NIR fluorescent DCPO glucose analogues and their application in cancer cell imaging

Abstract

Given the increased glucose uptake in cancer cells than normal cells, near-infrared (NIR) fluorescent glucose analogues have been previously synthesized and applied in cancer cell imaging. However, most NIR dyes usually have one or more charge in their structures, which may cause low cell membrane permeability and hamper their application in cell imaging. Here we report the synthesis and characterization of a series of DCPO-conjugated glucose analogues (N0–N4), which have no charge in their structures and have different lengths of the spacer arm. Experiments in different cancer cell lines showed the uptake of N0–N4 was dependent on the protein levels of GLUT-1. The distance between the dyes and glucose was adjusted by the length of PEG. Of these five glucose analogues, the length of the linker in N2 which contains a diethylene glycol was the most appropriate spacer arm, a longer or shorter linker exhibited reduced cellular uptake efficiency. Moreover, the uptake of DCPO-conjugated glucose analogues could be inhibited by phloretin, a GLUT-1 inhibitor or competitively inhibited by unlabeled D-glucose. Therefore, our study has reported a novel type of NIR-conjugated glucose analogues, whose cell permeability ensured the potential application for cancer cell bioimaging in the NIR region. We also demonstrated, for the first time, that the length of the linker between the dyes and glucose was also an important factor that will affect the delivery efficiency of the glucose analogues to cells.

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Introduction

Malignant cancer cells exhibit an altered metabolism, which includes an increase in cellular glycolysis, known as the Warburg effect. As a result, cancer cells are usually characterized with an increase in glucose uptake when compared to normal cells.¹ The transportation of glucose into cells is via glucose transporters (GLUTs), which are widely overexpressed in human cancers.² This characteristic has been successfully used in clinical practice known as 2-deoxy-2-(¹⁸F)fluoro-D-glucose (¹⁸F-FDG), a radiolabeled glucose analog. ¹⁸F-FDG has been used to visualize tumors and their metastases in vivo with positron emission tomography (PET). Besides the radiolabeled tracer, ways can be used to cancer screening.^3–5 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-2-deoxyglucose (2-NBDG, Fig. 1A), the first fluorescent tracer, has also been used for imaging and assessing glucose metabolism in some types of tumors in vivo.⁶ However, 2-NBDG has several drawbacks; including that it is only applicable in a non-physiological sugar-depleted environment and its tissue penetration is poor.

Fig. 1 Some of the previous glucose probes and the structure of DCPO.

Given the advantages of near-infrared (NIR) fluorescence, such as deep tissue penetration, minimum photo damage to biological samples, sensitively and noninvasively imaging, NIR glucose analogues have attracted great attention over the past decade.^7–10 By conjugation of 2-deoxyglucose or glucose to NIR dyes, several NIR glucose analogues have been synthesized and reported, including Cy5.5-2DG (Fig. 1), IRDye 800 CW 2-DG, CyNE 2-DG and Cy3-linked O-1-glycosylated glucose (Fig. 1).^11–13 However, these fluorochrome dyes have one or more charge in their structures, thus leading to the charged properties, which may cause low cell membrane permeability and hamper their application in cell imaging. DCPO (Fig. 1), a new type of NIR dye based on dicyanomethylene-4H-pyran (DCM), has no charge in its structure.^14–16 Moreover, it is more photostable than most of other NIR dyes and widely used in bioimaging.^17–20 Therefore, DCPO was chosen in our experiment to synthesize the novel fluorescence labeled glucose analogues.

