Fluorescent Hexose Conjugates Establish Stringent Stereochemical

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Fluorescent Hexose Conjugates Establish Stringent Stereochemical Requirement by GLUT5 for Recognition and Transport of Monosaccharides Olivier-Mohamad Soueidan, Thomas W Scully, Jatinder Kaur, Rashmi Panigrahi, Alexandr Belovodskiy, Victor Do, Carson D. Matier, M Joanne Lemieux, Frank Wuest, Chris Cheeseman, and F. G. West ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b01101 • Publication Date (Web): 16 Feb 2017 Downloaded from http://pubs.acs.org on February 17, 2017

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ACS Chemical Biology

Fluorescent Hexose Conjugates Establish Stringent Stereochemical Requirement by GLUT5 for Recognition and Transport of Monosaccharides Olivier-Mohamad Soueidan,a,b Thomas W. Scully,a Jatinder Kaur,c Rashmi Panigrahi,d Alexandr Belovodskiy,a Victor Do,b Carson D. Matier,a M. Joanne Lemieux,d Frank Wuest,c Chris Cheesemanb,* F. G. West,a,* a

Department of Chemistry, 11227 Saskatchewan Drive University of Alberta, Edmonton, AB, Canada T6G 2G2; Fax: +1 780 492 8231; Tel: +1 780 492 8187; E-mail: [email protected]

b

Department of Physiology, 7-55 Medical Sciences Building, University of Alberta, Edmonton, AB, Canada T6G 2H7; Fax: +1 780 492 8915; Tel: +1 780 9525875; E-mail: [email protected] c

Department of Oncology, Cross Cancer Institute, University of Alberta, Edmonton, AB, Canada T6G 1Z2

d

Department of Biochemistry,451 Medical Sciences Building, University of Alberta, Edmonton, AB, Canada T6G 2H7

ABSTRACT: The specificity characteristics of transporters can be exploited for the development of novel diagnostic therapeutic probes. The facilitated hexose transporter family (GLUTs) has a distinct set of preferences for monosaccharide substrates, and while some are expressed ubiquitously (e.g., GLUT1), others are quite tissue specific (e.g., GLUT5, which is overexpressed in some breast cancer tissues). While these differences have enabled the development of new molecular probes based upon hexose- and tissue-selective uptake, substrate design for compounds targeting these GLUT transporters has been encumbered by a limited understanding of the molecular interactions at play in hexose binding and transport. Four new fluorescently labeled hexose derivatives have been prepared and their transport characteristics were examined in two breast cancer cell lines expressing mainly GLUTs 1, 2 and 5. Our results demonstrate, for the first time, a stringent stereochemical requirement for recognition and transport by GLUT5. 6-NBDF, in which all substituents are in the D-fructose configuration, is taken up rapidly into both cell lines via GLUT5. On the other hand, inversion of a single stereocenter at C-3 (6-NBDP), C-4 (6-NBDT) or C-5 (6-NDBS) results in selective transport via GLUT1. An in silico docking study employing the recently published GLUT5 crystal structure confirms this stereochemical dependence. This work provides insight into hexose-GLUT interactions at the molecular level, and will facilitate structure-based design of novel substrates targeting individual members of the GLUT family and forms the basis of new cancer imaging or therapeutic agents.

Facilitated hexose transporters (GLUTs), of the gene family hSLC2A, are a group of mammalian transmembrane proteins responsible for the entry of monosaccharides into numerous cell types to provide the basic fuels for cellular metabolism. Due to the differing metabolic needs of specific cell and tissues types, the various GLUTs have different substrate affinities, transport kinetic properties and tissue-specific expression.1 The role of GLUTs in several conditions including some forms of cancer, obesity, and metabolic disease has recently been reported and scientific interest in these transporters is growing.2-7 Many of the fourteen members of this family mediate the transport of D-glucose, e.g. GLUT1 & 3, while a subset can mediate the transport of D-fructose, as exemplified by the fructose transporter GLUT5.8-11 However, despite the recently reported crystal structures of GLUTs 1, 3 & 5, the understanding of their molecular mechanisms for substrate recognition, binding and translocation is limited. The recent crystal structures suggest that transport is

achieved by a conformational shift of the protein such that the substrate binds first to the outward-open conformation, then the protein shifts through an occluded state in which neither end of the aqueous pore is fully open to solution. Finally, the protein reaches an inwardopen conformation exposing the substrate to the cytoplasm where it dissociates from the protein.12,13 The crystal structure of GLUT5 in both the outward-open and inward-open configurations has provided much needed information about the binding pocket and indicates that Trp420 is critical for D-fructose binding.14 Additionally, studies of hGLUT7 and hGLUT9 indicate a core of hydrophobic amino acids acts not only as part of the bindingpocket but is also responsible for the high degree of substrate specificity found in the GLUTs.11,15,16 Early work on the structural requirements of GLUT substrates demonstrated that hydroxyl groups at C-2 and C-6 of D-fructose appear to play a small role in substrate recognition, suggesting that structural modifications at

