Highly Specific near-Infrared Fluorescent Probe for the Real-Time


Highly Specific near-Infrared Fluorescent Probe for the Real-Time...

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A highly specific near-infrared fluorescent probe for the real-time detection of #-glucuronidase in various living cells and animals Yinzhu Jin, Xiangge Tian, Lingling Jin, Yonglei Cui, Tao Liu, Zhenlong Yu, Xiaokui Huo, JingNan Cui, Cheng-Peng Sun, Chao Wang, Jing Ning, Baojing Zhang, Lei Feng, and Xiaochi Ma Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04813 • Publication Date (Web): 05 Feb 2018 Downloaded from http://pubs.acs.org on February 5, 2018

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Analytical Chemistry

A highly specific near-infrared fluorescent probe for the realtime detection of β-glucuronidase in various living cells and animals Yinzhu Jin,†§ Xiangge Tian, †§ Lingling Jin,† Yonglei Cui, † Tao Liu,‡ Zhenlong Yu,† Xiaokui Huo, † Jingnan Cui, ‡ Chengpeng Sun, † Chao Wang, † Jing Ning, † Baojing Zhang, † Lei Feng,*†, ‡ Xiaochi Ma,* † †

College of Pharmacy, Academy of Integrative Medicine, Dalian Medical University, Lvshun South Road No 9, Dalian 116044, China



State Key Laboratory of Fine Chemicals, Dalian University of Technology, Ganjingzi District, Linggong Road No.2, Dalian 116024, China Correspondence Author: X.C. Ma and Lei Feng (E-mail: [email protected] and [email protected]). Tel.: +86 411 86110419, Fax: +8641186110408

ABSTRACT: β-glucuronidase (GLU) is an important biomarker for primary cancers and intestinal metabolism of drugs or endogenous substances; however, an effective optical probe for near-infrared (NIR) monitoring in vivo is still lacking. Herein, we design an enzyme-activatable off-on NIR fluorescent probe, HC-glu, based on a hemicyanine keleton, which is conjugated with a D-glucuronic acid residue via a glycosidic bond, for the fluorescent quantification and trapping of endogenous GLU activity in vitro and in vivo. The newly developed NIR probe exhibited prominent features including prominent selectivity, high sensitivity and ultrahigh imaging resolution. It has been successfully used to detect and image endogenous GLU in various hepatoma carcinoma cells, tumor tissues and tumor-bearing mouse models, for cancer diagnosis and therapy. Moreover, it could detect the in vivo activity of GLU in the intestinal tracts of animals including mice and zebrafish, where GLU performs a vital biological function and is mainly distributed. It could also evaluate real intestinal distribution and real-time variations of GLU in development and growth, all of which are very helpful to guide rational drug use in the clinic. Our results fully demonstrated that HC-glu may serve as a promising tool for evaluating the biological function and process of GLU in living systems. INTRODUCTION asthma during antigenic exposure when GLU is released from alveolar macrophages14. It has been reported that β-glucuronidase (GLU), as a member of the lysosomal glycosidase family, is mainly involved in the degradation high levels of GLU activity in intestinal tracts would result in greater risk in colorectal cancer with higher enzyme of glucuronic acid from glycosaminoglycans in levels than in healthy groups15,16. Additionally, it is also prokaryotic and eukaryotic organisms1. It is mainly reported that the GLU levels in human are significantly distributed in the cell membranes and extracellular elevated in numerous physiological diseases such as matrices of cancerous tissues, for the metastasis, invasion, cholelithiasis17, rheumatoid arthritis 18, hepatic disorders 19, apoptosis and proliferation of tumor cells2,3. It is also found that the GLU enzyme especially in the human urinary tract infection 20, renal diseases 21 and so on. Therefore, it is of great significance to develop a rapid and intestinal tract, is mostly responsible for the specific fluorescent probe for real-time endogenous GLU detoxification of reactive metabolites, which are related to detection in various disease diagnoses and therapies. a variety of diseases and the development of cancer4-6. For example, a considerable amount of bacterial GLU activity In recent years, some fluorescent probes have been is present in the intestinal tract, and can release the developed for monitoring the enzymatic activities of GLU aglycone that can in turn be reabsorbed and enter a cycle, in tissues or cell preparations, with the advantages of realthrough enterohepatic circulation. Therefore, bacterial time sampling capability and high sensitivity22-24. GLU activity in the intestinal tract exhibited various However, a common limitation for the present GLU significant biological functions closely related to the probes is wide application in living systems because the activity and toxicology of drugs and endogenous emission wavelengths are less than 650 nm or superior substances. fluorescence backgrounds, which results in matrix Additionally, in previous investigations, increased GLU activity compared to normal tissue has also been reported in a wide range of malignancies, such as liver cancer 7,8, colon carcinoma9, prostate cancer10, and renal carcinoma 8 particularly in necrotic regions11. Hence, GLU has been regarded as a tumor biomarker12,13, and the quantitative detection of GLU has become effective for the early-stage diagnosis, real-time elevating of tumor size and even clinical therapies. For example, local cleavage of glucuronides can occur in the lungs of patients with

