Construction of a novel asymmetric imidazole-cored AIE probe for ratiometric imaging of endogenous leucine aminopeptidase

Article abstract of DOI:10.1039/d1cc01940f

We report a rational strategy to deliberately construct the first asymmetric tetraarylimidazole-based AIE probe, integrating AIE behavior in synergy with ESIPT character to image endogenous LAP for the first time. It offered good sensitivity and selectivity, and concomitantly, was applied successfully for real-time tracking of LAP in the cisplatin-induced liver injury zebrafish model.

Full text of DOI:10.1039/d1cc01940f

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Construction of a novel asymmetric
imidazole-cored AIE probe for ratiometric
imaging of endogenous leucine aminopeptidase†
Cite this: Chem. Commun., 2021,
57, 6608
Received 12th April 2021,
Accepted 27th May 2021
ab
Xueyan Huang,
Qian Lei,ab Shuai Huang,ab Hongliang Zeng,c Bin Feng,ab
Qinghai Zeng,d Yibo Hud and Wenbin Zeng
*
ab
DOI: 10.1039/d1cc01940f
rsc.li/chemcomm
We report a rational strategy to deliberately construct the first living cells. This prompted us to develop novel LAP probes with a
asymmetric tetraarylimidazole-based AIE probe, integrating AIE self-calibration effect and robust fluorescence emission.
behavior in synergy with ESIPT character to image endogenous
Aggregation-induced emission (AIE) materials, different from
LAP for the first time. It offered good sensitivity and selectivity, and traditional dyes, exhibit intensive fluorescence upon aggregation
concomitantly, was applied successfully for real-time tracking of with strong resistance toward photobleaching. Furthermore, AIE
LAP in the cisplatin-induced liver injury zebrafish model.
fluorophores also possess flexible controllability, high sensitivity
and high selectivity, making them ideal candidates for enzyme
Leucine aminopeptidase (LAP), one of the most important detection.10,11 Prior to this, the implementation of the AIE strategy
exopeptidases, can selectively release leucine residues from in LAP detection was scarcely reported.12 Among the extensive
the N-terminus of protein or peptide substrates and plays a fluorescent scaffolds, tetraarylimidazole is a typical building block
vital role in numerous physiological and pathological of AIE, and related imidazole derivatives have been employed
processes.1,2 The abnormal catalytic function of LAP often successfully in the detection of various biomolecules in living
causes disorder of peptide activation, tumour cell proliferation, systems.13,14 These AIE fluorophores based on tetraarylimidazole
intrinsic resistance toward drug, invasion and angiogenesis.3,4 were synthesized by the one-pot method,15 in which the major
Increased LAP expression nearly correlates with many diseases, structural feature is the two identical aryl substituents at the 4,5-
such as hepatic dysfunction or liver cancer, therefore enabling positions of imidazole, largely limiting their capability for deriva-
it to serve as a potential biomarker for cancer diagnosis.5,6
tion (Scheme 1A). Therefore, efforts to construct these fluorophores
The invention of the light microscope has thoroughly acceler- with asymmetric substituents at the 4,5-positions become ground
ated the great cause of uncovering the microbial world.7 For the on- breaking in developing of novel AIE fluorophores, and in themean-
site dynamic monitoring of LAP, a fluorescence probe is regarded while, would spark more multi-functional fluorescent probes.