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these positions can be tolerated by GLUT5.17-23 In contrast, the hydroxyl groups at C-1, C-3 and C-4 are believed to form hydrogen bonds with specific amino acid residues forming the binding pocket. On the basis of these observations, we have previously reported new fluorinated Dfructose analogs as a specific substrate for GLUT5, some of which can function as PET imaging probes.24-26 After our success with the C-6 fluorinated compounds we extended this approach to fluorescently labeled compounds, which would provide valuable insight into the molecular recognition events involved in the GLUT substrate binding pocket. An understanding at the molecular level of how the protein and substrate interact is critical to determining how this then promotes the first step in the conformational change of the facilitated carrier, resulting ultimately in movement of the substrate across the cell membrane. However, surprisingly little is known about the structural constraints for hexoses themselves to be recognized and transported as substrates. Prior to this work, the stereochemical requirement for recognition and transport by GLUTs has not been systematically investigated. Here we show that GLUT5 requires all stereocenters to be in the D-fructose configuration for recognition and transport. Surprisingly, inversion of a single stereocenter around the furanose ring at C-3, C-4, or C-5 leads to preferred recognition and transport by GLUT1 rather than GLUT5. In silico docking studies based on reported crystal structures support the stereochemical requirements observed for GLUT5. Discovery of this exquisite stereospecificity will greatly assist in the future design of probe molecules and therapeutic agents targeting individual GLUTs.

RESULTS AND DISCUSSION To explore the stereochemical requirement for recognition and transport by GLUTs several key facts have to be established. Firstly, it has to be shown conclusively that the MCF-7 and EMT-6 cell lines being studied express the GLUT transporter of interest on their surfaces. Secondly, new fluorescently labeled hexose derivatives in which the stereochemistry about the furanose ring is systematically varied have to be synthesized. Thirdly, it has to be demonstrated that these compounds are entering the cells through a transport mediated process. Further it has to be proven that the compounds are entering cells specifically through the action of GLUTs and not via another transport pathway or through passive diffusion. Western Blot analysis was carried out to ensure that the cells expressed the GLUT transporters of interest on their surfaces (see Figure SI 1 in Supporting Information). The fluorescently labelled compound 6-NBDF (5) (NBD = 7nitrobenz-2-oxa-1,3-diazolyl), in which all substituents are in the D-fructose configuration, was prepared in a short procedure starting from the D-fructose derivative (1)27 (Scheme 1). Selective C-6 tosylation and subsequent displacement with sodium azide yielded 3 in good yield. This compound was then reduced to the corresponding amine

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which was treated with NBD-Cl to afford protected 6NBDF (4) in moderate Scheme 1. Synthetic route of 6-NBDF (5)

Reagents and conditions : a) TsCl, Pyr., 0 °C-r.t., 24 h, 70%, b) NaN3, DMF, 120 °C, 16 h, 75 %; c) Pd/C/H2, EtOH, r.t., 24 h then NBD-Cl, NaHCO3, MeOH, 24 h, 40%; d) HCl (1 M), CH3CN, 90 °C, 20 min then NaHCO3 (sat.) , 75%. Synthetic routes for 6-NBDP (6), 6-NBDT (7), and 6-NBDS (8) are found in the SI.

