Z-Gly-Pro-AMC is a fluorescent substrate. Z-Gly-Pro-AMC is hydrolyzed by prolyl endopeptidase to produce highly fluorescent 7-amido-4-methylcoumarin. (λex=380 nm, λem=465 nm).

Physicochemical Properties

Molecular Formula	C25H25N3O6
Molecular Weight	463.48
Exact Mass	463.174
CAS #	68542-93-8
PubChem CID	125702
Appearance	White to off-white solid powder
Density	1.358g/cm3
Boiling Point	770.8ºC at 760 mmHg
Melting Point	96-102ºC(lit.)
Flash Point	420ºC
Index of Refraction	1.635
LogP	3.359
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	7
Heavy Atom Count	34
Complexity	822
Defined Atom Stereocenter Count	1
SMILES	CC1=CC(=O)OC2=C1C=CC(=C2)NC(=O)[C@@H]3CCCN3C(=O)CNC(=O)OCC4=CC=CC=C4
InChi Key	YWOXKKRKNGWXEG-FQEVSTJZSA-N
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
Chemical Name	benzyl N-[2-[(2S)-2-[(4-methyl-2-oxochromen-7-yl)carbamoyl]pyrrolidin-1-yl]-2-oxoethyl]carbamate
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 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)

Biological Activity

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.

References

[1]. The development and validation of a combined kinetic fluorometric activity assay for fibroblast activation protein alpha and prolyl oligopeptidase in plasma. Clin Chim Acta. 2019 Aug;495:154-160.

Solubility Data

Solubility (In Vitro)

DMSO: 125 mg/mL (269.70 mM)

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. 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. 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 corn oil and mix evenly.  (Please use freshly prepared in vivo formulations for optimal results.)

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.