Fluorescent dye

1-NBDG was used as a fluorescent glucose analogue to conduct glucose uptake assay in COS-7 cells transiently transfected with pcDNAhSGLT1 and CHO–K1 cells stably expressing hSGLT2 for the screening of SGLT1 and SGLT2 inhibitors, respectively. Here we demonstrated that it was also feasible to use COS-7 cells transiently transfected with pcDNAhSGLT2 (hSGLT2/COS-7) for the screening of SGLT2 inhibitors. The method was also extended from 24-well format to 96-well format suitable for high-throughput screening.The validated method using 1-NBDG was applied to test 195 crude methanol extracts from approximately 60 species of plants in the Lauraceae family in search of novel natural SGLT inhibitors and two potent natural SGLT1 and SGLT2 inhibitors were identified from the leaves of Cinnamomum macrostemon (Yang et al., manuscript in preparation). The IC50 values of these two compounds (I and II) were in the nanomolar range and comparable to phlorizin, the most potent natural SGLT inhibitor with IC50 of 110 ± 10.2 nM for SGLT1 and 9.65 ± 1.83 nM for SGLT2 in our assay system[1].

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Glucose is an important energy source for cells. Glucose transport is mediated by two types of glucose transporters: the active sodium-coupled glucose cotransporters (SGLTs), and the passive glucose transporters (GLUTs). Development of an easy way to detect glucose uptake by the cell can be valuable for research. 1-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-1-deoxy-d-glucose (1-NBDG) is a newly synthesized fluorescent glucose analogue. Unlike 2-NBDG, which is a good substrate of GLUTs but not SGLTs, 1-NBDG can be transported by both GLUTs and SGLTs. Thus, 1-NBDG is useful for the screening of SGLT1 and SGLT2 inhibitors. Here we further characterized 1-NBDG and compared it with 2-NBDG. The fluorescence of both 1-NBDG and 2-NBDG was quenched under alkaline conditions, but only 1-NBDG fluorescence could be restored upon neutralization. HPLC analysis revealed that 2-NBDG was decomposed leading to loss of fluorescence, whereas 1-NBDG remained intact in a NaOH solution. Thus, after cellular uptake, 1-NBDG fluorescence can be detected on a plate reader simply by cell lysis in a NaOH solution followed by neutralization with an HCl solution. The fluorescence stability of 1-NBDG was stable for up to 5 h once cells were lysed; however, similar to 2-NBDG, intracellular 1-NBDG was not stable and the fluorescence diminished substantially within one hour. 1-NBDG uptake could also be detected at the single cell level and inhibition of 1-NBDG uptake by SGLT inhibitors could be detected by flow cytometry. Furthermore, 1-NBDG was successfully used in a high-throughput cell-based method to screen for potential SGLT1 and SGLT2 inhibitors. The SGLT inhibitory activities of 67 flavonoids and flavonoid glycosides purified from plants were evaluated and several selective SGLT1, selective SGLT2, as well as dual SGLT1/2 inhibitors were identified. Structure-activity relationship analysis revealed that glycosyl residues were crucial since the aglycon showed no SGLT inhibitory activities. In addition, the sugar inter-linkage and their substitution positions to the aglycon affected not only the inhibitory activities but also the selectivity toward SGLT1 and SGLT2[1].

Determination of the stability of 1-NBDG and 2-NBDG under alkaline, acidic conditions or at different pH. [1]
1-NBDG or 2-NBDG was diluted in 0.2 N NaOH, 0.2 N HCl or 20 mM Tris buffers with pH values of 6.8, 7.5, 8.0, 8.8 and 9.8. Fluorescence was detected in a black 96-well plate using a SpectraMax Paradigm Multi-Mode Microplate Reader (Molecular Devices) at an excitation wavelength of 458 nm and an emission wavelength of 528 nm (458/528 nm) for 1-NBDG and 475/550 nm for 2-NBDG.

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HPLC analysis[1]
Mightysil RP-18 GP column was used for HPLC analysis and the mobile phase was a gradient of water and acetonitrile from 100% water at 4 min to 100% acetonitrile at 30 min with a flow rate of 1 mL/min. Ten microliters of samples were injected and detected by a UV/Vis detector (set at 475 nm).

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Determination of intracellular stability of 1-NBDG and 2-NBDG[1]
SCC131 cells were seeded into 24-well plates at 1 × 105 cells/well and subjected to glucose uptake assay the following day. Cells were rinsed twice with 500 μL of warm choline buffer, followed by incubation with 200 μL of 160 μM 1-NBDG or 2-NBDG in sodium buffer for 2 h. Cells were then washed twice with warm choline buffer, kept in 500 μL PBS at 37 °C in the CO2 incubator for 0–5 h and lysed in 600 μL of a neutral buffer containing 1% sodium deoxycholate, 1% NP-40, 40 mM KCl and 20 mM Tris–HCl, pH 7.5. One hundred-microliter aliquots of cell lysates were transferred to a black 96-well plate for the detection of fluorescence intensity.

