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| 10mg |
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| Targets |
Tri-GalNAc(OAc)₃ targets the Asialoglycoprotein Receptor (ASGPR). ASGPR is an endocytic receptor highly expressed on hepatocytes that specifically recognizes and binds glycoproteins with terminal galactose or N-acetylgalactosamine residues. The trivalent structure of Tri-GalNAc enables multivalent binding to ASGPR, achieving high-affinity targeting.
Tri-GalNAc(OAc)3 TFA targets the asialoglycoprotein receptor (ASGPR), a C-type lectin receptor predominantly expressed on the sinusoidal surface of hepatocytes. ASGPR mediates the endocytosis of desialylated glycoproteins bearing terminal GalNAc residues. The tri-antennary GalNAc structure exhibits nanomolar affinity for ASGPR due to cooperative binding from its three carbohydrate recognition domains. This receptor is internalized via clathrin-mediated endocytosis, enabling the efficient delivery of conjugated therapeutic payloads into liver cells. ASGPR is considered an ideal target for liver-directed therapies because of its high expression level (approximately 500,000 receptors per hepatocyte), restricted expression to the liver, and rapid recycling capacity. |
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| ln Vitro |
- Protein Degradation Activity: As the ligand component of LYTACs, Tri-GalNAc significantly reduces target protein (e.g., EGFR, HER2) levels and inhibits downstream signaling pathways, such as EGFR-mediated Akt and MAPK signals.
- Antitumor Activity: In liver cancer cell models, Tri-GalNAc-based LYTACs degrade carcinogenic membrane proteins and inhibit tumor cell proliferation. As a targeting ligand rather than a therapeutic agent itself, Tri-GalNAc(OAc)3 TFA does not possess direct pharmacological activity in conventional cell-based assays. However, its biological activity is evaluated indirectly through ASGPR binding affinity and receptor-mediated uptake efficiency. Surface plasmon resonance (SPR) studies demonstrate that tri-antennary GalNAc clusters bind to ASGPR with dissociation constants (Kd) in the low nanomolar range (approximately 2-5 nM), representing a 50- to 100-fold improvement over mono-antennary GalNAc conjugates. The compound shows no intrinsic cytotoxicity in hepatocyte cell lines at concentrations up to 100 μM. |
| ln Vivo |
In vivo, Tri-GalNAc(OAc)3 TFA functions as a delivery vehicle that enables the liver-specific biodistribution of conjugated oligonucleotide therapeutics. Following subcutaneous or intravenous administration, the GalNAc conjugate preferentially accumulates in the liver via ASGPR-mediated uptake, with liver-to-kidney ratios exceeding 10:1 in rodent models. The compound significantly improves the therapeutic index of RNAi agents by reducing off-target accumulation in non-hepatic tissues. In non-human primate studies, GalNAc-siRNA conjugates have demonstrated >80% target gene knockdown in the liver at doses as low as 1-3 mg/kg, with sustained activity lasting up to three months after a single dose.
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| Enzyme Assay |
ASGPR binding affinity is typically assessed using a competitive binding assay with radiolabeled asialoorosomucoid (ASOR) or fluorescently labeled GalNAc probes. In this assay, varying concentrations of Tri-GalNAc(OAc)3 TFA are incubated with membrane preparations from ASGPR-expressing cells or purified recombinant ASGPR. After incubation, bound and free ligand are separated by filtration or centrifugation, and the radioactivity or fluorescence is measured. The inhibition constant (Ki) is calculated from competition curves. Surface plasmon resonance (SPR) can also be employed, where ASGPR is immobilized on a sensor chip and the GalNAc ligand is flowed over the surface to measure real-time binding kinetics (kon and koff).
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| Cell Assay |
- Protein Degradation Assay: Hepatocellular carcinoma cells are seeded and treated with Tri-GalNAc-conjugated LYTAC compounds. Target protein (e.g., EGFR, HER2) degradation is assessed by Western Blot.