interference by autofluorescence, poor tissue penetration, and misleading assessment of the real function of a target enzyme in vivo. Therefore, it is very necessary to develop a tool for detecting GLU bioactivity within in vivo models. As far as we known, near-infrared (NIR) fluorescent probes are more desirable due to excellent tissue penetration, low autofluorescence and low biological damage25-30. However, among the current methods available for GLU assay, none of the off-on NIR fluorescent probes for the in vivo monitoring of GLU have

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been reported. Therefore, it is imperative to develop a novel NIR fluorescent probe for highly selective and realtime evaluating the endogenous GLU in complex living biological samples.

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at room temperature under a nitrogen atmosphere for 48 h. After that, the solvent was removed under reduced pressure, the residue was dissolved in 10 mL of CH3OH, and K2CO3 (139 mg, 1 mmol) was added. This mixture was stirred at room temperature for 3 h and, then evaporated, and the resulting residue was purified by HPLC (utilizing a utilizing gradient from 0.03% trifluoroacetic acid (TFA) water (A) and acetonitrile (B). The gradient conditions were as follows: 0.0– 5.0 min, 20% B; 5.0– 25.0 min, 20%100% B; and 25.0– 30.0 min, 100% B. The flow rate was set at 1.0 mL/min to afford 20.5 mg of indigo solid (Yield 28.6%) The structures of these compounds were confirmed by 1H-, 13C-NMR and HRMS spectra (Figures S5-S7), and the data are shown as follows.

In the present work, according to the catalytic properties of GLU, HC-glu has been designed and synthesized as an NIR probe for detecting the bioactivity of GLU in complex biological samples with high selectivity and low cytotoxicity in vivo. Furthermore, HCglu could be successfully applied for in situ and in vivo visualization of GLU activity in a tumor-bearing mouse mode. Additionally, HC-glu has been successfully used for the real-time measurement of GLU activity in intestinal tracts of living mice and zebrafish.

HC-glu: 1H-NMR (500 MHz, DMSO-d6) δ 8.64 (d, J = 15.1 Hz, 1H), 7.74 (d, J = 7.1 Hz, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.54 (dd, J = 13.7, 8.1 Hz, 2H), 7.47 (dd, J = 16.4, 8.9 Hz, 2H), 7.17 (d, J = 2.0 Hz, 1H), 7.06 (dd, J = 8.6, 2.3 Hz, 1H), 6.61 (d, J = 15.0 Hz, 1H), 5.32 (d, J = 4.3 Hz, 1H), 5.05 (s, 1H), 5.00 (d, J = 7.2 Hz, 1H), 4.39 (t, J = 7.3 Hz, 2H), 3.52 – 3.48 (m, 1H), 3.20 – 3.15 (m, 1H), 2.73 (d, J = 5.7 Hz, 2H), 2.68 (t, J = 5.9 Hz, 2H), 2.54 (s, 1H), 1.90 – 1.80 (m, 4H), 1.76 (d, J = 6.4 Hz, 6H), 1.25 – 1.19 (m, 1H), and 0.98 (d, J = 7.4 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ 177.99, 170.74, 160.35, 160.22, 153.32, 145.08, 142.34, 141.09, 132.54, 128.77, 127.28, 127.06, 122.72, 116.11, 114.27, 113.86, 113.23, 104.63, 103.57, 100.40, 76.58, 73.59, 73.02, 72.05, 63.07, 50.51, 46.10, 28.29, 27.48, 27.36, 23.39, 20.88,19.85, and 10.94. HRMS (ESI positive) was calculated for C34H38NO8+ [M– I]+ and was found at 588.2595. All data are shown in Figures S5-S7.

