| Size | Price | Stock | Qty |
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| 50mg |
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| 100mg |
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| 250mg |
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| Targets |
This compound functions as a chemical linker for bioconjugation; it does not directly bind to biological receptors or enzymes.
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| ln Vitro |
ADC cytotoxins are connected to antibodies through an ADC connector to form ADCs [1].
This compound is a multifunctional PEG-based linker featuring an aminooxy group and a terminal propargyl moiety, separated by a 4-unit polyethylene glycol (PEG4) spacer. The aminooxy functionality enables chemoselective oxime ligation with aldehydes and ketones under mild conditions, forming stable oxime linkages. The propargyl group provides an alkyne handle for copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry with azides, allowing orthogonal conjugation strategies. The PEG4 spacer enhances solubility, flexibility, and biocompatibility, reducing steric hindrance in complex biological systems. Aminooxy-amido-PEG4-propargyl is highly valuable for inhibitor synthesis, site-specific drug conjugates, and bioorthogonal probe development in cancer, immunology, and metabolic disease research. It is a non-cleavable linker, meaning that once the conjugate is internalized into the target cell, the linker is not cleaved by intracellular enzymes or reducing agents. The conjugate must rely on the degradation of the antibody or protein for payload release. |
| ln Vivo |
As a linker, Aminooxy-amido-PEG4-propargyl is not designed to have direct in vivo activity. The in vivo efficacy is determined by the complete conjugate synthesized using this linker (e.g., ADC or imaging probe). For ADCs, a non-cleavable linker like this one offers advantages in plasma stability, as it is not susceptible to premature cleavage by proteases or reducing agents. However, non-cleavable linkers require that the entire conjugate (antibody + linker + payload) is internalized and degraded in the lysosome to release the active payload. This typically results in a lower bystander effect (payload release that can kill neighboring tumor cells) compared to cleavable linkers. The PEG4 spacer increases the hydrophilicity of the ADC, reducing aggregation and improving pharmacokinetics by reducing non-specific uptake. The oxime bond formed via the aminooxy group is stable in vivo. The propargyl group is used for conjugation; once reacted, it forms a stable triazole ring that is not cleaved in vivo.
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| Enzyme Assay |
As a chemical linker, Aminooxy-amido-PEG4-propargyl is characterized by chemical assays rather than biological binding assays. The purity (typically ≥95%) is confirmed by HPLC. The structure is verified by ¹H NMR spectroscopy and high-resolution mass spectrometry (HRMS). ¹H NMR (CDCl3 or DMSO-d₆) shows characteristic PEG peaks at delta 3.5-3.7 ppm (-O-CH2-CH2-O-), the aminooxy group (CONH-O) appears as a broad singlet at delta ~10-11 ppm, and the propargyl group (-C≡CH) appears as a triplet at delta ~2.5-3.0 ppm (depending on solvent) and a singlet at delta ~2.5 ppm for the terminal alkyne hydrogen. The molecular weight (304.34) is confirmed by ESI-MS. The aminooxy group is quantified by reaction with a standard aldehyde or ketone (e.g., salicylaldehyde) under mild acidic conditions (pH 4-6) to form an oxime, and the reaction progress is monitored by HPLC or UV-Vis spectroscopy. The propargyl group is quantified by CuAAC reaction with a standard azide (e.g., benzyl azide) in the presence of CuSO4 and sodium ascorbate, followed by HPLC analysis of the product. The PEG4 spacer length is confirmed by the characteristic NMR integration pattern. The chemical reactivity and specificity of the two orthogonal groups are tested separately. For the oxime ligation: the compound is reacted with a model aldehyde (e.g., 4-formylbenzoic acid) in 100 mM sodium acetate buffer (pH 4.5) or PBS (pH 6.8) at room temperature for 2-4 hours, and product formation is monitored by LC-MS. For CuAAC: the compound is reacted with a model azide (e.g., benzyl azide) in the presence of CuSO4 (0.5-1 mM) and sodium ascorbate (1-2 mM) in PBS or t-butanol/water at room temperature for 1-4 hours, and product formation is monitored by LC-MS.
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| Cell Assay |
As a linker, Aminooxy-amido-PEG4-propargyl is not used directly in cell-based assays. The final conjugate (e.g., ADC, fluorescent probe) is tested. A typical cell-based assay for an ADC built with this linker 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 concentration) and incubated for 72-120 hours. Cell viability is measured by CellTiter-Glo. The IC50 for antigen-positive cells is calculated. For fluorescence labeling, a fluorescent dye (e.g., Cy5, FITC) containing an azide group is clicked to the propargyl group of the linker via CuAAC. The resulting fluorescent conjugate is then added to cells (1-10 microM) for 1-24 hours, and cells are washed, fixed, and imaged by confocal microscopy to study uptake and localization. Alternatively, an aldehyde-containing targeting moiety is conjugated to the aminooxy group via oxime ligation. For FRET-based studies, the orthogonal conjugation allows labeling with two different fluorophores.