yield over two steps. Compound 4 was then deprotected under acidic conditions to give the desired product 5 in 75% yield. Through a set of similar steps, from known starting materials, 6-NBDP (6), 6-NBDT (7), and 6-NBDS (8) were also synthesised (see SI for experimental details). With these compounds in hand, each of them was evaluated for its ability to act as a GLUT substrate via 4 methodologies. Incubation of 5 (300 µM) with MCF-7 cells was observed to produce a steady increase in the fluorescence signal as shown in Figure 1 Panel A when analysed by a fluorescence plate reader (see SI, Figure SI 8 for corresponding studies with EMT-6). Challenging the uptake of these compounds with the natural substrates for the GLUTs allowed the determination of which GLUT is responsible for the transport of each compound. Inhibition experiments with co-incubation of D-glucose (50 mM) showed no statistical change from the original uptake experiment as calculated by a T-test; however, coincubation with D-fructose (50 mM) resulted in a significant drop (42±4%) in the observed fluorescence signal in MCF-7 cells as shown in Figure 1 Panel B. Efflux of 5 was measured and found to proceed rapidly, leaving only 25±2% of the initial fluorescence after 2 hours (Figure 1 Panel C). Inhibition of the uptake of [14C]-D-fructose and [14C]-D-glucose was then measured. Compound 5 was found to be an inhibitor of [14C]-D-fructose uptake but not of [14C]-D-glucose uptake. We observed that 5 caused an increase in the observed fluorescent signal in plate reader experiments and competitively inhibited the uptake of [14C]-D-fructose in MCF-7 and EMT-6 cell lines (see SI, Figure SI 6), but it was unclear whether it was binding and being translocated across the membrane or merely binding to the extracellular binding site of the GLUTs and preventing [14C]-D-fructose from binding and being trans-


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ported. To determine if 5 could be transported across the membrane, and to further implicate the transporter(s) involved, we carried out several qualitative confocal microscopy experiments to complement the quantitative experiments we had already performed. The observed images, which are presented in Figure 1 Panel D for MCF7 cells and in the accompanying SI for the EMT-6 (Figure SI 5), clearly show that 5 was internalized by both cell lines. (Full-size high resolution confocal microscopy images showing internalization by both MCF-7 and EMT-6 cells can be found in the SI.) Confocal fluorescence indicating uptake of 5 was inhibited by co-incubation with Dfructose (50 mM) but not by co-incubation with Dglucose (50 mM) in a similar fashion as was observed in fluorescent plate reader experiments over the same 30minute time frame. These results indicate that 5 was entering the cell and entry appeared to be mediated by GLUT5. To further support this we set about testing 6NBDF in cell cultures in which we could control the expression of GLUT5. CHO cells, which express a low level of GLUT5 naturally,28,29 were transfected with GLUT5 mRNA to increase the amount of GLUT5 present on the cell membrane (see SI, Figure SI 11 Panel A). This increase in GLUT5 expression caused a dramatic increase in the level of observed fluorescence between the transfected and non-transfected cells in fluorescence plate reader experiments (see SI, Figure SI 11 Panel B). As a second test for the role of GLUT5 and the correlation between GLUT5 levels and transport levels for 5 we moved our attention to a line of healthy breast cells which do not overexpress GLUT5, the MCF-10A cell line.30,31 Fluorescence plate reader experiments with MCF-10A cells showed significantly lower uptake than did the MCF-7 cells, which overexpress GLUT524 (See SI, Figure SI 11 Panel C). These correlations are key for the future

Figure 1. Panels A-C: Fluorescence plate reader measurements. Panel A: The observed fluorescence of MCF-7 cells incubated with 300 µm 6-NBDF (5) over time. Panel B: Re-

sponse of observed fluorescence of MCF-7 cells incubated with 5 and extracellular D-glucose (50 mM) or D-fructose (50 mM). Panel C: Decrease in observed fluorescence over time after removal of incubation solution. Panel D: Confocal images obtained after incubation of MCF-7 cells with 10 µM 5 for 30 min and the response to extracellular D-fructose (50 mM) or D-glucose (50 mM). Control images of MCF-7 and EMT-6 cells can be found in the accompanying SI. (FI = fluorescence intensity; RF = remaining fluorescence)

