Targeted pH-dependent fluorescent activity-based cathepsin probes

Article abstract of DOI:10.1039/c1cc12947c

Bifunctional, pH-activatable BODIPY dyes were developed and incorporated in mannose cluster-containing activity-based probes for cysteine proteases. Mannose receptor-dependent uptake of the probes in dendritic cells, followed by trafficking to acidic cellular compartments resulted in fluorescence as seen by live-cell imaging, and subsequent cathepsin inhibition.

Full text of DOI:10.1039/c1cc12947c

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Targeted pH-dependent fluorescent activity-based cathepsin probeswz
Sascha Hoogendoorn,a Kim L. Habets,b Solene Passemard,a Johan Kuiper,b
Gijsbert A. van der Marel,a Bogdan I. Floreaa and Herman S. Overkleeft*a
Received 19th May 2011, Accepted 29th June 2011
DOI: 10.1039/c1cc12947c
Bifunctional, pH-activatable BODIPY dyes were developed and
incorporated in mannose cluster-containing activity-based
probes for cysteine proteases. Mannose receptor-dependent
uptake of the probes in dendritic cells, followed by trafficking
to acidic cellular compartments resulted in fluorescence as seen
by live-cell imaging, and subsequent cathepsin inhibition.
by trafficking in the endocytic pathway towards the lysosomes.
Subsequently, multiple lysosomal cysteine proteases of the
cathepsin13 family are covalently and irreversibly addressed
by the ABP. We reasoned that incorporation of a pH-activatable
fluorophore in our construct would result in less background
because of selective fluorescence in the cellular compartments
of interest. Hence, we designed a set of bifunctional pH-sensitive
dyes that allowed orthogonal ligation to the mannose cluster
and DCG-04 amine (Fig. 1). We here report the development
of three different pH-activatable fluorescent activity-based
cathepsin probes that are selectively taken up by professional
antigen-presenting cells via the mannose receptor.
Fluorescent dyes are applied in many different areas of
chemical biology. Among these dyes, boron-dipyrromethene
(BODIPY) derivatives are frequently used due to their excellent
photochemical properties and relative stability under physio-
logical conditions.1,2 For example, covalent attachment of a
BODIPY dye to an activity-based probe (ABP) facilitates the
study of its target enzyme, by means of fluorescence scanning
and microscopy.3–7 Urano et al. recently developed a series of
acidic pH-activatable BODIPY dyes.8 Due to photoinduced
electron transfer (PeT) of the meso aniline substituent toward
the BODIPY fluorophore, fluorescence is quenched at neutral
or basic pH.9–11 Upon protonation of the aniline nitrogen,
fluorescence is restored.
We followed a modular synthetic approach, in which the
constructs are assembled from three building blocks in the two
final steps (Fig. 1A). The mannose cluster and cathepsin
inhibitor DCG-04 amine were synthesized as previously
reported.3,12 To obtain bifunctional pH-dependent BODIPY
dyes, the synthetic route of Urano and coworkers8 was
modified and extended, yielding azido-BODIPY-OSu inter-
