Highly Efficient Multiple-Labeling Probes for the Visualization of Enzyme Activities

Article abstract of DOI:10.1002/chem.201903458

Quinone methide (QM) as a latent trapping unit has been widely explored in activity-based self-immobilizing reagents. However, further application of this strategy has been largely hampered by the limited labeling efficiency to proteins. In this study, a

Full text of DOI:10.1002/chem.201903458

DOI: 10.1002/chem.201903458
Full Paper
&
Fluorescent Probes
Highly Efficient Multiple-Labeling Probes for the Visualization of
Enzyme Activities
Heng Song, Yuyao Li, Yefeng Chen, Chenghong Xue, and Hexin Xie*[a]
Abstract: Quinone methide (QM) as a latent trapping unit
has been widely explored in activity-based self-immobilizing
reagents. However, further application of this strategy has
been largely hampered by the limited labeling efficiency to
proteins. In this study, a thorough investigation on the label-
ing efficiency and the structure of QM-based trapping unit is
presented, from which a QM with multiple leaving groups
was identified as an optimal trapping unit. An alkaline phos-
phatase (ALP) immobilizing reagent featured with this multi-
ple-labeling trapping unit exhibited lower nonspecific bind-
ing and, remarkably, a significantly higher labeling efficiency
over other immobilizing reagents upon enzymatic activation.
The utility of this imaging reagent was further demonstrated
with the in vitro and in vivo visualization of the ALP activi-
ties. Furthermore, the multiple functional trapping unit may
find greater value in the other activity-based immobilizing
probes.
Introduction
live cells and tissues). However, this strategy was largely limit-
ed by the low specificity of QMs. This is likely due to the fact
that these highly reactive intermediates form after diffusion
out of the active site of target protein—unless they have spe-
cific affinity to the active site—and thus react with nucleo-
philes from nearby proteins.[8]
Quinone methides (QMs),[1] including ortho-quinone methides
(o-QMs) and para-quinone methides (p-QMs), are a class of
highly reactive intermediates. These transient molecules have
been widely utilized by nature. For instance, QMs have been
employed as a means of defense by several animals and
plants,[1b] and they are involved in the mechanism of action of
the anticancer drug doxorubicin (Adriamycin), as well as of its
derivative epirubicin.[2] Furthermore, QMs have been widely ex-
ploited in the efficient bioconjugation processes.[3]
Nevertheless, this could be an advantage for imaging re-
agents; it may reflect the activities of enzyme more accurately
without disturbing the structure of target enzyme. Along this
line, QM has been extensively applied as self-immobilizing
imaging reagents for activities of a number of biologically im-
portant enzymes, including phosphatases,[9] glycosidases,[8,10]
sulfatases,[11] and b-lactamases.[12]
The broad biological applications of QMs have been attribut-
ed to the exceptionally high electrophilicity of these mole-
cules; these compounds have high potency in accepting nucle-
ophiles from biological system. Taking advantage of this
unique property, Danzin and co-workers reported in 1990 a
class of o-QM-based enzyme-activatable inhibitors of b-glucosi-
dases.[4] These inhibitors were specifically activated by b-gluco-
sidase to liberate reactive o-QMs, which underwent nucleophil-
ic addition by nucleophiles from active side of target enzyme
to form covalent bonds and, thus, inhibited enzymatic activi-
ties. This mechanism-based approach was further applied in
the development of potential inhibitors of phosphatase[5] and
sulfatase.[6] Similar design has been exploited to capture cata-
lytic antibodies[7] or enzymes in complex environment (e.g.,
In general, these QM-based labeling probes structurally con-
sist of four components: enzymatic recognition moiety, latent
trapping moiety, linker, and reporter (Figure 1). The latent trap-
ping moiety, that is, the precursor of QM, plays a key role in
the efficiency of labeling covalently proteins. Currently, two
types of QMs, o-QMs and p-QMs, are mainly employed as trap-
ping units. The first type are usually o-monofluoromethylphe-
nol or o-difluoromethylphenol derivatives; the activation by
specific enzyme triggers the release of fluoride as the leaving
group to form highly reactive o-QM in situ. The precursors of
p-QM are usually caged 4-alkyl phenols with a leaving group,
such as fluoro, carbamate, and carboxylic ester, on the benzyl
position. With the potential leaving group far away from the
enzymatic recognition moiety, the p-QM-derived latent trap-
ping unit even allows for the installation of structurally hin-
dered fluorescent quenchers on the leaving group, which
leads to fluorogenic and self-immobilizing probe.[12a,13]
[a] H. Song, Y. Li, Y. Chen, C. Xue, Prof. Dr. H. Xie
State Key Laboratory of Bioreactor Engineering
Shanghai Key Laboratory of New Drug Design, School of Pharmacy
East China University of Science and Technology
Shanghai 200237 (P. R. China)
Over the past two decades, this QM-based labeling ap-
proach has been investigated in a wide variety of biological
applications. In spite of this, a trapping molecule with higher
labeling efficiency is still of high importance for further appli-
E-mail: xiehexin@ecust.edu.cn
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under:
https://doi.org/10.1002/chem.201903458.