The length of the conjugates' linker has been demonstrated to affect the efficiency and selectivity of the conjugates. Take folate conjugates as examples, the one with the shorter linker exhibited higher photo cytotoxicity and selectivity toward folate receptors positive cells (Fig. 2).²¹ In the case of glucose analogues, the fluorescent dyes were directly or indirectly conjugated with glucose (Fig. 1), however, to the best of our knowledge, whether the distance between dyes and glucose would affect the cellular uptake and targeting efficiency of glucose conjugates has never been studied. Therefore, different lengths of linker were taken into consideration when we designed DCPO-labeled glucose analogues. We expected to find out the best length of the spacer arm and the most promising glucose analogue. Glycosylation of protected glucose with propargylated alcohols and deprotection, the glucose was linked to alkynyl with different linkers.²² In order to connect with glucose, DCPO was modified with azide to obtain a novel DCPO derivative N₃-DCPO. We finally got five different DCPO-linked O-1-glycosylated glucose probes via click reaction (Fig. 3). The distance between glucose and DCPO was controlled by ethylene glycol chain.

Fig. 2 Folate-conjugated distyryl boron dipyrromethene based photosensitizers.

Fig. 3 Terminal probes and N₃-DCPO.

Experimental

General procedures

Melting points were taken on a Fisher Johns melting point apparatus, uncorrected and reported in degrees centigrade. ¹H NMR and ¹³C NMR spectra were recorded in CDCl₃ and DMSO-d₆ on a Bruker DRXe400 (400 MHz) spectrometer using TMS as internal standard. Chemical shifts were reported as δ (ppm) and spin–spin coupling constants as J (Hz) values. The mass spectra (MS) were recorded on a Finnigan MAT-95 mass spectrometer. UV-vis absorption spectra were recorded on a Varian Cary 100 spectrophotometer. Fluorescence spectra were measured with a Hitachi F-4500 Fluorescence spectrophotometer.

2-Hydroxy-4-methoxymethoxybenzaldehyde (2)

Into a solution of compound 1 2,4-dihydroxybenzaldehyde (5.0 g, 36.2 mmol) and K₂CO₃ (6.5 g, 47.1 mmol) in dry acetone (100 mL) was added MOMCl (3.3 mL, 38.7 mmol) dropwise at room temperature under nitrogen atmosphere. The resulting mixture was allowed to stir for 18 h. The reaction mixture was extracted with ethyl acetate, washed with water followed by brine, and dried over Na₂SO₄. The crude mixture was filtered, concentrated and purified by column chromatography (SiO₂, PE : EA = 4 : 1) to give the product as a white solid (4.2 g, 64.2%).¹H NMR (400 MHz, CDCl₃) δ 11.37 (s, 1H), 9.74 (s, 1H), 7.45 (d, J = 8.6 Hz, 1H), 6.65 (dd, J = 8.6, 2.2 Hz, 1H), 6.61 (d, J = 2.2 Hz, 1H), 5.22 (s, 2H), 3.48 (s, 3H).

4-Methoxymethoxy-2-(2-azidoethoxy)ethoxybenaldehyde (4)

Into a solution of compound 2 2-hydroxy-4-methoxymethoxybenzaldehyde (80 mg, 439.1 μmol) in dry DMF (2 mL) was added NaH (21 mg, 526.9 μmol) at 0 °C under nitrogen atmosphere. The mixture was allowed to stir for 15 min. 2-(2-Azidoethoxy)ethyl-4-methylbenzenesulfonate (3) was added dropwise under nitrogen atmosphere. The resulting mixture was allowed to stir for 20 h at room temperature. The reaction mixture was extracted with ethyl acetate, washed with water followed by brine, and dried over Na₂SO₄. The crude mixture was filtered, concentrated and purified by column chromatography (SiO₂, PE : EA = 8 : 1) to give the product as a pale yellow oil (80 mg, 61.7%).¹H NMR (400 MHz, CDCl₃) δ 10.35 (s, 1H), 7.80 (d, J = 8.6 Hz, 1H), 6.69 (d, J = 8.7 Hz, 1H), 6.62 (s, 1H), 5.22 (s, 2H), 4.28–4.19 (m, 2H), 3.96–3.88 (m, 2H), 3.76 (m, 2H), 3.48 (s, 3H), 3.44–3.37 (m, 2H).

