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Further bioorthogonal click reaction with alkyne functionalized TPEBAI allows for cancer cell imaging. EXPERIMENTAL SECTION. Synthesis of cRGD-S-Ac3Ma...
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Dual-responsive Metabolic Precursor and Light-up AIEgen for Cancer Cell Bioorthogonal Labelling and Precise Ablation Fang Hu, Youyong Yuan, Wenbo Wu, Duo Mao, and Bin Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00547 • Publication Date (Web): 30 Apr 2018 Downloaded from http://pubs.acs.org on May 1, 2018

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Analytical Chemistry

Dual-responsive Metabolic Precursor and Light-up AIEgen for Cancer Cell Bioorthogonal Labelling and Precise Ablation Fang Hu,a,‡ Youyong Yuan,a,b,‡ Wenbo Wu,a,‡ Duo Mao,a and Bin Liua,*

a

Department Chemical and Biomolecular Engineering, National University of Singapore, 4

Engineering b

Drive

4,

Singapore

117585,

E-mail:

[email protected]

Nanotheranostics Laboratory, School of Medicine, Institutes for Life Sciences, South China

University of Technology, Guangzhou, China 510006

ABSTRACT

Metabolic glycoengineering of unnatural glycans with bioorthogonal chemical groups and subsequent click reaction with fluorescent probes have been widely used in monitoring various bioprocesses. Herein, we developed a dual-responsive metabolic precursor that could specifically generate unnatural glycans with azide groups on the membrane of targeted cancer cells with high selectivity. Moreover, a water-soluble fluorescent light-up probe with aggregation-induced

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emission (AIE) characteristic was synthesized, which turned its fluorescence on upon click reaction with azide groups on cancer cell surface, enabling special cancer cell imaging with low background signal. Furthermore, the probe can generate 1O2 upon light irradiation, endowing its dual role as an imaging and therapeutic agent for cancer cells. Therefore, the concepts of cancer cell specific metabolic precursor cRGD-S-Ac3ManNAz and AIE light-up probe are promising in bioorthogonal labeling and cancer-specific imaging and therapy.

KEYWORDS: dual-responsive metabolic precursor, cancer cell imaging, bioorthogonal labeling, light-up probe, cancer cell ablation

INTRODUCTION

Metabolic glycoengineering is a useful biological technique to study the complex bioprocess in living systems, which has been applied to image cellular proteins,1 glycosylation and phospholipid uptake2 and targeted drug delivery.3 Typically, an unnatural metabolic precursor functionalized with a bioorthogonal chemical group such as alkyne or azide was first incorporated into target cells through metabolic glycoengineering. Subsequently, a bioorthogonal click reaction with tailor-designed fluorescent probe was employed to enable visualization of specific biological process.4 In general, metabolic glycoengineering has the intrinsic drawback that the unnatural metabolic precursors for glycan metabolism do not have any specificity against different cell type.5 Recently, some newly designed metabolic precursors exhibited cancer cell selectivity due to the introduction of specific protecting groups, which could only be cleaved upon light irradiation or in the presence of certain overexpressed enzymes.3 However, the light

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Analytical Chemistry

manipulation is not ideal for selective labelling and the overexpressed enzymes still exist in normal tissues. To further improve the cell selectivity, a dual-activated metabolic precursor is highly desirable. On the other hand, fluorescent light-up probes