as a preferential strategy due to its excellent spatial-temporal
Herein, we put forward an alternative method to construct
resolution, non-invasiveness and high specificity.8 Unfortunately, imidazole fluorophores with various aryl groups at 4,5-positions
existing probes often suffer from insufficient anti-interference (Scheme 1B) and further successfully developed a novel AIE fluor-
ability thereby producing false-negative or false-positive signals,9 escent probe ASSI-Leu for tracking endogenous LAP. The probe was
and in addition some probes also have strong background fluores- designed to purposefully incorporate the classic 2-(20-hydroxy-
cence or quenched fluorescence at high concentration in an phenyl) benzothiazole (HBT) skeleton into the 5-position aromatic
aqueous environment, which greatly limits their application in
a Xiangya School of Pharmaceutical Sciences, Central South University, Changsha,
410013, P. R. China. E-mail: wbzeng@hotmail.com, wbzeng@csu.edu.cn;
Fax: +86-731-82650459; Tel: +86-731-82650459
b Hunan Key Laboratory of Diagnostic and Therapeutic Drug Research for Chronic
Diseases, Changsha 410078, China
c Hunan Academic of Chinese Medicine, Inst Chinese Mat Med, Changsha,
P. R. China
d Dermatological Department, 3rd Xiangya Hospital, Central South University,
Changsha, 410078, P. R. China
Scheme 1 Strategies for the construction of tetraarylimidazole-based AIE
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
fluorophores.
d1cc01940f
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ring to avail both AIE and excited-state intramolecular proton
transfer (ESIPT) characters. Then the hydrogen bond donor was
elaborately blocked by a LAP substrate L-leucine amide which would
prevent the ESIPT process, thus, only enol emission can be
observed.16 However, upon exposure toward LAP, the block unit
Leu is cleaved from the fluorophore, accompanied by the fluores-
cence signal, which is ascribed to the combined AIE characteristics
and keto-emission behaviour. The relative change of the two indivi-
dual emissions could eliminate the interference of external environ-
ment factors, thereby affording ASSI-Leu as an efficient tool for
in situ ratiometric imaging of endogenous LAP (Scheme 2). This
strategy bestows the advantages of large Stokes’ shift, high sensitivity
and selectivity, favorable photostability and low cytotoxicity on the
novel probe. Benefiting from these advantages, the real-time tracking
of LAP activity in hepatoma cells and zebrafish models was achieved.
We employed ketone substrates and amidine derivatives as
raw materials by referring to Zhou’s contribution to the CBr4-
based synthesis.17 In the carbon tetrabromide-mediated tan-
Fig. 1 Related theoretical calculations of compound ASSI-OH. (A) Opti-
mized enol minimum-energy structure in the S1 state and the constructed
potential energy scan surface of both S0 and S1 states of ASSI-OH as a
function of O31–H58. The PESs’ illustration of enol form (B) and keto form
(C) as a function of dihedral angle C6–C1–C30–N32
.
Thus, ASSI-OH mainly existed in the enol form in an excited state.
dem cyclization, an imidazole derivative with asymmetrically Subsequently, a better understanding could be derived from the PES
substituted aryl groups at 4,5-positions of the imidazole of twisted intramolecular charge transfer (TICT). The calculated
ring was easily synthesized. HBT was incorporated into the result showed that the twisting process of the enol form of ASSI-
5-position of the imidazole ring to obtain the chromophore OH was obstructed, and the TICT process could not occur and
appeared as a luminous local excited state (Fig. 1B). However, for the
keto form, the TICT dark state dominated in governing the lumines-
cence behavior due to the lower energy of the TICT state (Fig. 1C),
which helps in explaining the phenomenon that only emission of
with both AIE and ESIPT properties. The detailed synthetic
routes of ASSI-Leu are described in Scheme S1 (ESI†).
Initially, we investigated the photophysical properties of
ASSI-OH in various solvents. The UV/Vis spectra exhibited the
absorption maximum at 365 nm, and there was no obvious keto- the enol form was observed during the solvent effect investigation.