4-Hydroxy-2-(2-azidoethoxy)ethoxybenaldehyde (5)

Into a solution of compound 4 (750 mg, 2.5 mmol) in dry MeOH (3 mL) was added H+ resin (1.5 g) at room temperature. The resulting mixture was allowed to stir for 30 h. The crude mixture was filtered, washed with DCM and MeOH, concentrated and purified by column chromatography (SiO₂, PE : EA = 2 : 1) to give the product as a pale yellow oil (140 mg, 22%). ¹H NMR (400 MHz, CDCl₃) δ 10.29 (s, 1H), 7.76 (d, J = 8.5 Hz, 1H), 6.95 (m, 1H), 6.51 (dd, J = 8.5, 2.1 Hz, 1H), 6.47 (d, J = 2.1 Hz, 1H), 4.26–4.18 (m, 2H), 3.95–3.88 (m, 2H), 3.80–3.71 (m, 2H), 3.46–3.36 (m, 2H).

N₃-DCPO (7)

Into a solution of compound 5 (950 mg, 3.8 mmol) and 6 (790 mg, 3.78 mmol) in dry CH₃CN (12 mL) was added piperidine (0.6 mL) dropwise at room temperature under nitrogen atmosphere. Then the AcOH (0.6 mL) was added in slowly. The resulting mixture was allowed to stir for 20 h at 70 °C. The reaction mixture was concentrated. DCM (2 mL), TFA (0.6 mL), EtO₂ (10 mL) was added in turn. The crude mixture was filtered, washed with water and EtO₂. The filter cake was product as a red solid (1.12 g, 67.1%). Mp: 184–186 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 10.26 (s, 1H), 8.71 (m, 1H), 7.93–7.83 (m, 2H), 7.70 (m, 1H), 7.66–7.54 (m, 2H), 7.29–7.17 (m, 1H), 6.88–6.79 (m, 1H), 6.47 (m, 2H), 4.17 (s, 2H), 3.91 (s, 2H), 3.83–3.75 (m, 2H), 3.51–3.44 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 161.76, 159.43, 159.17, 152.66, 151.94, 135.13, 134.65, 130.52, 125.97, 124.57, 118.75, 117.45, 117.12, 116.11, 115.52, 114.95, 108.74, 105.30, 99.96, 69.62, 68.69, 67.74, 58.45, 50.07. HRMS (ESI): m/z calcd for C₂₄H₁₉N₅NaO₄ [M + Na] 464.1335, found: 464.1318.

Probe N0–N4

Into a solution of compound 7 (1.1 equiv.) in solvent (water: t-BuOH = 1 : 1) was added 11-n glucose derivative (1 equiv.) at room temperature. CuI (5%) and DIPEA (10%) was added. The resulting mixture was allowed to stir for 1 h at 60 °C. The reaction mixture was concentrated and purified by column chromatography (SiO₂, DCM : MeOH = 10 : 1) to give the product.

N0: red solid (77.1%). Mp: 210–211 °C ¹H NMR (400 MHz, DMSO-d₆) δ 10.26 (s, 1H), 8.73 (d, J = 8.4 Hz, 1H), 8.09 (s, 1H), 7.89 (m, 2H), 7.72 (d, J = 8.4 Hz, 1H), 7.69–7.57 (m, 2H), 7.25 (d, J = 16.1 Hz, 1H), 6.87 (s, 1H), 6.48 (m, 2H), 5.02 (d, J = 4.5 Hz, 1H), 4.98–4.86 (m, 2H), 4.77 (m, 1H), 4.65–4.47 (m, 4H), 4.22 (m, 1H), 4.14 (d, J = 4.5 Hz, 2H), 4.01 (m, 2H), 3.93–3.84 (m, 2H), 3.73–3.65 (m, 1H), 3.49–3.42 (m, 1H), 3.08 (m, 3H), 2.96 (m, 1H).

