d-Glucose- and d-mannose-based antimetabolites. Part 4: Facile synthesis of mono- and di-acetates of 2-deoxy-d-glucose prodrugs as potentially useful antimetabolites.

2-Deoxy-d-glucose (2-DG), a compound known to interfere with d-glucose and d-mannose metabolism, has been tested as a potential anticancer and antiviral agent. Preclinical and clinical studies focused on 2-DG have highlighted several limitations related to 2-DG drug-like properties, such as poor pharmacokinetic properties. To overcome this problem, we proposed design and synthesis of novel 2-DG prodrugs that subsequently could be tested using a variety of biochemical and molecular methods. We narrowed here our focus to esters of 2-DG as potential prodrugs based on the hypothesis that ubiquitous esterases will regenerate 2-DG, leading to increased circulation time of drug and adequate organ and tumor penetration. Testing this hypothesis in vitro and, especially, in vivo requires significant amounts of respective pure mono- and previously unknown di-acetylated water-soluble derivatives of 2-DG. Development of their efficient and practical method of synthesis was imperative. We describe novel facile and scalable syntheses of seven selectively acetylated water-soluble derivatives of 2-DG and present a detailed 1H and 13C NMR analysis of all final products. X-ray diffraction analysis has been performed for compound WP1122 that was selected for detailed preclinical and subsequent clinical evaluation as potential anticancer or antiviral agent.

A Comprehensive Biological and Synthetic Perspective on 2-Deoxy-d-Glucose (2-DG), A Sweet Molecule with Therapeutic and Diagnostic Potentials.

Glucose, the primary substrate for ATP synthesis, is catabolized during glycolysis to generate ATP and precursors for the synthesis of other vital biomolecules. Opportunistic viruses and cancer cells often hijack this metabolic machinery to obtain energy and components needed for their replication and proliferation. One way to halt such energy-dependent processes is by interfering with the glycolytic pathway. 2-Deoxy-d-glucose (2-DG) is a synthetic glucose analogue that can inhibit key enzymes in the glycolytic pathway. The efficacy of 2-DG has been reported across an array of diseases and disorders, thereby demonstrating its broad therapeutic potential. Recent approval of 2-DG in India as a therapeutic approach for the management of the COVID-19 pandemic has brought renewed attention to this molecule. The purpose of this perspective is to present updated therapeutic avenues as well as a variety of chemical synthetic strategies for this medically useful sugar derivative, 2-DG.

A GLUT1 inhibitor-based probe significantly ameliorates the sensitivity of tumor detection and diagnostic imaging.

We report a non-antibody GLUT1 inhibitor probe NBDQ that is 30 times more sensitive than the traditional GLUT1 transportable tracer for cancer cell imaging and Warburg effect-based tumor detection. NBDQ reveals significant advantages in terms of tumor selectivity, fluorescence stability and in vivo biocompatibility in xenograft tumor imaging, including triple-negative breast cancer.

A Fluorescence-Based Assay for Measuring Glucose Uptake in Living Melanoma Cells.

Melanoma cells have high glycolytic capacity. Glucose uptake is a key rate-limiting step in glucose utilization. Here we describe a simple protocol for measuring direct glucose uptake in living melanoma cells by flow cytometry.

Cellular toxicity of the metabolic inhibitor 2-deoxyglucose and associated resistance mechanisms.

Most malignant cells display increased glucose absorption and metabolism compared to surrounding tissues. This well-described phenomenon results from a metabolic reprogramming occurring during transformation, that provides the building blocks and supports the high energetic cost of proliferation by increasing glycolysis. These features led to the idea that drugs targeting glycolysis might prove efficient in the context of cancer treatment. One of these drugs, 2-deoxyglucose (2-DG), is a synthetic glucose analog that can be imported into cells and interfere with glycolysis and ATP generation. Its preferential targeting to sites of cell proliferation is supported by the observation that a derived molecule, 2-fluoro-2-deoxyglucose (FDG) accumulates in tumors and is used for cancer imaging. Here, we review the toxicity mechanisms of this drug, from the early-described effects on glycolysis to its other cellular consequences, including inhibition of protein glycosylation and endoplasmic reticulum stress, and its interference with signaling pathways. Then, we summarize the current data on the use of 2-DG as an anti-cancer agent, especially in the context of combination therapies, as novel 2-DG-derived drugs are being developed. We also show how the use of 2-DG helped to decipher glucose-signaling pathways in yeast and favored their engineering for biotechnologies. Finally, we discuss the resistance strategies to this inhibitor that have been identified in the course of these studies and which may have important implications regarding a medical use of this drug.

