Synthesis and Evaluation of Ubiquitin–Dioxetane
Conjugate as a Chemiluminescent Probe for Monitoring Deubiquitinase
Activity
The removal of ubiquitin (Ub) from a modified protein or Ub chain is a process that occurs regularly by the ubiquitin–proteasome system. This process is known to be mediated by various deubiquitinating enzymes (DUBs) in order to control the protein’s half-life and its expression levels among many other signaling processes. Since the function of DUBs is also involved in numerous human diseases, such as cancer, there is an obvious need for an effective diagnostic probe that can monitor the activity of these enzymes. We have developed the first chemiluminescence probe for detection of DUBs activity. The probe was prepared by conjugation of the chemically synthesized C-terminally activated Ub(1-75) with a Gly-enolether precursor. Subsequent oxidation, under aqueous conditions, of the enolether conjuagate with singlet-oxygen furnished the dioxetane probe Ub-CL. This synthesis provides the first example of a dioxetane–luminophore protein conjugate. The probe’s ability to detect deubiquitinating activity was successfully validated with three different DUBs. In order to demonstrate the advantage of our new probe, comparison measurements for detection of DUB UCH-L3 activity were performed between the chemiluminescent probe Ub-CL and the well-known Ub-AMC probe. The obtained data showed significantly higher S/N, for probe Ub-CL (>93-fold) in comparison to that observed for Ub-AMC (1.5-fold). We anticipate that the successful design and synthesis of the turn-ON protein–dioxetane conjugate probe, demonstrated in this work, will provide the insight and motivation for preparation of other relevant protein–dioxetane conjugates.
<!>Author Contributions<!>
<p>Ubiquitination is a reversible
post-translational modification that involves the covalent attachment
of ubiquitin (Ub) monomer or Ub chains to a target protein.1 This process regulates the degradation of cellular
proteins by the Ub-proteasome system (UPS) and controls a protein's
half-life, therefore affecting numerous signaling pathways.2,3 While the conjugation of Ub to a target protein is performed by
the ubiquitination enzymes (E1, E2, and E3), the removal of Ub from
a ubiquitinated protein is mediated by a family of deubiquitinating
enzymes (DUBs).4−6 Since the function of DUBs is also involved in many
human diseases, there is an obvious need for an effective diagnostic
probe that can monitor the activity of these enzymes.7,8 Such a probe is ordinarily used to screen small molecule inhibitors
against DUBs for studying DUB activities and drug development.9 Indeed, optical molecular probes for detection
of DUB activity were developed years ago.10−13 The detection mode of these probes
is usually based on fluorescence, where 7-amino-4-methylcoumarin (AMC)
is used as a fluorescent dye.14−16 Such a ubiquitin–AMC conjugate
exhibits a typical turn-ON fluorescence response, following the DUB-mediated
hydrolysis.</p><p>Chemiluminescence modality has an inherent advantage
over fluorescence,
since irradiation by an external light source is not required.17,18 As a result, there is no interference by autofluorescence, and the
obtained background signal is extremely low.19 Among the known chemiluminescent luminophores, the triggerable phenoxy-dioxetanes,
discovered by Paul Schaap in 1987, are commonly used for chemiluminescent
probe design.20,21 Four years ago, a major breakthrough
was achieved by our group, with the development of new-generation
phenoxy-dioxetane luminophores.22,23 These new dioxetane
chemiluminescent luminophores exhibit up to 3000-fold enhancement
in light-emission quantum yield (Î¦CL) under physiological conditions.24,25 Our group and others have utilized these dioxetane luminophores
to prepare chemiluminescent probes for the detection and imaging of
various enzymes and chemical analytes.26−28</p><p>The most effective
chemiluminescent probes, in terms of sensitivity
and signal-to-noise ratio, were obtained by masking the phenolic group
of the luminophore with peptide substrates through a 4-aminobenzyl
alcohol self-immolative linker.29,30 Such probes produce
extremely low background signal, due to their high stability toward
spontaneous hydrolysis.31 The enzymatic
responsive group used to mask the phenoxy-dioxetane luminophores were
all composed of small molecules or short peptides.32−34 A responsive
group based on a full protein substrate, attached to the dioxetane
luminophore, has never been demonstrated. Here, we report the design,
synthesis, and evaluation of a new chemiluminescent probe, based on
a ubiquitin–dioxetane conjugate, for efficient detection of
DUB activity.</p><p>The general molecular structure and chemiexcitation
disassembly
pathway of the DUB chemiluminescent probe Ub-CL is presented
in Figure 1. Probe Ub-CL is composed of the protein sequence Ub(1-76)-X, where
X is the NH2 group of the self-immolative linker, p-amino-benzyl-alcohol (PABA). Proteolytic cleavage of the
specific peptide bond between Gly76 and the PABA linker,
followed by 1,6-elimination, releases the phenoxy-dioxetane luminophore I. This phenoxy-dioxetane then undergoes rapid chemiexcitation
disassembly to produce benzoate II and a green photon.</p><p>Molecular
structure and chemiexcitation disassembly pathway of
DUB chemiluminescent probe Ub-CL.</p><p>Phenoxy-dioxetane probes are regularly synthesized through oxidation
of the corresponded enolether precursor by singlet oxygen. Therefore,
we initially synthesized an enolether derivative, conjugated with
the ubiquitin segment Ub(1-75). The chemical synthesis of conjugate
Ub-Enolether 3 is presented in Figure 2. Fmoc-Gly was coupled with 4-aminobenzyl-alcohol
to generate amide 1a. Iodination of the benzylic position
of 1a with sodium iodide and trimethylsilyl chloride
yielded benzyl-iodide 1b. The latter was reacted with
previously synthesized phenol 1c under mild basic conditions
to afford ether 1d. The allyl and Fmoc protecting groups
in compound 1d were removed by Pd(PPh3)4. Subsequent addition of piperidine resulted with removal
of the Fmoc protecting group to generate Enolether 2.
