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| 50mg |
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| Targets |
This compound functions as an ADC linker, enabling conjugation of cytotoxic drugs to antibodies. The disulfide bond is the cleavable site, targeted by intracellular reducing agents such as glutathione.
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| ln Vitro |
ADC cytotoxins are connected to antibodies through an ADC connector to form ADCs [1].
4-Methyl-4-(methyldisulfanyl)pentanoic acid is a disulfide-containing ADC linker enabling selective cleavage and drug release. Its reactive acid group supports conjugation to antibodies for targeted cancer therapy applications. The disulfide bond is a key structural feature that is stable in the bloodstream (where glutathione levels are low, ~2-20 microM) but undergoes efficient reductive cleavage in the cytoplasm of target cells (where glutathione levels are high, ~1-10 mM). Upon internalization of the ADC into the target cell and trafficking to the endosome/lysosome, the disulfide bond is reduced by intracellular thiols such as glutathione, releasing the cytotoxic payload (typically attached via the sulfur atom of the linker). The free carboxylic acid group can be activated to an NHS ester or reacted with an amine-containing antibody using standard carbodiimide coupling reagents (e.g., EDC/HOBt). The methyl group on the carbon bearing the disulfide provides steric hindrance that can influence the rate of disulfide exchange and reduce premature cleavage in circulation compared to unsubstituted disulfide linkers. The linker has no inherent biological activity; its function is purely structural. When conjugated to an antibody and a cytotoxic payload, it forms an ADC that delivers the payload selectively to target cells. |
| ln Vivo |
As a linker, 4-Methyl-4-(methyldisulfanyl)pentanoic acid is not designed to have direct in vivo activity; it is a building block for ADCs. The in vivo efficacy of ADCs incorporating this linker (or similar disulfide-containing linkers) has been demonstrated in tumor xenograft models. For example, ADCs using a disulfide linker and a maytansinoid payload (e.g., DM1 or DM4) have shown potent anti-tumor activity in preclinical studies. The disulfide bond is stable in circulation but undergoes reductive cleavage in the intracellular environment, releasing the active payload. The steric hindrance provided by the methyl group adjacent to the disulfide bond may improve plasma stability compared to unhindered disulfides, reducing off-target release of the payload. The linker is designed to enable controlled release of the therapeutic agent within target cells, enhancing the efficacy of ADCs in cancer therapy while minimizing systemic toxicity. No clinical data specific to this linker is available.
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| Enzyme Assay |
As a chemical linker, 4-Methyl-4-(methyldisulfanyl)pentanoic acid is characterized chemically rather than in enzyme/receptor binding assays. The purity (>98%) is assessed by HPLC. The structure is confirmed by ¹H NMR spectroscopy and mass spectrometry (MS). ¹H NMR (CDCl3, 400 MHz) shows characteristic signals: the carboxylic acid proton appears as a broad singlet at delta ~11-12 ppm; the methylene groups adjacent to the acid (-CH2-CH2-COOH) appear as triplets at delta ~2.4 and 1.9 ppm; the methyl groups on the carbon bearing the disulfide appear as singlets at delta ~1.3 ppm; the S-CH3 group appears as a singlet at delta ~2.4 ppm. The molecular weight (194.31) is confirmed by ESI-MS. The disulfide bond content is quantified using an Ellman's assay (or similar thiol release assay) after reduction with DTT or TCEP. A sample of the compound is treated with excess DTT in a buffer (e.g., 0.1 M Tris-HCl, pH 8.0, containing 1 mM EDTA) at 37degC for 30-60 minutes to fully reduce the disulfide bond, generating two thiol groups. The released thiols are quantified by reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB, Ellman's reagent), which produces a yellow chromophore (5-thio-2-nitrobenzoic acid, TNB) that absorbs at 412 nm. The concentration of thiols is calculated using ε412 = 14,150 M-¹cm-¹. The reactivity of the carboxylic acid group is assessed by activation with EDC and NHS in DMSO or DMF, and the formation of the NHS ester is monitored by LC-MS.
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| Cell Assay |
4-Methyl-4-(methyldisulfanyl)pentanoic acid is not used directly in cell-based assays as it is a chemical linker. The complete ADC (antibody + linker + payload) is tested in cell-based assays. A typical assay involves testing the ADC on antigen-positive tumor cells. Target cells (e.g., antigen-expressing cancer cells) are seeded in 96-well plates at 5×103 cells per well in RPMI-1640 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 using CellTiter-Glo (luminescence-based ATP quantification) or MTT (absorbance-based). The IC₅0 is calculated by fitting the concentration-response data. Antigen-negative cells are used as a control to assess target specificity. To confirm that the disulfide bond is required for activity, the ADC can be reduced with DTT (1-10 mM) prior to addition to cells, which should dramatically enhance cytotoxicity by prematurely releasing the payload, or the assay can be performed in the presence of an endocytosis inhibitor that should block the ADC's activity, confirming that internalization and intracellular cleavage are required for activity. The free linker can be tested as a control to ensure it has no cytotoxicity (it should be non-toxic at concentrations up to 100 microM).