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| Animal Protocol |
The linker is not administered to animals directly; the final conjugate is tested. A typical in vivo protocol for an ADC built with a non-cleavable linker like this involves a murine xenograft model. Female athymic nude mice 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 a suitable vehicle and administered intravenously (i.v.) via the tail vein at doses of 1-10 mg/kg (based on antibody content). Control groups receive vehicle alone, non-targeting ADC, or unconjugated antibody. Tumor volumes are measured with calipers every 3-4 days, and body weights are recorded. Treatment is typically administered once weekly for 2-4 weeks. At the end of the study, tumors are excised, weighed, and analyzed for target expression and payload levels. For imaging probes, a conjugate (e.g., an antibody or peptide linked to a near-infrared dye via this linker) is injected intravenously into tumor-bearing mice, and fluorescence is monitored using an in vivo imaging system (IVIS) at various time points (1, 6, 24, 48, 72 hours).
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| ADME/Pharmacokinetics |
The pharmacokinetics (PK) are determined for the final conjugate, not the linker alone. For ADCs with non-cleavable linkers, the PK is characterized by the antibody, with the linker contributing to stability. ADC PK is biphasic: a distribution phase (alpha) with a half-life of hours and a terminal elimination phase (beta) with a half-life of 4-7 days for IgG1-based ADCs. Non-cleavable linkers are generally more stable in circulation than cleavable linkers because they are not susceptible to proteolytic or reductive cleavage. This reduces the premature release of the payload, leading to lower systemic toxicity and potentially improved therapeutic index. The half-life of the payload in circulation is therefore similar to the half-life of the ADC itself. For the conjugate, the PEG4 spacer increases the hydrodynamic volume, reducing clearance and improving circulation time compared to non-PEGylated conjugates. However, the small PEG4 spacer has a minimal effect compared to longer PEG chains. The volume of distribution (Vd) is low. Clearance (CL) is low, primarily via catabolism of the antibody.
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| Toxicity/Toxicokinetics |
The toxicity of the final conjugate, not the linker alone, is evaluated in preclinical studies. For ADCs using non-cleavable linkers, the off-target toxicity is generally lower than for cleavable linkers because the payload remains attached to the antibody until it is internalized and degraded, reducing systemic exposure to the free payload. However, if the ADC is internalized by non-target cells (e.g., via non-specific uptake), the payload can still be released, leading to toxicity. The PEG4 spacer reduces ADC aggregation, which can reduce immunogenicity and non-specific uptake. For laboratory handling of the linker, standard chemical safety precautions apply: use gloves, lab coat, eye protection. Avoid inhalation and skin contact. The compound is for research use only. Store at -20degC in a dry, dark environment. The aminooxy group may be sensitive to oxidation; store under inert atmosphere if possible. Avoid exposure to strong acids or bases.
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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 |
Aminooxy-amido-PEG4-propargyl is a non-degradable PEG-based ADC linker that can be used to synthesize antibody-drug conjugates (ADCs). It contains an Alkyne moiety that allows click chemistry with molecules containing an Azide moiety. The aminooxy group enables oxime ligation with aldehydes or ketones. The PEG4 spacer (4 ethylene glycol units) provides moderate water solubility and flexibility. This linker is classified as "non-cleavable," meaning that the linkage between the antibody and the payload is not specifically designed to be cleaved in the lysosome; instead, the entire conjugate is degraded, releasing the payload attached to a linker fragment. Non-cleavable linkers often have favorable stability in circulation. This compound is intended for research use only and is commercially available from chemical suppliers. The product is typically sold as a solid with ≥95% purity. It should be stored at -20degC, protected from light and moisture.
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| Molecular Formula |
C13H24N2O6
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| Molecular Weight |
304.339464187622
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| Exact Mass |
304.163
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| CAS # |
2253965-03-4
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| PubChem CID |
146026150
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| Appearance |
Colorless to light yellow viscous liquid
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| LogP |
-1.8
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
15
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| Heavy Atom Count |
21
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| Complexity |
292
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C#CCOCCOCCOCCOCCNC(=O)CON
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| InChi Key |
FHURZUINFBYFMV-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C13H24N2O6/c1-2-4-17-6-8-19-10-11-20-9-7-18-5-3-15-13(16)12-21-14/h1H,3-12,14H2,(H,15,16)
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| Chemical Name |
2-aminooxy-N-[2-[2-[2-(2-prop-2-ynoxyethoxy)ethoxy]ethoxy]ethyl]acetamide
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| Synonyms |
AminooxyamidoPEG4propargyl; Aminooxy amido PEG4 propargyl
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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, avoid exposure to moisture. |
| 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 | 3.2858 mL | 16.4290 mL | 32.8580 mL | |
| 5 mM | 0.6572 mL | 3.2858 mL | 6.5716 mL | |
| 10 mM | 0.3286 mL | 1.6429 mL | 3.2858 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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