| Size | Price | Stock | Qty |
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| 100mg |
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| 250mg |
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| 500mg |
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| Other Sizes |
| Targets |
The primary application of Fmoc-Val-Ala-PAB-PNP is as a cleavable linker in ADC synthesis, rather than having a direct biological target itself. The Val-Ala dipeptide sequence is specifically recognized by cathepsin B, a lysosomal protease that is overexpressed in various cancer cells and plays a key role in tumor progression and metastasis. Upon internalization of the ADC into target cancer cells via receptor-mediated endocytosis, the linker is cleaved by cathepsin B in the lysosomal compartment, releasing the cytotoxic payload specifically within the tumor microenvironment. The PNP ester moiety targets primary amines on drug molecules for conjugation, while the Fmoc group serves as a temporary protecting group during synthesis. This design ensures that the active drug is released only at the site of action, minimizing systemic toxicity.
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| ln Vitro |
ADC cytotoxins are connected to antibodies through an ADC connector to form ADCs [1].
In vitro, Fmoc-Val-Ala-PAB-PNP is evaluated as a component of complete ADC constructs rather than as an independent active compound. ADCs synthesized using this linker demonstrate selective cytotoxicity against antigen-positive cancer cell lines in cell viability assays. The Val-Ala sequence ensures efficient cleavage by cathepsin B in cellular assays, with drug release kinetics studied using HPLC or mass spectrometry to confirm payload liberation. The compound’s compatibility with various cytotoxic agents, including auristatins and maytansinoids, has been demonstrated in multiple in vitro conjugation studies. Stability studies confirm that the linker remains intact in plasma, preventing premature drug release and off-target toxicity. These in vitro evaluations are essential for optimizing ADC design and predicting therapeutic efficacy. |
| ln Vivo |
In vivo, Fmoc-Val-Ala-PAB-PNP is used in the synthesis of ADCs that have demonstrated significant antitumor activity in preclinical animal models. ADCs incorporating this cleavable linker have shown targeted tumor growth inhibition with reduced systemic toxicity compared to non-targeted chemotherapy. The Val-Ala-based linker design ensures that the cytotoxic payload is released specifically within the tumor microenvironment following cathepsin B-mediated cleavage, resulting in enhanced therapeutic index. In xenograft models, these ADCs have demonstrated dose-dependent efficacy with favorable tolerability profiles. The linker’s stability in circulation and efficient cleavage in tumors contribute to the overall pharmacokinetic and pharmacodynamic performance of the ADC constructs.
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| Enzyme Assay |
In vitro enzyme/receptor binding (cell-free) assays for evaluating Fmoc-Val-Ala-PAB-PNP focus on assessing the enzymatic cleavage efficiency of the Val-Ala sequence by cathepsin B. The linker is incubated with purified recombinant cathepsin B in buffered solutions at physiological pH (typically pH 5.5, mimicking the lysosomal environment). The release of the PNP-containing fragment is monitored by HPLC or LC-MS at various time points (e.g., 0, 1, 2, 4, 8, 24 hours). The reaction is typically performed at 37°C with enzyme concentrations ranging from 0.1-10 μg/mL and substrate concentrations from 1-100 μM. Kinetic parameters such as kcat, Km, and catalytic efficiency are determined. Appropriate controls include incubations without enzyme and with cathepsin inhibitors to confirm specificity.
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| Cell Assay |
In vitro cell-based assays for evaluating ADCs containing Fmoc-Val-Ala-PAB-PNP are conducted using antigen-positive and antigen-negative cancer cell lines. Cells are seeded in 96-well plates and treated with serial dilutions of the ADC (typically 0.001-100 nM) for 72-96 hours. Cell viability is assessed using standard colorimetric assays such as MTT, CellTiter-Glo, or resazurin reduction. The selectivity index is calculated by comparing IC₅₀ values between antigen-positive and antigen-negative cells. Internalization and intracellular trafficking of the ADC are evaluated using fluorescently labeled antibodies and confocal microscopy. Payload release is confirmed by LC-MS analysis of cell lysates. All experiments include vehicle controls and unconjugated antibody controls to establish specificity.