Cell-based screening method for SGLT1 and SGLT2 inhibitors using 1-NBDG [1]
COS-7 cells were transfected with pcDNAhSGLT1 or pcDNAhSGLT2 as previously described and plated into 96-wells the following day at 3–5 × 104 cells/well. Cells were allowed to attach overnight and reach 100% confluence, then subjected to glucose uptake assay in sodium buffer containing test compounds and 100 or 160 μM 1-NBDG at 37 °C for 1.5 h (SGLT1) or 2 h (SGLT2). Cells in 96-wells were rinsed with 150 μL of warm choline buffer and then incubated with 50 μL of sodium buffer containing 1-NBDG and test compounds. After incubation, cells were washed twice with cold choline buffer, examined under microscope to check cell conditions, then lysed in 75 μL of 0.2 N NaOH, and neutralized with 75 μL of 0.2 N HCl. Lysates (100 μL) were transferred to a black 96-well plate for fluorescence detection. Phlorizin was used as a positive control for SGLT inhibitor and 1-NBDG in choline buffer was used to measure sodium-independent glucose uptake via GLUTs. Cells in sodium buffer without 1-NBDG were used to measure autofluorescence (cell background).

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Detection of glucose uptake by flow cytometry[1]
Exponentially growing cells in 6-well plates were rinsed twice with 1 mL of warm choline buffer and then incubated with 500 μL of 100 μM 1-NBDG or 2-NBDG in sodium buffer or choline buffer for 30 min at 37 °C. Cells were then washed 3 times with warm choline buffer and harvested by trypsinization. After centrifugation, cells were resuspended in cold PBS and subjected to flow cytometric analysis by FACSCalibur with an excitation wavelength of 488 nm for FL1 channel. Phlorizin was used to inhibit glucose transport via hSGLT1. Flowing Software 2 was used for quantitative analysis.

[1]. Characterization of a fluorescent glucose derivative 1-NBDG and its application in the identification of natural SGLT1/2 inhibitors. J Food Drug Anal. 2021; 29(3): 521–532.
[2]. Development of a novel non-radioactive cell-based method for the screening of SGLT1 and SGLT2 inhibitors using 1-NBDG. Mol Biosyst. 2013 Aug;9(8):2010-20. doi: 10.1039/c3mb70060g.

Glucose is an important energy source for cells. Glucose transport is mediated by two types of glucose transporters: the active sodium-coupled glucose cotransporters (SGLTs), and the passive glucose transporters (GLUTs). Development of an easy way to detect glucose uptake by the cell can be valuable for research. 1-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-1-deoxy-d-glucose (1-NBDG) is a newly synthesized fluorescent glucose analogue. Unlike 2-NBDG, which is a good substrate of GLUTs but not SGLTs, 1-NBDG can be transported by both GLUTs and SGLTs. Thus, 1-NBDG is useful for the screening of SGLT1 and SGLT2 inhibitors. Here we further characterized 1-NBDG and compared it with 2-NBDG. The fluorescence of both 1-NBDG and 2-NBDG was quenched under alkaline conditions, but only 1-NBDG fluorescence could be restored upon neutralization. HPLC analysis revealed that 2-NBDG was decomposed leading to loss of fluorescence, whereas 1-NBDG remained intact in a NaOH solution. Thus, after cellular uptake, 1-NBDG fluorescence can be detected on a plate reader simply by cell lysis in a NaOH solution followed by neutralization with an HCl solution. The fluorescence stability of 1-NBDG was stable for up to 5 h once cells were lysed; however, similar to 2-NBDG, intracellular 1-NBDG was not stable and the fluorescence diminished substantially within one hour. 1-NBDG uptake could also be detected at the single cell level and inhibition of 1-NBDG uptake by SGLT inhibitors could be detected by flow cytometry. Furthermore, 1-NBDG was successfully used in a high-throughput cell-based method to screen for potential SGLT1 and SGLT2 inhibitors. The SGLT inhibitory activities of 67 flavonoids and flavonoid glycosides purified from plants were evaluated and several selective SGLT1, selective SGLT2, as well as dual SGLT1/2 inhibitors were identified. Structure-activity relationship analysis revealed that glycosyl residues were crucial since the aglycon showed no SGLT inhibitory activities. In addition, the sugar inter-linkage and their substitution positions to the aglycon affected not only the inhibitory activities but also the selectivity toward SGLT1 and SGLT2.[1]

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C12H14N4O8

342.264

342.0812

C, 42.11; H, 4.12; N, 16.37; O, 37.40

2376921-70-7

186689-07-6 (2-NBDG)

Typically exists as solids at room temperature

-1.1

187Ų

C1=C(C2=NON=C2C(=C1)[N+](=O)[O-])N[C@H]3[C@@H]([C@H]([C@@H]([C@H](O3)CO)O)O)O

ZXXYZPVBLJMGPU-UJDFUTJXSA-N

InChI=1S/C12H14N4O8/c17-3-6-9(18)10(19)11(20)12(23-6)13-4-1-2-5(16(21)22)8-7(4)14-24-15-8/h1-2,6,9-13,17-20H,3H2/t6-,9-,10+,11-,12-/m1/s1

(2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]oxane-3,4,5-triol

1-NBDG; 1NBDG; 1 NBDG; EX-A4323A; (2R,3S,4S,5R,6R)-2-(Hydroxymethyl)-6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)tetrahydro-2H-pyran-3,4,5-triol; 2376921-70-7; 1-NBDG; EX-A4323A; (2R,3S,4S,5R,6R)-2-(Hydroxymethyl)-6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)tetrahydro-2H-pyran-3,4,5-triol

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)