| Size | Price | Stock | Qty |
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| 1mg |
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| 5mg |
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| 100mg | |||
| Other Sizes |
| Targets |
Cathepsin B (cleavable site); TCO group is a reactant for IEDDA click chemistry.
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| ln Vitro |
ADC cytotoxins are connected to antibodies through an ADC connector to form ADCs [1].
Gly-Gly-Gly-PEG3-TCO is a cleavable (degradable) ADC (antibody-drug conjugate) linker containing a triglycine peptide (Gly-Gly-Gly) and a 3-unit polyethylene glycol (PEG3) spacer. The terminal trans-cyclooctene (TCO) group enables rapid and selective bioorthogonal conjugation via the inverse electron-demand Diels-Alder (IEDDA) reaction with a tetrazine-functionalized antibody or payload. This reaction is highly specific, fast, and proceeds under mild, physiologically compatible conditions. The triglycine peptide segment is designed to be selectively cleaved by specific lysosomal proteases, such as cathepsin B, which are overexpressed in many cancer cells. Upon internalization of the ADC into target cancer cells and trafficking to the lysosome, the protease cleaves the linker at the peptide bond, releasing the cytotoxic payload (e.g., MMAE, DM1) to exert its anti-tumor effect. The PEG3 spacer improves the hydrophilic character of the linker, which can help to maintain the solubility and stability of the ADC, as well as reduce aggregation. The linker itself has no inherent biological activity; it is a chemical tool for the controlled delivery of cytotoxins. |
| ln Vivo |
As an ADC linker, Gly-Gly-Gly-PEG3-TCO is not a therapeutic agent itself but a component used to create ADCs, which do have in vivo activity. The in vivo efficacy of an ADC incorporating this linker is typically evaluated in mouse xenograft models of human cancer. The ADC is administered intravenously, and tumor growth inhibition is measured. The linker's in vivo stability in circulation is crucial: it must be stable in the bloodstream to prevent premature release of the toxic payload (which would cause off-target toxicity), but it must be efficiently cleaved once inside the tumor cell. The PEG3 spacer reduces aggregation and improves pharmacokinetics (PK). The TCO group is not present in the final ADC; it is used to attach the linker to a tetrazine-modified component in a final conjugation step. Therefore, the activity data is generated for the complete ADC, not the linker itself. The triglycine linker is known to be cleaved by cathepsin B, which is an intracellular protease with elevated activity in many tumor types, providing a basis for tumor-selective drug release.
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| Enzyme Assay |
Gly-Gly-Gly-PEG3-TCO is a chemical linker, and as such, its primary characterization is chemical. The purity (>95%) is assessed by HPLC. The structure is confirmed by ¹H NMR and mass spectrometry (MS). The presence of the TCO group is confirmed by its characteristic reactivity with tetrazines, which can be monitored by HPLC or by UV-Vis spectroscopy (the reaction results in a color change). The cleavability of the linker by lysosomal proteases can be assessed in vitro in a biochemical assay. In this assay, the linker is conjugated to a model payload (e.g., a fluorophore) and a model antibody or just the payload and the cleavable dipeptide. This model ADC or linker-payload conjugate is then incubated with purified cathepsin B in a suitable buffer (e.g., 50 mM sodium acetate, pH 5.0, 1 mM DTT, 1 mM EDTA) at 37degC. The release of the free payload is monitored over time by HPLC or LC-MS. The release kinetics can be compared to a non-cleavable control linker. The specificity of cleavage can be confirmed by adding a cathepsin B inhibitor, such as CA-074, to the reaction, which should block the release. The rate of the IEDDA reaction between the TCO group and a tetrazine can also be measured in a test tube. A tetrazine compound is mixed with the TCO-linker in a buffer (e.g., PBS, pH 7.4, at room temperature). The reaction progress is typically monitored by a change in the UV-Vis spectrum (the tetrazine has a characteristic absorbance at ~520-550 nm, which disappears upon reaction with TCO) or by LC-MS to track the formation of the product. The second-order rate constant (k2) can be determined, which for TCO-tetrazine reactions is typically >1000 M-¹s-¹.
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| Cell Assay |
As a chemical linker, Gly-Gly-Gly-PEG3-TCO is not used directly in cell-based assays. Its function is assessed as part of a complete ADC. A typical cell-based assay for an ADC involves treating antigen-positive cancer cells (e.g., HER2-positive SK-BR-3 cells for an ADC targeting HER2) with the ADC. The ADC is composed of an antibody (e.g., trastuzumab), the linker, and a cytotoxic payload (e.g., MMAE). The cells are incubated with various concentrations of the ADC for 72-120 hours. After the incubation, cell viability is measured using a standard assay such as MTT or CellTiter-Glo. The IC50 (the concentration of ADC that kills 50% of the cells) is then calculated. To confirm the mechanism of action, the assay can be performed in the presence of a cathepsin B inhibitor (e.g., CA-074). If the linker is cleavable by cathepsin B, the inhibitor should block the ADC's cytotoxicity. As a control, the same assay is performed on antigen-negative cells (e.g., HEK-293) to confirm target specificity. For the TCO chemistry, the TCO group on the linker is not present in the final ADC; it is used to attach a tetrazine-modified payload to an antibody. The TCO is therefore not part of the final product and its properties are not relevant to the final biological activity, except for the efficiency of the conjugation step.
