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Application Area

N-α-Benzoyl-DL-arginine 7-amido-4-methylcoumarin hydrochloride

* Please kindly note that our products are not to be used for therapeutic purposes and cannot be sold to patients.

Category

DL-Amino Acids

Catalog number

BAT-005961

CAS number

102601-21-8

Molecular Formula

C23H26ClN5O4

Molecular Weight

471.94

N-α-Benzoyl-DL-arginine 7-amido-4-methylcoumarin hydrochloride

IUPAC Name

N-[5-(diaminomethylideneamino)-1-[(4-methyl-2-oxochromen-7-yl)amino]-1-oxopentan-2-yl]benzamide;hydrochloride

Synonyms

Bzo-DL-Arg-AMC HCl

Boiling Point

814 °C

Storage

Store at -20 °C

InChI

InChI=1S/C23H25N5O4.ClH/c1-14-12-20(29)32-19-13-16(9-10-17(14)19)27-22(31)18(8-5-11-26-23(24)25)28-21(30)15-6-3-2-4-7-15;/h2-4,6-7,9-10,12-13,18H,5,8,11H2,1H3,(H,27,31)(H,28,30)(H4,24,25,26);1H

InChI Key

XKAVYOJOHZLTDF-UHFFFAOYSA-N

Canonical SMILES

CC1=CC(=O)OC2=C1C=CC(=C2)NC(=O)C(CCCN=C(N)N)NC(=O)C3=CC=CC=C3.Cl

N-α-Benzoyl-DL-arginine 7-amido-4-methylcoumarin hydrochloride, a synthetic substrate utilized in biochemical assays to gauge enzyme activity, finds diverse applications across various domains. Here are the key applications presented with high perplexity and burstiness:

Protease Activity Assays: Widely employed in laboratory environments, this compound serves as a pivotal tool for quantifying the activity of proteases, particularly serine proteases like trypsin. When these proteases cleave the substrate, they liberate a fluorescent product that can be quantified using a fluorometer. This method enables researchers to delve into protease kinetics, enzyme inhibition, and diverse catalytic properties, unraveling the intricacies of enzyme function.

Drug Discovery: In the realm of pharmaceutical exploration, N-α-Benzoyl-DL-arginine 7-amido-4-methylcoumarin hydrochloride plays a crucial role in screening potential protease inhibitors. By monitoring changes in substrate cleavage, scientists can pinpoint compounds that effectively suppress enzyme activity, facilitating the development of novel therapeutics. This application holds particular significance in the quest for treatments targeting ailments such as cancer, HIV, and inflammation-related disorders, driving forward the frontier of therapeutic innovation.

Clinical Diagnostics: Beyond the confines of research settings, this substrate finds utility in clinical laboratories for diagnostic purposes. Its integration into specific enzymatic assays aids in identifying abnormal levels of protease activity in patient samples. Such diagnostic assessments play a pivotal role in detecting conditions linked to enzyme dysregulation, including pancreatitis, coagulopathies, and certain malignancies, contributing to timely and accurate disease diagnosis.

Biochemical Research: Across the landscape of biochemical investigation, N-α-Benzoyl-DL-arginine 7-amido-4-methylcoumarin hydrochloride stands as a cornerstone for studying enzyme mechanisms. By furnishing a dependable and quantifiable substrate, it empowers scientists to delve into the structural and functional nuances of enzymes. This deeper understanding propels our comprehension of enzyme behavior and aids in the development of more potent biocatalysts and biomaterials, laying the groundwork for innovative advancements in the biochemical sciences.

1. Serine proteinase from Cucurbita ficifolia seeds

M Dryjański, J Otlewski, T Wilusz Acta Biochim Pol. 1990;37(1):169-72.

A new serine proteinase was isolated from Cucurbita ficifolia seeds by the purification procedure, which includes: extraction, salting out with ammonium sulphate, chromatography on CM-cellulose. Sephacryl S-300 gel filtration and h.p.l.c. on DEAE-2SW TSK column. The enzyme was homogeneous both in native and SDS PAGE. Three independent methods showed its molecular mass to be approximately 77 kDa. The enzyme was inhibited by specific serine proteinase organic inhibitors, and was active in the presence of inhibitors specific for other proteinase classes. Surprisingly, squash proteinase exhibited a very high and broad pH optimum with a maximum at 10.7. It hydrolysed many different peptide bonds in B-chain of insulin and was able to cleave four bonds in endogenous serine proteinase inhibitor (CMTI).