usefulness of 5 and related analogs in imaging GLUT5 expressing breast cancers. Selective imaging, in a clinical or laboratory setting, would be driven by the relative expression levels of GLUT5 in cancerous and non-cancerous tissues. The clear relationship between the expression level of GLUT5 and the observed fluorescence of cells incubated with 5 suggests that it could be a first-generation molecular probe for the imaging of GLUT5 expressing tumors. Next, we turned our attention to the transport characteristics of 6, 7 and 8 in MCF-7 and EMT-6 cell lines. While utilizing the fluorescence plate reader the time dependent uptake of 6 and 7 was observed to be similar to 5 in MCF-7 cells while the uptake of 8 was significantly lower (Figure 2 Panel A). However, all four compounds demonstrated comparable uptake in EMT-6 cells (see SI, Figure SI 8). This indicates that the transport of these compounds is related to both the type and amount of GLUT transporters that are expressed on the cell surface. The uptake of 7 in MCF-7 cells was inhibited by 61±2% upon co-incubation with 50 mM D-glucose but no effect on the observed fluorescence signal was found with Dfructose co-incubation. Examination of 8 found no response to co-incubation with 50 mM D-fructose while 50 mM D-glucose reduced the observed fluorescence signal by 38±1%. A similar effect to co-incubation with either Dfructose or D-glucose was observed in EMT-6 cells as in MCF-7 cells. In all cases co-incubation with D-glucose showed a stronger reduction in fluorescence signal than with D-fructose. In MCF-7 cells 6 showed no significant response to the presence of D-fructose and was inhibited by 32±2% in the presence of 50 mM D-glucose (See SI, Figure SI 8). The efflux of 6-8 was found to be similar in MCF-7 and EMT-6 cells as shown in Figure 2 Panel B. We observed a slight decrease in observed fluorescence in some cases of EMT-6 with D-fructose co-incubation. This is likely due to the involvement of GLUT2 in the transport of the NBD-labelled compounds. The response to Dfructose was of very small magnitude and often within error of the uninhibited uptake. Finally, in studies of the inhibition of uptake of [14C]-D-fructose and [14C]-Dglucose 6-8 were found to be inhibitors of the uptake of [14C]-D-glucose but not of [14C]-D-fructose (see SI, Figure SI 7). Compounds 6-8 were all shown to be internalized by MCF-7 and EMT-6 cells via confocal microscopy using the same experimental procedure as with 5. Co-incubation with D-fructose (50 mM) resulted in little or no qualitative change in probe-derived fluorescence intensity, while co-incubation with D-glucose (50 mM) dramatically re-



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duced the amount of fluorescence observed. The same trend was found in both MCF-7 (Figure 2, Panel C) and EMT-6 (see SI, Figure SI 5) cell lines. These results indicate that 6-8 are transported into MCF-7 and EMT-6 cell lines via GLUT1, possibly with some assistance of GLUT2 in EMT-6 cells, and not through the GLUT5 pathway. Given the lack of any effect on [14C]-D-fructose uptake and a large effect on [14C]-D-glucose uptake when compounds 6-8 were used as inhibitors, we can conclude that these compounds interfere with a glucose transporter pathway, but not with the GLUT2 mediated glucose/fructose pathway. To implicate the role of GLUT-mediated transport in the uptake of these compounds, two additional experiments were conducted. When MCF-7 cells were incubated with 6 at 4 °C,32,33 the fluorescence intensity was dramatically reduced compared to incubation at 37 °C (see SI, Figure SI 12 Panel A). Moreover, incubation of MCF-7 cells under standard conditions with NBD-labeled serinol, a simple 3-carbon aminodiol, resulted in very low fluorescence barely above background (see SI, Figure SI 12 Panel B), indicating the importance of a hexose moiety for recognition and transport. Though 6, 7 and 8 all appear to be GLUT1 substrates and not GLUT5 substrates—and hence of no use for imaging GLUT5 expressing breast cancers, they do allow us to gain further insight into the binding pocket of GLUT5. It is apparent that the configuration at C-3, C-4, and C-5 is critical for GLUT5 specificity as each of these compounds differs from 5 in configuration at a single stereogenic center. The relative uptake levels of 6, 7 and 8, which are locked in the furanose form, were compared to the uptake of the known fluorescent D-glucose analog 2-NBD-Dglucosamine (2-NBDG) which exists exclusively in the pyranose form. 2-NBDG has been used as a probe of Dglucose uptake for a number of years, and is believed to be a GLUT1 substrate.34,35 Its transport shows that the GLUT1 pore can accommodate a large, nonpolar moiety (NBD). Comparison of 6-8 to 2-NBDG using fluorescence plate reader showed that these compounds produced a much more intense fluorescent signal than 2-NBDG in both MCF-7 and EMT-6 cells (Figure 3, Panel B; see SI Figure SI 10 for EMT-6). Although the furanose form of hexoses is not preferred by GLUT1,36 these probes (6-8) appear to be recognized and transported by this hexose transporter. Compounds 5-8 target GLUT5 5 or GLUT1 6-8 selectively and with higher affinity than the commonly used probe 2-NBDG, and thus may be valuable tools for monitoring hexose trafficking via these transporters.