mediates 3a–d (Scheme S1, ESIz). Condensation of BODIPYs
3a–d and DCG-04 amine followed by ligation to the mannose
cluster by copper(I) catalysed Huisgen 1,3-cycloaddition14,15
gave constructs 5a–d (Fig. 1A).
Depending on the choice of alkyl substituents on the aniline
nitrogen, the pKa and thus the pH-dependency of the fluoro-
phore can be tuned.8 A great advantage of these kind of
fluorophores is that they do not fluoresce when unprotonated,
making them convenient tools to study in-cell processes with
fluorescence microscopy. Since a (slightly) acidic pH is a
prerequisite for fluorescence, these fluorophores are ideally
suited for incorporation in probes that are transported to
acidic cellular compartments like lysosomes. In a previous
study, we have shown that lysosomal targeting of the epoxy-
succinate activity-based probe DCG-0412 can be accomplished
by attachment of a synthetic mannose cluster.3
To assess the fluorescence properties of the dyes, fluores-
cence-pH curves of probes 5a–d in a citric acid/phosphate
buffer system were measured (Fig. 2A and B). The resulting
curves for 5a–c (Fig. 2B) were shifted to the left as compared
to the curves reported by Urano et al.,8 indicative of a shift in
pKa values. Upon addition of a low amount (0.2%) of the
detergent sodium dodecyl sulphate (SDS) the values resembled
the expected pKa’s. Since the pH of the lysosomes in antigen
presenting cells such as macrophages is estimated to be
4.5–4.916 we expected that probe 5c, with pKa B 5.1 would
be the best candidate for imaging in living cells.
This mannose cluster binds to the mannose receptor, which
is predominantly present on professional antigen-presenting
cells such as dendritic cells and macrophages. Mannose
receptor-mediated internalization of the construct is followed
The ability of probes 5a–d to label lysosomal cysteine
proteases was first examined by incubation of mouse liver
lysates with various concentrations of the construct, followed
by resolution of the proteins on SDS-PAGE. We managed to
visualize the bands by overnight incubation of the gel in acidic
fixing solution (MeOH/H2O/acetic acid), thereby lowering the
pH to allow in-gel fluorescence scanning. Labeling is concen-
tration-dependent and the profile corresponds well with that
seen for our previously synthesized probe, with a MW-shift
of 3–4 kDa compared to BODIPY-DCG-043 (b-DCG-04;
Fig. S2, ESIz), consistent with the molecular weight of the
a Leiden Institute of Chemistry, Leiden University, P.O. Box 9052,
2300 RA Leiden, The Netherlands.
E-mail: h.s.overkleeft@chem.leidenuniv.nl; Tel: +31(0)715274342
b Dept of Biopharmaceutics LACDR, Leiden University,
P.O. Box 9052, 2300 RA Leiden, The Netherlands
w This article is part of the ChemComm ‘Glycochemistry and glyco-
biology’ web themed issue.
z Electronic supplementary information (ESI) available: Detailed
experimental procedures, spectral data for all new compounds and
supplemental figures. See DOI: 10.1039/c1cc12947c
c
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Chem. Commun., 2011, 47, 9363–9365 9363