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| Animal Protocol |
4-Methyl-4-(methyldisulfanyl)pentanoic acid is not administered to animals directly; the complete ADC is tested in vivo. A typical in vivo protocol for an ADC built with this linker involves a murine xenograft model. Female athymic nude mice (6-8 weeks old) are injected subcutaneously in the flank with 5-10×10⁶ antigen-positive tumor cells in 100 microL of PBS mixed 1:1 with Matrigel. When tumors reach a volume of approximately 150-200 mm3, mice are randomized into treatment groups (n=8-10 per group). The ADC is formulated in PBS (or a suitable vehicle) and administered intravenously (i.v.) via the tail vein at doses ranging from 1-10 mg/kg (based on antibody content). Control groups receive vehicle alone, non-targeting ADC (isotype control), or free payload. Tumor volumes are measured with a digital caliper every 2-3 days, and body weights are recorded as a general indicator of toxicity. Treatment is typically administered once weekly (QW) for 2-4 cycles. At the end of the study (when control tumors reach ethical limits, typically 2000 mm3), mice are euthanized, and tumors are excised and weighed. Tumor tissue may be processed for histology (H&E staining, Ki-67 IHC for proliferation, cleaved caspase-3 IHC for apoptosis). Blood is collected for pharmacokinetic analysis (measurement of ADC, total antibody, and free payload by ELISA or LC-MS).
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| ADME/Pharmacokinetics |
4-Methyl-4-(methyldisulfanyl)pentanoic acid is a linker and is not studied independently. The pharmacokinetics of the final ADC are measured. For the ADC, the PK is biphasic: a distribution phase (alpha) with a half-life of hours, and a terminal elimination phase (beta) with a half-life of several days (typically 5-10 days for an IgG1-based ADC). The linker influences the stability of the ADC in circulation. The disulfide bond can undergo thiol-disulfide exchange with plasma thiols (e.g., cysteine, glutathione, albumin), leading to premature release of the payload or transfer of the payload to plasma proteins. The methyl substitution adjacent to the disulfide provides steric hindrance that reduces the rate of disulfide exchange compared to unhindered disulfide linkers, improving the plasma half-life of the ADC. The volume of distribution (Vd) is low (similar to plasma volume) due to the large molecular weight of the antibody. Clearance (CL) is low, primarily via proteolytic catabolism. The free payload released from the ADC has its own PK properties, typically with a short half-life (hours).
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| Toxicity/Toxicokinetics |
The toxicity of 4-Methyl-4-(methyldisulfanyl)pentanoic acid is evaluated as part of the complete ADC. The linker itself is not toxic. For ADCs using disulfide linkers, the primary toxicities are related to the cytotoxic payload and include neutropenia, thrombocytopenia, peripheral neuropathy (for tubulin inhibitors), and hepatotoxicity (elevated liver enzymes). The disulfide bond may contribute to off-target toxicity if cleaved prematurely in circulation, releasing the payload into the bloodstream before reaching the tumor. This can be minimized by optimizing the linker design (e.g., adding steric hindrance, as in this compound). For laboratory handling, standard chemical safety precautions apply: use gloves, lab coat, and eye protection. The compound may have an unpleasant odor due to the disulfide/thiol groups. Avoid inhalation and contact with skin. The compound is for research use only, not for human therapeutic use. Store at -20degC in a dry, dark environment under inert atmosphere (argon or nitrogen) to prevent oxidation. Avoid co-storage with reducing agents (e.g., DTT, TCEP).
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| References | |
| Additional Infomation |
4-Methyl-4-(methyldisulfanyl)pentanoic acid is a cleavable ADC linker used in the synthesis of antibody-drug conjugates for targeted cancer therapy. The disulfide linkage is a classic "cleavable" linker strategy, enabling drug release in the reducing environment of the intracellular compartment. The compound is commercially available from various chemical suppliers for research use. The design of this linker (a sterically hindered disulfide) aims to balance circulatory stability with efficient intracellular cleavage. It is often used in combination with maytansinoid payloads (e.g., DM1, DM4) or other thiol-containing cytotoxic agents. The linker can be activated to its NHS ester form (via EDC/NHS chemistry) to facilitate conjugation to antibody lysine residues. The free carboxylic acid allows site-specific conjugation to an antibody that has been engineered to contain a cysteine residue (THIOMAB technology). This product is for research use only, not for diagnostic or therapeutic use.
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| Molecular Formula |
C₇H₁₄O₂S₂
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|---|---|
| Molecular Weight |
194.31
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| Exact Mass |
194.044
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| CAS # |
796073-55-7
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| PubChem CID |
11564634
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| Appearance |
Colorless to light yellow liquid
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| LogP |
2.641
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
11
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| Complexity |
134
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=C(CCC(C)(C)SSC)O
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| InChi Key |
GYFCEVXCVFNSCL-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C7H14O2S2/c1-7(2,11-10-3)5-4-6(8)9/h4-5H2,1-3H3,(H,8,9)
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| Chemical Name |
4-methyl-4-(methyldisulfanyl)pentanoic acid
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| Synonyms |
4Methyl4(methyldisulfanyl)pentanoic acid; 4 Methyl 4 (methyldisulfanyl)pentanoic acid
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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) |
DMSO : ~100 mg/mL (~514.64 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (12.87 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (12.87 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (12.87 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 5.1464 mL | 25.7321 mL | 51.4642 mL | |
| 5 mM | 1.0293 mL | 5.1464 mL | 10.2928 mL | |
| 10 mM | 0.5146 mL | 2.5732 mL | 5.1464 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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