| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| 25mg |
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| 50mg |
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| 100mg |
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| 250mg |
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| Targets |
Protease Cleavable Linker
Cleavable Linker |
|---|---|
| ln Vitro |
Protease-Specific Cleavage Activity: Conjugates containing Val-Lys(Boc)-PAB (e.g., PEG-A) can be specifically cleaved by cathepsin B in vitro. Incubated at pH 5.5 (simulating lysosomal environment) and 37℃ for 4 hours, the cleavage rate reaches 82%; no obvious response to other proteases (e.g., cathepsin D, trypsin) is observed, with cleavage rates all <10% [1]
- Cellular Target Binding and Internalization: After the conjugate specifically binds to target antigens on the surface of target cells via the carrier part, it mediates the entry of Val-Lys(Boc)-PAB and payload into the cell. Flow cytometry detection shows that after 6 hours of incubation, the internalization rate of the conjugate by target cells reaches 75%, which is significantly higher than that of non-target cells (internalization rate <12%) [1] - Intracellular Payload Release and Immune Activation: After internalization, cathepsin B in lysosomes cleaves the Val-Lys(Boc)-PAB linker to release free payload. Co-incubated with peripheral blood mononuclear cells (PBMC) for 72 hours, the concentration of IFN-γ in the cell culture supernatant increases from 25 pg/mL in the control group to 186 pg/mL, and the proliferation ratio of CD8+ T cells increases from 11% to 38% [1] |
| ln Vivo |
Tumor-Targeted Enrichment and Efficacy: After intravenous injection of the conjugate (PEG-A) containing Val-Lys(Boc)-PAB into tumor-bearing mice, the concentration of the conjugate in tumor tissue reaches a peak at 6 hours after administration, which is 3.8 times the plasma concentration; after 4 weeks of continuous administration, the tumor volume is reduced by 62% compared with the control group, and the number of CD8+ T cell infiltration in tumor tissue is increased by 2.3 times compared with the control group [1]
- Immune Response Regulation: In vivo experiments show that after conjugate treatment, the proportion of immune memory cells (CD44+CD62L+ T cells) in mouse spleen increases from 8% to 21%, and the concentration of IL-2 in peripheral blood increases from 32 pg/mL to 157 pg/mL. This effect depends on the efficient cleavage ability of Val-Lys(Boc)-PAB; if the linker cannot be cleaved, the immune activation effect completely disappears [1] |
| Enzyme Assay |
Cathepsin B Cleavage Efficiency Assay: Mix the Val-Lys(Boc)-PAB linker with recombinant cathepsin B in buffer, set the incubation conditions at pH 5.5 and 37℃, and sample at 0, 1, 2, 4, and 8 hours respectively. Separate the cleavage products from the uncleaved linker by high-performance liquid chromatography (HPLC), calculate the cleavage rate at different time points, and draw the cleavage kinetic curve to determine the enzymatic hydrolysis rate of the linker [1]
- Protease Specificity Verification Experiment: Prepare reaction systems containing cathepsin B, cathepsin D, trypsin, and elastase respectively, add equal amounts of Val-Lys(Boc)-PAB linker to each system, and incubate for 8 hours under their respective optimal reaction conditions. Detect the peak of the linker's cleavage products in each system by mass spectrometry, and analyze the response specificity of the linker to different proteases [1] |
| Cell Assay |
Cellular Internalization Efficiency Detection: Co-incubate fluorophore-labeled conjugates containing Val-Lys(Boc)-PAB with target cells (expressing carrier-targeted antigens) and non-target cells respectively, with incubation times set at 2, 4, 6, and 12 hours. After incubation, wash the cells with PBS to remove unbound conjugates, detect the intracellular fluorescence intensity by flow cytometry, and calculate the internalization rate at different time points [1]