EXPERIMENTAL SECTION

Materials and instruments. Lysozyme (Ls), αGlucosidase (α-Glc), N-acetyl glucosaminidase (NAG), Carbonic anhydrase (Cas), Proteinase K (Pak), Carboxylesterases (CE) including CE1 and CE2, βGalactosidase (β-Gla), β-Glucosidase (β-Glc), GLU from bovine liver, E. coli, E. coli K 12, E. coli Type IX-A , and E. coli Type VII-A were all obtained from Sigma-Aldrich (St. Louis, MO, USA). Bovine serum albumin (BSA), Glutamate, Glutamine, Glycine, Serine, Glutathione, Arginine, Lysine, Cysteine, Tryptophan, Glucose, Tyrosine, Bilirubin, and Myristic acid were purchased from ShanghaiYuan Ye (Shanghai, China). Bispnitrophenylphosphate (BNPP), α-galactose, loperamide (LPA) and baicalin were purchased from J&K Chemicals. LoVo, HepG2 and Hep3B cell lines were purchased from ATCC (Manassas, VA). All fluorescence tests were performed on a Synergy Neo Microplate Reader (Bio-Tek). NMR spectra were acquired using a Bruker 501. Accurate mass detection was measured on a Fourier transform ion cyclotron resonance mass spectrometer (LTQ Orbitrap XL). The supernatants were analyzed in HPLC-UV equipment (Waters e2695 equipped with 2998 PDA Detector). All other reagents and solvents used were of the highest grade commercially available.

Extinction coefficient and quantum yield. Briefly, in order to determine the fluorescence quantum yield (ф) of the HC (ф = 0.43, ε = 100225 M-1·cm-1) and HC-glu (ф = 0.03, ε =57015 M-1·cm-1) complex, OX1 in ethanol (ф= 0.15) was used as a standard.32 The values were calculated according to the following equation (1): фx/фs= [As/Ax][nx2/ns2][Dx/Ds]…………………………….(1) s: standard, x: sample, A: absorbance at the excitation wavelength, n: refractive index, and D:area under the fluorescence spectra on an energy scale. The extinction coefficient of HC and HC-glu were calculated by followed equation (2):

NMR spectra were recorded on a Bruker spectrometer using TMS as internal standard for 1H-NMR (400 MHz) and 13C-NMR (100 MHz). High-resolution mass data were measured on a Fourier transform ion cyclotron resonance mass spectrometer (LTQ Orbitrap XL). The UV–vis spectra and fluorescence emission/excitation spectra were measured on a Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek). A Waters2695 system equipped with a PDA detector was used to determine HC-glu and its metabolites.

A=εCL…………………………………………………………………….....(2) A: Absorption, ε: extinction coefficient, C: concentration of solution, and L: solution level thickness. General procedure for monitoring GLU activity. Based on the following procedure, all the evaluations of GLU activity were performed in the system of 100 mM potassium phosphate buffer (pH 7.0) with a final incubation volume of 0.2 mL. The system contained potassium phosphate buffer, and 2 μL of stock solution of HC-glu and GLU was mixed. After incubation at 37 °C for 30 min, the reaction was terminated by the addition of 100 μL of ice acetonitrile. The mixtures were then centrifuged at 20,000×g for 20 min at 4°C. Then, the further fluorescence analysis was conducted for the aliquots of supernatant. Control incubations without enzyme or without substrate were used to ensure that

Synthesis and structural characterization of compounds. Hemicyanine (HC) was synthesized as previously described (Figures S1-3).31 Next, the synthetic route of HC-glu is shown in Figure S4. Briefly, HC (54 mg, 0.1 mmol), bromo-2,3,4-tri-O-acetyl-α-Dglucopyranuronic acid methyl ester (200 mg, 0.5 mmol) and K2CO3 (28 mg, 0.2 mmol) were dissolved in dry CH3CN (10 mL) in a 25 mL round bottom flask and stirred

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Analytical Chemistry metabolite formation was enzyme-dependent. All assays were performed in duplicate.