- Cell Proliferation Inhibition Assay: In liver cancer cell models, cells are treated with Tri-GalNAc-LYTAC, and cell viability changes are evaluated using MTT or CCK-8 assays. Cellular uptake of Tri-GalNAc(OAc)3 TFA-conjugated payloads is evaluated using ASGPR-expressing hepatocyte cell lines such as HepG2 or primary human hepatocytes. Cells are incubated with fluorescently labeled GalNAc conjugates at 37°C for 1-4 hours. Uptake is quantified by flow cytometry or fluorescence microscopy. Competition experiments with excess free GalNAc or ASOR are performed to confirm ASGPR-specific uptake. Endocytosis pathway inhibitors (e.g., chlorpromazine for clathrin-mediated endocytosis, filipin for caveolae-mediated uptake) are used to determine the internalization mechanism. The acetylation status of the GalNAc moieties significantly influences cellular uptake kinetics due to altered hydrophobicity and membrane permeability. |
| Animal Protocol |
In animal studies, Tri-GalNAc(OAc)3 TFA-conjugated oligonucleotides are administered to rodents (typically C57BL/6 mice) via subcutaneous or intravenous injection at doses ranging from 0.3 to 10 mg/kg. Liver tissue and plasma are collected at various time points (1 hour to 90 days post-dose). Biodistribution is assessed by quantifying the oligonucleotide payload using hybridization ELISA or LC-MS/MS in liver, kidney, spleen, heart, and lung tissues. Target gene knockdown is measured by qRT-PCR or Western blot from liver homogenates. Pharmacodynamic endpoints include the reduction of target protein levels and downstream biomarkers. Non-human primate studies are conducted to establish the clinically relevant dose-response relationship.
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| ADME/Pharmacokinetics |
As a synthetic carbohydrate conjugate, Tri-GalNAc(OAc)3 TFA exhibits favorable pharmacokinetic properties that are largely dictated by the conjugated therapeutic payload. The GalNAc moiety itself undergoes rapid clearance from circulation with a half-life of less than 30 minutes in rodents, primarily via hepatic uptake. The compound shows minimal plasma protein binding (<20%) and limited distribution to extrahepatic tissues. For GalNAc-siRNA conjugates, the plasma half-life ranges from 2-8 hours in humans, with peak liver concentrations achieved within 2-4 hours post-dose. The acetal-protected (OAc) form is designed to enhance membrane permeability and is deacetylated by intracellular esterases following cellular uptake, trapping the active GalNAc ligand within hepatocytes.
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| Toxicity/Toxicokinetics |
Toxicology studies of Tri-GalNAc(OAc)3 TFA are typically conducted as part of the safety assessment of the complete GalNAc-conjugated therapeutic rather than as a standalone entity. In repeat-dose toxicology studies in rodents and non-human primates, GalNAc conjugates are generally well-tolerated at therapeutic doses. The no-observed-adverse-effect level (NOAEL) is typically established at exposures 10- to 30-fold above the clinical therapeutic dose. Common findings at high doses include mild to moderate injection site reactions, transient elevation of liver enzymes (ALT/AST), and occasional hepatocellular vacuolation. The GalNAc ligand itself is not considered immunogenic, and no significant complement activation or cytokine release has been observed in preclinical studies.
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| References | |
| Additional Infomation |
Selective protein degradation platforms have created new opportunities for therapeutic development and biological research. The first lysosome-targeting chimeras (LYTACs) promoted the degradation of extracellular and membrane proteins by bridging a target protein to the cation-independent mannose-6-phosphate receptor (CI-M6PR). In this study, we developed LYTACs that engage the asialoglycoprotein receptor (ASGPR), a liver-specific lysosome-targeting receptor, to degrade extracellular proteins in a cell-type-specific manner. We conjugated binders to a triantennary N-acetylgalactosamine (tri-GalNAc) motif that binds ASGPR, thereby driving protein downregulation. Degradation of the epidermal growth factor receptor (EGFR) by GalNAc-LYTAC attenuated EGFR signaling compared to inhibition with an antibody alone. Furthermore, we demonstrated that a LYTAC composed of a 3.4-kDa peptide binder linked to a tri-GalNAc ligand degrades integrins and reduces cancer cell proliferation. The use of a single tri-GalNAc ligand for site-specific conjugation on antibody scaffolds improved the pharmacokinetic profile of GalNAc-LYTACs in vivo. Thus, GalNAc-LYTACs represent a promising approach for cell-type-restricted protein degradation.