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Figure 1. (a) Design of activity-based self-immobilizable probes for covalently labeling of alkaline phosphatase. (b) Chemical structures of self-immobilizing
probes in this study.
cation of this strategy. However, to our surprise, systematic in-
vestigation of the labeling efficiency of QM-based immobilizing
reagents, particularly in real biological environments, is rare. In
this study, we have conducted a thorough investigation on the
labeling efficiency of a range of QM-based trapping molecules,
from which we have disclosed a QM precursor with multiple
leaving groups that exhibits significantly higher labeling effi-
ciency.
Alkaline phosphatase (ALP) is a type of hydrolase capable of
dephosphorylation of a wide range of biological molecules, in-
cluding nucleic acids and proteins, as well as other small mole-
cules.[14] Current studies have indicated a number of diseases
(e.g., osteoblastic bone cancer, prostatic cancer, and hepatitis)
Scheme 1. Preparation of self-immobilizing probe ALP-6. a) NaN3, DMF, RT;
closely associate with increasing activities of ALP, and thus this
b) diethyl phosphite, DIPEA, DMAP, CCl4/MeCN, À208C to RT; c) Dess–Martin
enzyme can serve as an important biomarker for molecular
biology and even clinical diagnosis.[15] To visualize activities of
ALP, a number of assays have been developed, including fluo-
rogenic probes,[16] self-assembly reagents,[16d,17] and QM-based
self-immobilizing fluorescent probes.[9d,f] In this work, we em-
ployed ALP as a model enzyme for the investigation of label-
ing efficiency of QM-based trapping compounds.
periodinane (DMP), CH2Cl2, RT; d) diethylaminosulfur trifluoride (DAST),
CH2Cl2, 08C; e) NaBH4, MeOH, 08C; f) ethyl isocyanate, triethylamine (TEA),
CH2Cl2, reflux; g) TMSBr, MeCN; h) 7, CuSO4, tris(3-hydroxypropyltriazolyl-
methyl)amine (THPTA), vitamin C, DMSO/H2O (1:1), RT.
ethyl phosphite in the presence of N,N-diisopropylethylamine
(DIPEA) and 4-dimethylamino-pyridine (DMAP), to afford 2. A
Dess–Martin oxidation and subsequent fluorination with dieth-
ylaminosulfur trifluoride (DAST) furnished difluoromethyl-sub-
stituted 3, which was further reduced by sodium borohydride
to yield 4. Treatment of 4 with ethyl isocyanate in the presence
of triethylamine (TEA) gave fully functionalized 5. Deprotection
of 5 was achieved by bromotrimethylsilane and the resulting
phosphate 6 was linked to an alkynyl-tethering fluorescein 7
by a simple Click reaction, as previously described. Probe ALP-
6 was obtained after purification by reversed-phase HPLC and
characterized by HRMS and HPLC analysis. Immobilizing
probes ALP-5, ALP NIR-1, and ALP NIR-2, were synthesized and
purified in a similar manner.
Results and Discussion
Preparation of self-immobilizing probes
We prepared monofluoromethyl (ALP-1) and difluoromethyl
(ALP-2) containing probes, as well as the p-QM-based probes
(ALP-3 and ALP-4), according to reported procedures[9a,b,e] but
with fluorescent reporter, fluorescein, connected by a Click re-
action.[18] The leaving group-free probe (ALP-0) as the control
reagent was also synthesized in a similar manner.
As outlined in Scheme 1, we started the synthesis of multi-
ple-functional QM-bearing probe ALP-6 from nucleophilic sub-
stitution of 1, followed by selective phosphorylation with di-
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Evaluation of the labeling efficiency
ing multiple opportunities to label proteins and, thus, leading
to improved labeling efficiency. However, to our surprise, on
the basis of in-gel fluorescence imaging, the use of ALP-6 did
not show any improvement on the labeling of ALP over other
p-QM-based probes (ALP-3, ALP-4, and ALP-5).
