Vinylboronic Acids as Efficient Bioorthogonal Reactants for Tetrazine Labeling in Living Cells

Article abstract of DOI:10.1021/acs.bioconjchem.7b00796

Bioorthogonal chemistry can be used for the selective modification of biomolecules without interfering with any other functionality present in the cell. The tetrazine ligation is very suitable as a bioorthogonal reaction because of its selectivity and hig

Full text of DOI:10.1021/acs.bioconjchem.7b00796

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Communication
Vinylboronic Acids as Efficient Bioorthogonal
Reactants for Tetrazine Labeling in Living Cells
Selma Eising, Nicole van der Linden, Fleur Kleinpenning, and Kimberly M. Bonger
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00796 • Publication Date (Web): 13 Feb 2018
Downloaded from http://pubs.acs.org on February 14, 2018
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Vinylboronic Acids as Efficient Bioorthogonal Reactants for Tetrazine Labeling in Living Cells.
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Selma Eising, Nicole G. A. van der Linden, Fleur Kleinpenning, Kimberly M. Bonger*
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Department of Biomolecular Chemistry, Institute for Molecules and Materials, Radboud University,
Heyendaalseweg 135, 6525 AJ Nijmegen, the Netherlands.
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Bioorthogonal chemistry can be used for the selective modification of biomolecules without interfering with
any other functionality present in the cell. The tetrazine ligation is very suitable as bioorthogonal reaction
because of its selectivity and high reaction rates with several alkenes and alkynes. Recently, we described
vinylboronic acids (VBAs) as novel hydrophilic bioorthogonal moieties that react efficiently with dipyridyl-s-
tetrazines and used them for protein modification in cell lysate. It is not clear, however, whether VBAs are
suitable for labeling experiments in living cells because of the possible coordination with, for example, vicinal
carbohydrate diols. Here, we evaluated VBAs as bioorthogonal reactants for labeling of proteins in living cells
using an irreversible inhibitor of the proteasome and compared the reactivity to that of an inhibitor containing
norbornene, a widely used reactant for the tetrazine ligation. No large differences were observed between the
VBA and norbornene probes in a two-step labeling approach with a cell-penetrable fluorescent tetrazine,
indicating that the VBA gives little or no side reactions with diols and can be used efficiently for protein labeling
in living cells.
In the last years, the development of reactions that are unaffected by any of the molecular
functionalities in a biological system has emerged as a major field of research in chemical biology.1–4 These
bioorthogonal reactions are used for tagging biomolecules with a high reaction rate by a two-step approach in
vitro and in vivo, without the need to attach these tags directly onto the biomolecule. The reactants should be
non-toxic to the cellular environment and should not influence the function of the labelled biomolecule of
interest, therefore a small hydrophilic reactant is often preferred. One of the most popular bioorthogonal
reactions is the inverse electron-demand Diels-Alder (iEDDA) reaction between electron-poor tetrazines5–8 and
electron-rich or strained alkenes or alkynes, e.g. trans-cyclooctene9,10, norbornene11, or cyclopropene12,13
.
Recently, we reported a new non-strained bioorthogonal reactant, vinylboronic acid (VBA), which reacts,
depending on its substituents, with second-order rate constants (k2) up to 27 M-1 s-1 with 3,6-dipyridyl-s-
tetrazines. These VBAs are one order of magnitude faster than the commonly used tetrazine reactant
norbornene (Figure 1A).14 We further showed that the VBA moiety is biocompatible with cellular components,
stable in cell lysate, and applicable for protein modification.
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Figure 1 – A) The reported tetrazine ligation with vinylboronic acids and norbornene, k2 values were determined in 5%
MeOH in PBS. B) Two step protein labeling protocol. Addition of an irreversible protein inhibitor containing a VBA is
followed by cycloaddition with a dipyridyl-s-tetrazine containing a fluorophore. C) The equilibria of carbohydrate diols with
a boronic acid.
