A two-photon tracer for glucose uptake.

Glucose is the principal energy source essential for cell growth. Fast-growing cancer cells exhibit a high rate of glycolysis; hence, the rate of glucose uptake is faster in these cells, primarily due to overexpression or enhanced intracellular translocation of glucose transporters (GLUTs) and increased activity of mitochondria-bound hexokinases in the tumor. To monitor glucose metabolism in living systems, a variety of tracers, such as [F]-2-fluoro-2-deoxyglucose (FDG), 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-2deoxy-d-glucose (2-NBDG; Scheme 1), and IR dye 800CW-2DG, have been developed. FDG is widely used in the in vivo analysis of glucose metabolism by positron emission tomography (PET), whereas 2-NBDG and IR dye 800CW-2DG are fluorescent probes that have been used for studying cellular metabolic functions involving GLUTs and in tumorimaging studies. Recently, we developed a new fluorescent probe, cyanine-3-linked O-1-glycosylated glucose (Cy3-Glc-a ; Scheme 1), which is a better glucose probe than 2-NBDG because it can be used without glucose starvation, produces a much brighter image, and can be applied for the screening of anticancer agents. In one-photon microscopy (OPM), the probes are excited with short-wavelength light ( 350–550 nm); this, however, limits their application in tissue imaging, owing to inherent problems such as shallow penetration depth (< 80 mm), interference by cellular autofluorescence, photobleaching, and photodamage. 11] To overcome these problems, it is crucial to use two-photon microscopy (TPM), which utilizes two near-infrared photons for excitation. TPM offers a number of advantages over OPM, including greater penetration depth (> 500 mm), localized excitation, and longer observation times. In particular, the extra penetration depth afforded by TPM is an essential element for application in tissue-imaging studies because the artifacts arising from surface preparation, such as damaged cells, can extend over 70 mm into the tissue interior. However, visualization of glucose uptake by live cells and tissues with two-photon (TP) tracers has not been reported so far. The requirements for a TP tracer to visualize glucose uptake include sufficient water solubility for staining cells and tissues, preferential uptake by cancer cells, a large TP crosssection for a bright TPM image, pH resistance, and high photostability. Our strategy was to link a-d-glucose with the fluorophore 2-acetyl-6-dimethylaminonaphthalene (acedan) through 3,6-dioxaoctane-1,8-diamine or a piperazine linkage (in AG1 and AG2, respectively; Scheme 1), so that the tracers are transported into the cells through the glucose-specific mechanism. Acedan is a polarity-sensitive fluorophore that has been successfully employed in the development of TP probes for the cell membrane, metal ions, and acidic vesicles. We now report that these tracers facilitate the visualization of glucose uptake in cancer cells and live tissues at a depth of 75–150 mm for more than 3000 s and can be used for screening anticancer agents. The preparation of AG1 and AG2 is shown in Scheme 2. 6-Acetyl-2-[N-methyl-N-(carboxymethyl)amino]naphthalene Scheme 1. Structures of fluorescent tracers AG1, AG2, 2-NBDG, and Cy3-Gly-a.