medium) for 30 min at 37 °C in a 5% CO2 incubator. Then, the living cells were co-incubated with HC-glu (50 μM) at37 °C for another 60 min. Then, the residual HC-glu was removed and washed three times using PBS, and the living cells were imaged on the confocal microscope (Leica TCS SP8). Cytotoxicity was assessed by the CCK8 assay (Roche Diagnosis, Indianapolis, IN). Briefly, HepG2, Hep3B and LoVo cells were seeded in a 96-well plate at a concentration of 6×103 mL−1 in DMEM medium with 10% FBS and incubated at 37 ◦C in a 5% CO2 incubator overnight. Then, these cells were transferred to fresh medium containing various concentrations of HC-glu (0, 1, 10, 50 and 100 μM). After 24 h incubation, the growth of cells was measured at 450 nm using the MultiModeMicroplate Reader (Biotek). Cell viability in the absence of HC-glu was considered 100%. Fluorescent imaging of cancer tissues and intestinal tracts in mice. The nude mice, 6–8 weeks old, used in this study were purchased from SPF Laboratory Animal Center of Dalian Medical University (DMU). The animal experiments were carried out, on the basis of the Care and Use of Laboratory Animals guidelines by the Animal Care and Ethical Committee of DMU. Mice were inoculated with hepatoma cells (HepG2) and colon cancer cells (LoVo) for 3 weeks until the cells became tumor; then, one group of nude mice was given an injection of HC-glu (0.15 mg/kg) into the tumor of HepG2 and the tumor of LoVo. Another group of nude mice was given intragastric administration of HC-glu (4.2 mg/kg). The fluorescence responses ex-vivo was detected among different organs including: (1) liver, (2) small intestine, (3) spleen, (4) lung, (5) heart, (6) kidney and (7) brain, were also carried out. Sample images were recorded at various reaction times by a NightOWL II LB983 small animal in vivo imaging system equipped with a Charge Coupled Device (CCD) camera, using an excitation laser of 630 nm and an emission filter of 670 - 730 nm. Confocal fluorescence imaging of cancer tissue slices. Frozen tissue slices were prepared from the cancer tissues of HepG2 and LoVo bearing nude mice, and para cancer tissue. For the experiment of fluorescence imaging of HCglu, the slice was first incubated with HC-glu (10 μM) for 0.5 h at 37°C, followed by washing three times with PBS. For immunofluorescence staining, the slices were washed in phosphate-buffered saline (PBS) and then permeabilized with 0.3% TritonX-100 for 20 min and blocked with 5% bovine serum albumin (BSA) in PBS for 60 min. Antibodies against GLU were added to the sample in the 1% blocking solution and incubated overnight at 4°C. Following three 10min washes with PBS, isothiocyanate- and rhodamine-conjugated secondary antibodies were added in 1% blocking solutions and incubated for 1 h. Subsequently, the stained samples, including HC-glu and immunofluorescence staining, were mounted with 4, 6-diamidino-2-phenylindole (DAPI)-containing Vecta shield solution (Vector Laboratories Inc.) to countersta in cell nuclei. After five additional 5 min washes, samples were examined with a Leica SP8DIVE confocal microscope at individual rational

The selectivity assays. To explore the selective sensing ability of HC-glu, the interferences of different glycosidase and, hydrolase enzymes, common metal ions and amino acids were tested in our standard incubation system. Briefly, HC-glu was incubated with series of enzymes including Ls, α-Glc, NAG, PaK, CE1b, CE1c, CE2, BSA, β-Gla, β-Glc and GLU at a final protein concentration of 50 μg/mL in the standard incubation system. Additionally, the influence of common metal ions including Ca2+, Zn2+, Mn2+, Mg2+, Fe3+, Cu2+, K+, Ni2+, Ba2+, Na+, and Sn4+ and endogenous substances such as Glutamic acid, Glutamine, Glycine, Serine, Glutathione, Arginine, Lysine, Cysteine, Tryptophan, Tyrosine, Glucose, bilirubin, and myristic acid on the probe reaction were also evaluated with the final concentration of 10 μM. All assays were conducted at 37°C for 30 min. To further confirm that the fluorescence changes were selectively mediated by GLU, HC-glu was incubated with GLU in the presence or absence of inhibitors including BNPP (CEs general inhibitor), LPA (CE2 selective inhibitor), α-Galactose and baicalin33,34; the inhibitors were used with the final concentration of 50 μM, 200 μM and 600 μM, respectively. The IC50 values were determined by incubating HC-glu (10 μM) with varying concentrations of the specific inhibitor agent (10-500 μM for baicalin). The inhibition percentage was calculated by comparing it to control samples that were incubated under identical conditions, without the presence of the inhibitors. Data were fit to log (inhibitor) vs. normalized response—variable slope equation using GraphPad Prism 6. Kinetic study. For estimating the hydrolysis kinetic parameters, HC-glu (0-600 μM) was incubated with different sources of GLU from bovine liver, E. coli, E. coli K 12, E. coli Type IX-A, and E. coli Type VII-A for 30min. Incubation times and protein concentrations were selected within the linear interval of metabolite turnover rates. To calculate the kinetic constants, the Michaelis– Menten model (eq.3) and Hill model (eq.4) were used: v=

௏೘ೌೣ ×[ௌ] ௄೘ ା[ௌ] ௏

v=ሺ௄೘ೌೣ ೙

…………………………………………………………………….(3)

×[ௌ]೙

…………………………………………………………………(4)