Figure 2. Results of fluorescence plate reader measurements. Panel A: 6-NBDP (6), 6-NBDT (7) and 6-NBDS (8) in MCF-7 cells. Panel B: Efflux of 6, 7 and 8 in MCF-7 cells. Panel C: Confocal images obtained after incubation of MCF-7 cells with 6, 7 and 8 (all at 10 µM) and the response to the presence of extracellular D-fructose (50 mM) or D-glucose (50 mM). (FI = fluorescence intensity; RF = remaining fluorescence)

Members of the GLUT family typically can transport more than one substrate, indicating that the binding site and pore are configured in such a way as to allow recognition and transport of several compounds.8,9 We have shown that a single inversion of the configuration at C-3, C-4, or C-5 appears to cause a complete change in which GLUT transporter these compounds interact with. In contrast, it must be emphasized that the presence of an NBD moiety did not prevent transport of 5–8. The NBD group is both sterically demanding and diversely functionalized


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in comparison to the C-6 hydroxyl group found in the natural hexose substrates, indicating a capacity for passage of unnatural moieties through the GLUT pore

Figure 3. Panel A: Structure of 2-NBDG. Panel B: Uptake comparison of 6-8 to 2-NBDG. MCF-7 cells were incubated with 300 µM of each probe for 30 min at 37 °C. (FI = fluorescence intensity)

when bound to a suitable hexose moiety. The pore appears to tolerate large, nonpolar groups so long as there is sufficient energy gained from substrate binding to drive the necessary protein conformational changes. The capability of suitably modified D-fructose analogs to deliver other payloads via GLUT5 clearly merits further study. The recent computational work of Kaback and coworkers37 indicates that the specificity of GLUTs is derived from a complex, and highly specific network of hydrogen bonding interactions at the protein’s extracellular pore. Their docking studies also show that the C-6 hydroxyl of D-fructose is less involved with H-bonding to GLUT5 and is located further from the pore than are the C-3, C-4 or C-5 groups. This is consistent with our observation that inversion of a single stereogenic center at C-3, C-4 or C-5 dramatically alters the selectivity of transport. We believe that the introduction of the large NBD fluorophore at C-6 is tolerated by the GLUT because the fluorophore is positioned just outside the sugar-binding pocket and the key hydrogen bonding network is minimally perturbed. We hypothesize that initiation of the shift from outward open to inward open GLUT conformation by substrate binding is smoothly followed by passage of the substrate through the pore with minimal interference by the trailing NBD moiety. Previous work carried out in our group as well as in experiments reported by the groups of Gambhir, Holman, and others have shown that modification at C-3, C-4 and C-5 of D-fructose are poorly tolerated for GLUT5 binding.21-26 Further, McQuade and coworkers have shown that GLUT5 will recognize 2,5-anhydro-Dmanitol derivatives, indicating that the anomeric center is not required for binding and transport.38 In this study, we have now shown that the binding events required for

GLUT5 to initiate its conformational change and transport a hexose molecule depends on stereospecific interactions in the binding site. The perturbation of even a single interaction prevents the transport event from occurring in the case of GLUT5. In contrast, GLUT1 transports all three diastereomeric compounds 6-8, each differing from the other as well as from the natural substrate Dglucose. It is clear from these results that GLUT5 has a highly stringent binding pocket while GLUT1 is much more permissive in substrate recognition and transport. The four compounds evaluated all showed similar efflux properties: rapid wash out of the compounds with approximately 25% of initial fluorescence remaining after two hours. This result indicates that these compounds are not trapped inside the cells. D-Fructose and D-glucose are phosphorylated by hexokinase and fructokinase once inside the cell, thus preventing them from being transported out of the cell.21,39 Replacement of the C-6 hydroxyl with NH-NBD removes the possibility of phosphorylation at this position. Alternative phosphorylation at the remaining primary hydroxyl at C-1 requires fructokinase, whose expression in these cell lines is minimal or absent.40 Assuming 5-8 cannot undergo phosphorylation, it is not surprising that substantial efflux occurs following the initial uptake. Breakdown of the compounds to nonfluorescent by-products is formally possible as an alternative mechanism for the diminution in fluorescence; however, we have seen no evidence for the occurrence of chemical or enzymatic processes on the timescale of these experiments that would consume the fluorophore. Further, the fluorophore may be cleaved from the hexose moiety and diffuse from the cell, leading to the observed decrease in fluorescence. Currently we have no evidence for these or any other breakdown pathways though we are working to determine how these chemical transformations may be occurring. Blind docking studies indicated that D-fructose and the four sugar derivatives 5-8 all bind to the same region of GLUT5, suggesting that this location has the highest binding affinity and provides the lowest energy conformer state for the above ligands (Figure 4). Interestingly, for compound 5, the preferentially transported substrate, the orientation in the pocket was opposite to that of other three substrates 6-8 for both the outward and inward facing conformations. These docking studies indicated that the furanose ring of 5 binds in the same position as the natural substrate D-fructose. Furthermore, the residues of GLUT5 in either outward open or inward-open conformations that appear to be interacting with different substrates were identified (see SI, Tables 1 and 2). Single alanine mutants of rat GLUT5 residues Y31 (Y32 in human GLUT5), Q287 (288), H386 (387), H418 (419) are known to strongly reduce D-fructose binding to GLUT5.14 The present docking study showed that only D-fructose and 5 interact with these residues in a similar hydrogen bonding network in both the outward and inward facing conformations. H418 (H419) was not observed to interact with 5 in the inward facing conformation. Furthermore, W419 (W420), which previously was shown to have al-