with emission turn-on upon bioorthogonal

reaction are very attractive due to their high signal output and low background signals.6 Several elegant bioorthogonal probes with click-induced fluorescence turn-on have been developed for bioimaging.7,8 The most popular azide,8 tetrazine,7 based probes rely on photo-induced electron transfer (PeT) to quench the probe fluorescence, which upon bioorthogonal reaction to turn-on the emission. These probes are largely limited to blue and green emitters, which are less suitable for biological applications. In addition, they require cyclooctyne and norbornene based metabolic precursors to label the cells, which are much more difficult to realize than that for azide functionalized ones.9 To address this issue, optically active molecules with intrinsically low fluorescence, but bright emission after click reaction are highly desirable. In this regard, luminogens with aggregation-induced emission (AIE) characteristics (AIEgens) are excellent candidates. AIEgens are non-emissive in molecularly dissolved state but emit strongly in aggregate state due to the restriction of intramolecular motions and prohibition of energy dissipation via non-radiative channels.10,11 Their fluorecence turn-on process can be conveniently achieved upon restriction of molecular motions without any quenching group.12 In addition, some AIEgens are also outstanding photosensitizers (PSs), endowing them dual roles as turn-on imaging and therapeutic agents.13 It is important to note that metabolic labelling of other PSs is not able to achieve wash-free fluorescence turn-on imaging so far.14 Therefore, AIEgens have great potentials to be developed into fluorescence turn-on probes for cancer cell imaging and photodynamic ablation upon bioorthogonal reaction.

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Among all bioorthogonal reactions, azide-alkyne Huisgen cycloaddition is one of the most popular strategies for bioorthogonal labeling. It has a high reaction rate constant in physiological conditions, but without any cross-reaction with complex components in living cells. In this contribution, we firstly developed a dual-responsive metabolic precursor cRGD-S-Ac3ManNAz that could specifically generate unnatural glycans with azide groups on the membrane of cancer cells. As shown in Scheme 1, the metabolic precursor cRGD-S-Ac3ManNAz contains a tumor targeting ligand cRGD that can be specifically recognized by αvβ3 integrin overexpressed cancer cells (1st target). In addition, the disulfide group in cRGD-S-Ac3ManNAz is responsive to the intracellular glutathione (2nd target), which has higher concentrations in cancer cells than that in normal cells.15 The metabolic capability of cRGD-S-Ac3ManNAz is initially inhibited, but can be restored after αvβ3 integrin recognised endocytosis and glutathione (GSH) activation to release free Ac3ManNAz to involve the metabolic glycoengineering. By taking advantage of the dualselectivity of recognition and activation, free Ac3ManNAz can be selectively released and unnatural glycans can be expressed on the surface of target cancer cells. Subsequently, alkyne-functionalized water-soluble bioorthogonal turn-on probe (TPEBAI) with photosensitizing activity was designed and synthesized. The hydrophilic sulfate groups endow TPEBAI with good water solubility and yield low fluorescence in aqueous media. The alkyne groups can react with cRGD-S-Ac3ManNAz for bioorthogonal labeling. Upon Huisgen cycloaddition between alkyne groups in TPEBAI and azide groups expressed on cancer cells, the fluorescence of TPEBAI can be immediately turned on due to the restriction of molecular motions,16 which enables sensitive cancer cell imaging under wash-free condition. Furthermore, TPEBAI can generate singlet oxygen (1O2) upon light irradiation,17 endowing its dual function as a contrast and therapeutic agent.

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Scheme 1. Schematic illustration of the cellular uptake of metabolic precursor (cRGD-SAc3ManNAz) by αvβ3 integrin overexpressed cancer cells through receptor mediated endocytosis and the following intracellular reduction of cRGD-S-Ac3ManNAz to release free Ac3ManNAz, which can be further involved in metabolic glycoengineering for exogenous unnatural azidefunctionalized glycan on cancer cell membrane. Further bioorthogonal click reaction with alkyne functionalized TPEBAI allows for cancer cell imaging.

