| Size | Price | Stock | Qty |
|---|---|---|---|
| 5g |
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| Other Sizes |
| Targets |
Apical membrane antigen 1 (AMA1) of Plasmodium falciparum. For the N-Me-Leu-8 peptide (synthesized using Fmoc-N-Me-Leu-OH):
- Binding affinity (KD) to 3D7 strain AMA1: 21.00 ± 1.73 nM (kinetic analysis) [1] - Binding affinity (KD) to W2mef strain AMA1: 16.8 ± 0.1 μM (steady-state analysis) [1] - Binding affinity (KD) to HB3 strain AMA1: 68.5 ± 0.5 μM (steady-state analysis) [1] |
|---|---|
| ln Vitro |
Inhibition of parasite invasion: The N-Me-Leu-8 peptide (containing N-methyl leucine) inhibited invasion of 3D7 Plasmodium falciparum parasites into erythrocytes more potently than the native R1 peptide. At 50 μg/mL, it showed complete inhibition. Titration assays revealed that N-Me-Leu-8 was a more potent invasion inhibitor than native R1, correlating with its enhanced AMA1 affinity. [1]
- AMA1 binding (ELISA): N-Me-Leu-8 peptide competed with R1-phage for binding to immobilized 3D7 AMA1. The IC50 (concentration reducing R1-phage binding by 50%) was lower than that of native R1, indicating improved binding. [1] - Proteolytic stability in mouse plasma: N-Me-Leu-8 peptide showed dramatically improved stability compared to native R1. After 80 min incubation in mouse plasma at 37°C, approximately 50% of the N-Me-Leu-8 peptide remained intact, whereas native R1 was almost completely degraded within 10 min. [1] |
| Enzyme Assay |
Surface Plasmon Resonance (SPR) binding analysis: The interaction kinetics of N-Me-Leu-8 peptide with AMA1 proteins from different P. falciparum strains (3D7, HB3, W2mef) were measured using a Biacore T100 biosensor at 25°C. AMA1 was immobilized on a CM5 sensor chip via standard amine coupling. The running buffer was HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.05% surfactant P20, pH 7.4). Peptide concentrations ranging from 5 to 320 nM (for 3D7) or 0.32 to 40.96 μM (for W2mef and HB3) were injected over immobilized AMA1 at a flow rate of 60 μL/min for 1 min, followed by dissociation for 5 min. Sensograms were processed using Scrubber software. Rate constants (ka and kd) were determined by global fitting to a 1:1 binding model including a mass transport term. KD was calculated as kd/ka. For rapidly dissociating interactions, steady-state affinity analysis was used. For N-Me-Leu-8 binding to 3D7 AMA1: ka = (2.74 ± 0.54)×10^7 1/Ms, kd = (2.84 ± 0.15)×10^-2 1/s, KD = 21.00 ± 1.73 nM. [1]
- ELISA-based competition binding assay: 96-well plates were coated with 2 μg/mL recombinant AMA1 (3D7 strain) overnight at 4°C. After blocking with 10% skim milk in PBS, varying concentrations of N-Me-Leu-8 peptide were added to wells together with a constant concentration of R1-displaying phage (~2×10^9 cfu/mL). After 1 h incubation at room temperature with shaking, unbound phage was removed by washing with PBS/0.05% Tween 20. Bound phage was detected with horseradish peroxidase-conjugated anti-M13 antibody (1:5000) using tetramethylbenzidine substrate, and absorbance read at 450 nm. The peptide concentration required to reduce R1-phage binding by 50% (IC50) was determined. [1] |
| Cell Assay |
Invasion inhibition assay (parasite growth inhibition): The 3D7 parasite line was cultured and synchronized to schizont stage by sorbitol lysis. Peptides (including N-Me-Leu-8) were diluted in PBS, and 50 μL was added in triplicate to sterile flat-bottomed microtiter wells. Then 50 μL of complete culture media and 100 μL of synchronized schizont-stage parasites (4% hematocrit, 0.3% parasitemia) were added. Plates were incubated for 40-42 h at 37°C in a humidified atmosphere of 94% N2, 1% O2, 5% CO2. After washing with ice-cold PBS, parasites were frozen and thawed, and relative parasitemia was determined by measuring parasite lactate dehydrogenase activity. Absorbance was read at 650 nm. Percent inhibition was calculated as: 100 − [(A650 peptide sample − A650 RBC only) / (A650 no peptide control − A650 RBC only) × 100]. N-Me-Leu-8 showed improved potency compared to native R1 in titration experiments (concentration-response curves shown in Fig. 3C). [1]