Over the last years, boronic acids have been used in living cells for various applications.15 The concept
that phenylboronic acid moieties can form reversible complexes with 1,2- and 1,3-cis-diols in aqueous
environments has been exploited in the development of chemosensors for carbohydrates (Figure 1C).16 The
rate of this condensation reaction is dependent on the pKa of the phenylboronic acid, where electron-
withdrawing substituents on the phenyl ring lower the pKa and thereby favor binding to the diols.17,18 Despite
the potential condensation reaction with vicinal diols present in and on the cell, many boronic acid-containing
compounds have been successfully used inside living cells such as protease inhibitors19, sensors for reactive
oxygen species20 and reactants in bioconjugation reactions.21–23 It is not clear, however, if or to what extent the
carbohydrate rich cellular environment interferes with the cellular uptake and distribution of these boronic-
acid containing compounds or with molecules containing our bioorthogonal VBA moiety.
To investigate the uptake and bioorthogonality of the VBA moiety in living cells, we chose a two-step
labeling strategy of the proteasome using a VBA-modified irreversible inhibitor, followed by visualization using
a dipyridyl-s-tetrazine functionalized with a fluorophore (Figure 1B). The proteasome is a large multi-subunit
proteolytic complex that is mainly responsible for the degradation of proteins into small peptides by the
ubiquitin-proteasome pathway.24–26 The proteolytic core contains seven distinct α and β subunits, whereof the
β1, β2 and β5 subunit are responsible for the proteolytic activities of the proteasome. These proteolytic
subunits act as N-terminal threonine proteases, where the nucleophilic hydroxyl attacks the peptide carbonyl
causing cleavage of the peptide bond. Recent progression in the development of inhibitors of the proteasome
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or of only one of the proteolytic subunits, is extensively reviewed.27–29 The two-step labeling strategy of the
proteolytic proteasome subunits using irreversible inhibitors followed by a bioorthogonal reaction with a
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fluorescent molecule has been previously performed using the copper-catalyzed alkyne-azide cycloaddition30,31
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strain-promoted alkyne-azide cycloaddition32, the Staudinger ligation30,33–36 and the tetrazine ligation30 and
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thereby serves as a well-established model system for our studies.
The (6-aminohexanoyl)3-(leucinyl)3-vinyl-(methyl)-sulfone (Ahx3L3VS) moiety is found to give strong
inhibition of the proteasome, without being selective for any of the three catalytic subunits.37 The three
leucines are recognized by the proteasome and the vinylsulfone moiety reacts covalently and irreversibly with
the γ-hydroxyl of the N-terminal threonine of the catalytic proteasome subunits. The long linker of the scaffold
is built up by aminohexanoic acids that are beneficial for efficient binding to the proteasome37 and were
thought to be an advantage for the two-step labeling of the proteasome as the bioorthogonal moiety could be
placed far away from the active site. Here, we describe the synthesis and application of proteasome inhibitor
VBA-Ahx3L3VS 7, containing a protected vinylboronic acid moiety that hydrolyzes quickly in aqueous solution to
the free boronic acid14. We compared this compound to Nor-Ahx3L3VS 8, which contains the commonly used
bioorthogonal norbornene moiety.
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The proteasome inhibitors were synthesized using modified literature procedures as depicted in
scheme 1 (SI – experimental section). In short, Boc-Leu-OH 1 was first reduced to its corresponding aldehyde 3
via Weinreb amide 2. A stabilized Wittig reaction gave trans-VS 4 containing a small amount of cis-VS that was
removed using column chromatography. Next, a double round of Boc-deprotection and coupling of Boc-Leu-OH
yielded Boc-L3VS 5. Finally, Boc-deprotection and then coupling of Fmoc-Ahx3-OH, yielded Fmoc-protected
Ahx3L3VS 6.