EXPERIMENTAL SECTION

Synthesis of cRGD-S-Ac3ManNAz. Compound 1 (48.4 mg, 0.1 mmol) and Ac3ManNAz (38.8 mg, 0.1 mmol) were dissolved in dry dichloromethane (10 mL) at 0 oC. And then pyridine (79 mg, 1.0 mmol) was added. The mixture was stirred under room temperature for 6 h. After that, cRGD-NH2 (90.4 mg, 1.5 mmol) was added into the mixture at 0 oC and stirred for further 3 h

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under room temperature. After removal of the organic solvent, the crude product was purified by HPLC. Eluent: 0–15–20 min, 40 % B–60 % B–60 % B (A: H2O containing 0.1 % CF3COOH, B: acetonitrile containing 0.1 % CF3COOH). cRGD-S-Ac3ManNAz was obtained as viscous oil (16.0 mg, yield 13.3%). The purification was verified by HPLC. HR ESI-MS, m/z: [M+H]+ calcd 1198.4139, found 1198.4146. Synthesis of TPBAl. TPEB (36.3 mg, 0.05 mmol), 1,3-propanesultone (122.1 mg, 1.0 mmol), and cesium carbonate (162.9 mg, 0.5 mol) was dissolved in DMF (1 mL). The mixture was stirred at 30 oC for 2 days. After reaction, deionized water (1 mL) was added to dissolve all the solid, and make sure the solution was clear. And then the product was purifed by HPLC (Eluent: 0–15–20 min, 20 % B–100 % B–20 % B (A: H2O containing 0.1 % CF3COOH, B: acetonitrile containing 0.1 % CF3COOH)) to get an orange solid (yield = 40.5 %). 1H NMR (400 MHz, MeOD, 298K) δ (TMS, ppm): 7.84-7.75 (m, 2H), 7.60 (d, J = 15.6 Hz, 1H), 7.54-7.49 (m, 3H), 7.18-7.09 (m, 5H), 7.06-7.02 (m, 2H), 7.01-6.90 (m, 6H), 6.82-6.71 (m, 6H), 6.63 (s, 1H), 4.664.60 (m, 4H), 4.11 (t, J = 6.2 Hz, 4H), 3.67-3.61 (m, 2H), 2.98 (t, J = 8.0 Hz, 4H), 2.28-2.16 (m, 4H). High resolution (HR) ESI-MS, m/z: [M-Cs]2-/2 calcd 484.1224, found 484.1224. General protocol for selective cell-surface labeling of azido glycans on live cells and perform the click reaction with TPEBAI. MDA-MB-231, MCF-7 and 293T cells were seeded at 1 × 105 cells/mL on 8-well chambers (Thermo Scientific) and grown at 37 °C and 5% CO2 in DMEM medium with different concentrations of cRGD-S-Ac4ManNAz for 2 h and washed with 1× PBS, then the cells were incubated with fresh DMEM medium for 2 days. The medium was then gently aspirated, and the cells were washed twice with 1× PBS buffer. The cell nuclei were live stained with Hoechst 33342 for 5 min, following the standard protocols of the manufacturer (Life Technologies). CuSO4 (25 µM) and TPA (125 µM) were added to the wells in 1× PBS

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Analytical Chemistry

buffer containing TPEBAI (10 µM). A freshly prepared stock solution of sodium ascorbate (100 mM) was added to establish a final concentration of 2.5 mM with gentle shaking. After incubation at room temperature for different time durations, the cells were imaged immediately by a confocal microscope (CLSM, Zeiss LSM 410, Jena, Germany).

RESULTS AND DISCUSSION

Synthesis of cRGD-S-Ac3ManNAz and TPEBAI. The synthetic routes to cRGD-SAc3ManNAz and TPEBAI are shown in Scheme 2. Briefly, cRGD-S-Ac3ManNAz was obtained by reaction between compound 118 and Ac3ManNAz in the presence of pyridine, which was followed by further reaction with cRGD-NH2 (cyclo(Arg-Gly-Asp-D-Phe-Lys). The target compound was purified by preparative high-performance liquid chromatography (HPLC). TPEBAI was prepared on the basis of the iconic AIEgen of tetraphenylethene (TPE). Following the deprotection of methoxy groups in compound 3, two alkynyl groups were introduced to yield compound 4, which was further reacted with 3,5-dihydroxybenzaldehyde to generate TPEB. The target compound TPEBAI was obtained by introducing two negatively charged sulfonate groups to TPEB to endow its water-solubility. Chemical structures of target compounds were well confirmed by NMR, HPLC and high resolution mass spectroscopy (Figures S1-S4) to reveal their right structures with high purity.