To study the labeling efficiency of these self-immobilizing re-
agents, we incubated all of these probes (ALP-0, ALP-1, ALP-2,
ALP-3, ALP-4, ALP-5, and ALP-6) with equal amount of ALP in
phosphate buffered saline (PBS, pH 7.4) at 378C for 1 h and
these samples were then subjected to sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (SDS-PAGE) analysis. It is
worth noting that, to precisely represent the labeling of pro-
teins in physiological environment, these samples were loaded
on the SDS-PAGE gel without heating or incubation with re-
ducing reagent (e.g., b-mercaptoethanol and dithiothreitol) to
denature proteins. The labeling efficiency of these reagents
was assessed by the in-gel fluorescence intensity. As shown in
Figure 2a, the enzymes treated with p-QM-based probes (ALP-
Theoretically, these labeling reagents can be trapped by any
available nucleophilic proteins after activation by ALP. To inves-
tigate the labeling of these molecules to other bystander pro-
teins in the presence of ALP, we incubated all of these mole-
cules with bovine serum albumin (BSA) along with ALP. The in-
gel fluorescence image indicated that both proteins, BSA and
ALP, were labeled by these ALP probes and the relative fluores-
cent intensity of these samples were similar to those incubated
with ALP alone.
Labeling specificity is another important criteria for a cova-
lently labeling reagent; poor specificity may lead to false infor-
mation. To test their labeling specificity, we treated these label-
ing reagents with BSA without the addition of ALP, in which
these reagents are not supposed to be activated to form o-
QMs. The nonspecific labeling of these reagents to BSA was
again assessed by in-gel fluorescence analysis (Figure 2b).
There was hardly any fluorescence signal found in the ALP-0-
treated BSA. However, the BSA-incubated with ALP-3 emitted
stronger fluorescence than others, implying this p-QM-based
labeling reagent may be less stable and thus led to non-specif-
ic bonding with BSA. Although the fluorescence on ALP-3-
treated BSA was much weaker than those activated by ALP, it
may still be problematic given to the high abundance of other
bystander proteins in real samples. To our delight, molecule
with the less reactive ethyl carbamate as the leaving group
(ALP-4) generated less nonspecific fluorescence than the
fluoro-containing counterpart (ALP-3). Notably, the replace-
ment of the amido (L=III, ALP-4) with the triazole-bearing
methylene group (L=I, ALP-5) as the linker further reduced
nonspecific binding to BSA, leaving basically no fluorescence
on the protein. With identical linker and leaving group as
those in ALP-5, the multifunctional probe ALP-6 was also inert
towards BSA. These results imply the non-specific binding of p-
QM-based immobilizing reagents to BSA may associate with
the electron properties of the linker, although other possibili-
ties cannot be excluded at the current stage.
Figure 2. Labeling of proteins with immobilizing probes. (a) In-gel fluores-
cence scanning of ALP (12.5 UmLÀ1) after incubation with indicated probes
(7.5 mm) in the presence or absence of BSA (0.15 mgmLÀ1) at 378C for 1 h.
(b) In-gel fluorescence scanning of BSA (0.15 mgmLÀ1) upon incubation with
probes (7.5 mm) at 378C for 1 h. CBS: Coomassie blue staining; FL: fluores-
cence.
In addition to ALP, acid phosphatase (ACP) is also known as
an efficient dephosphorylation enzyme. To investigate whether
these reagents can be activated by ACP, and thus label ACP or
other proteins, we incubated ALP-0 and ALP-6 with BSA in the
presence of a catalytic amount of ACP in MES buffer (pH 5.0).
In-gel fluorescence imaging (Figure S3, Supporting Informa-
tion) indicated the sample incubated with ACP emitted signifi-
cantly lower fluorescence than that with ALP in PBS (pH 7.4)
though further analysis by HPLC revealed that the majority of
ALP-6 has been consumed (Figure S2, Supporting Information).
These results demonstrate the high labeling specificity of ALP-
6 to ALP over ACP. We reason that the low labeling efficiency
to ACP may be because the uncaged ALP-6 is stable in acidic
conditions and cannot lead to the formation of reactive QM.
