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| 5mg |
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| 10mg |
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| Targets |
The biological target of Suc-Ala-Ala-Phe-AMC is the active site of chymotrypsin-like serine proteases and certain metalloendopeptidases. It is not a drug that modulates a target; rather, it is a tool used to measure the activity of these enzymes. Proteases that cleave the peptide bond at the C-terminus of the phenylalanine (Phe) residue release the fluorophore 7-amino-4-methylcoumarin (AMC).
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| ln Vitro |
In vitro, Suc-Ala-Ala-Phe-AMC itself is inactive; its value is as a reporter. When incubated with a protease that recognizes the Ala-Ala-Phe sequence, the enzyme cleaves the substrate, releasing the AMC fluorophore. The free AMC then emits a strong blue fluorescence when excited at 380 nm (emission at 460 nm). The rate of increase in fluorescence is directly proportional to the enzymatic activity in the sample. It is commonly used to measure chymotrypsin activity in purified enzyme preparations or complex biological samples.
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| ln Vivo |
Suc-Ala-Ala-Phe-AMC can be used in in vivo applications, specifically to assess the acrosome reaction in sperm cells. During the acrosome reaction, a specialized form of exocytosis, sperm release acrosomal proteases including acrosin (a chymotrypsin-like protease). The release of these proteases can be measured by incubating sperm with Suc-Ala-Ala-Phe-AMC; the hydrolysis of the substrate leads to fluorescent staining of the acrosome-reacted sperm, allowing for quantification by flow cytometry or fluorescence microscopy.
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| Enzyme Assay |
For non-cell-based in vitro enzyme assays, a standard protocol is used. A purified protease (e.g., chymotrypsin) is diluted in an assay buffer (e.g., 100 mM Tris-HCl, 10 mM CaCl2, pH 8.0). Suc-Ala-Ala-Phe-AMC is dissolved in DMSO to a stock concentration of 10 mM. The assay is performed in a black 96-well plate. A reaction mixture containing 100 uL of buffer, 50 uL of enzyme, and 50 uL of substrate (final concentration 100 uM) is prepared. The plate is incubated at 37degC for 10-30 minutes. Fluorescence is measured using a plate reader (excitation 360-380 nm, emission 440-460 nm).
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| Cell Assay |
For in vitro cell assays, this substrate is used to measure cellular protease activity. Cells (e.g., pancreatic acinar cells or neutrophils) are lysed in a buffer containing a non-ionic detergent. The cell lysate is clarified by centrifugation. The protein concentration of the supernatant is determined. A 50 ug aliquot of the lysate is then incubated with 100 uM Suc-Ala-Ala-Phe-AMC in 200 uL of assay buffer for 60 minutes at 37degC. The reaction is stopped by adding 25 uL of 1M sodium acetate (pH 4.3). Fluorescence is measured as described above.
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| Animal Protocol |
For in vivo assays of the acrosome reaction, mouse or human sperm are collected and washed. Sperm are then incubated in a capacitation medium for 0-4 hours to induce the acrosome reaction. Following incubation, the sperm are stained with a live/dead stain and then incubated with 50 uM Suc-Ala-Ala-Phe-AMC for 15 minutes at 37degC. After washing, the sperm are analyzed by flow cytometry. Acrosome-reacted sperm will fluoresce green (AMC emission), while non-reacted sperm will not. The percentage of acrosome-reacted sperm is calculated.
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| ADME/Pharmacokinetics |
As a peptide substrate, pharmacokinetic properties are not relevant for Suc-Ala-Ala-Phe-AMC, as it is not intended to be a systemic drug. It is water-soluble (as it is a succinylated peptide) and can be administered locally (e.g., in the reproductive tract for acrosome reaction assays). In vivo, the substrate is likely to be rapidly hydrolyzed by serum proteases and cleared from the circulation within minutes, which is a desired property for a probe in a localized assay.
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| Toxicity/Toxicokinetics |
Toxicity is not a primary consideration for Suc-Ala-Ala-Phe-AMC as it is a research reagent used at low concentrations. The compound is not intended for human therapeutic use. It is typically used at low micromolar concentrations (50-200 uM) in vitro. The released by-product, AMC (7-amino-4-methylcoumarin), is known to be moderately toxic at high concentrations but poses no significant hazard at the levels generated in standard assays. Standard lab safety precautions (gloves, lab coat) should be used.
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| Additional Infomation |
Suc-Ala-Ala-Phe-AMC is a widely used fluorogenic substrate in enzymology and cell biology. The AMC fluorophore has an excitation maximum of 380 nm and an emission maximum of 460 nm, making it compatible with standard FITC filter sets on fluorescence microscopes and flow cytometers. The "Succinyl" (Suc) group at the N-terminus enhances solubility and prevents non-specific degradation by aminopeptidases. This substrate is not a drug and has no clinical approval status.
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| Molecular Formula |
C29H32N4O8
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|---|---|
| Molecular Weight |
564.58638
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| Exact Mass |
564.222
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| CAS # |
71973-79-0
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| PubChem CID |
3265780
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| Appearance |
White to off-white solid powder
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| Density |
1.331g/cm3
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| Boiling Point |
1002.6ºC at 760 mmHg
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| Flash Point |
560.2ºC
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| Index of Refraction |
1.607
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| LogP |
2.887
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| Hydrogen Bond Donor Count |
5
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
12
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| Heavy Atom Count |
41
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| Complexity |
1040
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CC1=CC(OC2=C1C=CC(NC([C@@H](NC([C@@H](NC([C@@H](NC(CCC(O)=O)=O)C)=O)C)=O)CC3=CC=CC=C3)=O)=C2)=O
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| InChi Key |
HHPVJKZZYOXPLH-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C29H32N4O8/c1-16-13-26(37)41-23-15-20(9-10-21(16)23)32-29(40)22(14-19-7-5-4-6-8-19)33-28(39)18(3)31-27(38)17(2)30-24(34)11-12-25(35)36/h4-10,13,15,17-18,22H,11-12,14H2,1-3H3,(H,30,34)(H,31,38)(H,32,40)(H,33,39)(H,35,36)
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| Chemical Name |
4-[[1-[[1-[[1-[(4-methyl-2-oxochromen-7-yl)amino]-1-oxo-3-phenylpropan-2-yl]amino]-1-oxopropan-2-yl]amino]-1-oxopropan-2-yl]amino]-4-oxobutanoic acid
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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 Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| 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 : ~250 mg/mL (~442.80 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 | 1.7712 mL | 8.8560 mL | 17.7120 mL | |
| 5 mM | 0.3542 mL | 1.7712 mL | 3.5424 mL | |
| 10 mM | 0.1771 mL | 0.8856 mL | 1.7712 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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