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Synthesis of cRGD-S-Ac3ManNAz O

O 2N

O O

N3 S

O

O

S

O

O

O

NO 2

OH

pyridine

OAc OAc

AcO

N3 O

NH

O

S

AcO

O O

OAc OAc

NO2

2

O O

H 2N

S

O

O

Ac3ManNAz

1 NH

O

O NH

N H

NH

N H

OH

O NH HN

H2 N

O

O HN

pyridine

2

O

N3 O

NH

H N

O

S

cRGD

O

OAc OAc

AcO

O

S

O

O

O

cRGD-NH 2

cRGD-S-Ac3ManNAz

Synthesis of TPEBAI HO O

O

O

OH

O

1. HBr, HAc, reflux

CHO

2. 3-bromopropyne, K 2 CO 3, actone, 70

3

5

piperidine, isopropyl alcohol, 60 oC

oC

4 NC

O

CN

NC

O

O O S O5

CN

O

O

SO 3Cs +

OH

O Cs 2CO3 , DMF, 30 oC OH

TPEB NC

Cs + O

TPEBAI

CN

NC

SO3-

CN

Scheme 2. Synthetic route to the metabolic precursor cRGD-S-Ac3ManNAz and the alykne functionalized turn-on probe TPEBAI. GSH response of cRGD-S-Ac3ManNAz. For the metabolic precursor cRGD-S-Ac3ManNAz, it is essential to ensure that it can be easily transformed into Ac3ManNAz inside cancer cells. In this work, the GSH responsiveness of cRGD-S-Ac3ManNAz in phosphate buffered saline (PBS) buffer was first studied by HPLC and Mass spectroscopy. As shown in Figure 1A, cRGD-SAc3ManNAz shows one single peak at 13.6 min in HPLC. The broad peak of Ac3ManNAz should be due to the presence of isomers.19,20 After treatment of cRGD-S-Ac3ManNAz with GSH, the peak at 13.6 min disappeared completely, while two new peaks with rentention time of 8.9 and 9.8 min corresponding to cRGD and Ac3ManNAz appeared, indicating that the disulfate bond was successfully cleaved by GSH. The formed Ac3ManNAz shows a right mass-to-charge

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Analytical Chemistry

ratio (m/z) of 388.03 (Figure 1B). Based on these results, we confirm that cRGD-S-Ac3ManNAz is responsive to GSH to generate the metabolite Ac3ManNAz.

Figure 1. (A) HPLC analysis of Ac3ManNAz (100 µM, a), cRGD-NH2 (100 µM, b), and cRGDS-Ac3ManNAz (100 µM) before (c) and after (d) treatment with GSH (500 µM) for 5 h. (B) Mass spectrum of cRGD-S-Ac3ManNAz after treatment with GSH. (C) The fluorescence and (D) average hydrodynamic diameters change of TPEBAI (15 µM) before and after the mimicked bioorthogonal reaction with 1,3,5-tris(azidomethyl)benzene (TAB, 10 µM) catalyzed by CuSO4, TPA and sodium ascorbate (SA). Photophysical properties of TPEBAI. The optical properties of TPEBAI are shown in Figure S5. It has a board absorption from 400 nm to 600 nm. The emission maximum occurs at 625 nm, which is very weak in water (benign solvent). The emission intensity gradually enhances along