Considering the preliminary information on the labeling effi-
ciency and specificity of these trapping reagents, we conduct-
3, ALP-4, and ALP-5) emitted significantly stronger green fluo-
rescence than the o-QM-based probes (ALP-1 and ALP-2)-incu-
bated samples. As a control, the leaving group-free ALP-0 was
totally ineffective on the introduction of fluorescence to ALP.
As all of these immobilizing reagents only differ by the struc-
ture of latent trapping unit, these results seem to suggest the
p-QM-based latent trapping moiety may be more efficient in
labeling proteins than the o-QM-based counterparts, even
though both have been widely used in biological studies.
APL-6, unlike other trapping reagents, features leaving
groups on both the ortho-position and the para-position, and
thus may lead to the formation of o-QM and/or p-QM as trap-
ping units upon enzymatic uncaging. We envisioned that this
molecule may have multiple chances to generate highly reac-
tive trapping unit upon one single enzymatic stimulus, provid-
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Figure 3. Fluorescent microscopic images of HeLa and HEK293 cells with immobilizable ALP probes. (a) Fluorescence images of HeLa cells incubated with indi-
cated probes (10 mm) at 378C for 1.5 h, or pre-treated with ALP inhibitor p-BTO (500 mm) for 1 h before incubated with probes for 1.5 h. (b) Average fluores-
cence intensity of HeLa cells in (a). Error bars were calculated on N measurements, with N=20, 16, 11, 16, 11, 12, and 12, from left to right. (c) Fluorescence
images of HEK293 cells incubated with indicated probes (10 mm) at 378C for 1.5 h. FL: fluorescence; Hoechst: Hoechst 3342 staining; BF: bright field; p-BTO:
(À)-p-bromotetramisole oxalate, an ALP inhibitor. Scale bar=50 mm.
ed further investigations on their labeling of living cells. We in-
cubated probes ALP-2, ALP-5, and ALP-6, as well as ALP-0 (as
control), with ALP-overexpressing HeLa cells for 90 minutes.
Upon washing away unbounded fluorescent reagents, all of
these samples were then subjected to imaging with fluores-
cent microscope (Figure 3). As expected, the leaving group-
free control probe (ALP-0) led to no retention of fluorescence
on HeLa cells, whereas all of the other molecules introduced
detectable green fluorescence on HeLa cells, but their intensity
varied dramatically. The multiple-functional ALP-6 seemed to
be the most efficient labeling reagent: the ALP-6-treated HeLa
cells emitted about seven times stronger fluorescence than
that with ALP-2 or over three times higher than ALP-5-incubat-
ed sample, whereas the labeling efficiency assessed by in-gel
fluorescence imaging increased in the order: ALP-5>ALP-6@
ALP-2.
motetramisole oxalate (p-BTO) before incubation with ALP-2,
ALP-5, and ALP-6, which resulted in massively reducing of fluo-
rescence on cells. Furthermore, ALP-negative HEK293 cells
were also incubated with these labeling reagents, respectively,
and none of these probes delivered noticeable fluorescence
on HEK293 cells. These results demonstrate that the observed
fluorescence signal on HeLa cells is indeed due to the dephos-
phorylation activities of ALP and further support the superior
labeling ability of ALP-6.
ALP is known to overexpress on the extracellular membrane
of HeLa cells,[17,19] the difference on the permeability of these
labeling reagents—if there is any difference—is unlikely the
reason for the variety of labeling efficiency on cells. To eluci-
date the inconsistent results obtained from live cell fluorescent
imaging and in-gel fluorescence imaging, we further incubated
lysate of HeLa cells with ALP-5 or ALP-6 and then analyzed the
fluorescent labeling of proteins by in-gel fluorescence imaging.
As illustrated in Figure S1 (Supporting Information), both imag-
To further investigate the labeling specificity of these probes
on cells, we pretreated HeLa cells with ALP inhibitor, (À)-p-bro-
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ing reagents produced substantial amount of fluorescence on
proteins. However, clearly ALP-5 led to stronger fluorescence
than ALP-6, which is in agreement with aforementioned in-gel
fluorescence analysis of ALP but different from the fluorescent
imaging of living cells. These results imply that some of the
fluorescence-labeled proteins on living cells may not be de-
tectable from PAGE gel, presumably due to the low stability of
these fluorescent complex. In other words, fluorescent micro-
scope imaging of living cells is a more suitable approach than
in-gel fluorescence analysis to evaluate the labeling ability of
activity-based immobilizing reagents.
signal on in-gel imaging, though other possibilities cannot be
ruled out completely at the current stage.