೙ ೘ ା[ௌ] ሻ

Vmax represents the maximum rate and Km is the substrate concentration at the half-maximal rate. Kinetic constants were obtained using GraphPad Prism 6and are reported as the mean ± SE35. Cell culture and fluorescence imaging. HepG2 and Hep3B cells were cultured in DMEM medium, LoVo cells were grown in RPMI-1640 medium and both were supplemented with fetal bovine serum (FBS) of 10%in an atmosphere of 5% CO2 at 37 °C and incubated overnight. The cells were seeded on 20-mm glass polylysine-coated confocal cell culture dishes. After washing twice with FBS free culture medium, the adherent cells were incubated with/without 100 μM of baicalin (prepared in FBS-free

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718 nm for HC during the pH range from 7.0 - 12.0 upon excitation at 670 nm, and the high GLU activity remained over a pH range from 4.0 – 7.0. As expected, the fluorescence intensity of HC-glu toward GLU exhibited an enzyme and time linearity (Figure 1c and d, Figure S11), which was not influenced by adding common metallic ions or endogenous substances such as amino acids and bilirubin in biological systems (Figure S12). These results fully exhibited that HC-glu could serve as a turn-on NIR fluorescence probe for rapidly detecting GLU in aqueous solution under physiological conditions.

fluorescent conditions (ex: 633 nm, em: 690 -750 nm). Animal care and handing procedures were reviewed and approved by the Animal Care and Use Committee of Dalian Medical University. Fluorescence imaging of GLU in living zebrafish grown. To demonstrate further applications of HC-glu in living organisms, the NIR fluorescence imaging of zebrafish was carried out. The zebrafish were transferred to 90 mm Petri dishes filled with E3 embryo media. The components of the culture medium (E3M) with pH 7.5 included 15 mM of NaCl, 0.5 mM of KCl, 1 mM of MgSO4, 1 mM of CaCl2, 0.15 mM of KH2PO4, 0.05 mM of Na2HPO4, 0.7 mM of NaHCO336,37. The zebrafish were kept in the incubator at 28 °C replenishing fresh E3 Meach day. Before imaging, the 1-, 3-, 5- and 7-day-old zebrafish were transferred to 24-well microplates and incubated with 10 μM of HC-glu (containing 0.2% DMSO in E3M) for 3 h at 28°C. Afterward the zebrafish were washed with E3M to remove the remaining HC-glu and subjected to imaging according to the literature protocol38. Molecular modeling and docking studies. Molecular docking studies were performed to obtain the binding mode of HC-glu with GLU. The X-ray crystal structure of human GLU was used (PDB code 1BHG) for docking. The known substrate molecule p-nitrophenyl-β-Dglucuronide was docked into the active site of GLU first to adjust the docking parameters. Then, the HC-glu was docked into the active site to probe the interaction mode. The two-dimensional diagrams of the protein-ligand interaction were analyzed using the web-based online tool, Pose View (http://poseview.zbh.unihamburg.de/poseview)39.

Scheme 1 The chemical structure and the recognition mechanism of HC-glu for GLU detection.

RESULTS AND DISCUSSIONS Spectroscopic response of HC-glu to hemicyanine. The change of the spectroscopic properties of HC-glu after incubating with GLU were displayed in Figure 1a and b. HC-glu itself gave a strong absorption peak at 600 nm. Our probe HC-glu had a long-term stability PBS at 37 °C without any autohydrolysis. Due to the introduction of glucuronic acid as a polar group, our probe is watersoluble with the maximum concentration of 600 µM in PBS containing DMSO not exceeding 1% (v/v). After adding GLU, the maximum absorption peak is red shifted to approximately 670 nm. And a significant color variation (Figure 1) was observed from light blue to cyan, following with a remarkable fluorescence increase (approximately 40-fold) at 718 nm. The absorption and fluorescence spectra were identical with those of HC, which implies the release of HC mediated by GLU from the reaction system. Furthermore, HPLC analyses and electrospray ionization (ESI) mass spectra of the reaction products further verified the probe hydrolysis to yield HC. (Figures S8 and S9). The pH value is a key factor in the photophysical properties of the sensing probe and the reaction activity of the target enzymes. We subsequently evaluated the pH dependence of the emission profiles of HC and the biotransformation rate of GLU, as shown in Figure S10. There was excellent fluorescence response at