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Next, the behaviour of probes 5a–d in living cells was
investigated. Immature mouse dendritic cells (DC’s) were
incubated with 1 mM of probe and studied with live-cell
fluorescence microscopy. Brightly fluorescent vesicles appeared
inside the cells after 30 min of incubation, with increasing
fluorescence upon prolonged exposure to the probe (Fig. S1A,
ESIz). In contrast to the ‘‘always on’’ construct 5d, no background
signal was detected for the pH-activatable probes 5a–c and
therefore cells could be imaged without any additional washing
steps (Fig. S1C, ESIz). In accordance with the observed pKa
values, the signal for the methyl–methyl (5a) and methyl–ethyl
(5b) probes was less strong and took longer to appear. This
clearly indicates that the probes were indeed trafficked to
increasingly acidic cellular compartments over time. Subsequent
cell lysis and SDS-PAGE analysis showed a labeling profile of
cysteine proteases identical to that found in lysates (Fig. 3C
and Fig. S2B, ESIz). Dendritic cells, being professional
antigen-presenting cells, can take up macromolecules from
the environment by macropinocytosis.17 Therefore, incubation
times for follow-up experiments were set to two hours, to
diminish the amount of probe internalized by means other
than mannose receptor-mediated internalization. Indeed,
under these conditions, pre-incubation of the cells with mannan,
a naturally occurring polymannoside that binds the mannose
receptor, abolished the uptake of the probes and subsequent
labeling of cathepsins (Fig. 3B/C, cond. ii). Pre-incubation of
the cells with non-fluorescent azido-DCG-04 did not prevent
the probes from entering the cell, as seen by live-cell imaging.
However, no labeling of cathepsins was seen on SDS-PAGE,
indicating that the probes were taken up by the mannose
receptor, trafficked to the lysosomes, but unable to bind to, the
already blocked, cathepsins (Fig. 3B/C, cond. iii). Interestingly,
in-cell fluorescence remained even after prolonged washing of
the cells (Fig. 3B, cond. vi). To further investigate this, cells
were lysed after washing and analyzed by SDS-PAGE. As
shown in Fig. 3C, cond. vi (lane 8), labeling of cathepsins
was largely restored. Also, more cathepsins were labeled
after overnight washing compared to immediate lysis in the
control experiment (Fig. 3C, cond. n, lane 7 and Fig. S2B,
ESIz). Because of their size, the probes were probably
retained in the cells, where they could bind any newly formed
cathepsins.
Fig. 1 Synthesized pH-activatable mannose cluster-BODIPY-DCG-04
constructs (A) and a schematic overview of their expected internalization
and fluorescence properties in mannose receptor expressing cells (B).
In a next experiment, the intracellular pH was increased by
addition of ammonium chloride to the medium.18 Ammonium
chloride passively diffuses into the cell and acts as a weak base,
thereby increasing the pH up to 6 in lysosomes in a reversible
manner.16,19 Almost no in-cell fluorescence was detectable
for both the ethyl-ethyl (5c) and control probe (5d) after
pre-incubation with 10 mM NH4Cl, even after extensive
washing at normal pH, indicating that the levels of internalized
probe were low (Fig. 3B, cond. iv, Fig. S3, ESIz). This is in
accordance with the postulation of Tietze et al., that an
increased cellular pH decreases receptor recycling, which leads
to lower levels of cell surface receptors.20 SDS-PAGE analysis
on the other hand, revealed that not all cathepsin activity was
gone, as seen by, albeit weak, labeling on gel (Fig. 3C, cond. iv,
lane 6 and Fig. S2, ESIz). This finding illustrates the efficiency
with which the probes were internalized and processed in the
endocytic pathway.
Fig. 2 pH-dependency of compounds 5a–c. (A) Normalized absorption
and emission spectra of pH-activatable compounds 5a–c and the
‘‘always on’’ control 5d. (B) Fluorescence measurements as a function
of pH in citrate/phosphate buffer with or without 0.2% SDS.
(C) Apparent pKa values (50% fluorescence) as determined by the
curves shown in (B).
probes (Fig. 3A, Fig. S2, ESIz).3 In a competition experiment
with b-DCG-04 it was shown that the constructs competed for
the same set of cysteine proteases.
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Fig. 3 Biological evaluation of the constructs 5c–d. (A, C) 12.5% SDS-PAGE analysis; fluorescence scanning images (upper) and coomassie
staining for total proteins (lower) are shown. DC: dual color prestained protein marker (see also Fig. S4, ESIz). (A) Incubation of mouse liver
lysate shows concentration dependent labeling of cathepsins (lanes 2–6). The probes compete for the same set of cathepsins as the known cathepsin
inhibitor b-DCG-04 (red bands, lanes 7–9) and the bands are shifted consistent with the molecular weight of the probes. (B) Confocal microscopy
images of DC’s incubated for 2 h with probes 5c or 5d, under different conditions (i–vi) show mannose receptor dependent uptake of the constructs.
Scale bar (white) corresponds to 10 mm. (C) Cell lysis and analysis of cells treated as in (B) shows inhibition of cathepsin labeling by azido-DCG-04.
In summary, in the present study we showed that pH-
activatable fluorophores can be incorporated in large mannose
cluster containing ABPs, thereby facilitating their imaging
without influencing their uptake or distribution in dendritic
cells. We believe that these kind of molecules might find use in
studies concerning antigen cross-presentation and the role of
cysteine proteases herein. Moreover, the ease of access to our
panel of bifunctional, tunable, pH-activatable dyes should
allow their incorporation in bioconjugates quite different
from those presented here. In this fashion, we foresee that
alternative receptors can be targeted to specifically deliver
cargo to endocytic compartments with the aim to interfere
with a variety of subcellular targets.
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This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 9363–9365 9365