E64-RO

E-64

N-[N-(L-3-Trans-carboxirane-2-carbonyl)-L-leucyl]-agmatine

• Synonym(s): E-64, n-[n-( l-transepoxysuccinic acid)-l-leucyl]-agmatine, n-[n-(l-3-trans-carboxirane-2-carbonyl)-l-leucyl]-4-aminobutylguanidine, n-[n-(l-3-trans-carboxirane-2-carbonyl)-l-leucyl]-agmatine. ; note: another name for l-3-trans-carboxyoxiran-2-carboxylic acid acid is l-transepoxysuccinic acid. agmatine is 4-aminobutylguanidine., trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butane, L-trans-3-Carboxyoxiran-2-carbonyl-L-leucylagmatine, N-(trans-Epoxysuccinyl)-L-leucine 4-guanidinobutylamide
• Empirical Formula (Hill Notation): C₁₅H₂₇N₅O₅
• CAS Number: 66701-25-5
• Molecular Weight: 357.41
• Beilstein: 1405664
• MDL number: MFCD00080261
• UNSPSC Code: 12352200
• PubChem Substance ID: 329798998

Properties

• Quality Level: 100
• Assay: >95% (HPLC)
• form: powder
• mol wt: M_r 357.4
• packaging: pkg of 10 mg (10874523001); pkg of 25 mg (11585681001)
• manufacturer/tradename: Roche
• pH range: 2-10
• solubility: ethanol: water (1:1): soluble 20 mg/mL
• storage temp.: 2-8°C
• SMILES string: CC(C)C[C@H](NC(=O)[C@@H]1O[C@H]1C(O)=O)C(=O)NCCCCNC(N)=N
• InChI: 1S/C15H27N5O5/c1-8(2)7-9(20-13(22)10-11(25-10)14(23)24)12(21)18-5-3-4-6-19-15(16)17/h8-11H,3-7H2,1-2H3,(H,18,21)(H,20,22)(H,23,24)(H4,16,17,19)/t9-,10+,11+/m0/s1
• InChI key: LTLYEAJONXGNFG-HBNTYKKESA-N

Description

Specificity

The thiol protease inhibitor E-64 specifically inhibits papain and other cysteine proteases such as cathepsin B and L, bromelain, and ficin. Cathepsin A and D are not inhibited. L-lactate dehydrogenase from porcine heart is not inhibited, although the enzyme contains a functional thiol group.

Application

E-64 is an effective ligand for affinity purification of cysteine proteases. When coupled to a thiolated affinity matrix, binding is no longer irreversible, but specificity is retained.

E-64 is used for the inhibition of papain and other cysteine proteases such as cathepsin B and L, bromelain, and ficin during isolation and purification of proteins and enzymes.
It has been used in the affinity chromatography for the purification of hemoglobin receptors.
The inhibition of thiol proteases by E-64 appears to be of a non-competitive nature between the SH components. The inhibition is also irreversible, and is altered after gel filtration (Sephadex column) or dialysis after incubation of papain with E-64. The enzyme and inhibitor combine in an equimolar ratio.

Biochem/physiol Actions

E-64 is an irreversible, potent, and highly selective cysteine protease inhibitor. E-64 does not react with the functional thiol group of non-protease enzymes, such as L-lactate dehydrogenase or creatine kinase. E-64 will not inhibit serine proteases (except trypsin) like other cysteine protease inhibitors, leupeptin and antipain. The trans-epoxysuccinyl group (active moiety) of E-64 irreversibly binds to an active thiol group in many cysteine proteases, such as papain, actinidase, and cathepsins B, H, and L to form a thioether linkage. E-64 is a very useful cysteine protease inhibitor for use in in vivo studies because it has a specific inhibition, it is permeable in cells and tissues and has low toxicity.

Preparation Note

Working concentration: 0.5 to 1.0 μg/ml (1.4 to 28.0 μM)
Working solution: Soluble to 20 mg/ml (stock solution) in a 1:1 (v/v) mixture of ethanol and water (vortexing or slight warming in water bath (40 °C) may facilitate dissolution). Also soluble in a neutral water/methanol solution, in water, methanol, acetic acid, pyridine and DMSO. Sparingly soluble in ethanol and propanol. Insoluble in acetone, chloroform, ethyl ether and benzene.
Storage conditions (working solution): -15 to -25 °C
Solutions are stable for one month when stored in aliquots at -15 to -25 °C.

E-64 is soluble in water. A 20 mg/ml solution can be prepared in water (heat may be needed). A suggested stock solution is a 1 mM aqueous solution. The effective concentration for use as a protease inhibitor is 1 to10 μM. Aqueous stock solutions are stable for months at -20 °C. Diluted solutions are stable for days at neutral pH. E-64 is stable from pH 2-10, but is unstable in ammonia or in HCl. E-64 is also soluble in DMSO, a 10 mM solution can be prepared in dry DMSO and stored at -20 °C. Subsequent dilutions were in culture medium. Solutions for injection were prepared by dissolving E-64 in 0.9% sodium chloride or in a minimum amount of saturated sodium bicarbonate followed by dilution with 0.9% sodium chloride (after adjusting the pH to 7.0 with acetic acid).

Reconstitution

Soluble to 20 mg/ml in a 1:1 (v/v) mixture of ethanol and water. Solutions are stable for one month when stored in aliquots at -15 to -25 °C.

Other Notes

For life science research only. Not for use in diagnostic procedures.

Safety Information

• Pictograms: GHS08
• Signal Word: Warning
• Hazard Statements: H371
• Precautionary Statements: P260 - P264 - P270 - P308 + P311 - P405 - P501
• Hazard Classifications: STOT SE 2
• Storage Class Code: 11 - Combustible Solids
• WGK: WGK 2
• Flash Point(F): does not flash
• Flash Point(C): does not flash