- Quantitative Detection of Intracellular Payload Release: After co-incubating target cells with conjugates for 12 hours, collect and lyse the cells, and separate free payload from uncleaved conjugates in the cell lysate by high-performance liquid chromatography. Quantify the content of free payload through a standard curve, and calculate the intracellular cleavage efficiency of the Val-Lys(Boc)-PAB linker [1] - Immune Cell Proliferation and Cytokine Secretion Detection: Co-incubate conjugates with PBMC for 72 hours, label T cells with CFSE staining, analyze the proliferation ratio of CD4+ and CD8+ T cells by flow cytometry; simultaneously detect the concentrations of cytokines such as IFN-γ, IL-2, and TNF-α in the cell culture supernatant by ELISA [1] |
| Animal Protocol |
Efficacy Experiment in Tumor Xenograft Model: 6-8 week-old female nude mice are subcutaneously inoculated with 1×10^6 target tumor cells on the right flank, and randomly grouped when the tumor volume reaches 100-150 mm³. The treatment group is given the conjugate PEG-A containing Val-Lys(Boc)-PAB by intravenous injection at a dose of 10 mg/kg, once every 3 days for 4 consecutive weeks; the control group is given an equal volume of normal saline. Measure the tumor length, width, and mouse body weight twice a week, calculate the tumor volume by the formula V=length×width²/2, and draw the tumor growth curve; at the end of the experiment, dissect the tumor tissue, weigh it, and prepare paraffin sections [1]
- Pharmacokinetic and Tissue Distribution Experiment: Normal ICR mice are intravenously injected with the conjugate containing Val-Lys(Boc)-PAB (dose 5 mg/kg), and orbital venous blood (to separate plasma) and heart, liver, spleen, lung, kidney, and tumor tissues (if tumor-bearing mice) are collected at 0.5, 1, 2, 4, 8, 12, 24, and 48 hours after administration. Determine the concentrations of the conjugate, Val-Lys(Boc)-PAB cleavage fragments, and free payload in plasma and various tissues by liquid chromatography-tandem mass spectrometry (LC-MS/MS) [1] - Animal Experiment for Immune Response Evaluation: C57BL/6 mice are intravenously injected with the conjugate containing Val-Lys(Boc)-PAB (dose 8 mg/kg), and sacrificed at 3, 7, and 14 days after administration to collect spleen, lymph nodes, and peripheral blood. Analyze the proportion of immune cell subsets by flow cytometry, and detect the concentration of cytokines in serum by ELISA to evaluate the in vivo immune activation effect after the linker-mediated payload release [1] |
| ADME/Pharmacokinetics |
Plasma metabolic parameters: After intravenous injection of the Val-Lys(Boc)-PAB conjugate into mice, the elimination half-life (t1/2β) of the conjugate was 7.9 hours, the peak concentration (Cmax) was 13.2 μg/mL, and the area under the concentration-time curve (AUC0-∞) was 102.5 μg·h/mL; the t1/2 of the Val-Lys(Boc)-PAB cleavage fragment was 3.1 hours, and the Cmax was 2.8 μg/mL [1]
- Tissue distribution characteristics: 6 hours after administration, the concentration of the conjugate in tumor tissue reached the highest (42.6 μg/g), and the tumor/plasma concentration ratio was 3.8; the concentration in liver and kidney tissues was relatively low (8.5 μg/g and 6.3 μg/g, respectively), and the concentration in heart, lung and spleen tissues was all below 5 μg/g [1] - Excretion route: Within 48 hours after administration, approximately 68% Drug-related components are excreted in urine, of which 15% are Val-Lys(Boc)-PAB fragments, 30% are free payloads, and 23% are unlysed conjugates; approximately 12% are excreted in feces, while the remainder are retained in tissues [1]. |
| Toxicity/Toxicokinetics |
Acute toxicity: No mice were observed to die within 14 days after a single intravenous injection of the Val-Lys(Boc)-PAB conjugate at a dose of up to 50 mg/kg, nor were there any toxic symptoms such as sudden weight loss (weight change rate <5%), abnormal behavior or anorexia; median lethal dose (LD50) >50 mg/kg [1]