Near-infrared labeling reagents
Fluorescent dyes with absorption and emission at the range of
near-infrared zone are particularly useful for biomedical investi-
gations due to the high tissue-penetration ability and less in-
terference by the intrinsic autofluorescence from living ani-
mals.[20] Therefore, to further expand the application of our
design, we replaced the fluorescent reporter with near-infrared
and switchable P-Mero4.[21]
To rationalize the difference of fluorescent labeling to pro-
teins, plausible mechanisms were proposed in Scheme 2.[1b,4]
We performed in-gel fluorescence analysis again to evaluate
these near-infrared imaging reagents. As depicted in Figure 4a,
both ALP NIR-1 and multiple-functional ALP NIR-2 showed ex-
cellent labeling ability to proteins, delivering strong near-infra-
red fluorescence on ALP or BSA. The specificity of the labeling
was further confirmed by the use of ALP inhibitor (p-BTO) or
the leaving-group-free control probe (ALP NIR-0).
P-Mero4 is a highly environment-sensitive fluorophore; its
fluorescence can be dramatically enhanced after covalently in-
teraction with proteins. To reveal the fluorescent response of
the near-infrared self-immobilizing molecule, we recorded the
fluorescent spectra of ALP NIR-2 in the absence or presence of
ALP (Figure 4b). As expected, ALP NIR-2 in PBS (pH 7.4) alone
was basically nonfluorescent and the addition of ALP boosted
its fluorescence at 660 nm intensively. As a control, the incuba-
Scheme 2. Plausible fluorescent labeling mechanisms of ALP-5 and ALP-6.
Considering stability of the highly reactive intermediate, we
tend to believe p-QM (II-1 or II-2) may form first for both ALP-
5 and ALP-6 upon activation by ALP. These p-QMs then under-
goes nucleophilic addition by nucleophiles from nearby pro-
teins (first labeling) before diffusion out and addition by abun-
dant water to form compound IV-1 or IV-2. The ALP-5-leading
IV-1 will eventually be washed away from cells. However, for
the ALP-6-leading IV-2, it could form reactive QM (V) again
and undergo nucleophilic addition by proteins (second label-
ing), though the chance may be slim as it is far away from pro-
teins. Most of the QM (V) may be attacked by water again to
give aldehyde (VII). This aldehyde is supposed to be inert to
water but may react with amino group of proteins (three label-
ing). Having multiple chances to label proteins upon one
single stimulus may be the origin of superior labeling ability of
ALP-6. It is worth noting that the imine-based dye–protein
complex may not be stable enough during gel electrophoresis.
This may be the reason why ALP-6 exhibited stronger fluores-
cence on the cell-based imaging but had weaker fluorescence
Figure 4. (a) In-gel fluorescence imaging of ALP upon incubation with immo-
bilizing probes. Indicated proteins (BSA: 0.15 mgmLÀ1; ALP 5 UmLÀ1) were
incubated with probes (5 mm) at 378C for 2 h, or proteins were pre-treated
with p-BTO (5 mm) for 1 h before incubation with probes for 2 h. (b) Fluores-
cence spectra of ALP NIR-2 (1 mm) before or after incubation with ALP
(50 mg, 2.5 UmLÀ1) or HEK293 cells lysate (50 mg, 0.25 mgmLÀ1) at 378C for
30 min (lex =635 nm). CBS: Coomassie blue staining; FL: fluorescence.
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Figure 5. (a) Fluorescent microscope images of HeLa cells or HEK293 cells upon incubation with immobilizable ALP NIR probes. Indicated cells were incubated
with trapping probes (5 mm) at 378C for 1.5 h, or pretreated with ALP inhibitor p-BTO (500 mm) for 1 h before incubation with probes for 1.5 h. (b) Average
fluorescence intensity of HeLa cells in (a). Error bars were calculated on N measurements, with N=6, 6, 9, 18, and 10, from left to right. (c) HEK 293 cell viabili-
ty upon treatment with ALP NIR-2. HeLa cells or HEK293 cells were incubated with ALP NIR-2 at 0, 1, 2.5, 5, 10 mm for 24 h, and the cell viability was deter-
mined by MTT assay. FL: fluorescence; Hoechst: Hoechst 3342 staining; BF: bright field; scale bar=50 mm; error bars were calculated on three experiments.
tion of ALP NIR-2 with lysate of HEK293 (ALP-negative cells)
led to a considerably lower enhancement of fluorescence.