Scheme 1 – Synthesis of proteasome inhibitors 7-9. i) N,O-dimethyl hydroxylamine, NMM, EDC, CH2Cl2, 5 h, 95%. ii) LiAlH4,
Et2O, 0 ᵒC, 15 min, 81%. iii) Diethyl(methylsulfonylmethyl)phosphonate, NaH, THF, 0 ᵒC, 1 h, 35%. iv) (a) 4 M HCl in dioxane,
CH2Cl2, 2 h, (b) Boc-Leu-OH, EDC, HOBt, Et3N, CH2Cl2, o/n, 94%. v) Same as iv, yielding 5 in 98%. vi) (a) 4 M HCl in dioxane,
CH2Cl2, 2 h, (b) Fmoc-Ahx3-OH, EDC, HOBt, Et3N, CH2Cl2, o/n, 54%. vii) (a) piperidine, DMF, 15 min (b) VBA-NHS 12, DIPEA,
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DMF, 1 h, yielding 7 in 73%. viii) same as vii, only (b) with norbornene-NHS, yielding endo/exo-8 in 94%. ix) (a) DBU, DMF, 7
min, (b) BODIPY-FL NHS ester, HOBt, DIPEA, DMF, 1.5 h, yielding 9 in 44%. x) Ethyl chloroacetate, K2CO3, DMF, 16 h, 98%. xi)
Trimethylsilylacetylene, CuI, PdCl2(PPh3)3, DIPEA, toluene, 30 ᵒC, 24 h, 99%. xii) (a) LiOH, THF/H2O 1:1, 2 h, (b) N-
hydroxysuccinimide, EDC, DMF, 16 h, 88%. xiii) Pinacolborane, Ru(CO)ClH(PPh3)3, toluene, 50 ᵒC, 16 h, 74%.
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Next, we prepared the NHS ester of VBA and norbornene to ensure efficient coupling to scaffold 6,
after deprotection of the Fmoc. For VBA-NHS 12, we used the alkoxy-substituted phenylvinylboronic acid, as
we previously found that this electron rich VBA gave the highest reaction rate with dipyridyl-s-tetrazine14. First,
etherification of 4-iodophenol using ethyl chloroacetate followed by a Sonagashira coupling yielded TMS-
protected alkyne 10 in excellent yield. Deprotection of the TMS group and coupling of the acid with N-
hydroxysuccinimide yielded alkyne-NHS 11. Final hydroboration of the alkyne with pinacolborane yielded NHS-
boronic ester 12. Norbornene-NHS was prepared in one step from its corresponding alcohol in good yield. The
VBA-NHS 12 and the norbornene-NHS were finally coupled to the free amine of Fmoc-deprotected 6, yielding
VBA-Ahx3L3VS 7 and Nor-Ahx3L3VS 8 in good yields. In addition, the fluorescent BODIPY-Ahx3L3VS 9 was
prepared by direct coupling of the commercially available BODIPY-FL NHS ester to deprotected 6.
Having VBA-Ahx3L3VS 7 and Nor-Ahx3L3VS 8 in hand, we initially evaluated the selectivity and potency
of the inhibitors in cell lysate (Figure 2A, B). First, we established that a concentration of 0.3 µM of BODIPY-
Ahx3L3VS 9 was essential for visualization of all proteasome subunits in cell lysate (Figure S1). Then, compounds
7 or 8 were incubated for one hour at various concentrations in the protein lysate, after which BODIPY 9 was
added to label all residual unbound proteasomal subunits (Figure 2B). Using this set-up, we observed that low
micromolar concentrations of inhibitor 7 and 8 were essential for full inhibition of all subunits. In addition, we
observed a slight selectivity of VBA 7 for β5 compared to the other subunits, whereas norbornene 8 was more
selective for β2 as well as β5.
