Communications
DOI: 10.1002/anie.201001767
Proteomics
A Cleavable Linker Based on the Levulinoyl Ester for Activity-Based
Protein Profiling**
Paul P. Geurink, Bogdan I. Florea, Nan Li, Martin D. Witte, Joeri Verasdonck, Chi-Lin Kuo,
Gijs A. van der Marel, and Herman S. Overkleeft*
Activity-based protein profiling has recently come to the fore
as an attractive strategy to disclose enzyme activity in the
context of complex biological samples.[1] An activity-based
profiling probe (ABP) normally consists of a recognition
element targeted at a specific enzyme (family), and is
equipped with a reactive group through which the targeted
enzymes are covalently and irreversibly modified.[2,3] A third
essential element of ABPs is the affinity/identification tag,
which can be either incorporated into the ABP directly or
alternatively installed through a bioorthogonal process in so-
called two-step activity-based protein profiling strategies.[4]
Visualization/identification tags come in several types, includ-
ing fluorophores and immune epitope tags. The most popular
tag in this context is the biotin group. It is relatively small,
easy to manipulate, and has strong binding affinity to
streptavidin. As a consequence a range of biochemical tools
have become available over the decades for either the
visualization of biotinylated biomolecules or their pull-down.
The strong affinity of streptavidin for biotin makes the
release of biotinylated proteins captured by, for example,
streptavidin-coated beads difficult. Most effective procedures
rely on relatively harsh conditions, such as boiling of the
sample in denaturing conditions and often in the presence of
unmodified biotin. As a consequence, next to (denatured)
streptavidin, the eluted protein pool is often contaminated
with endogenously biotinylated biomolecules.
The introduction of a specific linker between biotin and
the ABP reactive group, which can be cleaved chemoselec-
tively so that only the ABP bound material is released, is an
Figure 1. a) Schematic representation of an activity-based enrichment
experiment using the cleavable linker based on a levulinoyl ester. After
attractive strategy to circumvent this problem (Figure 1a).
Examples of such cleavable linkers (CL) are the disulfide
labeling of the target enzyme and pull-down the linker is (chemo)-
linkage,[5] enzymatically (Tev) cleavable,[6] acid-cleavable,[7]
selectively cleaved with hydrazine. b) Structure of target proteasome
probe 1 containing the cleavable linker based on a levulinoyl ester.
diazobenzene-derived (cleavable with Na2S2O4),[8] and hydra-
zone-based linkers.[9] An ideal cleavable linker is stable
towards the various conditions (acidic, basic, reductive,
including generally applied buffer systems) to which the
biological sample may be exposed, depending on the nature
of the experiment, and can also withstand the reactive
(nucleophilic) species that are present in a cell extract. At
the same time, it is susceptible to mild cleavage conditions.
With this reasoning in mind our attention was focused on the
levulinoyl ester, a versatile protecting group often applied in
synthetic organic chemistry. It is acid stable and can be
removed selectively with respect to other esters by treatment
with hydrazine. A shortcoming is its intrinsic base-lability and
we reasoned that this drawback can be rectified by choosing
the alcohol with which the levulinolate group is condensed
such that it is both electron-rich (to reduce its leaving-group
properties) and sterically congested (to minimize intermo-
lecular nucleophilic attack). Altogether, we came to the
design of the epoxomicin-based,[10] levulinolate-modified
[*] P. P. Geurink, Dr. B. I. Florea, N. Li, Dr. M. D. Witte, J. Verasdonck,
C.-L. Kuo, Prof. Dr. G. A. van der Marel, Prof. Dr. H. S. Overkleeft
Leiden Institute of Chemistry and
The Netherlands Proteomics Center, Leiden University
Einsteinweg 55, 2300 RA, Leiden (The Netherlands)
Fax: (+31)71-527-4307
E-mail: h.s.overkleeft@chem.leidenuniv.nl
[**] Financial support for this work was provided by the Netherlands
Organisation for Scientific Research (NWO) and the Netherlands
Genomics Initiative (NGI).
Supporting information for this article is available on the WWW
6802
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2010, 49, 6802 –6805