Shiguang Chen‡
a,
Yanfen Fang‡a,
Qiwen Zhua,
Wanli Zhanga,
Xiongwen Zhang*a and
Wei Lu*ab
aSchool of Chemistry and Molecular Engineering, East China Normal University, 3663 North Zhongshan Road, Shanghai 200062, P. R. China. E-mail: wlu@chem.ecnu.edu.cn
bState Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, P. R. China
First published on 25th August 2016
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.
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.
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.
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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%. |
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Scheme 2 Synthesis of the 5 glucose derivatives (11-n). (a) BF3·Et2O, DCM, 54–65%; (b) NaOMe, MeOH, DCM, 86–94%. |
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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.
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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. |
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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.
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.
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.
Footnotes |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra18613k |
‡ These authors contributed equally. |
This journal is © The Royal Society of Chemistry 2016 |