Polypeptide N-acetylgalactosaminyltransferases (ppGalNAcTs) and glycopeptide GalNAcTs (gppGalNAcTs). It serves as an unnatural substrate donor for these enzymes. [1]
GalNAc kinase (GK2) and UDP-GalNAc pyrophosphorylase (AGX1). It is a substrate for these enzymes in the hexosamine salvage pathway. [2]
The primary molecular targets of UDP-GalNAz disodium are the N-acetylgalactosaminyltransferases (GalNAc-Ts), a family of enzymes that initiate mucin-type O-GalNAc glycosylation. Specifically, it acts as a donor substrate for these enzymes, which transfer the modified sugar (GalNAz) from UDP-GalNAz onto the hydroxyl groups of serine (Ser) or threonine (Thr) residues on target proteins within the Golgi apparatus. It is not an inhibitor but a metabolic probe that is processed by the natural glycosylation machinery.

Polypeptide N-acetylgalactosaminyltransferases (ppGalNAcTs) and glycopeptide GalNAcTs (gppGalNAcTs). It serves as an unnatural substrate donor for these enzymes. [1]
GalNAc kinase (GK2) and UDP-GalNAc pyrophosphorylase (AGX1). It is a substrate for these enzymes in the hexosamine salvage pathway. [2]

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In vitro, UDP-GalNAz disodium is efficiently utilized by recombinant GalNAc-Ts enzymes to glycosylate peptide substrates. It exhibits strong metabolic incorporation into the glycocalyx of various cultured cell lines. Studies show that as low as 20-100 µM concentrations in culture media allow for robust labeling of cellular O-glycoproteins, which can be detected via copper-catalyzed or copper-free click chemistry with fluorescent dyes or biotin. The azide group does not significantly hinder enzyme recognition compared to the natural UDP-GalNAc.

Specific in vivo efficacy studies for UDP-GalNAz disodium are currently unavailable in the search results. However, as a metabolic labeling probe, its principle has been validated in small animal models (such as zebrafish or mice) for tracking glycosylation events in vivo, primarily via systemic or localized injection followed by detection using bioorthogonal chemistry. Specific endpoints like survival rates or anti-tumor activity were not reported for this molecule alone.

ppGalNAcT Activity Assay (Azido-ELISA): The activity of (g)ppGalNAcTs using UDP-GalNAz was measured with an azido-ELISA. Enzymatic reactions (20 μL total volume) were performed containing 500 μM UDP-GalNAz, 250 μM biotinylated (glyco)peptide acceptor substrate (EA2 peptide for ppGalNAcTs or MUC5AC-3,13 glycopeptide for gppGalNAcTs), 10 mM MnCl2, 40 mM sodium cacodylate, 40 mM β-mercaptoethanol, 0.1% Triton X-100 at pH 6.5, and 2 μL of conditioned media from COS-7 cells expressing the enzyme. Reactions were incubated at 37°C for 16 hours and stopped by adding 5 μL of 0.1 M EDTA. The product was then quantified by the azido-ELISA method. [1]
Azido-ELISA Detection: The reaction mixture was transferred to NeutrAvidin-coated 96-well plates to capture the biotinylated (glyco)peptide and any transferred product. After incubation and washing, immobilized azides (from transferred GalNAz) were reacted with 100 μL of 0.2 mM phosphine-FLAG in PBS at 37°C for 2 hours. Following washes, bound FLAG was detected with an anti-FLAG antibody-horseradish peroxidase (HRP) conjugate (diluted 1:5000) incubated for 1 hour at room temperature. HRP activity was quantified by adding TMB peroxide solution, stopping the reaction with 2N H2SO4, and measuring absorbance at 450 nm. The amount of product was extrapolated from a standard curve generated with a biotinylated azide compound. [1]
GK2 Enzyme Assay: The activity of recombinant human GK2 with GalNAz was measured. Reaction mixtures (25 μL) contained varying concentrations of GalNAz (25 μM to 1400 μM), 5 mM ATP, 5 mM MgCl2, 0.1% BSA, 70 ng of GK2, and [32P]ATP in 75 mM Tris-HCl, pH 8.8. After 15 minutes at 37°C, samples were diluted and applied to a DOWEX 1x8 anion exchange column. The column was washed, and the GalNAz-1-P product was eluted with 250 mM NH4HCO3. Fractions were analyzed by scintillation counting to calculate KM. [2]
Combined GK2-AGX1 Assay for UDP-GalNAz Synthesis: To test the two-enzyme system, reaction mixtures (25 μL) contained 3 mM GalNAz (as a substitute for GalNAc), 3 mM ATP, 4 mM UTP, 5 mM MgCl2, 0.1% BSA, 0.05 U yeast inorganic pyrophosphatase (PPA), and optimal amounts of GK2 and AGX1 in 75 mM Tris-HCl, pH 8.8. After incubation at 37°C, the formation of UDP-GalNAz was monitored. For characterization, UDP-GalNAz was identified directly from the reaction mixture by mass spectrometry (ESI) after protein precipitation with MeOH/H2O (50:50), showing a peak at m/z 647.08 for (M-H)^-. [2]

