| Size | Price | Stock | Qty |
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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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| Other Sizes |
| Targets |
Protease Cleavable Linker
Protease-Specific Cleavage Activity: Prodrug conjugates containing Fmoc-Phe-Lys(Boc)-PAB can be specifically cleaved by intracellular proteases (mainly cathepsin B) in tumor cells in vitro. Incubated at pH 5.5 (simulating lysosomal environment) and 37℃ for 6 hours, the cleavage rate reaches 85%; no obvious response to non-target tissue proteases (e.g., trypsin, elastase) is observed, with cleavage rates all <8% [1] - Cellular Internalization and Payload Release: After the conjugate binds to antigens on the surface of metastatic tumor cells via the targeting carrier, it mediates the internalization of Fmoc-Phe-Lys(Boc)-PAB and payload into the cell. Flow cytometry detection shows that after 8 hours of incubation, the internalization rate of the conjugate by metastatic cancer cells reaches 82%, which is significantly higher than that of normal cells (internalization rate <10%); the content of free payload in the cell lysate accounts for 68% of the total conjugate, confirming efficient cleavage of the linker [1] - Anticancer Activity (Payload-Dependent): The released anticancer payload exhibits dose-dependent antiproliferative activity against metastatic tumor cells (e.g., breast cancer metastatic cells, lung cancer metastatic cells). After delivery mediated by Fmoc-Phe-Lys(Boc)-PAB, the cytotoxicity of the payload is 3.5 times higher than that of the free form, and the toxicity to normal cells is reduced by 40% [1] |
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| ln Vivo |
Tumor Metastasis-Targeted Enrichment and Efficacy: After intravenous injection of the conjugate containing Fmoc-Phe-Lys(Boc)-PAB into tumor-bearing mice (inoculated with breast cancer metastatic cells), the concentration of the conjugate in tumor tissue reaches a peak within 6 hours, which is 4.2 times the plasma concentration, and the concentration in metastatic lesions is 1.8 times that of the primary tumor; after 3 weeks of continuous administration, the number of lung metastatic lesions is reduced by 72% compared with the control group, and the volume of the primary tumor is reduced by 65% [1]
- Systemic Distribution and Targeted Payload Release: In vivo experiments show that the conjugate is mainly distributed in tumor tissues and metastatic lesions, with less distribution in liver and kidney tissues (only 12% and 15% of that in tumor tissues). The concentration of free payload in tumor tissues is 3.6 times that in plasma, confirming that Fmoc-Phe-Lys(Boc)-PAB specifically cleaves and releases the payload at the target site [1] |
| Enzyme Assay |
Cathepsin B Cleavage Efficiency Assay: Mix the Fmoc-Phe-Lys(Boc)-PAB linker with recombinant cathepsin B in buffer, set the incubation conditions at pH 5.5 and 37℃, and sample at 0, 2, 4, 6, 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 (6-hour cleavage rate reaches 85%) [1]
- Protease Specificity Verification Experiment: Prepare reaction systems containing cathepsin B, trypsin, elastase, and cathepsin D respectively, add equal amounts of Fmoc-Phe-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; obvious cleavage peaks only appear in the cathepsin B group [1] |
| Cell Assay |
Cellular Internalization Efficiency Detection: Co-incubate fluorophore-labeled conjugates containing Fmoc-Phe-Lys(Boc)-PAB with metastatic tumor cells and normal cells respectively, with incubation times set at 2, 4, 6, 8, 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 metastatic tumor 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 Fmoc-Phe-Lys(Boc)-PAB linker [1] - Cellular Antiproliferative Activity Detection: Seed metastatic tumor cells in 96-well plates, add conjugates containing Fmoc-Phe-Lys(Boc)-PAB, free payload, and blank control respectively. After 72 hours of incubation, detect cell viability by MTT assay, and compare the antiproliferative effects of different treatment groups [1] |
| Animal Protocol |
Efficacy Experiment in Tumor Metastasis Model: 6-8 week-old female nude mice are intravenously injected with 1×10^6 breast cancer metastatic cells to establish a lung metastasis model. Seven days after modeling, mice are randomly grouped. The treatment group is given the conjugate containing Fmoc-Phe-Lys(Boc)-PAB by intravenous injection at a dose of 5 mg/kg (calculated by payload equivalent), once every 4 days for 3 consecutive weeks; the control group is given an equal volume of normal saline or free payload. Measure the body weight of mice every week. At the end of the experiment, sacrifice the mice, count the number of lung metastatic lesions, dissect and weigh the primary tumor (if any), and prepare pathological sections of tumor tissues [1]
- Pharmacokinetic and Tissue Distribution Experiment: Normal ICR mice or tumor-bearing metastasis model mice are intravenously injected with a single dose of the conjugate containing Fmoc-Phe-Lys(Boc)-PAB (dose 5 mg/kg, calculated by payload equivalent). Orbital venous blood (to separate plasma) and heart, liver, spleen, lung, kidney, tumor tissue, and metastatic lesions are collected at 0.5, 1, 2, 4, 6, 8, 12, 24, and 48 hours after administration. Determine the concentrations of the conjugate, Fmoc-Phe-Lys(Boc)-PAB cleavage fragments, and free payload in plasma and various tissues by liquid chromatography-tandem mass spectrometry (LC-MS/MS) [1] |
| ADME/Pharmacokinetics |
Plasma metabolic parameters: After intravenous injection of the conjugate containing Fmoc-Phe-Lys(Boc)-PAB into mice, the elimination half-life (t1/2β) of the conjugate was 6.8 hours, the peak concentration (Cmax) was 10.5 μg/mL, and the area under the concentration-time curve (AUC0-∞) was 78.6 μg·h/mL; the t1/2 of the Fmoc-Phe-Lys(Boc)-PAB cleavage fragment was 2.5 hours, and the Cmax was 2.3 μg/mL [1]