¹³C NMR (101 MHz, DMSO-d₆) δ 161.77, 159.39, 159.05, 151.96, 143.59, 135.23, 134.32, 130.24, 126.03, 124.52, 118.95, 117.50, 117.15, 116.18, 115.55, 114.91, 108.83, 105.40, 102.10, 100.07, 76.90, 76.66, 73.31, 70.00, 69.07, 68.62, 67.71, 61.37, 61.07, 58.45, 49.39. HRMS (ESI): m/z calcd for C₃₃H₃₃N₅NaO₁₀ [M + Na] 682.2125; found: 682.2107. N1: red solid (82.4%). Mp: 128–131 °C ¹H NMR (400 MHz, MeOD) δ 8.81 (d, J = 8.3 Hz, 1H), 7.98 (s, 1H), 7.86–7.74 (m, 2H), 7.59 (d, J = 8.3 Hz, 1H), 7.46 (m, 2H), 6.99 (m, 1H), 6.66 (s, 1H), 6.50–6.35 (m, 2H), 4.66 (s, 2H), 4.47 (s, 2H), 4.23 (m, 1H), 4.15 (s, 2H), 4.07 (d, J = 4.5 Hz, 2H), 4.01–3.78 (m, 4H), 3.72–3.54 (m, 4H), 3.26 (m, 2H), 3.19–3.12 (m, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 161.95, 159.39, 159.09, 152.69, 151.94, 143.78, 135.15, 134.38, 130.30, 125.99, 124.53, 124.15, 118.90, 117.49, 117.13, 116.18, 115.41, 114.85, 108.89, 105.32, 102.94, 100.07, 76.83, 76.70, 73.33, 69.98, 69.05, 68.79, 68.62, 67.68, 63.40, 61.01, 58.36, 49.37. HRMS (ESI): m/z calcd for C₃₅H₃₇N₅NaO₁₁ [M + Na]: 726.2387; found: 726.2356.

N2: red solid (78.4%). Mp: 95–97 °C ¹H NMR (400 MHz, DMSO-d₆) δ 10.27 (s, 1H), 8.73 (d, J = 8.4 Hz, 1H), 8.03 (s, 1H), 7.88 (dd, J = 15.7, 7.9 Hz, 2H), 7.71 (d, J = 8.3 Hz, 1H), 7.67–7.56 (m, 2H), 7.24 (m, 1H), 6.86 (s, 1H), 6.48 (d, J = 8.0 Hz, 2H), 5.06–4.81 (m, 3H), 4.59 (m, 2H), 4.39 (s, 2H), 4.12 (dd, J = 11.5, 6.6 Hz, 3H), 4.00 (t, J = 5.1 Hz, 2H), 3.86 (m, 3H), 3.65 (d, J = 11.6 Hz, 1H), 3.59–3.39 (m, 8H), 3.17 (d, J = 4.9 Hz, 1H), 3.08 (m, 3H), 2.94 (m, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 161.81, 159.40, 159.08, 152.78, 151.96, 143.69, 135.20, 134.34, 130.28, 126.03, 124.55, 124.14, 118.95, 117.50, 117.14, 116.18, 115.53, 114.89, 108.83, 105.39, 102.94, 100.06, 76.84, 76.69, 73.32, 69.97, 69.56, 69.02, 68.85, 68.60, 67.76, 63.36, 61.01, 58.42, 49.35. HRMS (ESI): m/z calcd for C₃₇H₄₁N₅NaO₁₂ [M + Na]: 770.2649; found: 770.2616.