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| Animal Protocol |
In vivo animal studies with ADCs synthesized using Fmoc-Val-Ala-PAB-PNP are conducted in mouse xenograft models bearing human tumor cells expressing the target antigen. The ADC is administered via intravenous injection at doses ranging from 1-30 mg/kg, typically on a weekly or bi-weekly schedule. Tumor volume is measured by caliper twice weekly, and tumor growth inhibition (TGI) is calculated relative to vehicle controls. Body weight and clinical signs are monitored to assess tolerability. At study termination, tumors and major organs are collected for histopathological analysis and biomarker evaluation. Pharmacokinetic blood sampling is performed at multiple time points post-dose to assess ADC stability and exposure. Each group typically consists of 6-10 animals.
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| ADME/Pharmacokinetics |
Pharmacokinetic properties of Fmoc-Val-Ala-PAB-PNP are evaluated as part of complete ADC characterization rather than for the linker alone. ADCs incorporating this linker exhibit typical monoclonal antibody pharmacokinetics, including a prolonged circulation half-life (typically 3-7 days in rodents) and limited systemic release of the payload. The linker remains stable in plasma, with minimal premature cleavage observed in pharmacokinetic studies. The ADC is primarily cleared via proteolytic degradation and FcRn-mediated recycling pathways. The released payload, following cathepsin B cleavage in the tumor, has a much shorter half-life, limiting systemic exposure and reducing off-target toxicity. Tissue distribution studies confirm that the ADC accumulates preferentially in tumor tissue.
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| Toxicity/Toxicokinetics |
Toxicological data for ADCs containing Fmoc-Val-Ala-PAB-PNP are generated from preclinical safety studies in rodent and non-rodent species. The cleavable Val-Ala linker is designed to minimize systemic toxicity by ensuring that payload release occurs predominantly within the tumor microenvironment. At therapeutic doses, these ADCs demonstrate a favorable safety profile with limited off-target effects. Common adverse effects observed at high doses may include transient weight loss, liver enzyme elevations, and hematological changes, consistent with the mechanism of action of the cytotoxic payload. The no-observed-adverse-effect level (NOAEL) is determined in repeat-dose toxicology studies to establish a safe starting dose for clinical trials.
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| References | |
| Additional Infomation |
Fmoc-Val-Ala-PAB-PNP is a specialized research tool used exclusively in the development of antibody-drug conjugates for targeted cancer therapy. It belongs to the class of cathepsin B-cleavable linkers that enable selective payload release in the tumor microenvironment. The Fmoc protecting group allows for efficient solid-phase peptide synthesis, while the PNP ester facilitates straightforward conjugation to amine-containing drugs. The para-aminobenzyl (PAB) spacer ensures traceless drug release following enzymatic cleavage. This linker is not approved as a therapeutic agent itself but is a critical building block for ADC research and development. It is intended for research and manufacturing use only, not for human therapeutic application.
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| Molecular Formula |
C₃₇H₃₆N₄O₉
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|---|---|
| Molecular Weight |
680.70
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| Exact Mass |
680.248
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| CAS # |
1394238-92-6
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| PubChem CID |
100029276
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| Appearance |
White to light yellow solid powder
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| Density |
1.322±0.06 g/cm3(Predicted)
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| Boiling Point |
931.4±65.0 °C(Predicted)
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| LogP |
6.6
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
14
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| Heavy Atom Count |
50
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| Complexity |
1160
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| Defined Atom Stereocenter Count |
2
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| SMILES |
C[C@@H](C(=O)NC1=CC=C(C=C1)COC(=O)OC2=CC=C(C=C2)[N+](=O)[O-])NC(=O)[C@H](C(C)C)NC(=O)OCC3C4=CC=CC=C4C5=CC=CC=C35
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| InChi Key |
ZJHZWBDLYYJQAV-WYOOIXGGSA-N
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| InChi Code |
InChI=1S/C37H36N4O9/c1-22(2)33(40-36(44)48-21-32-30-10-6-4-8-28(30)29-9-5-7-11-31(29)32)35(43)38-23(3)34(42)39-25-14-12-24(13-15-25)20-49-37(45)50-27-18-16-26(17-19-27)41(46)47/h4-19,22-23,32-33H,20-21H2,1-3H3,(H,38,43)(H,39,42)(H,40,44)/t23-,33-/m0/s1
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| Chemical Name |
[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methyl (4-nitrophenyl) carbonate
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| Synonyms |
FmocValAlaPABPNP; Fmoc Val Ala PAB PNP
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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) |
DMSO : ~100 mg/mL (~146.91 mM)
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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 | 1.4691 mL | 7.3454 mL | 14.6908 mL | |
| 5 mM | 0.2938 mL | 1.4691 mL | 2.9382 mL | |
| 10 mM | 0.1469 mL | 0.7345 mL | 1.4691 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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