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tered fluorescence quenching during D-fructose binding,14 was found to be involved in interaction only in the case of 5 and 6 (outward conformation) and with 6 and 7 (inward conformation). However, in human GLUT5 the neighboring residue H419 was observed to be involved in interactions with all the substrates, suggesting its importance over W420. In contrast to 5, which interacts with the substrate binding pocket via an intricate hydrogen bonding network similar to D-fructose, 6-8 bind with predominantly hydrophobic interactions with few hydrogen bonds predicted (Figure 4 and see SI, Figures SI 3 and SI 4). In summary, four new fluorescently labeled hexose analogs have been synthesized and characterized for their ability to be recognized and transported by the facilitated hexose transporter proteins (GLUTs). We found that both MCF-7 and EMT-6 cells transport fructose analog 5 selectively via the GLUT5 pathway. We have also shown that C-3, C-4, and C-5 epimers of 5 were transported mainly

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This work enhances our understanding of how the GLUT transporters work at a molecular level concerning the recognition of substrates and the transport mechanism. The binding pocket of the GLUT proteins requires a highly organized hexose-binding event in order to initiate the protein conformation change. It also appears that once the GLUT is in flux between the outward and inward open conformations the pore is larger than a single hexose and a payload can enter along with the hexose. This knowledge should facilitate the structure-based design of novel substrates that can be recognized selectively by individual members of the GLUT family and carry a payload such as an imaging or pharmaceutical agent. The compounds reported herein also show promise as laboratory tools for monitoring D-fructose and D-glucose transport. Further studies are underway to determine the efficacy of these compounds in those roles. The fate of these compounds after they enter the intracellular environment has yet to be determined and future studies to determine their fate are underway in hopes that this knowledge will help us design the next generation of selective GLUT substrates.

METHODS

Figure 4. Varied orientation of the probes in the human GLUT5 outward-facing binding site. Views of human GLUT5 binding site in the outward-open conformation with (A) Dfructose, (B) 6-NBDF (5) or (C) 6-NBDP (6), showing residues participating in either hydrogen bonding or hydrophobic interactions.

by the GLUT1, and all showed greater uptake than the known D-glucose mimic 2-NBDG. Docking studies indicate that 5 interacts with GLUT5 with the sugar moiety oriented towards the GLUT pore, in the same orientation as D-fructose, while 6–8 oriented with the NBD moiety projecting into the pore, despite only a single stereochemical inversion on the furanose ring in each case. These experimental and computational results show that there is a stringent structural and stereochemical requirement for recognition by GLUT5. Modification of the configuration of the C-3, C-4, or C-5 centers in the furanose ring results in a change in transport pathway from GLUT5 to GLUT1.

General procedures. Reactions were carried out in flame-dried glassware under a positive argon atmosphere unless otherwise stated. Transfer of anhydrous solvents and reagents was accomplished with oven-dried syringes or cannulae. Solvents were distilled before use: dichloromethane (CH2Cl2) and dimethylformamide (DMF) from calcium hydride, tetrahydrofuran (THF) and ether (Et2O) from sodium/benzophenone ketyl and pyridine from KOH. Thin layer chromatography was performed on glass plates precoated with 0.25 mm silica gel. Flash chromatography columns were packed with 230–400 mesh silica gel. Optical rotations were measured at 22 ± 2 °C. Proton nuclear magnetic resonance spectra (1H NMR) were recorded at 400 MHz, 500 MHz or 700 MHz and coupling constants (J) are reported in hertz (Hz). Standard notation was used to describe the multiplicity of signals observed in 1H NMR spectra: broad (br), multiplet (m), singlet (s), doublet (d), triplet (t), etc. Carbon nuclear magnetic resonance spectra (13C NMR) were recorded at 100 MHz or 125 MHz and are reported (ppm) relative to the center line of the triplet from chloroform-d (77.0 ppm) or the center line of the heptuplet from methanol-d4 (49.0 ppm). Infrared (IR) spectra were measured with a FT-IR 3000 spectrophotometer. Mass spectra were determined on a high-resolution electrospray positive ion mode spectrometer. Synthesis of methyl 1,3-O-isopropylidene-6-O-(ptolylsulfonyl)-α α-D-fructofuranoside (2). The synthesis of methyl 1,3-O-isopropylidene-6-O-(p-tolylsulfonyl)-α-Dfructofuranoside 2 was modified from the procedure reported by Jung and co-workers.41 Compound 2 was prepared in two steps starting from D-fructose according to the following procedure. D-Fructose (5.4 g, 30 mmol) was added to a solution of anhydrous MeOH (30 mL) and TsOH·H2O (27 mg, 0.14 mmol) and the mixture was