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| ADME/Pharmacokinetics |
Proteolytic stability in mouse plasma: N-Me-Leu-8 peptide (0.4 mg) was dissolved in PBS (400 μL) containing 0.1% benzyl alcohol as internal standard. Equal volumes of mouse plasma solution (reconstituted from lyophilized plasma) and peptide solution were combined (total 800 μL) and incubated at 37°C. Samples (50 μL) were taken in triplicate at 0, 10, 20, 40, and 80 min, placed on ice, mixed with 20 μL of 0.5 M lysine monohydrochloride and 65 μL of acetonitrile, cooled, centrifuged, and the supernatant analyzed by reversed-phase HPLC. The ratio of peptide peak area to benzyl alcohol peak area was normalized to t=0. N-Me-Leu-8 exhibited dramatically improved stability: after 80 min, approximately 50% of the peptide remained intact, whereas native R1 was almost completely degraded (<20% remaining) by 10 min. [1]
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| References | |
| Additional Infomation |
Background: The R1 peptide is a 20-residue inhibitor of malaria parasite invasion that binds to AMA1. However, it is rapidly degraded by proteases and shows strain-specific activity. N-methylation, including at Leu-8 using Fmoc-N-Me-Leu-OH, was explored to improve stability, affinity, and breadth of specificity. [1]
- Mechanism of action: N-Me-Leu-8 peptide retains the ability to bind AMA1 and block parasite invasion. The increased affinity (slower dissociation rate) suggests that N-methylation stabilizes the peptide-AMA1 complex. The peptide likely targets a conserved hydrophobic pocket on AMA1, as it competes with inhibitory monoclonal antibodies 1F9 and 4G2dc1. [1] - Structural impact: NMR studies showed that N-methylation at Leu-8 does not cause long-range structural changes; backbone chemical shift differences are local. The solution structure of N-Me-Leu-8 remained similar to that of native R1, with two turn regions. However, inclusion of N-methyl NOEs suggested some local stabilization. [1] - Strain specificity: While native R1 binds only to 3D7 and D10 strains, N-Me-Leu-8 showed weak but measurable binding to HB3 and W2mef AMA1 (KD ~68.5 μM and ~16.8 μM respectively), indicating partial improvement in cross-strain recognition. |
| Molecular Formula |
C22H25NO4
|
|---|---|
| Molecular Weight |
367.4382
|
| Exact Mass |
367.178
|
| CAS # |
103478-62-2
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| PubChem CID |
7015835
|
| Appearance |
White to off-white solid powder
|
| Density |
1.2±0.1 g/cm3
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| Boiling Point |
537.3±29.0 °C at 760 mmHg
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| Melting Point |
113-116ºC
|
| Flash Point |
278.7±24.3 °C
|
| Vapour Pressure |
0.0±1.5 mmHg at 25°C
|
| Index of Refraction |
1.581
|
| LogP |
5.44
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| Hydrogen Bond Donor Count |
1
|
| Hydrogen Bond Acceptor Count |
4
|
| Rotatable Bond Count |
7
|
| Heavy Atom Count |
27
|
| Complexity |
512
|
| Defined Atom Stereocenter Count |
1
|
| SMILES |
CC(C)C[C@@H](C(=O)O)N(C)C(=O)OCC1C2=CC=CC=C2C3=CC=CC=C13
|
| InChi Key |
BUJQSIPFDWLNDC-FQEVSTJZSA-N
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| InChi Code |
InChI=1S/C22H25NO4/c1-14(2)12-20(21(24)25)23(3)22(26)27-13-19-17-10-6-4-8-15(17)16-9-5-7-11-18(16)19/h4-11,14,19-20H,12-13H2,1-3H3,(H,24,25)/t20-/m0/s1
|
| Chemical Name |
(2S)-2-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]-4-methylpentanoic acid
|
| 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)
|
| Solubility (In Vitro) |
DMSO : ~100 mg/mL (~272.15 mM)
|
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.80 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 (6.80 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. 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 (6.80 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 | 2.7215 mL | 13.6077 mL | 27.2153 mL | |
| 5 mM | 0.5443 mL | 2.7215 mL | 5.4431 mL | |
| 10 mM | 0.2722 mL | 1.3608 mL | 2.7215 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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