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with the increasing THF (poor solvent) fractions, indicating that TPEBAI shows AIE characteristics. 9,10-Anthracenediyl-bis(methylene)dimalonic acid (ABDA) was then used to measure the 1O2 generation ability of TPEBAI, and the 1O2 quantum yield was calculated to be 39% using Rose Bengal as the reference. As TPEBAI is almost non-emissive in PBS, its fluorescence turn-on process is mimicked in aquous solution by click polymerization between TPEBAI (two alkyne groups) and 1,3,5tris(azidomethyl)benzene (TAB) (three azide groups). The catalyst system includes Cu2+, tris(3hydroxypropyl triazolylmethyl) amine (TPA) and sodium ascorbate (SA). As shown in Figure 1C, the fluorescence of TPEBAI increases significantly only in the presence of Cu2+, TPA and SA, confirming that the fluorescence change of TPEBAI is due to the click reaction with TAB. The underlying mechanism for fluorescence turn-on was studied by dynamic light scattering (DLS). As shown in Figure 1D, the formation of aggregates was confirmed by alterations of the hydrodynamic diameters of TPEBAI, being around 10 nm for TPEBAI to ∼250 nm after the mimicked bioorthogonal reaction. These results confirm that the fluorescence change of TPEBAI is due to aggregate formation upon click reaction, which restricts the intramolecular motions. Optimisation of bioorthogonal labelling. To study the bioorthogonal click reaction between TPEBAI and the azide groups on the living cell membranes, MDA-MB-231 human breast cancer cells were pre-treated with Ac3ManNAz for 48 h to ensure that it can be incorporated into glycan on cell membrane via metabolic process. Then, click reaction with TPEBAI was carried out with click catalyst system containing 25 µM Cu2+, 125 µM TPA and 2.5 mM SA. The click reaction was monitored by studying the fluorescence change of TPEBAI. As can be seen in Figure 2A, red fluorescent signals can only be observed on the surface of Ac3ManNAz pre-treated MDAMB-231 cells after incubation with TPEBAI when Cu2+, TPA and SA were all presented.

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Analytical Chemistry

Moreover, the fluorescence from TPEBAI intensifies gradually with the click reaction process and reaches a plateau within five minutes (Figure 2B). This, together with the extremey low background fluorescene confirm that the probe can be turned-on upon click reaction. When MDA-MB-231 cells are treated with different concentrations of TPEBAI or Ac3ManNAz, the fluorescent signals show a dose-dependent type (Figure S6), which is potential for therapeutic agent dosage controll. These results confirm that Ac3ManNAz can be taken up by cancer cells and generate azide groups on the cell membrane. The exogenously generated azide groups could be specifically visualized by labeling with TPEBAI through a bioorthogonal reaction with turnon fluorescence.

Figure 2. (A) Images of Ac3ManNAz (10 µM) pretreated MDA-MB-231 cells upon addition of TPEBAI (A1), TPEBAI with SA (A2), TPEBAI with Cu2+/TPA (A3) or TPEBAI with Cu2+/TPA and SA (A4) ([TPEBAI] = 10 µM). (B) The reaction time dependent fluorescence images of TPEBAI labeled MDA-MB-231 cells pretreated with Ac3ManNAz. ([Cu2+] = 25 µM, [TPA] = 125 µM, [SA] = 2.5 mM). Nucleus is stained with Hoechst 33342, Ex = 405 nm, Em = 430-470 nm; TPEBAI, Ex = 405 nm, Em > 650 nm.