- Tissue distribution characteristics: 6 hours after administration, the concentration of the conjugate in tumor tissue reached the highest (44.1 μg/g), and the tumor/plasma concentration ratio was 4.2; the concentration in lung metastases was 39.8 μg/g, the concentrations in liver and kidney tissues were 5.3 μg/g and 6.6 μg/g, respectively, and the concentrations in heart and spleen tissues were all below 3 μg/g [1] - Excretion pathway: Within 48 hours after administration, approximately 72% of the drug components are excreted in the urine, of which 18% are Fmoc-Phe-Lys(Boc)-PAB cleavage fragments, 35% are free effective payloads, and 19% are uncleaved conjugates; approximately 15% is excreted in feces, while the remaining components remain in tumor tissue and metastases [1] |
| Toxicity/Toxicokinetics |
Acute toxicity: After a single intravenous injection of the conjugate containing Fmoc-Phe-Lys(Boc)-PAB (at a dose up to 40 mg/kg, calculated on a payload equivalent basis) into mice, no mice were observed to die within 14 days, nor were there any signs of toxicity such as sudden weight loss (weight change rate <5%), abnormal behavior, or anorexia; the median lethal dose (LD50) was >40 mg/kg [1]
- Repeated-dose toxicity: Mice were injected intravenously with the conjugate once every 4 days for 3 consecutive weeks (at a dose of 5 mg/kg, calculated on a payload equivalent basis). 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 there was no obvious pathological damage in the heart, liver, spleen, lungs, kidneys and other major organs, and no obvious inflammatory infiltration was seen around the tumor tissue and metastatic lesions [1] - Plasma protein binding rate: In vitro experiments showed that the plasma protein binding rate of the conjugate containing Fmoc-Phe-Lys(Boc)-PAB was 75%-80%, and the plasma protein binding rate of the Fmoc-Phe-Lys(Boc)-PAB cleavage fragment was 18%-22% [1] |
| References | |
| Additional Infomation |
Structural characteristics: Fmoc-Phe-Lys(Boc)-PAB is composed of fluorenemethyloxycarbonyl (Fmoc), L-phenylalanine (Phe), L-lysine (Lys, with the side chain amino group protected by Boc), and p-aminobenzyl alcohol (PAB) linked by amide bonds, with a molecular weight of 687.8 Da. The Phe-Lys dipeptide fragment in the structure is the recognition site of cathepsin B[1] - Mechanism of action: As the core linker of anticancer prodrug conjugates, Fmoc-Phe-Lys(Boc)-PAB is stable in the physiological environment (pH 7.4) and the cleavage rate is <4% after incubation at room temperature for 72 hours; after entering the lysosomes of metastatic tumor cells, the linker will be recognized by cathepsin B and cleave the Phe-Lys peptide bond, releasing PAB and its linked anticancer payload, thereby achieving targeted delivery and release of the payload at the target site and reducing the toxic side effects of non-target sites[1] - Application scenario: This linker is mainly used to prepare anticancer prodrug conjugates targeting metastatic tumor cells. By specifically binding to the surface antigens of metastatic cells via a targeting carrier (such as an antibody or peptide ligand), the precise delivery of anticancer drug payloads is mediated, which is applicable to the treatment of metastatic tumors such as breast cancer, lung cancer, and colon cancer [1]. - Synthesis method: Fmoc-Phe-Lys(Boc)-PAB can be prepared by solid-phase synthesis. After synthesis, it is coupled with the targeting carrier molecule and the anticancer payload to construct a stable targeted anticancer prodrug conjugate, thereby improving the therapeutic effect of anticancer drugs on metastatic tumors and reducing systemic toxicity [1].
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| Molecular Formula |
C42H48N4O7
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|---|---|
| Molecular Weight |
720.85313129425
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| Exact Mass |
720.35
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| Elemental Analysis |
C, 69.98; H, 6.71; N, 7.77; O, 15.54
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| CAS # |
206133-42-8
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| PubChem CID |
155908176
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| Appearance |
Solid powder
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| LogP |
6.2
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| Hydrogen Bond Donor Count |
5
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
18
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| Heavy Atom Count |
53
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| Complexity |
1160
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| Defined Atom Stereocenter Count |
2
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| SMILES |
CC(C)(C)OC(=O)NCCCC[C@@H](C(=O)NC1=CC=C(C=C1)CO)NC(=O)[C@H](CC2=CC=CC=C2)NC(=O)OCC3C4=CC=CC=C4C5=CC=CC=C35
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| InChi Key |
FSNKYAMDHPYUFM-BCRBLDSWSA-N
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| InChi Code |
InChI=1S/C42H48N4O7/c1-42(2,3)53-40(50)43-24-12-11-19-36(38(48)44-30-22-20-29(26-47)21-23-30)45-39(49)37(25-28-13-5-4-6-14-28)46-41(51)52-27-35-33-17-9-7-15-31(33)32-16-8-10-18-34(32)35/h4-10,13-18,20-23,35-37,47H,11-12,19,24-27H2,1-3H3,(H,43,50)(H,44,48)(H,45,49)(H,46,51)/t36-,37-/m0/s1
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| Chemical Name |
tert-butyl ((S)-5-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropanamido)-6-((4-(hydroxymethyl)phenyl)amino)-6-oxohexyl)carbamate
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| Synonyms |
Fmoc-Phe-Lys(Boc)-PAB; FBN33428; FBN-33428; FBN 33428;
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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 | 1.3873 mL | 6.9363 mL | 13.8725 mL | |
| 5 mM | 0.2775 mL | 1.3873 mL | 2.7745 mL | |
| 10 mM | 0.1387 mL | 0.6936 mL | 1.3873 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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