Modular continuous flow synthesis of orthogonally protected 6-deoxy glucose glycals.

An efficient, modular continuous flow process towards accessing two orthogonally protected glycals is described with the development of reaction conditions for several common protecting group additions in flow, including the addition of benzyl, naphthylmethyl and tert-butyldimethylsilyl ethers. The process affords the desired target compounds in 57-74% overall yield in just 21-37 minutes of flow time. Furthermore, unlike batch conditions, the flow processes avoided the need for active cooling to prevent unwanted exotherms and required shorter reaction times.

Oleanane Triterpenoids from the Leaves of Gymnema inodorum and Their Insulin Mimetic Activities.

During an effort to find insulin mimetic compounds, the leaves of Gymnema inodorum were shown to have a stimulatory effect on glucose uptake in 3T3-L1 adipocyte cells. Bioassay-guided fractionation on a 70% ethanol extract of G. inodorum was applied to yield two new (1 and 2) and two known (8 and 9) oleanane triterpenoids with a methyl anthranilate moiety together with five further new oleanane triterpenoids (3-7). The chemical structures of all isolates were determined based on their spectroscopic data, including IR, UV, NMR, and mass spectrometric analysis. The isolated compounds (1-9) were determined for their stimulatory activities on glucose uptake in differentiated 3T3-L1 adipocyte cells using 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-d-glucose (2-NBDG) as a fluorescent-tagged glucose probe. Three compounds (3, 5, and 9) showed stimulatory effects on the uptake of 2-NBDG in 3T3-L1 adipocyte cells. Chemicals with a methyl anthranilate moiety have been considered as crucial contributors of flavor odor in foods, and quantitative analysis showed the content of compound 8 to be 0.90 ± 0.01 mg/g of the total extract. These results suggest that the leaves of G. inodorum have the potential to be used as an antidiabetic functional food or tea.

One-stop radiotherapeutic targeting of primary and distant osteosarcoma to inhibit cancer progression and metastasis using 2DG-grafted graphene quantum dots.

The application of radiotherapy (RT) to treat osteosarcoma (OS) has been limited, but this is starting to change as the ability to target radiation energy to niches improves. Furthermore, lung cancer from highly metastatic OS is a major cause of death, so it is critical to explore new strategies to tackle metastasis. In this study, we designed a nanoscale radiosensitizer by grafting 2-deoxy-d-glucose (2DG) onto graphene quantum dots (GQD) to achieve OS targeting and boost RT efficacy. Combining the use of 2DG-grafted GQDs (2DG-g-GQD) with RT produced a significant increase in oxidative stress response and DNA damage in the 143B OS cell line compared with RT alone. Moreover, 2DG-g-GQDs selectively associated with 143B cells, and demonstrated the inhibition of migration in a scratch assay. We also demonstrated remarkable improvement in their ability to inhibit tumour progression and lung metastasis in an OS xenograft mouse model. Our results show that the use of 2DG-g-GQDs as OS-targeting radiosensitizers improves their therapeutic outcome and exhibits potential for use in low-dose precision RT for OS.

5-Hydroxymethylfurfural accumulation plays a critical role on acrylamide formation in coffee during roasting as confirmed by multiresponse kinetic modelling.

This study aims to investigate in depth the mechanism of acrylamide formation in coffee during roasting. For this purpose, a comprehensive kinetic model including the elementary steps for acrylamide formation was proposed. The changes in sucrose, reducing sugars, free amino acids, asparagine, acrylamide, 3-deoxyglucosone, methylglyoxal, glyoxal, and 5-hydroxymethylfurfural were monitored in coffee during roasting at 200, 220 and 240 °C. Dominant pathways of complex reactions leading to acrylamide were unravelled by means of multiresponse kinetic modelling approach. The results of the model indicated that sucrose degrades into glucose and a reactive fructofuranosyl cation. Interestingly, glucose takes part mostly in the formation of intermediates, glyoxal and especially 3-deoxyglucosone rather than acrylamide formation. On the other hand, fructofuranosyl cation contributed mostly to the formation of 5-hydroxymethylfurfural which was found to be the most important intermediate precursor of acrylamide formed in coffee during roasting.

In Vivo Epithelial Metabolic Imaging Using a Topical Fluorescent Glucose Analog.

