. J. Agard, J. A. Prescher,
C. R. Bertozzi, J. Am. Chem. Soc. 2004, 126, 15046; d) N. K.
Devaraj, R. Weissleder, S. A. Hilderbrand, Bioconjugate Chem.
2008, 19, 2297.
[4] a) V. Aucagne, D. A. Leigh, Org. Lett. 2006, 8, 4505; b) P. V.
Chang, J. A. Prescher, M. J. Hangauer, C. R. Bertozzi, J. Am.
Chem. Soc. 2007, 129, 8400; c) P. Kele, G. Mezo, D. Achatz, O. S.
Wolfbeis, Angew. Chem. 2009, 121, 350; Angew. Chem. Int. Ed.
2009, 48, 344; d) L. Nurmi, J. Lindqvist, R. Randev, J. Syrett,
D. M. Haddleton, Chem. Commun. 2009, 2727; e) B. C. Sanders,
F. Friscourt, P. A. Ledin, N. E. Mbua, S. Arumugam, J. Guo, T. J.
4434 www.angewandte.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2012, 51, 4431 –4434