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  • protease inhibitor Introduction Glucose is the major carbon


    Introduction Glucose is the major carbon source in most organisms. Cancer protease inhibitor in particular require a steady source of energy to maintain growth and proliferation, and preferentially protease inhibitor use glycolysis for their energy supply even under aerobic conditions. Glucose uptake in mammals is mainly mediated by glucose transporters (GLUTs), and their expression is increased in various cancers. Therefore, the visualization of glucose uptake is useful for detection of tumors, and positron emission tomography (PET) with the radiolabeled glucose analogue 2-[fluorine-18]-fluoro-2-deoxy-d-glucose (F-FDG) is widely used for imaging tumors in clinical medicine. Its ability to noninvasively map glucose metabolism is important not only in current clinical decision-making, but also in the preclinical evaluation of new therapies. However, although this method is quite effective for tumor imaging, it cannot measure glucose uptake at the single-cell level. Furthermore, fluorescence optical imaging has several advantages compared to other imaging modalities, including relative low cost, high temporal and spatial resolutions, lack of ionizing radiation, and ready translation to minimally invasive and endoscopic and intraoperative imaging.6, 7, 8 The representative fluorescent glucose analogue 2-NBDG (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose) has been developed as a fluorescent tracer for measurement of glucose uptake into cells via GLUT, and enables sensitive measurement of glucose uptake by individual living cells.9, 10 2-NBDG consists of a small fluorophore (molecular weight: 164) attached to d-glucosamine at the C-2 position, and it fluoresces at 540 nm when excited at 465 nm. Some other fluorescent glucose analogues have also been reported, but there are no activatable fluorescent glucose analogues for detecting GLUT activity. Such activatable analogues would have a number of advantages for endoscopic or intraoperative detection of cancers, including highly sensitive cancer visualization with a minimal background signal from nontarget tissues,13, 14 even though always-on type fluorescent substrates have already been developed, e.g., for imaging bile duct cancer during confocal endoscopy in a hamster model. Here, we describe the development of a fluorescent glucose analogue as a basic platform for off/on-type activatable fluorescent glucose analogues targeting GLUTs.
    Results and discussion
    Conclusions In conclusion, we have developed a platform for activatable fluorescent glucose analogues, 2-Me-4-OMe TGG, by utilizing the xanthene-based fluorophore, TokyoGreen. 2-Me-4-OMe TGG was rapidly taken up into MIN6 cells, and the strong inhibition of this 2-Me-4-OMe TGG uptake by phloretin indicates that the uptake is specific, and most probably mediated by GLUTs. It is interesting that fluorescein-conjugated glucosamines were not taken up into MIN6 cells, but TokyoGreen conjugates showed high cellular uptake. Since MIN6 cells express the neutral solute carriers GLUT2 and GLUT1, we think that the lower negative charge of the TokyoGreen conjugates may facilitate their interaction with GLUTs. Activatable fluorescent probes of this kind have not been developed before, probably because of the difficulty in their molecular design. For fluorescence off/on control of the molecules, we optimized the TokyoGreen structure of the fluorescent glucosamine conjugate based on the PeT mechanism. The developed platform, 2-Me-4-OMe TGG, showed stronger fluorescence in the anionic form than in the neutral form of its xanthene moiety. 2-Me-4-OMe TGG also has a more appropriate pKa for the intracellular environment (pH 7.4) compared to the acidic state (pH ∼ 6.5) seen in the extracellular environment of tumors.25, 26 Further optimization of its pKa should lead to the development of very useful activatable glucose analogues. It would also be weakly fluorescent in the extracellular environment of gastric cancers, because the environmental pH in the stomach is ∼1.5. Moreover, this platform could be developed to target intracellular enzymatic activities such as esterases with an O-acetyl group, β-galactosidase (for ovarian cancers) with a O-β-galactopyranosyl group, and bioreductases with a 4-nitrobenzyl or 4-nitrofuryl O-protecting group, as shown in Fig. 5a. As a proof of concept, we designed and synthesized 2-Me-4-OMe TGG masked with an O-β-galactopyranosyl group (Scheme S7, Figs. S8 and S9) and applied it to the fluorescence imaging of MIN6 cells. The probe was fluorescently activated by β-galactosidase in the cells (Figs. S10 and S11) and the cells could be visualized without any washout process of excess probe (Fig. S12), though some improvement of the uptake efficiency may still be needed. Such activatable fluorescent glucose analogues would be useful to detect cancers without the need for any washout process to remove excess analogues, and might be applicable for the rapid detection of cancers with high S/N during endoscopy and/or surgical operation. The appropriate target intracellular enzyme (or the target pH change) would depend upon the target cancer, and further investigations of biological applications of 2-Me-4-OMe TGG are in progress.