emission in the fluorescence spectra (Fig. S1, ESI†), which did not
Then, we studied the AIE characteristics of ASSI-OH in the
seem to tally with the optical characteristics of the traditional DMSO/water mixture with different water fractions (fw). In pure
ESIPT dyes. To gain further insights into this photophysical DMSO solution, it showed blue fluorescence which was assigned to
enol emission. With an increase in the water percentage to 60%,
the fluorescence intensity had a remarkable enhancement at
554 nm (Fig. S2, ESI†), evidently suggesting that ASSI-OH was
AIE-active. This phenomenon could be ascribed to that the intra-
behavior, we performed the TD/DFT calculations at the PBE0/
def2-SVP level to optimize enol geometry and adopted the method
of constructing a potential energy surface (PES) to explore the
ability of ASSI-OH to experience the ESIPT process. A single point
calculation along the relaxed S1 paths yielded the unrelaxed S0 molecular rotation and vibration were restricted in the aggregation
energy profiles. As shown in Fig. 1A, there was an energy barrier of state; meanwhile, the intramolecular hydrogen bonds were easy to
0.638 kcal molꢀ1 in the S1 state for the conversion of enol into form, thereby reducing the barrier of enol–keto conversion to the
keto species, suggesting the low efficiency of the IPT reaction. active ESIPT process. As the fw was above 60%, the reduction in the
fluorescence could be explained by the fact that those aggregates
reached a critical point and precipitated out from the solution with
weaker emission. Combined with the above solvation behavior, this
novel asymmetrically substituted imidazole-based fluorophore
exhibited unique properties. Concretely, it showed AIE perfor-
mance at the keto emission location in the aggregation state due
to the restraint of the TICT and activated ESIPT processes, while
only enol emission was observed in the solution owing to the high
energy barrier. This aggregation-assisted ESIPT process and the
behavior of showing AIE properties at the position of keto emission
are synchronous and consistent.
Subsequently, the optical responses of ASSI-Leu toward LAP in an
aqueous solution containing 10% DMSO at 37 1C were system-
atically investigated. After incubation with LAP, the absorbance of
ASSI-Leu sharply enhanced (Fig. S3, ESI†). And as anticipated, a
significant ratiometric fluorescence signal (I554 nm/I417 nm) was
observed in the titration experiment of the enzymatic reaction
(Fig. 2A). With the increase of LAP concentration, the fluorescence
Scheme 2 Schematic illustration of probe ASSI-Leu for detecting LAP
and the performance in living cells.
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while the peak of m/z 522.15 ([M-H]+) corresponding to ASSI-OH
dominantly appeared. This unequivocally supported the LAP-
triggered cleavage of the probe causing the release of the free
fluorophore. Also, according to the distance-dependent photo-
nic properties of the fluorophore, dynamic light scattering
(DLS) was performed, and the results manifested the formation
of nanoparticles with an average size of 200 nm after incuba-
tion with LAP at 37 1C for 30 min (Fig. S9, ESI†). These results
confirmed the cleavage of ASSI-Leu by LAP and the aggregation
of the released ASSI-OH. Moreover, these were further indicated
by inhibitor experiments (Fig. S4, ESI†).
From the above observations, ASSI-Leu exhibited good spe-
cificity and sensitivity for LAP in aqueous solution and there-
fore is expected to realize the ratiometric imaging of
endogenous LAP. First, HepG2 cells (a human hepatoma cell
line with overexpressed LAP) were selected to evaluate the
biocompatibility via cell viability assay. The results showed that
the cells retained high cell viability upon 24 h incubation with
various concentrations of the probe (2.5–40 mM) (Fig. S10, ESI†),
indicating the negligible cytotoxicity of ASSI-Leu. Next, the
Fig. 2 (A) Emission spectra of ASSI-Leu (5 mM) after 40 min upon treat-
ment with increasing concentrations of LAP (0–0.25 U mLꢀ1) in aqueous
solution (10% DMSO, v/v) at 37 1C. (B) Plots of fluorescence ratio (I554 nm
/
I
417 nm) versus LAP concentration. (C) Linear fitting curve of fluorescence
ratio (I554 nm/I417 nm) toward the concentration of LAP from 0–0.05 U mLꢀ1
.