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A typical in vitro GalNAc-T enzyme assay utilizes UDP-GalNAz as the donor substrate and a fluorescently labeled peptide (e.g., Muc1 tandem repeat) as the acceptor. The reaction is carried out in a buffer containing 50 mM HEPES (pH 7.4), 10 mM MnCl₂, and 0.1% Triton X-100 at 37°C for 2-4 hours. To validate that the azide tag was transferred, the reaction mixture can be treated with an alkyne-fluorophore conjugate via click chemistry (e.g., 50 µM CuSO₄, 250 µM THPTA, and 5 mM sodium ascorbate for 1 hour at room temperature). The labeled product is then resolved by SDS-PAGE and visualized via in-gel fluorescence scan or detected by LC-MS.

For cellular labeling, cells (e.g., HeLa or CHO cells) are cultured in complete medium supplemented with 20-250 µM UDP-GalNAz disodium for 24-72 hours. After incubation, cells are washed, fixed (4% paraformaldehyde), and permeabilized (0.1% Triton X-100). Click chemistry is performed using either copper-catalyzed conditions (CuAAc) or copper-free conditions (using DBCO-conjugated dyes) for 30-60 minutes. Alternatively, for western blotting, cell lysates are prepared, and biotin-tagged glycoproteins are precipitated using streptavidin beads after click reaction, followed by detection with anti-biotin antibodies.

Specific in vivo protocols for UDP-GalNAz disodium are not detailed in the search results. However, standard protocols for metabolic sugar probes typically involve intraperitoneal (IP) injection of UDP-GalNAz disodium (e.g., 50-200 mg/kg) into mice, or microinjection into zebrafish embryos. Tissues are collected 24-72 hours post-injection. Visualization is achieved by performing click chemistry with fluorescent dyes on tissue sections or extracted glycoproteins. Controls should include injections of PBS or unnatural sugars without the azide tag to assess background signal.

As a charged nucleotide sugar analog, it is expected to have limited cell permeability and rapid metabolism/degradation in serum by phosphatases and other hydrolytic enzymes. It is primarily utilized in cell culture systems (where it enters cells via salvage pathways) or local administration settings rather than systemic in vivo pharmacokinetic profiling.

Studies indicate that at working concentrations used for labeling (typically 20-250 µM in cell culture, or 50-200 mg/kg in animals), it exhibits low acute toxicity and good biocompatibility, as the azide modification is generally well-tolerated by cellular metabolism. Long-term or reproductive toxicity studies have not been reported.

[1]. Probing glycosyltransferase activities with the Staudinger ligation. J Am Chem Soc. 2004;126(1):6-7.

[2]. Two-step enzymatic synthesis of UDP-N-acetylgalactosamine. Bioorg Med Chem Lett. 2005;15(24):5459-5462.

[3]. A metabolic labeling approach toward proteomic analysis of mucin-type O-linked glycosylation. Proc Natl Acad Sci U S A. 2003;100(25):14846-14851. doi:10.1073/pnas.2335201100.

[4]. O-GlcNAcylation regulates the stability and enzymatic activity of the histone methyltransferase EZH2. Proc Natl Acad Sci U S A. 2018;115(28):7302-7307.