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stirred for 16 h at room temperature. 2,2Dimethoxypropane (30 mL) was then added to the reaction mixture. After stirring for 40 min at room temperature the reaction was neutralized with anhydrous NaHCO3. The mixture was filtered and concentrated under reduced pressure, then purified through a silica gel column using hexane:EtOAc (gradient from 50:50 to 30:70) as eluant to afford methyl 1,3-O-isopropylidene-α-Dfructofuranoside 1 as a pale yellow oil (3.8 g, 55%).27 Compound 1 was dissolved in anhydrous pyridine (20 mL). The resulting solution was cooled to 0 °C and tosyl chloride (4.08 g, 21.4 mmol) was added as a single portion. The mixture was slowly warmed up to room temperature and stirred for an additional 24 h. The mixture was concentrated under reduced pressure, then purified through a silica gel column to afford the desired product 2 as a pale yellow oil (8.3 g, 70%).40 Rf 0.4 (Hexane:EtOAc 50:50); [α]D20 +26.19° (c 0.5, CH2Cl2); IR (cm−1) 3516, 2991, 2941, 1363, 1177, 1098; 1H NMR (500 MHz, CDCl3) δ 7.83 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 8.2 Hz, 2H), 4.26-4.13 (m, 3H), 3.99 (d, J = 0.7 Hz, 1H), 3.87 (br, 2H), 3.85 (d, J = 11.0 Hz, 1H), 3.28 (s, 3H), 2.72 (d, J = 10.0 Hz, 1H), 2.45 (s, 3H), 1.42 (s, 3H), 1.25 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 144.9, 132.7, 129.8 (2 CH), 128.0 (2 CH), 102.1, 98.5, 84.4, 79.1, 77.7, 69.7, 61.5, 48.8, 27.6, 21.6, 19.4; HRMS (ESI) calcd for C17H24NaO8S [M + Na]+ 411.1084; found 411.1082. Synthesis of methyl 1,3-O-isopropylidene-6-azidoα-D-fructofuranoside (3). NaN3 (2.76 g, 42.5 mmol) was added as a single portion to a solution of compound 2 (3.3 g, 8.5 mmol) in anhydrous DMF (20 mL). The reaction mixture was stirred at 120 °C for 16 h. The mixture was filtered and concentrated under reduced pressure, then purified through a silica gel column to afford the desired product 3 as a pale yellow oil (1.6 g, 75%). Rf 0.50 (Hexane:EtOAc 50:50); [α]D20 +65.26° (c 1.15, CH2Cl2); IR (cm−1) 3448, 2991, 2940, 2101, 1376, 1271, 1221, 1099; 1H NMR (500 MHz, CDCl3) δ 4.16 (ddd, J = 7.3, 5.0, 2.6 Hz, 1H), 4.04 (d, J = 0.6 Hz, 1H), 3.96 (d, J = 12.2 Hz, 1H), 3.93 (d, J = 12.2 Hz, 1H), 3.90 (dd, J = 9.9, 3.4 Hz, 1H), 3.54 (dd, J = 13.0, 7.5 Hz, 1H), 3.44 (dd, J = 13.0, 5.5 Hz, 1H), 3.33 (s, 3H), 2.65 (d, J = 10.0 Hz, 1H), 1.46 (s, 3H), 1.39 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 102.2, 98.7, 86.1, 79.6, 78.5, 61.7, 52.7, 48,8, 27.6, 19.5; HRMS (ESI) calcd for C10H17N3NaO5 [M + Na]+ 282.106; found 282.1055. Synthesis of methyl 1,3-O-isopropylidene-6-[N-(7nitrobenz-2-oxa-1,3-diazole-4-yl)amino]-α α-Dfructofuranoside (4). Palladium on carbon (0.7 g, 10 wt% Pd/C) was added as a single portion to a solution of compound 3 (0.95 g, 3.6 mmol) in EtOH (10 mL). The round bottom flask was then equipped with a hydrogenfilled balloon. After 24 h stirring at rt, the reaction mixture was filtered through a short Celite pad to remove particulates and then concentrated under reduced pressure to give the amine intermediate as a pale yellow oil that was used without further purification in the next reaction. An aqueous solution of NaHCO3 (0.3 M, 23 mL) was added to a solution of the amine intermediate along with NBDCl (0.87 g, 4.4 mmol) in MeOH (40 mL). After stirring at rt for 24 h in the dark, the mixture was concen-