(D) Time dependence of I554 nm/I417 nm for ASSI-Leu in the presence (red)
and absence (gray) of LAP. lex = 365 nm.
intensity at 417 nm decreased progressively, and a red-shifted potential to detect endogenous LAP was investigated in HepG2
fluorescence peak at 554 nm emerged concurrently, which corre- cells. After being treated with ASSI-Leu for 30 min, bright yellow
sponded to the evolution of ASSI-OH. Moreover, the fluorescence fluorescence could be captured in the 490–560 nm emission
intensity at 554 nm demonstrated more than a 7-fold increase upon channel upon excitation at 405 nm (Fig. S11, ESI†). This
reaction with 0.1 U mLꢀ1 LAP for 1 h. However, this obvious benefited from the aggregation of released ASSI-OH by the
fluorescence enhancement could be suppressed in the presence of enzymatic transformation in living cells, which allows in situ
the LAP inhibitor bestatin, substantiating that the signal is gener- LAP imaging due to the synergistic effect of AIE and the
ated by LAP catalysis in a specific manner (Fig. S4, ESI†). In addition, activated ESIPT process. Notably, the fluorescence signal
the fluorescence signal ratio (I554 nm/I417 nm) was observed to increase increased in a dose-dependent manner. Then, the time-course
gradually, presenting evident concentration dependence (Fig. 2B). assay was conducted to study the real-time ratiometric imaging
Notably, it exhibited good linearity with LAP in 0–0.05 U mLꢀ1 ability. As illustrated in Fig. 3, when incubated with
(R2 = 0.997) and the detection limit was determined to be ASSI-Leu for 10 min, channel one gave strong blue fluorescence
2.983 ꢁ 10ꢀ3 U mLꢀ1, indicative of high sensitivity (Fig. 2C). Hence, (410 nm–470 nm) while channel two showed weak yellow
the curve was promising to provide convenience for LAP detection. fluorescence. The measured fluorescence ratio from channel
Concurrently, time-dependent fluorescence measurement was two to channel one was about 0.79. With the prolongation of
carried out for exploring the kinetics of the hydrolysis reaction by incubation, the fluorescence intensity of channel one gradually
LAP. A concomitant ratiometric fluorescence signal increase was decreased accompanied by a remarkable fluorescence enhance-
observed in the initial stage and took about 40 min to reach the ment in channel two, and the ratio improved to be 2.47 after 1 h
plateau, while the signal without LAP remained unchanged (Fig. 2D of incubation.
and Fig. S5, ESI†). Clearly, it implicated the potential of ASSI-Leu
Subsequently, HepG2 cells were exposed to bestatin for
for fast and effective detection of LAP. Then, the selectivity experi- 1 hour and then treated with ASSI-Leu for another 30 min;
ment was conducted by incubating the probe with a series of the fluorescence intensity in channel two was significantly
important biomolecules. As illustrated in Fig. S6 (ESI†), none of suppressed and the fluorescence ratio decreased to 0.72 (Fig.
these species could trigger the change of the ratiometric fluores- S12, ESI†). The above results undeniably demonstrated the
cence signal, indicating the satisfactory specificity to LAP over all ability of the probe to real-time track endogenous LAP, which
other interferents, which is valuable in biological applications. granted it the potential for clinicopathological analysis. Nota-
Next, we investigated the pH effect on the probe. The fluorescence bly, it was found that the fluorescence signal was mainly
signal of the probe itself remained consistently unchanged in the located at a certain site rather than being evenly distributed,
test range, while the reaction system could achieve the maximum which prompted us to further carry out a subcellular colocaliza-
fluorescence ratio enhancement in neutral media, indicating that tion study. As shown in Fig. S13 (ESI†), there was a substantial
ASSI-Leu can be used for the superior detection of LAP under overlap between the yellow fluorescence of the probe and the
physiological conditions (Fig. S7, ESI†).
red fluorescence of LysoTracker red. The change of emission
To confirm the activation effect of LAP on ASSI-Leu, we intensity in the linear region of interest (ROI) exhibited a
injected the mixture upon incubation with LAP to an electron synchronous trend, and the Pearson’s correlation coefficient
spray ionization-mass spectrometer (ESI-MS). As shown in was 0.67 (Fig. S14 and S15 ESI†). This result suggested that the
Fig. S8 (ESI†), the peaks of m/z 740.29 ([M-H]+) were quite weak, probe may mainly accumulate in lysosomes.