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Figure 2 – A) Schematic figure of the competition assay of VBA 7 or norbornene 8 using BODIPY 9 in cell lysate and living
HeLa cells. B) SDS-PAGE analysis of the competition assay in cell lysate (10 µL of 1 mg/mL), which was first incubated with 7
or 8 (indicated concentration) for 1 h at 37 ᵒC and next with 9 (0.3 µM) for 1 h at 37 ᵒC. C) Competition assay in living HeLa
cells, which were first incubated with 7 or 8 (indicated concentration) for 3 h at 37 ᵒC, whereupon the cells were lysed and
the lysate (10 µL of 1 mg/mL) was incubated with 9 (0.3 µM) for 1 h at 37 ᵒC. In-gel fluorescence (top) was measured at 488
nm followed by colloidal staining (bottom) as a loading control of the protein lysates.
We continued to evaluate the inhibition of the proteasome with the Ahx3L3VS probes 7 and 8 in living
cells. Here, performing a similar competition experiment as above, a 100× higher concentration of both 7 and 8
was essential to inhibit the proteasome subunits completely. Despite the moderate cell permeability of the
probes this indicates that, similarly to the norbornene handle, the VBA moiety did not hamper membrane
permeability of the inhibitor (Figure 2C). Elevated concentrations for complete proteasome inhibition and the
observed reduced labeling of the β1 subunit in living cells was also observed with BODIPY 9 (Figure S2) and
reported previously using a different Ahx3L3VS probe.37 Aside, significant cell death was visible when
norbornene 8 was used at concentrations > 100 µM, while VBA 7 did not show any toxicity up to 1 mM
concentration.
Next, we investigated whether we could visualize the proteasome subunits with a two-step labeling
strategy using VBA 7 or norbornene 8 followed by the tetrazine ligation with dipyridyl-s-tetrazine 13, containing
a BODIPY-FL fluorophore, in cell lysate as well as in living cells (Figure 3A). After inhibition of the subunits using
VBA 7 and norbornene 8 in cell lysate at concentrations that showed full subunit inhibition as established
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above (3 ꢀM), about 3 ꢀM of tetrazine 13 was necessary to give a complete reaction (Figure S3). In living cells,
after inhibition of the proteasome with 300 ꢀM VBA 7 and 100 ꢀM norbornene 8, a comparable concentration
of 10 ꢀM tetrazine 13 was necessary to visualize the subunits (Figure 3B and 3C). To our delight, similar labeling
intensities of the proteasome subunits were observed using the probes VBA 7 and norbornene 8 in lysate and
in living cells, as evidenced by SDS-PAGE gel.
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In addition, we evaluated the stability of the VBA and norbornene handles in living cells and the
efficiency of the tetrazine ligation by monitoring the protein labeling in the course of time. After inhibition of
the proteasome with VBA 7 and norbornene 8 (300 ꢀM and 100 ꢀM, respectively), the cells were lysed and the
subsequent tetrazine ligation was performed with tetrazine 13 for various amounts of time. Complete labeling
of the subunits was observed within 2-5 minutes, indicating that both functionalities were intact and the
reaction was very efficient (Figure S4).
Finally, we used the same two-step labeling protocol to visualize the proteasome in living HeLa cells by
confocal microscopy (Figure 3B and D). Again, similar intensities were observed using VBA 7 or norbornene 8
followed by addition of 3 µM of tetrazine-BODIPY 13, indicating successful labeling of the proteasome. For
comparison, the labeling pattern of BODIPY 9 was measured, which was similar to that observed for 7 and 8,
indicating that the VBA, norbornene and BODIPY labels did not significantly influence the cellular distribution of
the inhibitors (Figure S5). Performing the ligation with a higher concentration of tetrazine 13 using the same
microscopy settings resulted in higher labeling intensities, however, in this case also increased background
signal was observed (Figure S6).
Figure 3 – A) Structure of the cell-permeable tetrazine-BODIPY 13. B) Schematic figure of the two-step labeling of the
proteasome. C) SDS-PAGE fluorescence analysis of a concentration range of tetrazine 13 in the two-step labeling of the
proteasome subunits with VBA 7 (300 µM) or norbornene 8 (100 µM) in living cells. D) Confocal microscopy of the two-step
labeling of the proteasome. HeLa cells were first incubated with DMSO, VBA 7 (300 µM) or norbornene 8 (100 µM) for 3 h
at 37 ᵒC, after which tetrazine 13 (3 µM) was applied. Scale bar = 50 µm.
