| Size | Price | |
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| 500mg | ||
| 1g | ||
| Other Sizes |
| Targets |
Carrier-mediated transport (CMT) systems at the blood-brain barrier (BBB) - nicotinic acid (niacin) is known to be carried to the brain by nicotine receptors. The compound itself is a prodrug that delivers the monosaccharide analogue for metabolic incorporation into sialoglycoproteins. [1]
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| ln Vitro |
- SH-SY5Y (human neuroblastoma) cells incubated with compound 2 (50 μM, 48 h) showed robust expression of N-azidoacetyl-neuraminic acid (NeuAz) on cell surface sialoglycoproteins, as detected by flow cytometry using Cu-AAC biotinylation with propiolamide-biotin reagent 10 and FITC-avidin staining. The incorporation levels were comparable to the parent non-hybrid compound 1 (Ac₄ManNAz). [1]
- Far-Western blotting (HRP-avidin) of total cell lysates from SH-SY5Y cells treated with compound 2 (50 μM, 48 h) showed robust biotinylation of sialoglycoproteins after Cu-AAC with reagent 10, indicating successful metabolic incorporation of NeuAz. Silver staining confirmed equal protein loading. [1] - Cell viability studies using MTT assay on SH-SY5Y cells treated with compound 2 (0-500 μM) showed that 80-85% of cells were viable at 250 μM. Annexin-V/propidium iodide staining confirmed similar viability compared to vehicle control. [1] |
| ln Vivo |
- Metabolic glycan engineering of brain tissue: Intravenous (i.v.) administration of compound 2 (0.26 mmol/kg, corresponding to 112 mg/kg of compound 1, once daily for 3 days) via tail vein in C57BL/6J mice resulted in robust expression of NeuAz-carrying sialoglycoproteins in both heart and brain tissues, as detected by HRP-avidin blotting after Cu-AAC biotinylation. In contrast, the parent non-hybrid compound 1 showed expression only in heart but not in brain. Similar results were obtained in BALB/cByJ mice. [1]
- Dose-dependent study: Mice injected i.v. with compound 2 at three concentrations (molar equivalents corresponding to 75, 115, and 150 mg/kg of compound 1, once daily for 3 days) showed robust expression in brain with moderate dose dependency, characteristic of saturable carrier-mediated transport. [1] - Effect of number of doses: Mice injected i.v. with compound 2 (0.26 mmol/kg once daily for 7 days) showed a significant increase in biotinylation levels in brain tissues compared to 3-day treatment, indicating that saturable CMT could be overcome by increasing the number of doses. [1] - Perfusion control: Trans-cardinal perfusion with PBS prior to tissue harvest confirmed that the NeuAz observed in brain tissues originated from brain cells and not from contaminating hematopoietic cells. [1] |
| Enzyme Assay |
- Cu-AAC (Copper(I)-catalyzed Azide-Alkyne Cycloaddition) biotinylation for detection of NeuAz: Tissue lysates or cell lysates containing NeuAz-labeled glycoproteins were subjected to Cu-AAC reaction with propiolamide-biotin reagent 10. The reaction mixture contained CuSO₄ (1 mM), sodium ascorbate (5 mM), THPTA ligand (2 mM), and reagent 10 (20 μM). The reaction was carried out at room temperature for 2-3 h. Biotinylated proteins were then precipitated, resolved by SDS-PAGE, transferred to membranes, and detected using HRP-conjugated avidin. [1]
- Neuraminidase treatment: Brain and heart tissue lysates from mice treated with compound 2 were incubated with neuraminidase from Clostridium perfringens (0, 25, 50, and 100 units) in G1 reaction buffer (50 mM sodium citrate, pH 6.0) at 37°C for 12 h prior to Cu-AAC biotinylation. Neuraminidase treatment resulted in a reduction in biotinylation levels as a function of enzyme concentration, confirming the presence of NeuAz on sialic acids. [1] |
| Cell Assay |
- Flow cytometry for cell surface NeuAz expression: SH-SY5Y cells were incubated with compound 2 (50 μM, 48 h), harvested, fixed, subjected to Cu-AAC biotinylation with propiolamide-biotin reagent 10, and stained with FITC-conjugated avidin. Fluorescence was measured by flow cytometry. Robust NeuAz expression was observed, with mean fluorescence intensity comparable to that of compound 1. [1]
- Confocal microscopy: SH-SY5Y cells treated with compound 2 (50 μM, 48 h) were fixed, biotinylated with reagent 10 via Cu-AAC, stained with AlexaFluor-594-conjugated avidin (red), and counterstained with DAPI (blue, nuclei). Confocal microscopy confirmed cell surface labeling of NeuAz. [1] - Far-Western blotting of cell lysates: SH-SY5Y cells treated with compound 2 (50 μM, 48 h) were lysed, and lysates were subjected to Cu-AAC biotinylation with reagent 10. Proteins were resolved by SDS-PAGE, transferred to membranes, and detected with HRP-avidin. Silver staining of the gel served as loading control. [1] - MTT cell viability assay: SH-SY5Y cells were treated with compound 2 at concentrations ranging from 0 to 500 μM for 48 h. MTT reagent was added, and formazan absorbance was measured at 570 nm. The percentage of viable cells was calculated relative to vehicle control. 80-85% viability was observed at 250 μM. [1] - Annexin-V/propidium iodide apoptosis assay: SH-SY5Y cells treated with compound 2 (100 and 250 μM, 48 h) were stained with FITC-conjugated annexin-V and propidium iodide, then analyzed by flow cytometry. Results showed no significant increase in apoptosis compared to vehicle control. [1] |