trated under reduced pressure, and then purified through a silica gel column to afford the desired product 4 as an orange solid (0.58 g, 40%). Rf 0.60 (Hexane:EtOAc 50:50); mp 83-86 °C; [α]D20 +59.44° (c 0.4, CH2Cl2); IR (cm−1) 3353, 2992, 2930, 1583, 1307, 1103; 1H NMR (500 MHz, CDCl3) δ 8.52 (d, J = 8.5 Hz, 1H), 6.89 (t, J = 5.0 Hz, 1H), 6.31 (d, J = 8.5 Hz, 1H), 4.42 (ddd, J = 4.3, 5.0, 2.4 Hz, 1H), 4.16 (s, 1H), 4.06 (d, J = 12.5 Hz, 1H), 4.06 (d, J = 8.6 Hz, 1H), 4.01 (d, J = 12.5 Hz, 1H), 3.83 (br, 2H), 3.36 (s, 3H), 2.81 (d, J = 11.0 Hz, 1H), 1.55 (s, 3H), 1.54 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 144.2, 144.1, 143.8, 136.4, 124.2, 101.6, 99.3, 99.3, 84., 79.3, 78.3, 61.8, 49.0, 45.7, 28.2, 19.1; HRMS (ESI) calcd for C16H20N4NaO8 [M + Na]+ 419.1173; found 419.1168. Synthesis of 6-NBDF (5). An aqueous solution of HCl (1 M, 3 mL) was added to a solution of compound 4 (0.10 g, 0.25 mmol) in CH3CN (6 mL) at room temperature. The reaction mixture was heated at 90 °C for 20 min and then cooled to room temperature. The mixture was neutralized with a saturated solution of NaHCO3 and then concentrated under reduced pressure. The residue was purified through a silica gel column, using the dry-loading technique, to afford the desired product 5 (0.65 g, 75%) as an orange solid in a 3:1 mixture of anomers (β anomer is major in this case). Compound (5): Rf 0.24 (DCM:MeOH 90:10); [α]D20 +59.07° (c 1.37, MeOH); IR (cm−1) 3357, 2931, 1619, 1596, 1481, 1357, 1330, 1295, 1132, 1039; HRMS (ESI) calcd for C12H14N4NaO8 [M + Na]+ 365.0704; found 365.0702.

β-anomer: 1H NMR (500 MHz, CD3OD) δ 8.51 (d, J = 8.5 Hz, 1H), 6.44 (d, J = 8.5 Hz, 1H), 4.14-4.03 (m, 3H), 3.75 (br, 2H), 3.52 (d, J = 11.5 Hz, 1H), 3.49 (d, J = 11.5 Hz, 1H); 13 C NMR (125 MHz, CD3OD) δ 145.3, 144.4, 143.9, 137.0, 121.9, 102.3, 98.8, 79.1, 76.6, 75.6, 62.7, 46.1.

α-anomer: 1H NMR (500 MHz, CD3OD) δ 8.51 (d, J = 8.5 Hz, 1H), 6.41 (d, J = 8.5 Hz, 1H), 4.27 (ddd, J = 5.5, 6.0, 3.8 Hz, 1H), 4.07-4.01 (m, 2H), 3.75 (br, 2H), 3.72 (d, J = 11.5 Hz, 1H), 3.55 (d, J = 11.5 Hz, 1H); 13C NMR (125 MHz, CD3OD) δ 145.3, 144.4, 143.9, 136.8, 121.8, 105.0, 98.7, 82.1, 79.3, 77.8, 63.3, 45.1.

ASSOCIATED CONTENT Supporting Information 1

Experimental procedures, characterization data, copies of H 13 and C NMR spectra and additional biological data. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT



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This work was funded by a Collaborative Health Research Project grant from NSERC (Canada) and Canadian Institutes of Health Research (CHRP J 365459-2009 and CPG 3654592009, respectively), and by and an operating grant from the Canadian Breast Cancer Foundation. The authors also wish to thank the Canadian Glycomics Network for a Catalyst Grant (CD-9) and K. Wong for technical support.

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Fluorescent Hexose Conjugates Establish Stringent Stereochemical

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