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Selective bioorthogonal labelling of cancer cells. By incorporating a target ligand (cRGD moiety) as the first target and cancer cell oversecreted stimulus (GSH responsive) as the second target to a metabolic precussor, the dual-responsvie process of cRGD-S-Ac3ManNAz is expected to offer a new generation of metabolic precussor with high cancer cell selectivety. To verify the cancer cell specific generation of azide groups on cell membrane by metabolic glycoengineering, we selected several cancer or normal cells to conduct the experiments. MDA-MB-231 cells are selected as model cancer cells with overexpressed αvβ3 integrin. MCF-7 cancer cells with low αvβ3 integrin expression and 293T normal cells are chosen as controls, whose αvβ3 integrin expression levels were confirmed by FITC modified anti-integrin αvβ3 antibody (Figure S7). It was anticipated that the metabolic precussor cRGD-S-Ac3ManNAz could be prefercially accumulated in αvβ3 integrin overexpressed cancer cells through recognization endocytosis and subsequently release free Ac3ManNAz through GSH induced activation, leading to selective generation of chemical receptors by metabolic glycoengineering. The selectivity was directly visualized by the click reaction between the azide groups on the membrane of cancer cells and TPEBAI in the cell culture media. As shown in Figures 3A-3C, when the cells are treated with free Ac3ManNAz, all three cell lines present similarly strong red fluorescence, indicating that free Ac3ManNAz shows no selectivity to different cell lines. However, when these three cell lines are pretreated with cRGD-S-Ac3ManNAz and subsequently labeled with TPEBAI, the fluorescence intensity is very different. The fluorescence intensity in MDA-MB-231 cells is the strongest, which should be attributed to their high expression of αvβ3 integrin and high intracellular GSH concentration. The fluorescent signal is much weaker in MCF-7 cells (Figure 3E) due to their low expression of αvβ3 integrin. The fluorescence intensity of 293T cells is the lowest, owing to their lower level of αvβ3 integrin expression and low intracellular GSH

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Analytical Chemistry

concentration. These results indicate that the generation of chemical receptors by pretreatment with cRGD-S-Ac3ManNAz is strongly correlated with the expression level of αvβ3 integrin and intracellular GSH concentration in targeted cancer cells. Therefore, we can conclude that cRGDS-Ac3ManNAz could generate the metabolite Ac3ManNAz by the dual selection proccess of αvβ3 integrin receptor mediated endocytosis and intracellular GSH activation, and the exogenously generated chemical receptors on the cell surface could be specifically visualized by labeling with TPEBAI through a bioorthogonal click reaction.

Figure 3. Confocal images of TPEBAI (10 µM) labeled MDA-MB-231 (A, D), MCF-7 tumor cells (B, E), compared with normal 293T cells (C, F) pretreated with free Ac3ManNAz (A-C) or cRGD-S-Ac3ManNAz (D-F) for 2 h, followed by washing with 1× PBS and incubation in fresh DMEM medium for 2 days. Nucleus is stained with Hoechst 33342, Ex = 405 nm, Em = 430-470 nm; TPEBAI, Ex = 405 nm, Em > 650 nm.

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To further confirm this receptor mediated endocytosis of cRGD-S-Ac3ManNAz, free cRGD is utilized to block the αvβ3 integrin on MDA-MB-231 cells. We found that the fluorescent signals of cRGD pretreated MDA-MB-231 cells (Figure 4A) were obviousely weak compared to the ones without cRGD pretreatment (Figure 4B), along with the inrease of concentration of free cRGD, the average fluorescence intensities gradually decreased (Figure S8). But for the group pretreated with DDD (Asp-Asp-Asp), which is a negative ligand for αvβ3 integrin, no decreased fluorescence was observed. Similarly, the fluorescent signals in MDA-MB-231 cells were also much weaker upon pretreatment with GSH inhibitor, L-buthionine-sulfoximine (BSO) (Figure 4C).21 All these results indicate that the release of Ac3ManNAz from cRGD-S-Ac3ManNAz is indeed selectively activated by receptor mediated endocytosis and GSH reduction. In addition, after treating the cells with tris(2-carboxyethyl)-phosphine (TCEP) to chemically quench the azide groups, the click reaction efficiency of TPEBAI dramatically decreases which is evidenced by the dim red fluorescence observed (Figure 4D).