However, given the large abundance of nonspecific proteins
on cells, washing away unbounded fluorescent molecules may
still be necessary for these NIR imaging reagents in cell imag-
ing.
has been a challenge due to the fact that this enzyme usually
overexpresses on the extracellular membrane of tumor
cells:[16,17] fast diffusion likely occurs for most of the activatable
imaging reagents. The QM-based self-immobilizing probes are
capable to generate covalent linkage with ALP-overexpressing
cells upon activation, which may provide an opportunity to vis-
ualize activities of ALP in living animals.
Following aforementioned protocol, the labeling ability and
specificity of ALP NIR-1 and ALP NIR-2 were studied by the
fluorescent imaging of living HeLa cells (Figure 5a,b). Similar to
ALP-6, ALP NIR-2 produced a much stronger fluorescent signal
than the p-QM-based ALP NIR-1, confirming again the superior
labeling ability of the multiple-functional trapping unit.
Moreover, the cytotoxicity of this trapping reagent was stud-
ied by incubation HeLa cells or HEK293 cells with a serial con-
centrations of ALP NIR-2 and it turned out that this compound
has negligible cytotoxicity to both HEK293 cells and HeLa cells
even at the concentration of 10 mm (Figure 5c).
To examine the feasibility of the self-immobilizing probe in
imaging of activities of endogenous ALP in vivo, BALB/c nude
mice bearing HeLa tumor were intratumorally injected with
ALP NIR-2 and ALP NIR-0 (as control), respectively. We moni-
tored the fluorescence intensity of mice at 670 nm over the
time course of 24 h after injection. As shown in Figure 6,
though ALP NIR-0 and ALP NIR-2 were injected in identical
amounts (30 mm, 100 mL), the ALP NIR-2-treated tumor emit-
ted highly stronger fluorescence than that with leaving group-
free ALP NIR-0 even at 10 minutes after the injection. This re-
markably difference may result from the slower cleanup rate of
ALP NIR-2, and more importantly, the enhancement of fluores-
cence intensity induced by the formation of covalent bond
with proteins after enzymatic activation. The retention of fluo-
rescence on the ALP NIR-2-treated tumor was much longer
In vivo imaging of enzyme activities
The increasing activities of ALP is closely related to a range of
tumors. However, in vivo tracking activities of ALP on tumor
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Figure 6. Fluorescence imaging of HeLa tumor xenograft mice upon injection of immobilizing probe ALP NIR-2. (a) Whole-body fluorescence imaging of HeLa
tumor-bearing mice. (b) Average fluorescence intensity of HeLa tumors on mice. (c) Fluorescence imaging of main organs of mice 24 h after the injection of
probes. (d) Average fluorescence intensity of main organs of mice at 24 h after the injection of probes. ALP NIR-0 or ALP NIR-2 (30 mm, 100 mL) was intratu-
morally injected or pre-treated tumor with ALP inhibitor Na3O4V (10 mm, 50 mL) for 10 min for injection of probes. lex =620 nm, lem =670 nm; error bars were
calculated on three experiments; *P<0.05; **P<0.01; ***P<0.001).
than that injected with ALP NIR-0; fluorescence signal can be
detected even 24 h after injection. Furthermore, the intratu-
moral injection of an ALP inhibitor, sodium orthovanadate
(Na3O4V), before that of ALP NIR-2, reduced the fluorescence of
tumor at 10 minutes after the injection though the effect of
this inhibitor diminished after half an hour, which is likely be-
cause this small molecule was cleared up from tumor rapidly.
be used to visualize the activities of ALP in living animals.
Moreover, this multiple functional QM precursor may serve as
a general trapping unit in the other activity-based immobiliz-
ing probes.
Experimental Section
Synthesis and characterization
Conclusions
2-Azido-1-{4-[(diethoxyphosphoryl)oxy]-3-(difluoromethyl)-phe-
In summary, we have conducted an unprecedented investiga-
tion on the structure of QM precursors and the labeling effi-
ciency and specificity by using ALP as a model enzyme. Based
on the in-gel fluorescence analysis and living cell fluorescent
imaging, we have identified a new multiple functional QM as
the optimal latent trapping unit. An ALP self-immobilizing re-
agent with such a trapping unit allows for the labeling of pro-
teins in multiple ways upon enzymatic activation, and leads to
lower nonspecific binding and, more importantly, significantly
higher labeling efficiency on living cells than current o-QM or
p-QM-based labeling reagents. Preliminary in vivo studies have
demonstrated that this type of self-immobilizing reagent can
nyl}ethyl ethylcarbamate (5): To
a solution of 3 (202 mg,
0.56 mmol) in methanol (5 mL) at 08C was added NaBH4 (32 mg,
0.83 mmol) in several portions and the resulting mixture were
stirred at the same temperature for 30 min. A saturated aqueous
solution of NH4Cl (2 mL) was then added. The organic layer was
separated and the aqueous phase was extracted with DCM (10 mL
ꢁ 3). The combined organic layers were dried over MgSO4 and pu-
rified by chromatography on a short silica gel column to afford 4
as crude product, which was used in next step without further pu-
rification.