Figure 1. Absorption (a) and fluorescence emission spectra (b) of HC-glu (10 μM ) before (red) and after (blue) incubating with GLU (bovine liver) at 37°C for 30 min in buffer (pH = 7.0); (c, d) Fluorescence spectra and linear relationship of HC-glu (10 μM) upon addition of increasing concentrations of GLU (0-50 μg/mL) in buffer– acetonitrile (v/v = 2:1, pH = 7.0) at 37 °C for 30 min. Selectivity of HC-glu. In order to detect GLU activity in complex biological systems, the selectivity of HC-glu was systemically investigated among different hydrolases. The significant fluorescence enhancements of HC-glu (10 μM) after adding GLU (50 μg/mL) were observed, while no significant fluorescence change was detected in the presence of several hydrolases including Ls, α-Glc, β-Glc, NAG, β-Gla, Cas, PaK, CE1b, CE1c, CE2 and BSA. (Figure 2a) Recently, an NIR fluorescent probe was developed which could sensitively and rapidly detect the endogenous β-Gla in living cells.27 However, our results indicated that β-Gla had no catalytic activity for our probe HC-glu. To further investigate the selectivity of

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Analytical Chemistry HC-glu toward GLU, the chemical inhibition study was performed by adding different selective inhibitors of hydrolases (BNPP, LPA, α-galactose and Baicalin) in our standard incubation system. Baicalin as a selective inhibitor of GLU, could significantly inhibit the deglycosylation of HC-glu mediated by GLU with an IC50 value of 200 μM, (Figures 2b and S13), while extremely weak influence were observed in the reactions catalyzed by GLU in the presence of other selective inhibitors of hydrolases. These results fully demonstrated that GLU has a selective catalytic reaction for our probe HC-glu, and it could be used for sensing the real bioactivity of GLU in complex biological systems. In the process of biological analysis, the accurate measurement of enzymatic parameters was vital in the quantitative applications for activity-sensitize probes. Therefore, we also determined the kinetic parameters of GLU by using HC-glu in different enzyme sources from bovine liver, E. coli, E. coli K 12, E. coli Type IX-A, and E. coli Type VII-A which are given in Figures S14-15 and Table S1. The kinetics data was processed by the Hill and Michaelis–Menten equations, the kinetic parameters both Km and Vmax are calculated and listed in Table S1. Additionally, the interaction between human GLU and HC-glu were illustrated by a docking study. It embedded in the active site of human GLU neatly. According to the best docking conformation, the hydroxyl groups of HCglu at the position formed hydrogen bond interaction with the carboxyl oxygen of Glu 451 (2.62 Å). Its hydroxyl group at the C-5 position was involved in hydrogen bonding, with the side chain carboxyl oxygen of Asp 207. The high binding constant of HC-glu toward GLU could be explained by these two strong hydrogen bonds. The predictial binding mode of HC-glu is shown in Figure S16. Additionally, His 385, Tyr 508, Arg 600, and Lys 606 could also stabilize the binding of HC-glu in the active site of GLU (Figure S16),which is similar to other substrates of GLU.40,41 The docking studies elucidate the molecular basis of the underlying interaction between HC-glu and GLU.

concentrations up to 100 μM by CCK8 assay (Figure S17). Then, as shown in Figures 3 and S18, following incubation of HepG2, LoVo and Hep3B cells with HC-glu (10 μM) at 37 °C for 60 min, these cells were each subjected to image capture excited at 633 nm. In addition, these cells exhibited a specific signal in red channels (690 - 750 nm), which indicates the excellent cell permeability of our probe and its biotransformation catalyzed by endogenous GLU. Furthermore, under the same imaging condition, Baicalin as an inhibitor for pretreating HepG2 cells exhibited a sharply suppressed fluorescence compared to the control group, as described in Figure 3. This fluorescence variation can be attributed to catalysis by intracellular GLU; in other words, HC-glu had an ultrahigh imaging capability for the endogenous GLU. Similarly, HC-glu also exhibited a good real-time imaging application in both LoVo and Hep3B cells, respectively. Furthermore, after the confocal fluorescence images in living LoVo cells from 5 – 35 min (Figure S19), our results indicated that the probe HC-glu could be applied to imaging intracellular GLU for long time periods without photobleaching. All types of cells untreated with HC-glu manifested no fluorescent background under the same imaging conditions.

Figure 2. (a) Fluorescence responses of HC-glu (10 μM) toward various species of enzymes with the concentration of 50 μg/mL at 37 °C for 30 min. (b) Chemical inhibitions of HC-glu hydrolyzation by BNPP, LPA, α-Galactose and Baicalin in GLU (bovine liver) incubation systems.