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| Animal Protocol |
Gly-Gly-Gly-PEG3-TCO is used in the production of ADCs, which are then tested in animal models. There is no animal protocol for the linker itself. A typical in vivo protocol for an ADC involves its administration to immunocompromised mice bearing subcutaneous xenografts of human cancer cells (e.g., SK-BR-3 for HER2-targeted ADC). When the tumors reach a volume of approximately 150-200 mm3, the ADC is administered intravenously (i.v.) via the tail vein at doses of 1-10 mg/kg (based on the antibody content). A control group receives the vehicle (PBS) or a non-targeting ADC. Tumor volumes are measured with a caliper every 2-3 days. At the end of the study (typically after 2-4 weeks), the mice are euthanized, and the tumors are excised and weighed. Blood samples may be collected during the study for pharmacokinetic analysis. The linker contributes to the overall stability and PK of the ADC. If the linker is cleavable by cathepsin B, it should provide potent tumor growth inhibition (TGI) at well-tolerated doses. A non-cleavable control linker would be expected to have a different efficacy and safety profile.
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| ADME/Pharmacokinetics |
The pharmacokinetic (PK) properties of ADCs incorporating Gly-Gly-Gly-PEG3-TCO depend largely on the antibody portion of the conjugate. The linker itself contributes to the overall hydrophilicity and can influence the rate of clearance. ADC pharmacokinetics typically exhibit two phases: a rapid initial distribution phase (alpha phase) and a slower terminal elimination phase (beta phase) with a half-life of several days (e.g., 5-10 days for an IgG-based ADC). The linker's stability in circulation is critical: it must not release the payload prematurely. Premature release would lead to higher clearance of the payload and potential off-target toxicity. The PEG3 spacer helps to reduce aggregation, which can lead to rapid clearance by the reticuloendothelial system (RES). The TCO group is not part of the final ADC; it is a reactive handle used during synthesis.
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| Toxicity/Toxicokinetics |
Gly-Gly-Gly-PEG3-TCO is a chemical linker and is not intended for in vivo use as a standalone entity. Its toxicity is assessed as part of the final ADC. The linker's components (amino acids, PEG, TCO) are generally considered to have low inherent toxicity. However, premature release of the payload due to linker instability can cause dose-limiting toxicities, such as neutropenia or peripheral neuropathy, which are typically the dose-limiting toxicities of the ADC. The triglycine sequence is cleaved by cathepsin B, an enzyme that is present in all lysosomes but is more active in certain cancer cells; thus, the linker is designed to release the payload more efficiently in the tumor than in normal tissues, which improves the therapeutic index.
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| References | |
| Additional Infomation |
Gly-Gly-Gly-PEG3-TCO is a specialized linker for the synthesis of ADCs via bioorthogonal conjugation. ADCs are targeted cancer therapeutics that combine the specificity of monoclonal antibodies with the potent cell-killing ability of cytotoxic drugs. The IEDDA click chemistry using TCO and tetrazine is one of the fastest bioorthogonal reactions available, with rate constants up to 10⁶ M-¹s-¹. This allows for highly efficient conjugation in dilute solutions. The use of a cleavable linker is a key strategy in ADC design to achieve a "bystander effect," where the released payload can diffuse out of the target cell and kill neighboring tumor cells that may not express the target antigen. This is particularly important for heterogeneous tumors. The PEG3 linker is of a specific length; other lengths (PEG4, PEG8, etc.) are also available to fine-tune the properties. This product is for research use only.
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| Molecular Formula |
C23H41N5O8
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|---|---|
| Molecular Weight |
515.600346326828
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| Exact Mass |
515.295
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| CAS # |
2353409-81-9
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| PubChem CID |
146026033
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| Appearance |
Off-white to light yellow oil
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| LogP |
-1.1
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| Hydrogen Bond Donor Count |
5
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
19
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| Heavy Atom Count |
36
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| Complexity |
677
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1C/C=C\CCC(C1)OC(=O)NCCOCCOCCOCCNC(=O)CNC(=O)CNC(=O)CN
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| InChi Key |
GUKWYRQLMOYPGS-UPHRSURJSA-N
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| InChi Code |
InChI=1S/C23H41N5O8/c24-16-20(29)27-18-22(31)28-17-21(30)25-8-10-33-12-14-35-15-13-34-11-9-26-23(32)36-19-6-4-2-1-3-5-7-19/h1-2,19H,3-18,24H2,(H,25,30)(H,26,32)(H,27,29)(H,28,31)/b2-1-
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| Chemical Name |
[(4Z)-cyclooct-4-en-1-yl] N-[2-[2-[2-[2-[[2-[[2-[(2-aminoacetyl)amino]acetyl]amino]acetyl]amino]ethoxy]ethoxy]ethoxy]ethyl]carbamate
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| Synonyms |
GlyGlyGlyPEG3TCO; Gly Gly Gly PEG3 TCO
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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 | 1.9395 mL | 9.6974 mL | 19.3949 mL | |
| 5 mM | 0.3879 mL | 1.9395 mL | 3.8790 mL | |
| 10 mM | 0.1939 mL | 0.9697 mL | 1.9395 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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