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In summary, we evaluated the use of the vinylboronic acid functionality for bioorthogonal labeling
with tetrazines in living cells. The synthesized proteasome inhibitor containing a vinylboronic acid was
successfully used for the two-step labeling of the proteasome in cell lysate and in living cells without showing
diminished labeling efficiency with a dipyridyl-s-tetrazine compared to the proteasome inhibitor functionalized
with a norbornene moiety. We have to emphasize that the potential condensation of the VBAs to vicinal diols
would not be visible on SDS-PAGE gel or on confocal microscopy, as the boronic acid is released after the
tetrazine ligation. However, if significant condensation would occur, a higher concentration of VBA 7 would be
essential to fully inhibit the proteasome subunits. Our results show that three times more VBA 7 compared to
norbornene 8 was needed for complete inhibition of all subunits in vitro as well as in living cells, indicating that
the vinylboronic acid handle does not significantly hamper cellular uptake of the probes into the cell.
The number of bioorthogonal reactions that are suitable for labeling inside living cells is limited,
because of the requirement of toxic reagents (e.g. copper(I)-catalyzed alkyne-azide cycloaddition) or possible
side reactions of the reactants (e.g. the thiol-ene or thiol-yne side reactions of free intracellular cysteines with
strained alkenes or alkynes). We have shown that the tetrazine ligation with vinylboronic acid is little or not
affected by possible side reactions that can occur in the cell with biomolecules such as carbohydrates, making
the reaction a valuable addition to the bioorthogonal toolbox.
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Associate Content
Supporting Information
Experimental details for the synthesis and proteasome inhibition in cell lysate and in living cells, full
spectroscopic data for all new compounds, and additional figures.
Author information
Corresponding Author
*E-mail: k.bonger@science.ru.nl
Notes
The authors declare no competing financial interest.
Acknowledgements
This work was financially support from the Netherlands Research Institute for Chemical Biology (NRSCB) and
the Institute of Molecules and Materials (IMM) of the Radboud University in Nijmegen. We thank Prof. Dr.
F.P.J.P. Rutjes for useful discussions and proofreading this manuscript.
References
(1) Shih, H.-W., Kamber, D. N., and Prescher, J. A. (2014) Building better bioorthogonal reactions. Curr. Opin. Chem. Biol. 21,
103–111.
(2) Shieh, P., and Bertozzi, C. R. (2014) Design Strategies for Bioorthogonal Smart Probes. Org. Biomol. Chem. 12, 9307–
9320.
(3) King, M., and Wagner, A. (2014) Developments in the field of bioorthogonal bond forming reactions - past and present
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trends. Bioconjug. Chem. 25, 825–839.
(4) Chen, X., and Wu, Y.-W. (2016) Selective chemical labeling of proteins. Org. Biomol. Chem. 14, 5417–5439.
(5) Knall, A.-C., and Slugovc, C. (2013) Inverse electron demand Diels-Alder (iEDDA)-initiated conjugation: a (high) potential
click chemistry scheme. Chem. Soc. Rev. 42, 5131–5142.
4
5
6
7
8
9
(6) Kozma, E., Demeter, O., and Kele, P. (2017) Bioorthogonal Fluorescent Labelling of Biopolymers via Inverse Electron
Demand Diels-Alder Reactions. ChemBioChem 18, 486–501.
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(7) Šečkutė, J., and Devaraj, N. K. (2013) Expanding room for tetrazine ligations in the in vivo chemistry toolbox. Curr. Opin.
Chem. Biol. 17, 761–767.
(8) Oliveira, B. L., Guo, Z., and Bernardes, G. J. L. (2017) Inverse electron demand Diels–Alder reactions in chemical biology.
Chem. Soc. Rev. 46, 4895–4950.
