# US 5,334,506 A · Chromogenic method of detecting endoproteases

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- Number: US 5,334,506 A
- Document ID: US5334506A
- Status: Granted
- Kind: A
- Application number: 07/785,020
- Filed: 1991-10-30
- Granted: 1994-08-02
- Earliest claimed: 1986-08-28
- Assignees: Behringwerke Aktiengesellschaft
- Inventors: Werner Stuber; Dieter Schnaitmann
- Web page: https://patentgrep.com/patent/US5334506A

## CPC

- C07K 5/0823: {and Pro-amino acid; Derivatives thereof}
- C07K 5/0806: {the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala}
- C07K 5/0812: {and aromatic or cycloaliphatic}
- C07K 5/0825: {and Glp-amino acid; Derivatives thereof}
- Y02P 20/55: Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## Abstract

Compounds of the formula I ##STR1## in which X denotes a hydrogen atom, a group which irreversibly masks the terminal amino group, or a protecting group which is conventional in peptide chemistry, such as, for example, Boc-, Z- or Fmoc-, A and B may be identical or different and denote an alpha- beta- or gamma-amino acid which comprises 2 to 15 carbon atoms and up to 4 nitrogen atoms, 2 sulfur atoms and 6 oxygen atoms and whose side chain may be substituted, and B, if appropriate, denotes a dipeptide formed from these amino acids, C denotes arginine, Lysine, tyrosine, phenylalanine or tryptophane, and their homologs, R.sub.1 and R.sub.2 are identical or different and denote a hydrogen atom or an alkyl radical having up to 4 carbon atoms, R.sub.3 to R.sub.8 are identical or different and denote hydrogen, an alkyl radical, an alkoxy radical or a halogen radical, Y denotes oxygen, and An.sup.- denotes an anion, and their water-soluble salts, and a process for their preparation are described. These compounds or salts can be used for detecting or determining enzymes.

## Claims

### Claim 1

A method for the detection and determination of endoproteases comprising:
a) incubating a solution suspected of containing an endoprotease with a solution containing a chromogenic peptide substrate for said endoprotease, said chromogenic peptide substrate comprising a compound of the formula I ##STR5## in which X denotes a hydrogen atom, a group which irreversibly masks the terminal amino group, or a conventional protecting group selected from the group consisting of carbobenzoxy, t-butoxycarbonyl and 9-fluorenyloxycarbonyl,
A and B may be identical or different and denote an alpha-, beta- or gamma-amino acid which comprises 2 to 15 carbon atoms and up to 4 nitrogen atoms, 2 sulfur atoms and 6 oxygen atoms, and B may denote a dipeptide formed from these amino acids,
C denotes arginine, lysine, tyrosine, phenylalanine or tryptophane, and their homologs,
R.sub.1 and R.sub.2 may be identical or different and denote a hydrogen atom or an alkyl radical having up to 4 carbon atoms,
R.sub.3 to R.sub.8 may be identical or different and denote hydrogen, an alkyl radical, an alkoxy radical or a halogen radical,
Y denotes oxygen, and
An.sup.- denotes an anion,
and its water-soluble salts, and
b) determining the amount of chromophore released by said endoprotease from said chromogenic peptide substrate.

### Claim 2 (depends on 1)

The method of claim 1, in which X is a hydrogen atom and the amino acid A is present in the D-form, if A is a chiral amino acid.

### Claim 3 (depends on 1)

The method of claim 1, wherein
R.sub.1 and R.sub.2 are identical or different and are selected from the group consisting of methyl, ethyl, propyl and hydrogen, and
R.sub.7 is selected from a group consisting of hydrogen, methyl, ethyl, propyl, methoxy and ethoxy.