Next, the amine functional group of Enolether 2 was coupled
with the chemically synthesized Ub(1-75)-MeNbz35 (compound 1, see Supporting Information for synthesis) to yield the desired ubiquitin–enolether
conjugate Ub-Enolether 3.</p><p>Synthetic route for the
preparation of conjugate Ub-Enolether 3.</p><p>With Ub-Enolether 3 in hand, we sought oxidation
conditions,
appropriate for enolether–protein conjugates, which can be
performed by singlet oxygen.36,37 Since singlet oxygen
is a highly reactive reagent, that undergoes quenching to some extent,
by polar solvents like water,38 the oxidation
procedure of the enolether precursor is usually performed in a nonpolar
organic solvent like methylene chloride. The enolether precursor of
probe Ub-CL is composed of the Ub protein, conjugated
with the enolether small molecule. Such a protein conjugate can be
mainly solubilized in aqueous solvents. Therefore, we initially sought
to develop a procedure aimed to establish oxidation conditions for
general enolethers, by singlet oxygen, in aqueous solvent, which can
also be applied to other similar systems.</p><p>Enolether 2 was selected as a model compound for developing
oxidation conditions in the aqueous solvent, PBS 7.4. Thus, the oxidation
of Enolether 2 was evaluated in PBS 7.4, using polystyrene-bound
Rose Bengal as a photosensitizer, and irradiation with white light,
while oxygen is bubbled through the reaction solvent. The reaction
progress was monitored by RP-HPLC (Figure 3A). Within 45 min under such conditions,
more than 50% of Enolether 2 was oxidized to form the
desired Dioxetane 2a. In addition, gradual decomposition
of the dioxetane to its corresponded Benzoate 2b is also
observed. After keeping the reaction time of 60 min, Dioxetane 2a appeared as the major product of the reaction. Further
extending the reaction time resulted in substantial formation of Benzoate 2b. Therefore, oxidation under these conditions for 60 min
yielded the optimal results in our hands.</p><p>(A) RP-HPLC chromatograms
showing the reaction progress, over 60
min, for oxidation of Enolether 2 to Dioxetane 2a in PBS 7.4 as a solvent, polystyrene-bound Rose Bengal
as a photosynthesizer, oxygen bubbling and irradiation with white
light. The reaction progress was monitored at wavelength of 285 nm.
(B) Oxidation of Ub-Enolether 3 by singlet oxygen to
its corresponded dioxetane, probe Ub-CL in TRIS buffer,
pH 7.5. The Mass obtained for dioxetane probe Ub-CL (9071)
is suitable to oxidation of the S methionine group to SO2.</p><p>The establishment of conditions
for the oxidation of enolethers
to dioxetanes with singlet oxygen, under aqueous conditions, is of
high importance, since protein–enolether conjugates are likely
to be not soluble/compatible in/with organic solvents. Applying the
established oxidation conditions on Ub-Enolether 3, and
subsequent filtration of the polystyrene-bound Rose Bengal photosensitizer,
afforded the desired probe Ub-CL (Figure 3B). The obtained probe was used for measurements,
in its current status, with no further purification steps.</p><p>With
probe Ub-CL in hand, we sought to evaluate its
ability to detect the catalytic activity of various DUBs. The probe
was incubated in a suitable buffer and its chemiluminescence light
emission profile was measured with three different DUBs: UCH-L3, UCH-L1,
and USP-2 (Figure 4). Probe Ub-CL in the presence of USP-2 showed only
slight light emission enhancement (2.5-fold), but still significantly
higher than the background signal (Figure 4C). Remarkably, the total light emission
signal produced by Ub-CL upon incubation with UCH-L1
(Figure 4B) and UCH-L3
(Figure 4A) was significantly
higher than the background signal (in the absence of the DUB), with
S/N values of 45 and 300, respectively. The substantially higher catalytic
activity observed for UCH-L3 DUB, toward activation of probe Ub-CL, was observed before with other assays.10,39,40 Nevertheless, probe Ub-CL was able to clearly detect the activity of all three DUBs evaluated
in this assay. It appears that probe Ub-CL is poorly
turned over by USP-2; thus, this probe may be best suited for the
UCH family DUBs.</p><p>Chemiluminescence kinetic profile (left) and total light
emission
(right) of probe Ub-CL [10 Î¼M] in TRIS PH 7.5,
DTT [0.2 mM] with and without (A) UCH-L3 [0.8 nM], (B) UCH-L1 [50
nM], and (C) USP-2 [12 nM]. The total light emission was measured
over 15 min at 37 °C. Error bars represent the mean of three
different replicate measurements.</p><p>As mentioned above, turn-ON chemiluminescent probes have an inherent
advantage over fluorescent probes. The request for an external light
excitation source in fluorescence generates a substantial noise signal.