Figure 3. Confocal fluorescence images of HepG2 cells (af), and LoVo cells (g-i) treated with HC-glu (50 μM) at 37 °C for 60 min; bright and fluorescent fields of HepG2 (a, d) and LoVo cells (g, j) untreated with HC-glu; bright and fluorescent fields of HepG2 cells (b, e) and LoVo cells (h, k) treated with HC-glu; bright and fluorescent images from HepG2 cells (c, f) and LoVo cells (i, l) pretreated with Baicalin (100 μM)and stained with HC-glu.

Fluorescence imaging of HC-glu in living cells. We further explored whether HC-glu could be applied to the imaging of endogenous GLU activity in living cells. First, HC-glu did not show the cytotoxicity at various

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In vivo imaging of mouse. Near-infrared light usually has low background autofluorescence interference from animal tissue and causes little damage to the biosample; thus, many NIR fluorescent probes with excellent photophysical properties were developed for in vivo imaging42,43. Thus, investigation of the potential application of HC-glu for the imaging of living animal models including mouse and zebrafish was performed. The fluorescence imaging property of HC-glu was investigated with a living animal imaging instrument. First, the tumor imaging ability in vivo of HC-glu was examined using expressed GLU HepG2 and LoVo cells bearing xenograft models. A tumor-bearing mouse xenograft model was established by subcutaneous inoculation of HepG2 and LoVo cells in both flanks of nude mice. Both HepG2 and LoVo had been proven to express some GLU. As shown in Figure 4, the accumulation of intratumorally injected HC-glu (0.15 mg/kg) and PBS (20 μL) for 30 min in both tumors was clearly visualized under whole-body in vivo fluorescence imaging with emission at 670 -730 nm, which is extremely practical for cancer diagnosis in clinic. Our results indicated that a significant fluorescence was observed rapidly in the tumor regions of HepG2 and LoVo.

Moreover, this strong fluorescence may remain for at least 2h, which might provide some convenience for cancer treatments. Meanwhile, the tumor region of LoVo tumor-bearing mice displayed a much weaker fluorescence than that of HepG2 (Figure 4), consistent with the results of imaging in cell lines, which proved that the fluorescence signal of tumor tissue dependent on the GLU activity. On the condition of our present results, we continued to investigate the capability of HC-glu in imaging GLU bioactivity in para-carcinoma (normal) and tumor tissue area, which is vital for cancer pathological diagnosis. Fresh tumor tissues of HepG2 and LoVo were collected from tumor-bearing models of mice. The tumor sections with a thickness of 10 μm, were prepared by a cryostat microtome, and these samples were subjected to HC-glu, immunohistochemical and Hematoxylin-Eosin staining. Our results strongly indicated that the remarkable fluorescence image mediated by GLU is well consistent with the immunohistochemical results by Masson and Sirius Red staining (Figure 4). Taken together, these findings suggested that HC-glu is a useful approach for imaging GLU activity in tissue sections based on their distinctive GLU activity, which can be used for surgical diagnosis and therapy of various cancers.

Figure 4. In vivo imaging of GLU in tumor-bearing nude mice. (a and b) In vivo imaging of GLU activity in tumor (LoVo and HepG2) bearing nude mice after tumor injection of HC-glu (0.15 mg/kg) for 2h (a. bright field; b. fluorescence field); (c-e) The fluorescence microscope image of HC-glu for the tissue slices of normal (paracarcinoma tissue), LoVo and HepG2 tumor tissues, respectively; (f-h) The immunofluorescence images of HC-glu bioactive in normal (para-carcinoma tissue, f) and tumor samples (LoVo and HepG2). (i-k) Hematoxylin-Eosin staining in normal (i) and tumor tissues (LoVo and HepG2), respectively.

Figure 5. In vivo real-time image of GLU in intestinal tracts after oral administration of HC-glu. (a-f) The fluorescence variation of GLU in intestinal tracts at 0min, 15 min, 30 min, 45 min, 60 min and 120 min. g. After administration of HC-glu at 60 min, the distribution of intestinal GLU (a: duodenum; b: jejunum; c: cecum); h. The fluorescent imaging of ex vivo-dissected organs after administration at 60 min (1: liver; 2: heart; 3: lung; 4: spleen; 5: kidney; 6: intestine; and 7: muscles). Additionally, a considerable amount of bacterial GLU activity is present in the small intestines, which can