(9) Blackman, M. L., Royzen, M., and Fox, J. M. (2008) Tetrazine ligation: fast bioconjugation based on inverse-electron-
demand Diels-Alder reactivity. J. Am. Chem. Soc. 130, 13518–13519.
(10) Selvaraj, R., and Fox, J. M. (2013) trans-Cyclooctene - a stable, voracious dienophile for bioorthogonal labeling. Curr.
Opin. Chem. Biol. 17, 753–760.
(11) Devaraj, N. K., Weissleder, R., and Hilderbrand, S. A. (2008) Tetrazine-based cycloadditions: application to pretargeted
live cell imaging. Bioconjug. Chem. 19, 2297–2299.
(12) Patterson, D. M., Nazarova, L. A., Xie, B., Kamber, D. N., and Prescher, J. A. (2012) Functionalized cyclopropenes as
bioorthogonal chemical reporters. J. Am. Chem. Soc. 134, 18638–18643.
(13) Yang, J., Šečkutė, J., Cole, C. M., and Devaraj, N. K. (2012) Live-cell imaging of cyclopropene tags with fluorogenic
tetrazine cycloadditions. Angew. Chem. Int. Ed. 51, 7476–7479.
(14) Eising, S., Lelivelt, F., and Bonger, K. M. (2016) Vinylboronic Acids as Fast Reacting, Synthetically Accessible, and Stable
Bioorthogonal Reactants in the Carboni–Lindsey Reaction. Angew. Chem. Int. Ed. 55, 12243–12247.
(15) Hall, D. G. (2006) Structure, Properties, and Preparation of Boronic Acid Derivatives. Overview of Their Reactions and
Applications. Boronic Acids Prep. Appl. Org. Synth. Med.
(16) Wu, X., Chen, X.-X., and Jiang, Y.-B. (2017) Recent advances in boronic acid-based optical chemosensors. Analyst 142,
1403–1414.
(17) Hall, D. G. (2011) Boronic acids, Preparation and Applications in Organic Synthesis, Medicine and Materials second.
WILEY-VCH, Weinheim.
(18) Yan, J., Springsteen, G., Deeter, S., and Wang, B. (2004) The relationship among pKa, pH, and binding constants in the
interactions between boronic acids and diols—it is not as simple as it appears. Tetrahedron 60, 11205–11209.
(19) Diaz, D. B., and Yudin, A. K. (2017) The versatility of boron in biological target engagement. Nat. Chem. 9, 731–742.
(20) Lippert, A. R., van de Bittner, G. C., and Chang, C. J. (2011) Boronate oxidation as a bioorthogonal reaction approach for
studying the chemistry of hydrogen peroxide in living systems. Acc. Chem. Res. 44, 793–804.
(21) Chalker, J. M., Wood, C. S. C., and Davis, B. G. (2009) A convenient catalyst for aqueous and protein Suzuki-Miyaura
cross-coupling. J. Am. Chem. Soc. 131, 16346–16347.
(22) Spicer, C. D., Triemer, T., and Davis, B. G. (2012) Palladium-mediated cell-surface labeling. J. Am. Chem. Soc. 134, 800–
803.
(23) Akgun, B., Li, C., Hao, Y., Lambkin, G., Derda, R., and Hall, D. G. (2017) Synergic “click” Boronate/Thiosemicarbazone
System for Fast and Irreversible Bioorthogonal Conjugation in Live Cells. J. Am. Chem. Soc. 139, 14285–14291.
(24) Kleiger, G., and Mayor, T. (2014) Perilous journey: a tour of the ubiquitin–proteasome system. Trends Cell Biol. 24, 352–
359.
(25) Inobe, T., and Matouschek, A. (2014) Paradigms of protein degradation by the proteasome. Curr. Opin. Struct. Biol. 0,
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Page 14 of 15
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156–164.
(26) Schreiber, A., and Peter, M. (2014) Substrate recognition in selective autophagy and the ubiquitin-proteasome system.
Biochim. Biophys. Acta - Mol. Cell Res. 1843, 163–181.