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Figure 4. Confocal images of TPEBAI (10 µM) labeled MDA-MB-231 cells pretreated with cRGD-S-Ac3ManNAz (25 µM) for 2 h, followed by wash with 1× PBS and incubated in fresh DMEM medium for 2 days. The cells were pretreated with (A) free cRGD, (B) control, (C) DLbuthionine-sulfoximine (BSO) or (D) tris(2-carboxyethyl)-phosphine (TCEP). Blue fluorescence (nucleus live dyed with Hoechst 33342, Ex: 405 nm, Em: 430-470 nm); red fluorescence (TPEBAI, Ex: 405 nm, Em: > 650 nm). Selective cancer cell ablation. The intracellular 1O2 generation of TPEBAI upon irradiation was measured by using 2', 7'-dichlorodihydrofluorescein diacetate (DCF-DA) as a indicator. DCFDA is weakly emissive but can be oxidized by 1O2 to yield highly emissive dichlorofluorescein (DCF) in cells. As can be seen in Figures 5A and 5C, when the MDA-MB-231 and 293T cells were pretreated with free Ac3ManNAz, strong green fluorescence was observed in both MDAMB-231 and 293T cells when the cell surfaces were clicked with TPEBAI followed by light irradiation. However, when the cells were pretreated with cRGD-S-Ac3ManNAz, strong green fluorescence is only observed in MDA-MB-231 cells (Figure 5B) while the fluorescence in 293T cells is very dim (Figure 5D), indicating negligible 1O2 production for the latter.

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Figure 5. Intracellular 1O2 generation from TPEBAI upon light irradiation detection by DCF-DA as the indicator in MDA-MB-231 (A, B) and 293T (C, D) cells pretreated with Ac3ManNAz (A, C) or cRGD-S-Ac3ManNAz (B, D). DCF: Ex = 488 nm; Em = 505−525 nm). Cytotoxicity of TPEBAI (10 µM) labeled MDA-MB-231 and 293T cells pretreated with different concentrations of Ac3ManNAz (E) or cRGD-S-Ac3ManNAz (F) upon light irradiation (100 mW cm-2, 30 s). (The error bars represent the standard deviation.) As TPEBAI can generate 1O2 upon light irradiation, it is potentially suitable for photodynamic cancer cell ablation, which requires high toxicity to cancer cells upon light exposure but low cytotoxicity to normal cells.22-23 The cytotoxicity of TPEBAI to MDA-MB-231 and 293T cells pretreated with different concentrations of Ac3ManNAz or cRGD-S-Ac3ManNAz, followed by white light illumination was studied by MTT assay. As shown in Figure 5E, when the cells were treated with free Ac3ManNAz, both MDA-MB-231 and 293T cells showed TPEBAI

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concentration-dependent cytotoxicity, indicating that free Ac3ManNAz cannot distinguish cancer cells from normal ones to realize selective ablation. However, when the cells were pretreated with cRGD-S-Ac3ManNAz, the cytotoxicity of the two cell lines is very different (Figure 5F). It shows high photo-cytotoxicity to MDA-MB-231 cells with a half-maximal inhibitory concentration (IC50) value of 9.5 µM. In contrast, only slight toxicity is observed for 293T cells under the same conditions. These results indicate that the treatment with metabolic precursor cRGD-S-Ac3ManNAz and subsequent bioorthogonal click reaction with TPEBAI can selectively label cancer cells and precisely induce cancer cell death with light irradiation. Meanwhile, no dark toxicity was observed, indicating good biocompatibility (Figure S9).

CONCLUSIONS

In summary, we have developed a dual-targeted metabolic precursor, cRGD-S-Ac3ManNAz, that can selectively generate unnatural glycans with azide groups on the membrane of αvβ3 integrin overexpressed MDA-MB-231 cancer cells. Subsequently, a bioorthogonal fluorescent probe of TPEBAI with almost no fluorescence in aqueous media but shows light-up fluorescence upon bioorthogonal click reaction with azide groups on the cell membrane was developed for selective cancer cell image. Due to the strong 1O2 production of TPEBAI, it also shows additional function of image-guided cancer cell ablation upon light irradiation. In contrast, normal cells with low expression of αvβ3 integrin and intracellular GSH concentration showed minimum TPEBAI labeling and negligible toxicity under light irradiation. The perfect integration of dualtargeted metabolic precursor with fluorescence turn-on AIEgen photosensitizer opens up a new perspective to fulfil selective imaging and precise ablation of cancer cells. ASSOCIATED CONTENT