In chemiluminescence, the excited state of the emitter is formed through
breakage of energetic chemical bonds. When the molecule has high chemical
stability, this mode of excitation practically produced zero noise
signal. In order to demonstrate the advantage of our DUB chemiluminescent
probe, we performed comparison measurements of the S/N ratio produced
by probe Ub-CL, and the commercially available fluorescent
probe Ub-AMC. The probes were incubated with and without
DUB UCH-L3 in TRIS pH 7.5, under similar conditions, and the produced
optical signal was measured over 25 min (Figure 5). Expectedly, both probes produced a typical
turn-ON response, upon reaction with UCH-L3, for either the chemiluminescence
or the fluorescence mode of action. However, the background signal
produced by fluorescent probe Ub-AMC is considerably
higher than the background signal observed for our chemiluminescent
probe Ub-CL. In addition, probe Ub-CL exhibited
a faster response to UCH-L3, with a signal intensity of up to 93-fold
higher than the signal intensity without the DUB. In contrast, probe Ub-AMC produced only a 1.5-fold increase over the background
signal without the DUB at a similar time slot. The significantly higher
signal-to-noise ratio, obtained for probe Ub-CL, clearly
demonstrates the superior detection capability of the chemiluminescence
modality over a fluorescent one.</p><p>(A) Chemiluminescence (left) and fluorescence
(right) kinetic profiles
of Ub-CL [50 nM] and Ub-AMC [1 Î¼M]
in TRIS pH 7.5, DTT [0.2 mM] at 37 °C with and without UCH-L3
[0.8 nM]. (B) Signal to noise ratios obtained for probes Ub-CL (left) and Ub-AMC (right) with and without UCH-L3.
The values were calculated at peak-max for chemiluminescence, after
3 min, since the starting measurement. Error bars represent the mean
of three different replicate measurements.</p><p>The dioxetane–Ub conjugate, described herein, acts as a
turn-ON chemiluminescent probe for detection of DUBs activity. The
activation mechanism is based on the catalytic cleavage of Ub at a
specific site, and results in the release of a phenoxy-dioxetane luminophore
that undergoes rapid chemiexcitation to emit light. As shown in Figure 3, the oxidation of
the Ub-enolether precursor with singlet oxygen, under aqueous conditions,
was incomplete and also generates some benzoate decomposition product.
However, unlike the fluorescence assay, in the chemiluminescence modality,
such side products do not produce any noise signal. Thus, the obtained
dioxetane–Ub conjugate can directly be used, without further
purification, after removal of the polymer-immobilized photosensitizer
by filtration. The exact concertation of the active dioxetane species
of the Ub-Cl probe can be extrapolated by a light-emission
calibration curve, obtained for an analogous known chemiluminescent
probe. Importantly, the dioxetane functional group of the Ub conjugate
was found to be highly stable over several days of storage (see Figure S7 in the Supporting Information), and the light-emission signal, produced by the
dioxetane–ubiquitin probe, has remained quantitively similar
over the evaluated time period.</p><p>In summary, we have developed
the first chemiluminescence probe
for detection of DUBs activity. The probe was prepared by conjugation
of the chemically synthesized C-terminal of Ub1–75 protein with the NH2-Gly-enolether precursor. Subsequent
oxidation, under aqueous conditions, of the enolether precursor with
singlet oxygen furnished the synthesis dioxetane probe Ub-CL. This synthesis provides the first example of a dioxetane–luminophore
conjugate with a protein. The probe ability to detect DUB's
activity was successfully validated with three different DUBs. In
order to demonstrate the advantage of our new probe, comparison measurements
for detection DUB UCH-L3 activity were performed between the chemiluminescent
probe Ub-CL and a commercially available fluorescent
probe Ub-AMC. The obtained data showed significantly
higher S/N, observed for probe Ub-CL (93-fold) in comparison
to that observed for probe Ub-AMC (1.5-fold). The successful
synthesis and demonstration of this protein–dioxetane conjugate
example, as a turn-ON probe, open a door for preparation of other
relevant protein–dioxetane conjugates, such as those for Ub-like
modifiers and for other biomacromolecules, e.g., nucleic acids.</p><p>Synthetic procedures,
characterization data (NMR and
MS) for all new compounds and chemiluminescence control experiments
(PDF)</p><p>bc1c00413_si_001.pdf</p><!><p># Sara Gutkin and Satish Gandhesiri contributed equally.</p><!><p>The authors declare no
competing financial interest.</p>