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| Other Sizes |
| Targets |
Bis-PEG4-NHS ester does not have a biological target in the traditional sense of a receptor or enzyme. Its "target" is the primary amine groups (-NH2) present on proteins, peptides, and other amine-containing molecules. The NHS ester moiety reacts with these amines under mild conditions (pH 7-9) to form a stable amide bond, thereby crosslinking two amine-containing molecules or conjugating a drug to an antibody. As a PEG-based linker, its role is to provide a physical bridge between two functional moieties, facilitating the creation of bioconjugates, such as ADCs and PROTACs, without inherent pharmacological activity of its own.
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| ln Vitro |
Bis-PEG4-NHS ester itself does not possess intrinsic biological activity in cell-based assays; its activity is defined by its ability to conjugate to and crosslink biomolecules. In vitro, the compound is used to crosslink proteins via their primary amine groups. For example, it can be used to create protein-protein conjugates or to attach a drug to a carrier protein. The efficiency of the conjugation reaction can be assessed by SDS-PAGE, mass spectrometry, or ELISA to confirm the formation of the desired conjugate. The PEG4 spacer ensures that the conjugated proteins retain solubility and are less likely to aggregate, which is crucial for subsequent functional assays of the conjugated product.
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| ln Vivo |
Bis-PEG4-NHS ester is not intended for direct in vivo administration as a therapeutic agent. Its in vivo relevance is as a linker in the construction of ADCs and other bioconjugates that are then administered in vivo. The PEG4 spacer in the final ADC construct can influence the pharmacokinetics of the drug by increasing its hydrodynamic volume and reducing renal clearance, thereby extending the half-life of the conjugated therapeutic. The non-degradable nature of this linker means that the drug-antibody linkage is stable in circulation, ensuring that the cytotoxic payload is not released prematurely.
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| Enzyme Assay |
The non-cellular in vitro assay for Bis-PEG4-NHS ester involves characterizing its conjugation efficiency. A typical protocol involves dissolving the NHS ester in anhydrous DMSO or DMF. A target protein or amine-containing molecule is prepared in a reaction buffer (e.g., PBS, pH 7.4). The NHS ester solution is added to the protein solution at a molar ratio ranging from 2:1 to 20:1 (linker to protein) and incubated at room temperature for 1-4 hours. The reaction is quenched by the addition of Tris-HCl or glycine, which reacts with any remaining NHS ester. The conjugated product is then purified by size-exclusion chromatography or dialysis to remove excess linker and by-products. The degree of labeling is determined by MALDI-TOF mass spectrometry or by measuring the absorbance of the conjugate.
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| Cell Assay |
Cellular assays for Bis-PEG4-NHS ester are not applicable as the compound itself does not act on cells. Instead, the activity is assessed on the functional consequences of the conjugates it helps create. For instance, after using Bis-PEG4-NHS ester to conjugate a drug to an antibody, the resulting ADC can be tested in cell viability assays (e.g., MTT or CellTiter-Glo) on target antigen-expressing cancer cells. The cells are treated with varying concentrations of the ADC for 48-72 hours, and cell viability is measured to determine the IC50. The specificity of the ADC can be confirmed by comparing its activity on target-positive vs. target-negative cell lines.
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| Animal Protocol |
In vivo animal studies are performed with the final ADC or bioconjugate synthesized using Bis-PEG4-NHS ester, not with the linker itself. A typical protocol involves administering the ADC intravenously to tumor-bearing mouse models (e.g., xenograft models). Mice are dosed with the ADC at various concentrations (e.g., 1-10 mg/kg) on a schedule such as once weekly for 2-3 weeks. Tumor volume and body weight are measured regularly to assess efficacy and toxicity. Pharmacodynamic markers, such as target engagement and downstream signaling, may also be evaluated in tumor tissue samples collected at the end of the study.
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| ADME/Pharmacokinetics |
Bis-PEG4-NHS ester is a chemical linker and does not have pharmacokinetic properties as an active drug. However, when used to create ADCs, the PEG4 linker influences the overall PK of the conjugate. The hydrophilic PEG spacer increases the hydrodynamic volume of the ADC, reducing renal clearance and extending the half-life in circulation. The non-degradable nature of the PEG4 linker ensures that the conjugate remains intact in the bloodstream, preventing premature release of the payload. For the linker itself, it is expected to be rapidly hydrolyzed or conjugated and cleared from the system, but specific PK data are not available.
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| Toxicity/Toxicokinetics |
Bis-PEG4-NHS ester is a chemical reagent for research use only and is not intended for therapeutic or diagnostic use in humans. As a reactive NHS ester, it is moisture-sensitive and should be stored under anhydrous conditions at 2-8°C. Toxicity data for the compound itself are limited, but standard laboratory safety precautions should be followed when handling it, as it can react with proteins and may cause irritation. In the context of ADC development, the toxicity is primarily attributed to the cytotoxic payload, not the PEG linker.
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| Additional Infomation |
Bis-PEG4-NHS ester is a key building block in the field of bioconjugation and drug delivery. Its homobifunctionality allows for the creation of symmetric conjugates, while the PEG4 spacer provides an optimal balance between solubility and flexibility. This linker is part of a broader family of PEG-based linkers used in the synthesis of ADCs and PROTACs. The compound is commercially available and is a standard reagent in many research laboratories. It is not a drug itself and has no clinical trial or regulatory approval status. Its value lies in its utility as a tool for creating more complex therapeutic molecules.
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| Molecular Formula |
C20H28N2O12
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|---|---|
| Molecular Weight |
488.4425
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| Exact Mass |
488.164
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| CAS # |
1314378-11-4
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| PubChem CID |
75535130
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| Appearance |
White to off-white solid powder
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
605.1±65.0 °C at 760 mmHg
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| Flash Point |
319.8±34.3 °C
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| Vapour Pressure |
0.0±1.7 mmHg at 25°C
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| Index of Refraction |
1.538
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| LogP |
-4.59
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
12
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| Rotatable Bond Count |
19
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| Heavy Atom Count |
34
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| Complexity |
662
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
BQLKLYYHZOLCLV-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C20H28N2O12/c23-15-1-2-16(24)21(15)33-19(27)5-7-29-9-11-31-13-14-32-12-10-30-8-6-20(28)34-22-17(25)3-4-18(22)26/h1-14H2
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| Chemical Name |
(2,5-dioxopyrrolidin-1-yl) 3-[2-[2-[2-[3-(2,5-dioxopyrrolidin-1-yl)oxy-3-oxopropoxy]ethoxy]ethoxy]ethoxy]propanoate
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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 | 2.0473 mL | 10.2367 mL | 20.4733 mL | |
| 5 mM | 0.4095 mL | 2.0473 mL | 4.0947 mL | |
| 10 mM | 0.2047 mL | 1.0237 mL | 2.0473 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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