4
5
6
7
8
(27) Kisselev, A. F., Van Der Linden, W. A., and Overkleeft, H. S. (2012) Proteasome inhibitors: An expanding army attacking
a unique target. Chem. Biol. 19, 99–115.
9
(28) Micale, N., Scarbaci, K., Troiano, V., Ettari, R., Grasso, S., and Zappalà, M. (2014) Peptide-Based Proteasome Inhibitors
in Anticancer Drug Design. Med. Res. Rev. 34, 1001–1069.
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15
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(29) Cromm, P. M., and Crews, C. M. (2017) The Proteasome in Modern Drug Discovery: Second Life of a Highly Valuable
Drug Target. ACS Cent. Sci. 3, 830–838.
(30) Willems, L. I., Li, N., Florea, B. I., Ruben, M., van der Marel, G. A., Overkleeft, H. S., Van Der Marel, G. A., Overkleeft, H.
S., van der Marel, G. A., Overkleeft, H. S., Van Der Marel, G. A., and Overkleeft, H. S. (2012) Triple Bioorthogonal Ligation
Strategy for Simultaneous Labeling of Multiple Enzymatic Activities. Angew. Chem Int. Ed. 51, 4431–4434.
(31) Kaschani, F., Verhelst, S. H. L., Van Swieten, P. F., Verdoes, M., Wong, C. S., Wang, Z., Kaiser, M., Overkleeft, H. S.,
Bogyo, M., and Van Der Hoorn, R. A. L. (2009) Minitags for small molecules: Detecting targets of reactive small molecules in
living plant tissues using “click chemistry.” Plant J. 57, 373–385.
(32) Van Der Linden, W. A., Li, N., Hoogendoorn, S., Ruben, M., Verdoes, M., Guo, J., Boons, G. J., Van Der Marel, G. A.,
Florea, B. I., and Overkleeft, H. S. (2012) Two-step bioorthogonal activity-based proteasome profiling using copper-free click
reagents: A comparative study. Bioorganic Med. Chem. 20, 662–666.
(33) Ovaa, H., Van Swieten, P. F., Kessler, B. M., Leeuwenburgh, M. A., Fiebiger, E., Van den Nieuwendijk, A. M. C. H.,
Galardy, P. J., Van der Marel, G. A., Ploegh, H. L., and Overkleeft, H. S. (2003) Chemistry in living cells: Detection of active
proteasomes by a two-step labeling strategy. Angew. Chem. Int. Ed. 42, 3626–3629.
(34) Geurink, P. P., Florea, B. I., Van der Marel, G. a, Kessler, B. M., and Overkleeft, H. S. (2010) Probing the proteasome
cavity in three steps: bio-orthogonal photo-reactive suicide substrates. Chem. Commun. 46, 9052–9054.
(35) van Swieten, P. F., Samuel, E., Hernández, R. O., van den Nieuwendijk, A. M. C. H., Leeuwenburgh, M. A., van der Marel,
G. A., Kessler, B. M., Overkleeft, H. S., and Kisselev, A. F. (2007) A cell-permeable inhibitor and activity-based probe for the
caspase-like activity of the proteasome. Bioorganic Med. Chem. Lett. 17, 3402–3405.
(36) Verdoes, M., Florea, B. I., Hillaert, U., Willems, L. I., van der Linden, W. A., Sae-Heng, M., Filippov, D. V., Kisselev, A. F.,
van der Marel, G. A., and Overkleeft, H. S. (2008) Azido-BODIPY acid reveals quantitative staudinger-bertozzi ligation in two-
step activity-based proteasome profiling. ChemBioChem 9, 1735–1738.
(37) Kessler, B. M., Tortorella, D., Altun, M., Kisselev, A. F., Fiebiger, E., Hekking, B. G., Ploegh, H. L., and Overkleeft, H. S.
(2001) Extended peptide-based inhibitors efficiently target the proteasome and reveal overlapping specificities of the
catalytic β-subunits. Chem. Biol. 8, 913–929.
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