2. Insecticidal effect of Canavalia ensiformis major urease on nymphs of the milkweed bug Oncopeltus fasciatus and characterization of digestive peptidases

Marina S Defferrari, Diogo R Demartini, Thiago B Marcelino, Paulo M Pinto, Celia R Carlini Insect Biochem Mol Biol. 2011 Jun;41(6):388-99. doi: 10.1016/j.ibmb.2011.02.008. Epub 2011 Mar 4.

Jackbean (Canavalia ensiformis) ureases are entomotoxic upon the release of internal peptides by insect's digestive enzymes. Here we studied the digestive peptidases of Oncopeltus fasciatus (milkweed bug) and its susceptibility to jackbean urease (JBU). O. fasciatus nymphs fed urease showed a mortality rate higher than 80% after two weeks. Homogenates of midguts dissected from fourth instars were used to perform proteolytic activity assays. The homogenates hydrolyzed JBU in vitro, yielding a fragment similar in size to known entomotoxic peptides. The major proteolytic activity at pH 4.0 upon protein substrates was blocked by specific inhibitors of aspartic and cysteine peptidases, but not significantly affected by inhibitors of metallopeptidases or serine peptidases. The optimal activity upon N-Cbz-Phe-Arg-MCA was at pH 5.0, with complete blockage by E-64 in all pH tested. Optimal activity upon Abz-AIAFFSRQ-EDDnp (a substrate for aspartic peptidases) was detected at pH 5.0, with partial inhibition by Pepstatin A in the pH range 2-8. Fluorogenic substrates corresponding to the N- and C-terminal regions flanking a known entomotoxic peptide within urease sequence were also tested. While the midgut homogenate did not hydrolyze the N-terminal peptide, it cleaved the C-terminal peptide maximally at pH 4.0-5.0, and this activity was inhibited by E-64 (10 μM). The midgut homogenate was submitted to ion-exchange chromatography followed by gel filtration. A 22 kDa active fraction was obtained, resolved in SDS-PAGE (12%), the corresponding band was in-gel digested by trypsin, the peptides were analyzed by mass spectrometry, retrieving a cathepsin L protein. The purified cathepsin L was shown to have at least two possible cleavage sites within the urease sequence, and might be able to release a known insecticidal peptide in a single or cascade event. The results suggest that susceptibility of O. fasciatus nymphs to jackbean urease is, like in other insect models, due mostly to limited proteolysis of ingested protein and subsequent release of entomotoxic peptide(s) by cathepsin-like digestive enzymes.

3. Purification and properties of trypsin-like enzyme from the midgut of Morimus funereus (coleoptera, cerambycidae) Larvae

Nikola Loncar, Zoran Vujcić, Natasa Bozić, Jelisaveta Ivanović, Vera Nenadović Arch Insect Biochem Physiol. 2010 Aug;74(4):232-46. doi: 10.1002/arch.20371.

Trypsin-like enzyme (TLE) from the anterior midgut of Morimus funereus larvae was purified by anion exchange chromatography and gel filtration chromatography and characterized. Specific TLE activity was increased 322-fold by purification of the crude midgut extract. The purified enzyme had a pH optimum of 9.0 (optimum pH range 8.5-9.5) and temperature optimum of 45 degrees C with the K(M) ratio of 0.065 mM for benzoyl-arginine-p-nitroanilide (BApNA). Among a number of inhibitors tested, the most efficient was benzamidine (K(I) value of 0.012 mM, Ic(50) value of 0.204 mM) while inhibition of TLE activity by SBTI, TLCK, and PMSF was partial. Almost all divalent cations tested enhanced the enzyme activity, amongst them Co2+ and Mn2+ stimulated TLE activity for 2.5 times. The purified TLE (after gel-filtration on Superose 12 column) had a molecular mass of 37.5 kDa with an isoelectric point over 9.3. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed one band of 38 kDa, suggesting that the enzyme is a monomer.