| Animal Protocol |
- Intravenous (i.v.) administration: Male C57BL/6J or BALB/cByJ mice (4-6 weeks old) were anesthetized with ketamine/xylazine (80 mg/kg and 10 mg/kg, i.p.). Compound 2 was dissolved in 90% DMSO (DMSO:water = 9:1) as a stock solution (50 mg/mL). Mice were injected via the tail vein once daily for 3 or 7 days at doses of 0.17, 0.26, 0.34, and 0.69 mmol/kg (corresponding to 75, 115, 150, and 300 mg/kg equivalent of compound 1). Each group consisted of 4-6 mice. [1]
- Tissue harvest and processing: On day 4 or day 8, mice were anesthetized and euthanized by cervical dislocation. Tissues (heart and brain) were harvested, washed with ice-cold PBS, homogenized in PBS using a homogenizer at 15,000 rpm, and washed multiple times to remove blood residuals. Pellets were lysed in lysis buffer (10 mM Tris, 150 mM NaCl, 1.0% NP-40, protease inhibitor cocktail, pH 7.4). Lysates were clarified by centrifugation (16,000g, 30 min, 4°C) and used for protein estimation and Cu-AAC biotinylation. [1] - Trans-cardial perfusion: In some experiments, blood was replaced by trans-cardial perfusion of ice-cold PBS (20 mL over 20 min by cardiac puncture with outflow through the jugular vein) prior to tissue harvest to eliminate contamination from blood cells. [1] |
| ADME/Pharmacokinetics |
The study notes that carrier-mediated transport (CMT) systems at the BBB are saturable, and the moderate increase in NeuAz expression as a function of concentration supported this characteristic mechanism. [1]
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| Toxicity/Toxicokinetics |
Histopathology: Independent histopathological analysis of liver sections from mice treated with compound 2 (0.26 mmol/kg, i.v. once daily for 3 days, harvested on day 4) did not show any visible changes compared to untreated and vehicle-treated controls. [1]
- Serum biochemistry: Levels of creatinine and activity of liver enzymes (ALT, AST) in serum of mice treated with compound 2 were found to be within the standard range. [1] - Behavioral observations: No adverse effects in terms of weight loss or abnormal behavior were noticeable in mice treated with compound 2 during the study period. [1] |
| References | |
| Additional Infomation |
Compound 2 is a carbohydrate-neuroactive hybrid (CNH) molecule designed to piggyback non-natural monosaccharide analogues across the blood-brain barrier (BBB) using carrier-mediated transport systems. Nicotinic acid (vitamin B₃, niacin) was chosen as the neuroactive carrier because it is known to be carried to the brain by nicotine receptors. [1]
- The compound is a prodrug: the peracetylated groups are hydrolyzed intracellularly, and ManNAz is metabolically converted to N-azidoacetyl-neuraminic acid (NeuAz), which is then incorporated into sialoglycoproteins via the sialic acid biosynthetic pathway. [1] - Unlike the parent non-hybrid compound 1 (Ac₄ManNAz), which showed no expression in brain after i.p. or i.v. administration, compound 2 successfully delivered the monosaccharide analogue to the CNS and enabled metabolic glycan engineering of brain tissue. This strategy represents a non-invasive tool for investigating brain glycosylation and could potentially be developed for brain mapping applications. [1] - The compound was synthesized by attaching nicotinic acid to the C-1 position of Ac₄ManNAz via an ester linkage. This position was chosen to create a bioleachable ester that could be cleaved intracellularly to release the active monosaccharide. [1] |
| Molecular Formula |
C20H23N5O10
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|---|---|
| Molecular Weight |
493.42
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| CAS # |
2102591-12-6
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| Appearance |
Typically exists as solids at room temperature
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| Synonyms |
6'Nicotinate Ac4ManNAz; 6'-Nicotinate Ac4ManNAz; Compound 2
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0267 mL | 10.1334 mL | 20.2667 mL | |
| 5 mM | 0.4053 mL | 2.0267 mL | 4.0533 mL | |
| 10 mM | 0.2027 mL | 1.0133 mL | 2.0267 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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