| Size | Price | Stock | Qty |
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| 100mg |
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| 250mg |
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| 500mg | |||
| Other Sizes |
| Targets |
Primary amines (e.g., on antibody lysine residues) via NHS ester reactions. This compound functions as an ADC linker scaffold; it has no direct biological target.
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|---|---|
| ln Vitro |
ADC cytotoxins are connected to antibodies through an ADC connector to form ADCs [1].
m-C-tri(CH2-PEG1-NHS ester) is a non-cleavable one-unit polyethylene glycol (PEG) linker employed for the synthesis of antibody-drug conjugates (ADCs). It is a trifunctional linker bearing three reactive NHS esters per molecule. This architecture enables the attachment of up to three payload molecules per conjugation site on an antibody. The NHS ester groups react efficiently with primary amines on antibody lysine residues at neutral or slightly basic pH (pH 7.0-8.5) to form stable amide bonds, releasing N-hydroxysuccinimide. The PEG1 spacers provide a short, flexible, hydrophilic tether between the central benzene core and the payload, reducing steric hindrance and improving solubility. The linker is non-cleavable, meaning that the payload remains attached to the antibody until the entire ADC is internalized into the target cell and degraded in the lysosome. This can result in a lower bystander effect but offers high stability in circulation, reducing off-target toxicity. The linker has no inherent biological activity; its function is to enable high-DAR ADCs for targeted cancer therapy. By using a trifunctional linker, a DAR of up to 3 can be achieved per conjugation site. Depending on the number of accessible lysine residues on the antibody and the conjugation conditions, overall DARs can be significantly higher (e.g., 6-9) when multiple linkers are attached. |
| ln Vivo |
As a linker, m-C-tri(CH2-PEG1-NHS ester) is not designed to have direct in vivo activity. The in vivo efficacy is determined by the complete ADC synthesized using this linker. A high-DAR ADC may exhibit enhanced potency due to more payload delivered per antibody molecule. However, high DAR can also increase the risk of ADC aggregation, leading to rapid clearance and potential toxicity. The non-cleavable nature of this linker means that the payload is released only after lysosomal degradation of the antibody. The payload (typically a cytotoxic agent such as a tubulin inhibitor or DNA-damaging agent) may require release in its active form; if the payload is attached via a stable bond, the released payload may still be active after degradation. The PEG1 spacers provide minimal steric protection but improve solubility compared to non-PEGylated trifunctional linkers. The meta-substitution on the benzene ring may influence the geometry and flexibility of the linker, potentially affecting conjugation efficiency and ADC stability.
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| Enzyme Assay |
As a chemical linker, m-C-tri(CH2-PEG1-NHS ester) is characterized by chemical assays. The purity (typically >95%) is confirmed by HPLC. The structure is verified by ¹H NMR spectroscopy and mass spectrometry (MS). ¹H NMR (DMSO-d₆) shows: the NHS ester methylene protons as a singlet at delta ~2.8 ppm (for the -CH2-CH2- of the NHS ring), the PEG1 protons (-O-CH2-CH2-O-) as triplets at delta ~3.6 ppm, the benzene core aromatic protons as multiplets at delta ~7.0-7.5 ppm, and the amide NH (if present) as a broad singlet. The molecular weight (627.55) is confirmed by ESI-MS. The NHS ester content is quantified by reaction with a primary amine standard (e.g., benzylamine or glycine) and measuring the release of NHS. The compound is treated with an excess of benzylamine in DMSO or PBS (pH 7.4) for 30 minutes, and the amount of released NHS is measured by HPLC or by a colorimetric assay using the Marfey's reagent derivatization. Alternatively, the number of NHS esters per molecule can be calculated by ¹H NMR integration of the NHS peaks relative to the aromatic protons. The reactivity and hydrolysis rate of the NHS esters are assessed by incubating the compound in PBS (pH 7.4) at room temperature and monitoring the disappearance of the starting material by LC-MS over time. The half-life of NHS ester hydrolysis is typically 1-2 hours. The PEG1 spacer is confirmed by the characteristic NMR integration pattern.
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| Cell Assay |
As a linker, m-C-tri(CH2-PEG1-NHS ester) is not used directly in cell-based assays. The complete ADC is tested. For an ADC built with this linker, a typical cell-based assay involves testing the ADC on antigen-positive and antigen-negative tumor cells. Target cells are seeded in 96-well plates at 5×103 cells per well in medium containing 10% FBS. After overnight attachment, the ADC is added at varying concentrations (typically 0.001-100 nM based on antibody content) and incubated for 72-120 hours. Cell viability is measured by CellTiter-Glo or MTT assay. The IC50 for antigen-positive cells is calculated. Antigen-negative cells serve as a control for target specificity. The potency of the high-DAR ADC (potentially DAR 6-9) is compared to a low-DAR or standard ADC. The linker itself (or a control linker without payload) is tested to confirm it has no cytotoxicity (CC50 > 100 microM). To confirm that the mechanism of action requires internalization, an endocytosis inhibitor can be used.