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by employing an alternative synthesis strategy. Owing to the
incorporation of the HBT skeleton, the fluorophore exhibited
AIE-assisted ESIPT behaviour, and its unique optical properties
were rationalized in detail by theoretical analysis. To the best of
our knowledge, the obtained probe ASSI-Leu is the first imple-
ment of combining the AIE mechanism with ESIPT character in
ratiometric tracking of LAP accurately. The probe offered large
Stokes’ shift, appreciable biocompatibility, and high sensitivity
and selectivity. Remarkably, it has been successfully used in
Fig. 3 (A) Confocal fluorescence images of HepG2 cells incubated with hepatoma cells and drug-induced liver injury zebrafish models,
ASSI-Leu (10 mM) for 10 min, 30 min and 60 min. Channel one was
collected from 410 nm to 470 nm (blue); channel two was collected from
indicating the potential for facilitating diagnosis in complex
biosystems. It is worth mentioning that the novel building
block ASSI-OH and its synthesis strategy are anticipated to
490 nm to 560 nm (yellow), lex = 405 nm. The images of the ratio were
generated with ImageJ software. (B) The relative fluorescence intensity
stimulate more fluorescent probes bearing multi-functional
sites as the potential biological vehicle. Related studies are
being progressively carried out as well in our laboratory.
The authors gratefully acknowledge the financial support
from the National Natural Science Foundation of China
(81971678 and 81671756), the Science and Technology Founda-
tion of Hunan Province (2020SK3019, 2019SK2211 and
2019GK5012), and the Research Innovation Fund for Postgrad-
uates of Central South University (2018zzts877).
ratio from channel one to channel two (Y/B). Data represent the mean
standard error (n = 3). Scale bar is 10 mm.
Encouraged by the above results, we further employed the
zebrafish model to validate the feasibility of ASSI-Leu to image
LAP in vivo. It was documented that the LAP level is significantly
associated with the drug induced damage in the case of overdose
cisplatin treatment.18 To strengthen this matter, 7 day old zebrafish
were pre-treated with 0.1 mg mLꢀ1 cisplatin for 12 h, and then loaded
with the probe for confocal imaging. The results clearly showed a
strongly emerged yellow signal in channel two as time prolongs and
the synchronous attenuation of the blue signal in channel one
Conflicts of interest
(Fig. 4A–C), which exhibited a high signal-to-noise ratio. Fig. 4E There are no conflicts to declare.
illustrates the change of fluorescence ratio between channel two and
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Taken together, we can reasonably conclude that ASSI-Leu possessed
high selectivity for the specific tracking of LAP in living organisms,
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Fig. 4 Confocal fluorescence images for visualizing endogenous LAP in
the cisplatin-induced zebrafish acute liver injury model using ASSI-Leu.
The zebrafish models were pretreated with 0.1 mg mLꢀ1 cisplatin and then
incubated with ASSI-Leu (10 mM) for 10 min (A), 30 min (B) and 60 min (C),
respectively. (D) The pretreated zebrafish models were incubated with
bestatin (100 mM) for 12 h and then cultured with ASSI-Leu (10 mM). (E)
Fluorescence ratios (Y/B) of the corresponding fluorescence images.
Channel one: 410–470 nm; channel two: 490–560 nm, lex = 405 nm.
Data represent the mean standard error (n = 3). Scale bar is 100 mm.
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