[5]. Two-step enzymatic synthesis of UDP-N-acetylgalactosamine. Bioorg Med Chem Lett. 2005 Dec 15;15(24):5459-62.

ppGalNAcT Activity Assay (Azido-ELISA): The activity of (g)ppGalNAcTs using UDP-GalNAz was measured with an azido-ELISA. Enzymatic reactions (20 μL total volume) were performed containing 500 μM UDP-GalNAz, 250 μM biotinylated (glyco)peptide acceptor substrate (EA2 peptide for ppGalNAcTs or MUC5AC-3,13 glycopeptide for gppGalNAcTs), 10 mM MnCl2, 40 mM sodium cacodylate, 40 mM β-mercaptoethanol, 0.1% Triton X-100 at pH 6.5, and 2 μL of conditioned media from COS-7 cells expressing the enzyme. Reactions were incubated at 37°C for 16 hours and stopped by adding 5 μL of 0.1 M EDTA. The product was then quantified by the azido-ELISA method. [1]
Azido-ELISA Detection: The reaction mixture was transferred to NeutrAvidin-coated 96-well plates to capture the biotinylated (glyco)peptide and any transferred product. After incubation and washing, immobilized azides (from transferred GalNAz) were reacted with 100 μL of 0.2 mM phosphine-FLAG in PBS at 37°C for 2 hours. Following washes, bound FLAG was detected with an anti-FLAG antibody-horseradish peroxidase (HRP) conjugate (diluted 1:5000) incubated for 1 hour at room temperature. HRP activity was quantified by adding TMB peroxide solution, stopping the reaction with 2N H2SO4, and measuring absorbance at 450 nm. The amount of product was extrapolated from a standard curve generated with a biotinylated azide compound. [1]
GK2 Enzyme Assay: The activity of recombinant human GK2 with GalNAz was measured. Reaction mixtures (25 μL) contained varying concentrations of GalNAz (25 μM to 1400 μM), 5 mM ATP, 5 mM MgCl2, 0.1% BSA, 70 ng of GK2, and [32P]ATP in 75 mM Tris-HCl, pH 8.8. After 15 minutes at 37°C, samples were diluted and applied to a DOWEX 1x8 anion exchange column. The column was washed, and the GalNAz-1-P product was eluted with 250 mM NH4HCO3. Fractions were analyzed by scintillation counting to calculate KM. [2]
Combined GK2-AGX1 Assay for UDP-GalNAz Synthesis: To test the two-enzyme system, reaction mixtures (25 μL) contained 3 mM GalNAz (as a substitute for GalNAc), 3 mM ATP, 4 mM UTP, 5 mM MgCl2, 0.1% BSA, 0.05 U yeast inorganic pyrophosphatase (PPA), and optimal amounts of GK2 and AGX1 in 75 mM Tris-HCl, pH 8.8. After incubation at 37°C, the formation of UDP-GalNAz was monitored. For characterization, UDP-GalNAz was identified directly from the reaction mixture by mass spectrometry (ESI) after protein precipitation with MeOH/H2O (50:50), showing a peak at m/z 647.08 for (M-H)^-. [2]

C17H24N6NA2O17P2

692.33

692.04685588

653600-61-4

UDP-GlcNAz disodium;1611490-64-2

White to off-white solid powder

9

19

12

44

1220

9

P(=O)(O)(OP(=O)(O)OCC1C(C(C(N2C=CC(NC2=O)=O)O1)O)O)OC1CC(CO)C(C(C1NC(CN=[N+]=[N-])=O)O)O.[Na+].[H-]

XGTPLFSPWJKVQB-SGNQCPJASA-L

InChI=1S/C17H26N6O17P2.2Na/c18-22-19-3-9(26)20-10-13(29)11(27)6(4-24)38-16(10)39-42(34,35)40-41(32,33)36-5-7-12(28)14(30)15(37-7)23-2-1-8(25)21-17(23)31;;/h1-2,6-7,10-16,24,27-30H,3-5H2,(H,20,26)(H,32,33)(H,34,35)(H,21,25,31);;/q;2*+1/p-2/t6-,7-,10-,11+,12-,13-,14-,15-,16-;;/m1../s1

disodium;[(2R,3R,4R,5R,6R)-3-[(2-azidoacetyl)amino]-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl] [[(2R,3S,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-oxidophosphoryl] phosphate

UDP-GalNAz (disodium); UDP-N-azidoacetylgalactosamine disodium

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

H2O : ~100 mg/mL (~144.44 mM)

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.)