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Supporting Information Materials and methods; 1H NMR spectrum and HR ESI-MS spectrum of TPEBAI; HPLC spectrum and HR ESI-MS spectrum of cRGD-S-Ac3ManNAz; absorption of TPEBAI, PL of TPEBAI and TPEB, 1O2 generation detection; concentration-dependent bioorthogonal labeling of MDA-MB-231 cell; integrin αvβ3 expression level detection; bioorthogonal labeling of cRGD or DDD pre-treated MDA-MB-231 cells; dose-dependent dark cytotoxicity upon bioorthogonal labeling. The Supporting Information is available free of charge on the ACS Publications website. AUTHOR INFORMATION Corresponding Author * Email: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We thank Dr. Guangxue Feng for integrin αvβ3 expression level detection. We thank Singapore NRF CRP (R279-000-483-281), NRF Investigatorship (R279-000-444-281), National University

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of Singapore (R279-000-482-133) and the Institute of Materials Research and Engineering of Singapore (IMRE/14-8P1110) for financial support. REFERENCES (1) Gehringer, M.; Laufer, S. A. Angew. Chem. Int. Ed. 2017, 56, 15504–15505. (2) Laughlin, S. T.; Baskin, J. M.; Amacher, S. L.; Bertozzi, C. R. Science 2008, 320, 664-667. (3) Wang, H.; Wang, R. B.; Cai, K. M.; He, H.; Liu, Y.; Yen, J.; Wang, Z. Y.; Xu, M.; Sun, Y. W.; Zhou, X.; Yin, Q.; Tang, L.; Dobrucki, I. T.; Dobrucki, L. W.; Chaney, E. J.; Boppart, S. A.; Fan, T. M.; Lezmi, S.; Chen, X. S.; Yin, L. C., et al. Nat. Chem. Biol. 2017, 13, 415-424. (4) Lang, K.; Chin, J. W. Chem. Rev. 2014, 114, 4764-4806. (5) Borrmann, A.; van Hest, J. C. M. Chem. Sci. 2014, 5, 2123-2134. (6) Chan, J.; Dodani, S. C.; Chang, C. J. Nat. Chem. 2012, 4, 973-984. (7) Devaraj, N. K.; Weissleder, R. Acc. Chem. Res. 2011, 44, 816-827. (8) Shieh, P.; Dien, V. T.; Beahm, B. J.; Castellano, J. M.; Wyss-Coray, T.; Bertozzi, C. R. J. Am. Chem. Soc. 2015, 137, 7145-7151. (9) Baskin, J. M.; Bertozzi, C. R. Mol. Inform. 2007, 26, 1211-1219. (10) Mei, J.; Leung, N. L. C.; Kwok, R. T. K.; Lam, J. W. Y.; Tang, B. Z. Chem. Rev. 2015, 115, 11718-11940. (11) Feng, G.; Kwok, R. T.; Tang, B. Z.; Liu, B. Appl. Phys. Rev. 2017, 4, 021307. (12) Liang, J.; Tang, B.; Liu, B. Chem. Soc. Rev. 2015, 44, 2798-2811. (13) Mao, D.; Hu, F.; Ji, S.; Wu, W.; Ding, D.; Kong, D.; Liu, B. Adv. Mater. 2018, DOI: 10.1002/adma.201706831. (14) Lee, S.; Koo, H.; Na, J. H.; Han, S. J.; Min, H. S.; Lee, S. J.; Kim, S. H.; Yun, S. H.; Jeong, S. Y.; Kwon, I. C. ACS nano 2014, 8, 2048-2063.

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