N3: red solid (80.6%). Mp: 87–89 °C ¹H NMR (400 MHz, MeOD) δ 8.61 (d, J = 8.3 Hz, 1H), 7.96 (s, 1H), 7.70 (t, J = 7.7 Hz, 1H), 7.54 (d, J = 16.0 Hz, 1H), 7.42 (d, J = 8.3 Hz, 1H), 7.35 (t, J = 7.7 Hz, 1H), 7.26 (d, J = 8.5 Hz, 1H), 6.79 (d, J = 16.0 Hz, 1H), 6.42 (s, 1H), 6.36 (d, J = 8.5 Hz, 1H), 6.30 (s, 1H), 4.65 (t, J = 4.6 Hz, 2H), 4.49 (s, 2H), 4.30 (d, J = 7.8 Hz, 1H), 4.02 (dd, J = 16.6, 11.2 Hz, 5H), 3.87 (d, J = 11.9 Hz, 3H), 3.75–3.64 (m, 4H), 3.63–3.52 (m, 8H), 3.38 (d, J = 11.9 Hz, 4H), 3.28 (s, 1H), 3.21 (t, J = 8.4 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 161.76, 159.35, 159.06, 152.68, 151.91, 143.71, 135.15, 134.34, 130.28, 125.98, 124.51, 124.12, 118.89, 117.46, 117.10, 116.15, 115.49, 114.88, 108.78, 105.34, 102.94, 100.00, 76.83, 76.69, 73.33, 69.98, 69.70, 69.65, 69.54, 69.04, 68.84, 68.60, 67.77, 67.66, 63.36, 61.01, 58.42, 49.36, 48.56. HRMS (ESI): m/z calcd for C₃₉H₄₅N₅NaO₁₃ [M + Na] 814.2912; found: 814.2888.

N4: red solid (84.2%). Mp: 86–88 °C ¹H NMR (400 MHz, MeOD) δ 8.66 (d, J = 8.2 Hz, 1H), 7.97 (s, 1H), 7.74 (t, J = 7.6 Hz, 1H), 7.59 (d, J = 16.0 Hz, 1H), 7.47 (d, J = 8.3 Hz, 1H), 7.39 (t, J = 7.6 Hz, 1H), 7.32 (d, J = 8.5 Hz, 1H), 6.85 (d, J = 16.0 Hz, 1H), 6.48 (s, 1H), 6.40 (d, J = 8.5 Hz, 1H), 6.34 (s, 1H), 4.67 (t, J = 4.5 Hz, 2H), 4.51 (s, 2H), 4.32 (d, J = 7.7 Hz, 1H), 4.11–3.97 (m, 5H), 3.88 (s, 3H), 3.77–3.57 (m, 16H), 3.31 (d, J = 6.8 Hz, 2H), 3.22 (t, J = 8.4 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 161.76, 159.35, 159.06, 152.68, 151.91, 143.71, 135.15, 134.34, 130.28, 125.98, 124.51, 124.12, 118.89, 117.46, 117.10, 116.15, 115.49, 114.88, 108.78, 105.34, 102.94, 100.00, 76.83, 76.69, 73.33, 69.98, 69.70, 69.65, 69.54, 69.04, 68.84, 68.60, 67.77, 67.66, 63.36, 61.01, 58.42, 49.36, 48.56. HRMS (ESI): m/z calcd for C₄₁H₄₉N₅NaO₁₄ [M + Na] 858.3174; found: 858.3133.

Cell cultures

Human breast cancer cells MCF-7, gastric cancer cells MKN-45 and cervical cancer cells HeLa were purchased from Cell Bank of China Science Academy (Shanghai, China) and maintained in MEM, RPMI-1640 or DMEM medium (Hyclone or Gibco), respectively, supplemented with 10% FBS (Biological Industries) and antibiotics (100 units per mL penicillin and 100 μg mL⁻¹ streptomycin) (Thermo Scientific). All cell lines were cultured in an incubator with humidified 5% CO₂ at 37 °C.

Cellular uptake of compounds by cancer cells

Exponentially growing cells were seeded in a 96-well plate with an amount of 10 000–12 000 cells per well overnight, and then treated with different compounds with indicated concentration in the presence or absence of phloretin (Sigma) or glucose (Shanghai RichJoint Chemical Reagents Co., Ltd) for 24 h. In all experiments, the final DMSO solvent concentration was ≤0.2% (v/v). Before observation, the cells were washed three times with cold PBS. Images of cells were obtained with Olympus microscope (IX73). The fluorescence intensity was quantitated by Image J.