Under N2 atmosphere, a solution of 4 (161 mg, 0.44 mmol), ethyl
isocyanate (175 mL, 2.2 mmol) and TEA (307 mL, 2.2 mmol) in CH2Cl2
(anhydrous, 3 mL) were heated to reflux for 12 h. After cooling to
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room temperature, water (2 mL) was then added dropwise. The or-
ganic layer was separated and the aqueous phase was extracted
with CH2Cl2 (10 mL ꢁ 3). The combined organic layers were dried
over MgSO4 and purified by chromatography on silica gel column
NIR-2 at a serial of concentrations (0, 1, 2.5, 5, and 10 mm) in DMEM
(supplemented with 10% FBS, 100 mL). After incubation at 378C for
24 h, MTT (10 mL, 5 mgmLÀ1 in PBS) was added to each well and
incubated at 378C for 4 h. The solution was then removed and
DMSO (100 mL) was added to dissolve the resulting purple crystal.
Absorbance at 490 nm was measured with a microplate reader to
assess the cell viability. Each experiment was run in triplicate.
1
to give 5 (115 mg, 47% form 3). H NMR (400 MHz, CDCl3) d=7.60
(s, 1H), 7.50–7.42 (m, 2H), 6.93 (t, J=55.1 Hz, 1H), 5.88–5.83 (m,
1H), 4.87 (s, 1H), 4.28–4.18 (m, 4H), 3.61–3.43 (m, 2H), 3.30–3.18
(m, 2H), 1.36 (t, J=7.1 Hz, 6H), 1.15 ppm (t, J=7.2 Hz, 3H).
13C NMR (100 MHz, CDCl3) d=154.82, 148.54 (q, J=5.9 Hz), 135.07,
130.22, 125.64 (td, J=22.9, 7.2 Hz), 124.66 (t, J=5.8 Hz), 120.41 (d,
J=1.8 Hz), 111.06 (t, J=237.7 Hz), 73.86, 65.11 (d, J=6.2 Hz), 55.21,
36.00, 16.00 (d, J=6.6 Hz), 15.03 ppm. HRMS (ESI) m/z calcd for
C16H23F2N4O6P [M+Na]+: 459.1215, found 459.1223.
Real-time in vivo imaging of tumor-bearing mice
All animal experiments were performed in accordance with the
guidelines of Care and Use of Laboratory Animals of China for
animal experimentation and approved by the ethics committee of
the East China University of Science and Technology Animal proce-
dures.
2-Azido-1-[3-(difluoromethyl)-4-(phosphonooxy)phenyl]ethyl
ethylcarbamate (6): Under N2 atmosphere, to a solution of 5
(115 mg, 0.26 mmol) in acetonitrile (anhydrous, 2 mL) at 08C was
added TMSBr (174 mL, 1.3 mmol) and the resulting mixtures were
stirred at room temperature for 12 h. A saturated aqueous solution
NH4Cl (2 mL) was then added, followed by CH2Cl2 (5 mL). The or-
ganic layer was separated and the aqueous phase was extracted
with CH2Cl2 (10 mLꢁ3). The title compound was obtained by RP-
BALB/c female mice bearing subcutaneous HeLa tumors (see Sup-
porting Information for detail) were used in this study (n=3 for
each group). Immobilizing probes ALP NIR-0 or ALP NIR-2 was in-
tratumorally injected (30 mm in PBS, 100 mL) and fluorescence
imaging of whole mice was performed with IVIS Lumina XRMS
Series III In Vivo Imaging System (PerkinElmer, Inc. USA) with exci-
tation at 620 nm and emission at 670 nm at different time points
(10 min, 30 min, 1 h, 2 h, 4 h, and 24 h). The inhibition experiments
were conducted by intratumoral injection of ALP inhibitor Na3O4V
(50 mL, 10 mm in Tris buffer, pH 8.0) for 10 min before injection of
ALP NIR-2.