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Analytical Chemistry release the aglycone that can in turn be reabsorbed and enter a cycle called “enterohepatic circulation”, an important physiological process in humans. This process usually results in delayed elimination of drug molecules to induce small intestinal mucosal injuries in clinic44,45. For example, GLU of intestinal brush border can convert conjugated bilirubin to the unconjugated form for reabsorption in the gut. Therefore, it is very necessary to evaluate the real-time GLU activity and its distribution within the small intestines in vivo. Herein, HC-glu as an NIR probe was also investigated to monitor the real time intestinal GLU bioactivity in both mouse and zebrafish models for in vivo fluorescence endoscopy of endogenous GLU. Inspired by the distinct “light-up” NIR emission, we next examined its capability for real-time in vivo visualization of GLU activity in the intestinal tracts of mice. As shown in Figure 5, after 15 min post-oral administration of HC-glu, (4.2 mg/kg), the NIR fluorescence signal was already clearly visible in intestinal tracts, which suggested its rapid activation. The intensity gradually increased and reached a maximum level after 2h post-administration. In sharp comparison, for the unpretreated normal nude mouse, there is minimal lightup fluorescence under the same conditions of imaging. Our findings clearly indicated that the GLU mainly distributed in the middle and tail end of small intestine (Figure 5). After oral administration of HC-glu, the realtime bioactivity of GLU could be clearly detected in the intestinal tract rather than other organs including liver, kidney, spleen, lung, heart and muscles, all of which suggested that our probe could be successfully used to distinguish the intestinal tract from other tissues mentioned above, without fluorescent interference from other organs (Figure 5). Fluorescence imaging variation of GLU in living zebrafish growth. As a common animal model, zebrafish have been applied extensively for studying molecular mechanism of embryonic and tissue development, and even human health study including cancer, cardiovascular disease, and drug discovery. Its genome has been fully sequenced, and it has well-understood, easily observable and testable developmental behaviors. However, the GLU in zebrafish especially its change during their individual growth development had never been illustrated. Based on the preferred fluorescent characteristic of HC-glu, it was used to visualize the endogenous GLU following the growth development in zebrafish. In the present study, after zebrafish growing in E3 embryo media for different times from 1 to 7 days, the GLU in zebrafish were evaluated using our probe HC-glu by fluorescence imaging. As shown in Figure 6 and Figure S20, the 1-, 3-, 5- and 7-day-oldzebrafish were treated with HC-glu, and the control group of zebrafish shows no fluorescent background in NIR region. Interestingly, after treatment of HC-glu, negligible fluorescent intensity was detected in 1-day-old (embryos) and 3-day-old zebrafish was almost undetectable, but zebrafish for 5- and 7-day-old zebrafish exhibited stronger fluorescence intensity in the intestinal tract after 3 h treatment of HC-glu (10μM).

with the growth and development of zebrafish. Meanwhile, the fluorescence intensity induced by GLU is tissue selective in zebrafish and, especially in the intestinal tract, indicated prominently strong fluorescence, consistent with the fluorescent imaging of HC-glu in mice. These results indicated that GLU is a key enzyme, mainly distributed in the intestinal tract. Furthermore, its expression level in the intestinal tract is closely related to the growth period of zebrafish, following improvements in intestinal function. This realtime observation in vivo has not been reported to the best of our knowledge.

Figure 6. Fluorescence detection of GLU in living zebrafish at different periods from 1 - 7 days. (a, d, g, j) Fluorescent fields of zebrafish incubated with HC-glu (10 μM) for 3 h. (b, e, h, k) The bright fields of the above corresponding samples. (c, f, i, l) The merged images of bright and fluorescence fields for the different growth periods of zebrafish. Scale bar, 500 μm.

CONCLUSION In summary, a novel NIR probe HC-glu for the prominently selective and sensitive monitoring of endogenous GLU in living cells and in vivo has been developed for the first time. In vitro experiments suggested that HC-glu can be applied for fluorescent visualization for GLU in living cells without cytotoxicity and autofluorescence and preferred cell penetration. More importantly, HC-glu has been successfully applied to the imaging of real-time endogenous GLU in tumorbearing mice and the intestinal tracts of mice and zebrafish models. Our results demonstrated that HC-glu could be served as a useful tool for investigating the biological function and changing process of GLU in living systems.

All of this implied that intestinal GLU would increase

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ASSOCIATED CONTENT AUTHOR INFORMATION *Corresponding Author E-mail addresses: [email protected] (L. Feng) & [email protected] (X. C. Ma)

Author Contributions §

These authors contributed equally.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT The authors thank the National Natural Science Foundation of China (No. 81622047, 81473334 and 21572029), Dalian Outstanding Youth Science and Technology Talent (2015J12JH201), distinguished professor of Liaoning Province for financial support and State Key Laboratory of Fine Chemicals (KF1603).

Supporting Information The detailed experimental procedures, structural characterization of compounds and some imaging experiments. This material is available free of charge via the Internet at http://.

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Analytical Chemistry For TOC only: :

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