Western blot

Cells were lysed with RAPI buffer (Thermo Scientific) and incubated at 4 °C for 30 min. The lysate was then centrifuged at 14 000 g for 20 min to remove insoluble materials. Protein extracts were separated on SDS-PAGE and electroblotted onto PVDF membranes according to standard procedures. The membranes were blocked with 5% nonfat dry milk and incubated with primary antibody Glut-1 (Proteintech). α-Tubulin (Santa Cruz) was used as a loading control. Western blotting were visualized with HRP conjugated secondary antibodies (MultiSciences) followed by enhanced chemiluminescence detection (Millipore).

Results and discussion

The synthesis of five glucose analogues. The synthesis procedure could be divided to two parts, which are the synthesis of fluorescent N₃-DCPO and glucose derivatives, respectively. N₃-DCPO was synthesized from 2,4-Dihydroxybenzaldehyde (1) by selective production with MOMCl, condensation with 2-(2-azidoethoxy)ethyl-4-methylbenzenesulfonate (3), deproduction, and condensation with 4-dicyanomethylene-2-methyl-4H-pyran (6) (Scheme 1). Compound 3 and 6 were synthesized according to the literatures.^22–25 The five different glucose derivatives were prepared as the method mentioned in previous articles (Scheme 2).^21,26 After the click reaction between N₃-DCPO and the glucose derivatives separately, we got five glucose analogues N0–N4 (Scheme 3).

Scheme 1 Synthesis of the fluorescent dye N₃-DCPO (7). (a) MOMCl, K₂CO₃, acetone, 64%; (b) NaH, DMF, 62%; (c) resin, MeOH, 22%; (d) Piperidine, AcOH, MeCN, 67%.

Scheme 2 Synthesis of the 5 glucose derivatives (11-n). (a) BF₃·Et₂O, DCM, 54–65%; (b) NaOMe, MeOH, DCM, 86–94%.

Scheme 3 Synthesis of the five probes (N0–N4). Click reaction between fluorescent dye (7) and the glucose derivatives (11-n). CuI, DIPEA, water, t-BuOH.

We first tested the optical properties of the compounds in PBS buffer (50 μM, pH 7.4, 50% DMSO). The five novel probes exhibited similar UV-vis spectra to N₃-DCPO with one main absorption at 580 nm (Fig. 4). The emission at 670 nm appeared upon excitation at 580 nm among all five probes, which was the same as N₃-DCPO exhibited (Fig. 5). These results indicated that the conjugate of glucose to N₃-DCPO had little effect on the wavelengths of the dyes. According to Fig. 4 and 5, the six DCPO analogues had different molar absorption or emission. The order of molar absorption intensity was N0 > N₃-DCPO > N4 > N1 > N3 > N2. The order of molar emission intensity was N3 > N3, N4 > N2 > N0 > N₃-DCPO. We guessed the hydrogen bond between hydroxyl of glucose and phenolic hydroxyl group affected the absorption and emission.

Fig. 4 Absorption spectra of the probes. 50 μM in PBS buffer (pH 7.4, 50% DMSO) at room temperature. UV-vis spectra of each probes exhibited the same maximum absorption wavelength at 580 nm.

Fig. 5 Emission spectra of the probes. 50 μM in PBS buffer (pH 7.4, 50% DMSO) at room temperature. The spectra of each probes exhibited the same maximum emission wavelength at about 670 nm.

Human cancer cells with different GLUT-1 expression levels were employed in our experiment. Photos were taken on a fluorescence inversion microscope system and the fluorescence intensity was quantitated by Image J. As shown in Fig. 6A, breast cancer cells MCF-7 and cervical cancer cells HeLa expressed the lowest and highest level of GLUT-1, respectively. Then the uptake of the five compounds was compared among these three cell lines. As expected, the fluorescence of all five compounds in HeLa cells was the strongest, which was in line with the highest levels of GLUT-1 in HeLa cells, suggesting the uptake of the compounds was related with the protein levels of GLUT-1 in cancer cells. We presumed that the distance between the dye and glucose might affect the cellular uptake efficiency of the glucose analogues. As expected, among these five compounds, compound N2 showed the best cellular uptake by all three cell lines (Fig. 6B and C), indicating the length of the linker in N2 was the most appropriate spacer arm, longer or shorter linker exhibited less efficiency. Therefore, compound N2 was chosen for the following experiments.