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| Animal Protocol |
The ADC built with m-C-tri(CH2-PEG1-NHS ester) is administered to animals. A typical in vivo protocol for a high-DAR ADC involves a murine xenograft model. Female athymic nude mice (6-8 weeks old) are injected subcutaneously with 5-10×10⁶ antigen-positive tumor cells in 100 microL of PBS mixed 1:1 with Matrigel. When tumors reach 100-200 mm3, mice are randomized into treatment groups (n=8-10). The ADC is formulated in PBS or 0.9% saline and administered intravenously (i.v.) via the tail vein at doses of 1-5 mg/kg (based on antibody content). Control groups receive vehicle alone, non-targeting ADC, unconjugated antibody, or free payload. Treatment is typically administered once weekly (QW) for 2-3 weeks. Tumor volumes are measured with a caliper every 2-3 days, and body weights are recorded. At the end of the study, tumors are excised, weighed, and analyzed for payload concentrations by LC-MS. Blood is collected for pharmacokinetic analysis. The high-DAR ADC may show improved efficacy but also increased toxicity compared to lower-DAR ADCs.
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| ADME/Pharmacokinetics |
The pharmacokinetics (PK) of the ADC are determined, not the linker alone. For ADCs built with trifunctional linkers resulting in high DAR, the PK may be affected by increased hydrophobicity and potential aggregation, leading to faster clearance (shorter half-life) compared to lower-DAR ADCs. The PEG1 spacers improve solubility and may mitigate aggregation to some extent. The volume of distribution (Vd) for ADCs is typically low, similar to plasma volume. Clearance (CL) is primarily via catabolism of the antibody. The half-life may be reduced if the ADC is recognized as "aggregated" or "damaged" and cleared by the reticuloendothelial system. The non-cleavable nature of the linker means that the payload is not prematurely released in circulation, so the free payload in plasma should be minimal. The half-life of the ADC in mice may range from 2-7 days.
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| Toxicity/Toxicokinetics |
The toxicity of the ADC, not the linker alone, is evaluated in preclinical toxicology studies. High-DAR ADCs have been associated with increased toxicity due to on-target/off-tumor effects (payload delivered to normal tissues expressing the target antigen) and off-target/off-tumor effects (non-specific uptake of aggregated or hydrophobic ADC). Common toxicities include neutropenia, thrombocytopenia, peripheral neuropathy, and hepatotoxicity. The trifunctional linker itself is not toxic. The non-cleavable nature may reduce systemic toxicity compared to cleavable linkers because the payload remains attached until internalization. However, if the ADC is cleared by the liver, the payload can be released in hepatocytes, potentially causing hepatotoxicity. For laboratory handling, m-C-tri(CH2-PEG1-NHS ester) should be handled with standard chemical safety precautions: use gloves, lab coat, eye protection. The NHS esters are reactive with amines (including those in skin and mucous membranes) and can cause irritation and sensitization. Avoid inhalation and contact with skin. The compound is for research use only. Store at -20degC in a dry, dark environment under inert atmosphere to prevent hydrolysis.
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| References |
[1]. Beck A, et al. Strategies and challenges for the next generation of antibody-drug conjugates. Nat Rev Drug Discov. 2017 May;16(5):315-337.
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| Additional Infomation |
This linker is a non-cleavable 1 unit PEG ADC linker used in the synthesis of antibody-drug conjugates (ADCs). It features three reactive NHS ester groups on a central meta-substituted benzene scaffold. The NHS ester groups react with primary amines (e.g., antibody lysine residues) to form stable amide bonds, allowing for the attachment of up to three payload molecules per conjugation site. This enables high drug-to-antibody ratios (DAR). The PEG1 spacers improve solubility and reduce steric hindrance. The meta-substitution pattern on the benzene ring provides optimal geometry for multi-payload attachment. This compound is for research use only and is not for human therapeutic use. It is commercially available from chemical suppliers. The linker should be stored at -20degC in a desiccator, protected from light and moisture. Avoid exposure to water which will hydrolyze the NHS esters.
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| Molecular Formula |
C26H33N3O15
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|---|---|
| Molecular Weight |
627.551328420639
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| Exact Mass |
627.191
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| CAS # |
173414-89-6
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| PubChem CID |
12125692
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| Appearance |
Colorless to off-white solid powder
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| LogP |
-2.8
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
15
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| Rotatable Bond Count |
21
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| Heavy Atom Count |
44
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| Complexity |
997
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CC(COCCC(=O)ON1C(=O)CCC1=O)(COCCC(=O)ON2C(=O)CCC2=O)COCCC(=O)ON3C(=O)CCC3=O
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| InChi Key |
IMRQCGBFYSSHHS-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C26H33N3O15/c1-26(14-39-11-8-23(36)42-27-17(30)2-3-18(27)31,15-40-12-9-24(37)43-28-19(32)4-5-20(28)33)16-41-13-10-25(38)44-29-21(34)6-7-22(29)35/h2-16H2,1H3
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| Chemical Name |
(2,5-dioxopyrrolidin-1-yl) 3-[3-[3-(2,5-dioxopyrrolidin-1-yl)oxy-3-oxopropoxy]-2-[[3-(2,5-dioxopyrrolidin-1-yl)oxy-3-oxopropoxy]methyl]-2-methylpropoxy]propanoate
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| Synonyms |
mCtri(CH2PEG1NHS ester); m C tri(CH2 PEG1 NHS ester)
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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 | 1.5935 mL | 7.9675 mL | 15.9350 mL | |
| 5 mM | 0.3187 mL | 1.5935 mL | 3.1870 mL | |
| 10 mM | 0.1593 mL | 0.7967 mL | 1.5935 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.
Products are for research use only; We do not sell to patients
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