Tri-GalNAc(OAc)3 TFA is a critical enabling technology for liver-targeted oligonucleotide therapeutics. The GalNAc conjugation platform has been validated clinically with multiple FDA-approved drugs including inclisiran (PCSK9 siRNA), lumasiran (PH1 siRNA), and givosiran (ALAS1 siRNA). The tri-antennary design was optimized through structure-activity relationship studies to achieve maximal ASGPR affinity while maintaining synthetic accessibility. The acetylated (OAc) prodrug strategy enhances oral bioavailability and cellular penetration while the TFA salt form facilitates handling and formulation. This compound represents a cornerstone of modern RNA therapeutics, demonstrating that receptor-mediated delivery can achieve potent and durable target knockdown in the liver with an extended dosing interval of once every 3-6 months. |
| Molecular Formula |
C81H129F3N10O38
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|---|---|
| Molecular Weight |
1907.93
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| Exact Mass |
1906.842
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| Elemental Analysis |
C, 50.99; H, 6.82; F, 2.99; N, 7.34; O, 31.86
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| CAS # |
1159408-65-7
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| Related CAS # |
1159408-64-6
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| PubChem CID |
162394061
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| Appearance |
White to off-white ointment
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| Hydrogen Bond Donor Count |
11
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| Hydrogen Bond Acceptor Count |
42
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| Rotatable Bond Count |
69
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| Heavy Atom Count |
132
|
| Complexity |
3220
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| Defined Atom Stereocenter Count |
15
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| SMILES |
CC(=O)N[C@@H]1[C@H]([C@H]([C@H](O[C@H]1OCCCCC(=O)NCCCNC(=O)CCOCC(COCCC(=O)NCCCNC(=O)CCCCO[C@H]2[C@@H]([C@H]([C@H]([C@H](O2)COC(=O)C)OC(=O)C)OC(=O)C)NC(=O)C)(COCCC(=O)NCCCNC(=O)CCCCO[C@H]3[C@@H]([C@H]([C@H]([C@H](O3)COC(=O)C)OC(=O)C)OC(=O)C)NC(=O)C)N)COC(=O)C)OC(=O)C)OC(=O)C.C(=O)(C(F)(F)F)O
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| InChi Key |
ZQQFLUVDYMPTBZ-QBHSIBJISA-N
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| InChi Code |
InChI=1S/C79H128N10O36.C2HF3O2/c1-46(90)87-67-73(120-55(10)99)70(117-52(7)96)58(40-114-49(4)93)123-76(67)111-34-16-13-22-61(102)81-28-19-31-84-64(105)25-37-108-43-79(80,44-109-38-26-65(106)85-32-20-29-82-62(103)23-14-17-35-112-77-68(88-47(2)91)74(121-56(11)100)71(118-53(8)97)59(124-77)41-115-50(5)94)45-110-39-27-66(107)86-33-21-30-83-63(104)24-15-18-36-113-78-69(89-48(3)92)75(122-57(12)101)72(119-54(9)98)60(125-78)42-116-51(6)95;3-2(4,5)1(6)7/h58-60,67-78H,13-45,80H2,1-12H3,(H,81,102)(H,82,103)(H,83,104)(H,84,105)(H,85,106)(H,86,107)(H,87,90)(H,88,91)(H,89,92);(H,6,7)/t58-,59-,60-,67-,68-,69-,70+,71+,72+,73-,74-,75-,76-,77-,78-;/m1./s1
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| Chemical Name |
[(2R,3R,4R,5R,6R)-5-acetamido-6-[5-[3-[3-[3-[3-[3-[5-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetyloxy-6-(acetyloxymethyl)oxan-2-yl]oxypentanoylamino]propylamino]-3-oxopropoxy]-2-[[3-[3-[5-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetyloxy-6-(acetyloxymethyl)oxan-2-yl]oxypentanoylamino]propylamino]-3-oxopropoxy]methyl]-2-aminopropoxy]propanoylamino]propylamino]-5-oxopentoxy]-3,4-diacetyloxyoxan-2-yl]methyl acetate;2,2,2-trifluoroacetic acid
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| Synonyms |
Tri-GalNAc(OAc)3 TFA; 1159408-65-7; Tri-GalNAc(OAc)3 (TFA); orb2279003
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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 Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| 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 | 0.5241 mL | 2.6206 mL | 5.2413 mL | |
| 5 mM | 0.1048 mL | 0.5241 mL | 1.0483 mL | |
| 10 mM | 0.0524 mL | 0.2621 mL | 0.5241 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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