The demanding metabolic needs of cancer cells are met by aerobic glycolysis. While whole-body PET imaging methods exist for evaluating this metabolic response, these are not ideal for local, more detailed regions such as mucosal surfaces. Fluorescence imaging of glucose analogs with similarities to radiolabeled deoxyglucose used in PET, namely, fluorescent 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG), offers such an alternative, particularly as this glucose analog may be delivered by local topical delivery. In this chapter, methods for in vivo epithelial imaging in a preclinical hamster model for oral cancer and oral epithelial dysplasia are described. Outlined are methods for preparation and in vivo delivery of 2-NBDG by topical application to the oral mucosa followed by fluorescence imaging to compare fluorescence responses between neoplasia and control mucosa or to monitor changes in fluorescence signal with time in both groups.

Kinetic study on the generation of furosine and pyrraline in a Maillard reaction model system of d-glucose and l-lysine.

A kinetic model for Maillard reaction (MR) model system of d-glucose and l-lysine was established; activation energy (Ea) of each step was calculated. Potential generation pathways of furosine and pyrraline were a combination of either 3-deoxyglucosone (3-DG) or methylglyoxal (MG) with l-lysine. Ea value for furosine generated through 3-DG pathway was 81.70 ± 14.01 kJ mol-1, which was significantly higher than that through MG pathway (52.08 ± 4.48 kJ mol-1). As for pyrraline, Ea for the 3-DG pathway (53.45 ± 4.02 kJ mol-1) was significantly lower than that through the MG pathway (110.22 ± 18.77 kJ mol-1). Results of the kinetic study indicated that furosine was preferred to be generated through the MG pathway since MG is more likely to react with each other and form a furan ring as a precursor of furosine. Pyrraline was more easily to be generated from the 3-DG pathway through cyclization of 1,4-dicarbonyl compounds to pyrrole.

An In Vitro Method to Analyze Glucose Uptake in Podocytes.

Podocytes are terminally differentiated, insulin-sensitive cells of the glomerular filtration barrier, with a central role in filtration barrier maintenance. Podocyte injury is one of the earliest features observed in diabetic nephropathy (DN) and plays a key role in the development of albuminuria. Several factors are associated with diabetes-mediated podocyte injury, including dysregulated metabolic pathways and insulin sensitivity (Lay & Coward, Nephrol Dial Transplant 29:1127-1133, 2014); thus, reliable assays to study these responses are key in understanding podocyte alterations in DN. Here, we detail an in vitro method to analyze glucose uptake in conditionally immortalized human podocytes (Saleem, J Am Soc Nephrol 13:630-638, 2002; Coward, Diabetes 54:3095-3102, 2005); this assay is useful for detecting changes in podocyte metabolism, nutrient sensing, and insulin sensitivity.

Antibacterial activity of 1,2-dicarbonyl compounds and the influence of the in vitro assay system.

The antibacterial activities of the dicarbonyl compounds glyoxal (GO), methylglyoxal (MGO), 3-deoxyglucosone (3-DG) were assessed against Gram-positive and Gram-negative pathogenic and food spoilage bacteria, both in agarised and liquid assay system. The kinetics of dicarbonyls' degradation at different antimicrobial assay conditions were studied, to determine the possible interference of the nutrient medium. In agarised assay system, GO and MGO exhibited antimicrobial activity, with higher efficacy against Gram-positive strains than Gram-negative ones. The nutrient medium reacted quickly both with GO and MGO, interfering with the antibacterial potential and the degradation kinetics indicated first-order reactions. In liquid assay system, both GO and MGO inhibited the target bacteria at concentrations significantly lower than those estimated in agarised assay system. Moreover, to the best of our knowledge, the antibacterial activity of GO and MGO against Listeria innocua, Pseudomonas fluorescens, Salmonella enterica and Bacillus cereus has not been previously reported.

Single-Cell Analysis for Glycogen Localization and Metabolism in Cultured Astrocytes.

Cerebral glycogen is principally localized in astrocytes rather than in neurons. Glycogen metabolism has been implicated in higher brain functions, including learning and memory, yet the distribution patterns of glycogen in different types of astrocytes have not been fully described. Here, we applied a method based on the incorporation of 2-NBDG, a D-glucose fluorescent derivative that can trace glycogen, to investigate glycogen's distribution in the brain. We identified two types of astrocytes, namely, 2-NBDGI (glycogen-deficient) and 2-NBDGII (glycogen-rich) cells. Whole-cell patch-clamp and fluorescence-activated cell sorting (FACS) were used to separate 2-NBDGII astrocytes from 2-NBDGI astrocytes. The expression levels of glycogen metabolic enzymes were analyzed in 2-NBDGI and 2-NBDGII astrocytes. We found unique glycogen metabolic patterns between 2-NBDGI and 2-NBDGII astrocytes. We also observed that 2-NBDGII astrocytes were mainly identified as fibrous astrocytes but not protoplasmic astrocytes. Our data reveal cell type-dependent glycogen distribution and metabolism patterns, suggesting diverse functions of these different astrocytes.