Fig. 6 The uptake of compounds by cancer cells is related with GLUT-1 expression levels. (A) Western blots for the expression levels of GLUT-1 in different cancer cell lines. α-Tubulin was used as a loading control. (B) Fluorescent image of cells exposed to different compounds (25 μM) for 24 h. After washout of the compounds, the cells were observed under microscope (40). The data are representative of three independent experiments. Scale bar is 20 μm. (C) Fluorescence intensity was quantitated by Image J. Data presented are the mean ± SD of three independent experiments.

To further determine whether the uptake of compounds was via GLUT-1, glucose was added into MCF-7 cells together with compound N2 as a competitive substrate for GLUT-1. As expected, glucose competitively inhibited the transport of compound N2 into cancer cells, which was evidenced by the decreased fluorescence of MCF-7 cells. Moreover, the application of phloretin, a GLUT-1 inhibitor further confirmed the uptake of compound N2 was via GLUT-1. Phloretin-treated MCF-7 cells could only take up a tiny amount of compound N2 compared with vehicle-treated cells (Fig. 7). Together, these results indicated that the delivery of compound N2 is GLUT-1 dependent. Considering GLUT-1 is usually overexpressed in tumor cells, compound N2 might be used as a fluorescence probe for tumor detection in vivo.

Fig. 7 Inhibition of compound N2 cellular uptake by glucose or GLUT-1 inhibitor phloretin. MCF-7 cells were pretreated with glucose (100 mM) or phloretin (100 μM) for 1 h and subsequently treated with compound N2 (25 μM) for 24 h. Then cells were observed under microscope (40) after washout of the compounds. The data are representative of three independent experiments. Scale bar is 20 μm.

In order to determine the relationship between cellular uptake and compound concentration, cancer cells were exposed to different concentration of compound N2 (3.125, 6.25, 12.5, 25 and 50 μM) for 24 h. As shown in Fig. 8, the uptake of compound N2 by all three cell lines were in a dose-dependent manner. Of note, the uptake of compound N2 in HeLa cells was detectable at concentration at least as low as 3.125 μM, which should be attributed to the high expression of GLUT-1 in HeLa cells. Meanwhile, with the decreased expression levels of GLUT-1 in MKN-45 and MCF-7 cells, the concentration required to detect cellular uptake increased to 12.5 and 25 μM, respectively. These results demonstrated the uptake of compound N2 was in a dose-dependent manner, suggesting that compound N2 could also be used as a fluorescence probe to determine the cellular uptake of glucose. And the concentration required for the experiments depends on the GLUT-1 expression levels.

Fig. 8 Dose-dependent uptake of compound N2 by cancer cells. Cancer cells were treated with the indicated concentration of compound N2 for 24 h, and then were observed under microscope (40) after washout of the compounds. The data are representative of three independent experiments. Scale bar is 20 μm.

Conclusions

In summary, we have synthesized a series of DCPO-conjugated glucose analogues (N0–N4), whose transportation were via GLUT1 and could be inhibited by GLUT1 inhibitor phloretin or by unlabeled D-glucose. We also found out the spacer arm of N2 exhibited the best length between the dye and glucose. It was the very first time that the influence of the length between glucose and dye on cellular uptake was studied. Together, DCPO as the dye that has no charge and exhibits good optical properties ensures the potential application of N2 in cancer cell bioimaging in vitro and in vivo.

Acknowledgements

This work was supported by the State Key Laboratory of Fine Chemicals (KF1517).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra18613k

‡ These authors contributed equally.

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