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| Targets |
Z-Gly-Pro-AMC targets prolyl endopeptidase (post-proline cleaving enzyme), a protease that specifically cleaves peptide bonds on the carboxyl side of proline residues. The compound serves as a fluorogenic substrate for this enzyme, releasing the fluorescent AMC moiety upon enzymatic cleavage.
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| ln Vitro |
Guidelines (This is our suggested protocol; it should be adjusted based on your unique requirements as it just offers as a guideline). Assay Z-Gly-Pro-AMC[1]: 1. Prepare a 5 μL plasma sample and pre-incubate it for 15 minutes at 37 °C with 10 μL of either a 250 nM FAP inhibitor, 250 nM PREP inhibitor, or 10 μL of 0.0025% (v/v) DMSO. 2. To get a final concentration of 266 μM, add 35 μL of pre-heated Z-Gly-Pro-AMC (380 μM diluted in buffer). 3. For thirty minutes at 37 °C, fluorescence is measured kinetically.
The in vitro activity of Z-Gly-Pro-AMC is its function as a fluorogenic substrate for prolyl endopeptidase. Upon enzymatic cleavage by the protease, the substrate releases the highly fluorescent 7-amino-4-methylcoumarin (AMC), which can be quantified by fluorescence spectroscopy. The fluorescence intensity is proportional to enzyme activity, enabling the sensitive and quantitative measurement of prolyl endopeptidase activity. The compound is used in biochemical experiments to monitor and quantify protease activity. |
| ln Vivo |
Z-Gly-Pro-AMC is not used as a therapeutic agent. Its in vivo utility is limited to research applications involving the measurement of prolyl endopeptidase activity in biological samples. The compound is not intended for systemic therapeutic use.
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| Enzyme Assay |
In vitro enzyme assays for Z-Gly-Pro-AMC involve incubating the fluorogenic substrate with prolyl endopeptidase enzyme preparations in appropriate buffer conditions. The enzyme cleaves the substrate, releasing fluorescent AMC, which is measured fluorimetrically with excitation at 380 nm and emission at 465 nm. The fluorescence intensity is proportional to enzyme activity. Inhibitors can be evaluated by measuring the reduction in fluorescence increase in the presence of test compounds.
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| Cell Assay |
For in vitro cellular experiments, Z-Gly-Pro-AMC is typically used in cell-free systems for enzyme activity assays. The substrate can be added to cell lysates or purified enzyme preparations to assess prolyl endopeptidase activity. The fluorogenic readout provides a sensitive and quantitative measure of enzyme activity.
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| Animal Protocol |
In vivo animal experiments with Z-Gly-Pro-AMC are not typically performed, as the compound is used as an in vitro enzyme substrate. However, the substrate can be used to measure prolyl endopeptidase activity in samples collected from animal models.
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| ADME/Pharmacokinetics |
Pharmacokinetic properties of Z-Gly-Pro-AMC have not been characterized, as it is a research-use enzyme substrate rather than a drug candidate. The compound has a molecular weight of 463.48 g/mol and should be stored under appropriate conditions, protected from light.
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| Toxicity/Toxicokinetics |
Toxicological data for Z-Gly-Pro-AMC has not been systematically evaluated, as it is intended for research applications only and not for therapeutic use. Standard laboratory safety precautions should be observed when handling this chemical reagent.
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| References | |
| Additional Infomation |
Fluorescent substrates for proline lyase
Z-Gly-Pro-AMC is a fluorogenic substrate for prolyl endopeptidase that releases fluorescent AMC upon enzymatic cleavage. It is used to monitor and quantify protease activity in biochemical experiments and has no clinical or therapeutic applications. |
| Molecular Formula |
C25H25N3O6
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|---|---|
| Molecular Weight |
463.48
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| Exact Mass |
463.174
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| CAS # |
68542-93-8
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| PubChem CID |
125702
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| Appearance |
White to off-white solid powder
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| Density |
1.358g/cm3
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| Boiling Point |
770.8ºC at 760 mmHg
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| Melting Point |
96-102ºC(lit.)
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| Flash Point |
420ºC
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| Index of Refraction |
1.635
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| LogP |
3.359
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
34
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| Complexity |
822
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| Defined Atom Stereocenter Count |
1
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| SMILES |
CC1=CC(=O)OC2=C1C=CC(=C2)NC(=O)[C@@H]3CCCN3C(=O)CNC(=O)OCC4=CC=CC=C4
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| InChi Key |
YWOXKKRKNGWXEG-FQEVSTJZSA-N
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| InChi Code |
InChI=1S/C25H25N3O6/c1-16-12-23(30)34-21-13-18(9-10-19(16)21)27-24(31)20-8-5-11-28(20)22(29)14-26-25(32)33-15-17-6-3-2-4-7-17/h2-4,6-7,9-10,12-13,20H,5,8,11,14-15H2,1H3,(H,26,32)(H,27,31)/t20-/m0/s1
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| Chemical Name |
benzyl N-[2-[(2S)-2-[(4-methyl-2-oxochromen-7-yl)carbamoyl]pyrrolidin-1-yl]-2-oxoethyl]carbamate
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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: (1). This product requires protection from light (avoid light exposure) during transportation and storage. (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. |
| 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: 125 mg/mL (269.70 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.49 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 20.8 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: ≥ 2.08 mg/mL (4.49 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 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: ≥ 2.08 mg/mL (4.49 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 | 2.1576 mL | 10.7880 mL | 21.5759 mL | |
| 5 mM | 0.4315 mL | 2.1576 mL | 4.3152 mL | |
| 10 mM | 0.2158 mL | 1.0788 mL | 2.1576 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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