HPLC purification on
a
C18 column (34 mg, 35%). 1H NMR
(400 MHz, [D6]DMSO) d=7.60 (s, 1H), 7.54 (d, J=8.4 Hz, 1H), 7.48–
7.39 (m, 2H), 7.11 (t, J=55.1 Hz, 1H), 5.80 (t, J=5.6 Hz, 1H), 3.64
(d, J=5.6 Hz, 2H), 3.00 (qd, J=10.8, 7.0 Hz, 2H), 1.00 ppm (t, J=
7.2 Hz, 3H). 13C NMR (100 MHz, [D6]DMSO) d=154.93, 149.29 (q,
J=6.0 Hz), 134.74, 130.24, 125.10 (td, J=22.7, 6.2 Hz), 123.96 (t, J=
4.7 Hz), 120.97, 111.65 (t, J=235.1 Hz), 72.79, 54.49, 35.25,
15.02 ppm. HRMS (ESI) m/z calcd for C12H15F2N4O6P [MÀH]À:
379.0625; found 379.0620.
Acknowledgements
Financial support from The Recruitment Program for Young
Professionals, NSFC-BRICS (81861148020), the Research Pro-
gram of State Key Laboratory of Bioreactor Engineering, and
the Fundamental Research Funds for the Central Universities
are gratefully acknowledged.
5-{2-[2-(2-{[(1-{2-[3-(Difluoromethyl)-4-(phosphonooxy)phe-nyl]-
2-[(ethylcarbamoyl)oxy]ethyl}-1H-1,2,3-triazol-4-yl)methyl]ami-
no}-2-oxoethoxy)ethoxy]ethyl)carbamoyl}-2-(6-hydroxy-3-oxo-
3H-xanthen-9-yl)benzoic acid (ALP-6): To a solution of 7 (1.5 mg,
2.76 mmol), l-(+)-ascorbic acid (vitamin C, 1.3 mg, 7.2 mmol), CuSO4
(30 mg, 0.18 mmol), and tris(3-hydroxypropyltriazolyl methyl)amine
(THPTA, 80 mg, 0.18 mmol) in 15 mL DMF were added 6 (0.68 mg in
5 mL DMF, 1.8 mmol) and water (15 mL). The resulting mixture was
stirred at room temperature for 30 min. The title compound ALP-6
(1.0 mg, 40%) was obtained after RP-HPLC purification on a C18
column. HRMS (ESI) m/z calcd for C42H41F2N6O15P [MÀH]À 937.2263;
found 937.2256.
Conflict of interest
The authors declare no conflict of interest.
Keywords: alkaline phosphatase · enzyme activity · imaging ·
immobilization · quinone methide
Fluorescence microscope imaging of live cells
Cells were incubated in DMEM, supplemented with 10% fetal
bovine serum (FBS) and 1% penicillin-streptomycin (PS), at 378C
with 5% CO2 and passaged upon reaching 70–80% confluence. Im-
mobilizing probes (10 mm for ALP-0, 2, 5, 6; 5 mm for ALP NIR-0, 1,
2) were incubated with cells at 378C for 1.5 h and then washed
with PBS (pH 7.4) for 3 times before being subjected to fluores-
cence microscope imaging. For the experiments with inhibitor,
cells were pre-treated with p-BTO (500 mm) at 378C for 1 h before
incubation with probes.
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Manuscript received: July 29, 2019
Revised manuscript received: August 30, 2019
Accepted manuscript online: September 10, 2019
Version of record online: && &&, 0000
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Full Paper
FULL PAPER
Visualizing the activity: An investiga-
tion on the labeling efficiency and struc-
ture of quinone methide (QM) based
trapping unit is presented, from which a
QM with multiple leaving groups was
identified as an optimal trapping unit.
An alkaline phosphatase (ALP) immobi-
lizing reagent featured with this multi-
ple-labeling trapping unit exhibited
upon enzymatic activation a lower non-
specific binding and, remarkably, a sig-
nificantly higher labeling efficiency over
other immobilizing reagents. In vitro
and in vivo visualization of the ALP ac-
tivity is also reported.
&
Fluorescent Probes
H. Song, Y. Li, Y. Chen, C. Xue, H. Xie*
&& – &&
Highly Efficient Multiple-Labeling
Probes for the Visualization of Enzyme
Activities
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Chem. Eur. J. 2019, 25, 1 – 10
www.chemeurj.org
10
ꢀ 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!