Pam3CSK4-Biotin is a biotinylated Pam3CSK4 acting as a novel and potent Toll-like receptor 1/2 (TLR1/2) agonist.
Physicochemical Properties
| Molecular Formula | C103H192N14O17S2 |
| Molecular Weight | 1962.85 |
| Exact Mass | 1961.40313 |
| CAS # | 1429504-10-8 |
| PubChem CID | 90488959 |
| SequenceShortening | XSKKKX |
| Appearance | Typically exists as solid at room temperature |
| LogP | 17.5 |
| Hydrogen Bond Donor Count | 16 |
| Hydrogen Bond Acceptor Count | 22 |
| Rotatable Bond Count | 98 |
| Heavy Atom Count | 98 |
| Complexity | 3160 |
| Defined Atom Stereocenter Count | 9 |
| SMILES | C([C@@H]1SC[C@]2([H])NC(N[C@@]21[H])=O)CCCC(=O)NCCCCCC(=O)NCCCCCC(=O)NCCCC[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)CCCCCCCCCCCCCCC)CSCC(COC(=O)CCCCCCCCCCCCCCC)OC(=O)CCCCCCCCCCCCCCC |
| InChi Key | GVXJPWKMFMNHRE-XHHKIZCASA-N |
| InChi Code | InChI=1S/C103H192N14O17S2/c1-4-7-10-13-16-19-22-25-28-31-34-37-42-67-93(122)110-88(79-135-78-81(134-95(124)69-46-39-36-33-30-27-24-21-18-15-12-9-6-3)77-133-94(123)68-45-38-35-32-29-26-23-20-17-14-11-8-5-2)101(129)115-86(76-118)100(128)113-83(60-50-54-71-105)98(126)111-82(59-49-53-70-104)97(125)112-84(61-51-55-72-106)99(127)114-85(102(130)131)62-52-58-75-109-91(120)65-44-41-56-73-107-90(119)64-43-40-57-74-108-92(121)66-48-47-63-89-96-87(80-136-89)116-103(132)117-96/h81-89,96,118H,4-80,104-106H2,1-3H3,(H,107,119)(H,108,121)(H,109,120)(H,110,122)(H,111,126)(H,112,125)(H,113,128)(H,114,127)(H,115,129)(H,130,131)(H2,116,117,132)/t81?,82-,83-,84-,85-,86-,87-,88-,89-,96-/m0/s1 |
| Chemical Name | (2S)-6-[6-[6-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]hexanoylamino]hexanoylamino]-2-[[(2S)-6-amino-2-[[(2S)-6-amino-2-[[(2S)-6-amino-2-[[(2S)-2-[[(2R)-3-[2,3-di(hexadecanoyloxy)propylsulfanyl]-2-(hexadecanoylamino)propanoyl]amino]-3-hydroxypropanoyl]amino]hexanoyl]amino]hexanoyl]amino]hexanoyl]amino]hexanoic acid |
| Synonyms | Pam3CSK4 Biotin; 1429504-10-8; orb1984076; AKOS024458267; PD079725; (2S,3Z,5S,6Z,8S,9Z,11S,12Z,14S,15Z,17R)-5,8,11-tris(4-aminobutyl)-4,7,10,13,16-pentahydroxy-2-(4-((Z)-(1-hydroxy-6-((Z)-(1-hydroxy-6-((Z)-(1-hydroxy-5-((3aR,6S,6aS)-2-hydroxy-3a,4,6,6a-tetrahydro-1H-thieno[3,4-d]imidazol-6-yl)pentylidene)amino)hexyli |
| HS Tariff Code | 2934.99.9001 |
| 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) |
Biological Activity
| Targets |
- Toll-like receptor 2 (TLR2)-TLR1 heterodimer
- No IC50/Ki/EC50 values for Pam3CSK4-Biotin were reported in the study. The parent compound Pam3CSK4 is a TLR2-TLR1 agonist with an EC50 of 43.2 ± 5.6 nM for equine TLR2-TLR1 activation [1] |
| ln Vitro |
- TLR2-TLR1 signaling activation: 1. NF-κB reporter gene activation: HEK-Blue™ cells expressing human TLR2-TLR1 were treated with Pam3CSK4 (100 ng/mL) or LTA (1 μg/mL) for 24 hours. Both ligands induced NF-κB-dependent SEAP (secreted embryonic alkaline phosphatase) activity, which was abolished by clathrin/dynamin inhibitors (chlorpromazine, dynasore) [1] 2. Cytokine induction: Human primary monocytes treated with Pam3CSK4 (100 ng/mL) for 24 hours showed increased TNF-α secretion (ELISA), which was blocked by endocytosis inhibitors [1] |
| Enzyme Assay |
- NF-κB reporter gene assay for TLR2-TLR1 activation [1]: 1. Cell culture: HEK-Blue™ cells (expressing TLR2-TLR1 and SEAP reporter) were seeded in 96-well plates (1×10⁵ cells/well). 2. Drug treatment: Cells were incubated with Pam3CSK4 (10–1000 ng/mL) or LTA (0.1–10 μg/mL) for 24 hours at 37°C, 5% CO₂. 3. Activity detection: SEAP activity in cell supernatants was measured using QUANTI-Blue™ reagent, with absorbance read at 655 nm. Relative activity was normalized to vehicle control. |
| Cell Assay |
- Monocyte endocytosis and cytokine induction assay [1]: 1. Cell isolation: Human peripheral blood monocytes were isolated via CD14⁺ magnetic beads and cultured in RPMI 1640 with 10% FBS. 2. Drug treatment: Monocytes were pre-treated with endocytosis inhibitors (chlorpromazine, dynasore, chloroquine) for 45 minutes, then stimulated with Pam3CSK4 (100 ng/mL) or LTA (1 μg/mL) for 4–24 hours. 3. Analysis: TNF-α mRNA expression (qPCR) and protein secretion (ELISA) were measured. Internalization of Pam3CSK4 was visualized via confocal microscopy using Alexa Fluor-labeled Pam3CSK4 (not biotinylated) [1] |
| References | [1]. TLR2 ligands induce NF-κB activation from endosomal compartments of human monocytes. PLoS One. 2013 Dec 12;8(12):e80743. |
| Additional Infomation |
- Key findings: The study demonstrated that TLR2 ligands (Pam3CSK4 and LTA) induce NF-κB activation from endosomal compartments in human monocytes. This process requires clathrin/dynamin-dependent endocytosis and CD14-mediated internalization [1] - Biotinylated analog not used: The study did not utilize Pam3CSK4-Biotin but focused on unmodified Pam3CSK4. Biotinylation of Pam3CSK4 is a common modification for tracking ligand-receptor interactions (e.g., streptavidin-based detection), but its specific effects were not investigated here [3,6] - Mechanistic insight: Endosomal localization of TLR2-TLR1 is critical for signaling, as cell-surface TLR2 alone cannot activate NF-κB without internalization [1] - Species relevance: The findings are specific to human monocytes and HEK-Blue™ cells expressing human TLR2-TLR1 [1] |
Solubility Data
| 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 |
| 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 (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *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). 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)] *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. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL 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). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.5095 mL | 2.5473 mL | 5.0946 mL | |
| 5 mM | 0.1019 mL | 0.5095 mL | 1.0189 mL | |
| 10 mM | 0.0509 mL | 0.2547 mL | 0.5095 mL |