- Repeated-dose toxicity: Mice were intravenously injected twice a week for 4 consecutive weeks with the conjugate (dose 10 mg/kg). At the end of the experiment, blood routine and liver and kidney function indicators (ALT, AST, creatinine, urea nitrogen) were all within the normal physiological range; pathological examination showed that no obvious pathological damage, inflammatory infiltration or cell necrosis were found in the heart, liver, spleen, lungs, kidneys and other major organs [1] - Plasma protein binding rate: In vitro experiments showed that the plasma protein binding rate of the Val-Lys(Boc)-PAB conjugate was 68%-72%, and the plasma protein binding rate of the Val-Lys(Boc)-PAB cleavage fragment was 23%-27% [1] |
| References | |
| Additional Infomation |
Structural features: Val-Lys(Boc)-PAB is composed of L-valine (Val), L-lysine (Lys, with the side chain amino group protected by Boc) and p-aminobenzyl alcohol (PAB) linked by an amide bond, with a molecular weight of 419.5 Da. The Val-Lys dipeptide fragment in the structure is the recognition site of cathepsin B [1] - Mechanism of action: As the core linking unit of the conjugate, Val-Lys(Boc)-PAB is stable in the physiological environment (pH 7.4), and the cleavage rate is <5% after incubation at room temperature for 72 hours; after the linker enters the target cell lysosome, it will be recognized by cathepsin B and cleave the Val-Lys peptide bond, releasing PAB and its linked load, thereby achieving targeted intracellular release of the load and reducing the toxicity of non-target sites [1] - Application scenarios: This linker is mainly used to prepare immunomodulatory conjugates related to tumor immunotherapy (such as PEG-A in the patent). By targeting and delivering immunomodulators (such as cytokines and immune checkpoint modulators), the immune response of the tumor microenvironment can be enhanced while reducing systemic immune-related side effects [1]. - Synthesis method: Val-Lys(Boc)-PAB can be prepared by solid-phase synthesis. After synthesis, it can be coupled with carrier molecules and immunomodulatory loads to construct stable targeted immunomodulatory conjugates, suitable for therapeutic research on various tumors expressing specific antigens [1].
|
| Molecular Formula |
C23H38N4O5
|
|---|---|
| Molecular Weight |
450.571626186371
|
| Exact Mass |
450.28
|
| Elemental Analysis |
C, 61.31; H, 8.50; N, 12.43; O, 17.75
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| CAS # |
1432969-86-2
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| PubChem CID |
117841397
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| Appearance |
Solid powder
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| LogP |
1.8
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| Hydrogen Bond Donor Count |
5
|
| Hydrogen Bond Acceptor Count |
6
|
| Rotatable Bond Count |
13
|
| Heavy Atom Count |
32
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| Complexity |
598
|
| Defined Atom Stereocenter Count |
2
|
| SMILES |
CC(C)[C@@H](C(=O)N[C@@H](CCCCNC(=O)OC(C)(C)C)C(=O)NC1=CC=C(C=C1)CO)N
|
| InChi Key |
BPDGVDDLTIMMLY-OALUTQOASA-N
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| InChi Code |
InChI=1S/C23H38N4O5/c1-15(2)19(24)21(30)27-18(8-6-7-13-25-22(31)32-23(3,4)5)20(29)26-17-11-9-16(14-28)10-12-17/h9-12,15,18-19,28H,6-8,13-14,24H2,1-5H3,(H,25,31)(H,26,29)(H,27,30)/t18-,19-/m0/s1
|
| Chemical Name |
tert-butyl ((S)-5-((S)-2-amino-3-methylbutanamido)-6-((4-(hydroxymethyl)phenyl)amino)-6-oxohexyl)carbamate
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| Synonyms |
Val-Lys(Boc)-PAB;
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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)
|
| Solubility (In Vitro) |
DMSO : ~125 mg/mL (~277.43 mM）
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.62 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 20.8 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: ≥ 2.08 mg/mL (4.62 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 20.8 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: ≥ 2.08 mg/mL (4.62 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 | 2.2194 mL | 11.0971 mL | 22.1941 mL | |
| 5 mM | 0.4439 mL | 2.2194 mL | 4.4388 mL | |
| 10 mM | 0.2219 mL | 1.1097 mL | 2.2194 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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