Synthesis and Comparative Structure-Activity Study of Carbohydrate-Based Phenolic Compounds as α-Glucosidase Inhibitors and Antioxidants.

Twenty-one natural and unnatural phenolic compounds containing a carbohydrate moiety were synthesized and their structure-activity relationship (SAR) was evaluated for α-glucosidase inhibition and antioxidative activity. Varying the position of the galloyl unit on the 1,5-anhydro-d-glucitol (1,5-AG) core resulted in changes in the α-glucosidase inhibitory activity and notably, particularly strong activity was demonstrated when the galloyl unit was present at the C-2 position. Furthermore, increasing the number of the galloyl units significantly affected the α-glucosidase inhibition, and 2,3,4,6-tetra-galloyl-1,5-AG (54) and 2,3,4,6-tetra-galloyl-d-glucopyranose (61) exhibited excellent activities, which were more than 13-fold higher than the α-glucosidase inhibitory activity of acertannin (37). Moreover, a comparative structure-activity study suggested that a hemiacetal hydroxyl functionality in the carbohydrate core and a biaryl bond of the 4,6-O-hexahydroxydiphenoyl (HHDP) group, which are components of ellagitannins including tellimagrandin I, are not necessary for the α-glucosidase inhibitory activity. Lastly, the antioxidant activity increased proportionally with the number of galloyl units.

[13C6,D8]2-deoxyglucose phosphorylation by hexokinase shows selectivity for the ß-anomer.

A non-radioactive 2-deoxyglucose (2DG) analog has been developed here for hyperpolarized magnetic resonance investigations. The analog, [13C6,D8]2DG, showed 13% polarization in solution (27,000-fold signal enhancement at the C1 site), following a dissolution-DNP hyperpolarization process. The phosphorylation of this analog by yeast hexokinase (yHK) was monitored in real-time with a temporal resolution of 1 s. We show that yHK selectively utilizes the ß anomer of the 2DG analog, thus revealing a surprising anomeric specificity of this reaction. Such anomeric selectivity was not observed for the reaction of yHK or bacterial glucokinase with a hyperpolarized glucose analog. yHK is highly similar to the human HK-2, which is overexpressed in malignancy. Thus, the current finding may shed a new light on a fundamental enzyme activity which is utilized in the most widespread molecular imaging technology for cancer detection - positron-emission tomography with 18F-2DG.

The human milk protein-lipid complex HAMLET disrupts glycolysis and induces death in Streptococcus pneumoniae.

HAMLET is a complex of human α-lactalbumin (ALA) and oleic acid and kills several Gram-positive bacteria by a mechanism that bears resemblance to apoptosis in eukaryotic cells. To identify HAMLET's bacterial targets, here we used Streptococcus pneumoniae as a model organism and employed a proteomic approach that identified several potential candidates. Two of these targets were the glycolytic enzymes fructose bisphosphate aldolase (FBPA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Treatment of pneumococci with HAMLET immediately inhibited their ATP and lactate production, suggesting that HAMLET inhibits glycolysis. This observation was supported by experiments with recombinant bacterial enzymes, along with biochemical and bacterial viability assays, indicating that HAMLET's activity is partially inhibited by high glucose-mediated stimulation of glycolysis but enhanced in the presence of the glycolysis inhibitor 2-deoxyglucose. Both HAMLET and ALA bound directly to each glycolytic enzyme in solution and solid-phase assays and effectively inhibited their enzymatic activities. In contrast, oleic acid alone had little to no inhibitory activity. However, ALA alone also exhibited no bactericidal activity and did not block glycolysis in whole cells, suggesting a role for the lipid moiety in the internalization of HAMLET into the bacterial cells to reach its target(s). This was verified by inhibition of enzyme activity in whole cells after HAMLET but not ALA exposure. The results of this study suggest that part of HAMLET's antibacterial activity relates to its ability to target and inhibit glycolytic enzymes, providing an example of a natural antimicrobial agent that specifically targets glycolysis.

Pyrraline Formation Modulated by Sodium Chloride and Controlled by Encapsulation with Different Coating Materials in the Maillard Reaction.

Advanced glycation end products (AGEs), which are present in heat-processed foods, have been associated with several chronic diseases. Sodium chloride (NaCl) modulates the formation of furfurals and acrylamide in the Maillard reaction; however, the effects of NaCl on AGE formation are inconsistent. In this study, we investigated the effects of NaCl on pyrraline formation using glucose-lysine model systems. NaCl, especially at 0.50%, promoted Maillard browning and pyrraline formation, with a simultaneous increase in the 3-deoxyglucosone concentration. To reduce the rate of pyrraline formation, NaCl coated with different gums and starches were used. The results showed that NaCl encapsulation is an effective approach to mitigate pyrraline and 3-deoxyglucosone formation. The content of NaCl in the microparticles were 284 ± 12, 269 ± 6, 258 ± 8, 247 ± 10, 273 ± 16, and 288 ± 15 mg/g (coated with waxy maize starch, normal maize starch, HYLON VII high amylose maize starch, gelatinized resistant starch, xanthan gum, and gum arabic, respectively). The heat resistance of the coating material was negatively correlated with the pyrraline and 3-deoxyglucosone formation, whereas the solubility of the coating material had the opposite results. Coating the material with gum had little effects on the reduction of pyrraline and 3-deoxyglucosone.

Effect of Liposomal Encapsulation on the Chemical Exchange Properties of Diamagnetic CEST Agents.

Exogenous chemical exchange saturation transfer (CEST) contrast agents such as glucose or 2-deoxy-d-glucose (2-DG) have shown high sensitivities and significant potential for monitoring glucose uptake in tumors with MRI. Here, we show that liposome encapsulation of such agents can be exploited to enhance the CEST signal by reducing the overall apparent exchange rate. We have developed a concise analytical model to describe the liposomal contrast dependence on several parameters such as pH, temperature, irradiation amplitude, and intraliposomal water content. This is the first study in which a model has been constructed to measure the exchange properties of diamagnetic CEST agents encapsulated inside liposomes. Experimentally measured exchange rates of glucose and 2-DG in the liposomal system were found to be reduced due to the intermembrane exchange between the intra- and extraliposomal compartments because of restrictions in water transfer imposed by the lipid membrane. These new theoretical and experimental findings will benefit applications of diamagnetic liposomes to image biological processes. In addition, combining this analytical model with measurements of the CEST signal enhancement using liposomes as a model membrane system is an important new general technique for studying membrane permeability.

In vivo and in vitro evaluation of 177Lu-labeled DOTA-2-deoxy-D-glucose in mice. A novel radiopharmaceutical agent for cells imaging and therapy.

OBJECTIVE: Incorporation of lutetium-177 (177Lu) into suitable molecules that are implicated in cancer pathology represents a promising approach for the diagnosis and treatment of cancer. The goal of the present study was to develop a novel 177Lu labeled radiopharmaceutical agent for both radioimaging and targeted radionuclide therapy. ANIMALS AND METHODS: Given the synthetic versatility of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) ligand as a metal chelator and high demand of sugar molecules such as deoxyglucose (DG) in cancer cells, we carried out the radiosynthesis of a novel radiopharmaceutical agent, namely, 177Lu-DOTA-DG, and utilized it for imaging of cancer and also for the targeted radiation therapy of cancer tissues. RESULTS: In this study, we developed an efficient radiochemical synthesis of 177Lu-DOTA-DG and evaluated its pharmacological properties in vitro/in vivo. Our results showed DOTA-DG can be labeled with 177Lu with excellent radiochemical yield at 90oC in 30min. The resulting 177Lu-DOTA-DG exhibited high degree of stability without significant radiolysis up to 120h in human serum and phosphate buffer. Favorable pharmacokinetics profile was demonstrated by rapid blood clearance in 4T1 murine tumor mice and heterogeneous whole body biodistribution of 177Lu-DOTA-DG. Further, Comet assay experiments indicated that cancer cells treated with 177Lu-DOTA-DG showed significant higher degree of DNA damage compared to cells treated with 177Lu3+ or non-treated cells. CONCLUSION: This study showed that there is a great potential of using 177Lu-DOTA-DG as an imaging and therapeutic agent for cancer diagnosis and treatment. Furthermore, this study provides valuable information for developing novel 177Lu-labeled radiopharmaceuticals.