# US 5,334,703 A · Chromogenic substrate

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- Number: US 5,334,703 A
- Document ID: US5334703A
- Status: Granted
- Kind: A
- Application number: 07/859,717
- Filed: 1992-06-11
- Granted: 1994-08-02
- Earliest claimed: 1989-12-12
- Assignees: Chromonogenix AB
- Inventors: Salo Arielly
- Web page: https://patentgrep.com/patent/US5334703A

## CPC

- C07K 11/00: Depsipeptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K 5/101: {the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu}
- 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

The invention relates to, novel peptide derivatives with the formula: R.sub.1 -A.sub.1 -A.sub.2 -A.sub.3 -A.sub.4 -R.sub.2 or its salt, wherein R.sub.1 =H or a protective group, A.sub.1 =H, Ile, Leu or Val, A.sub.2 =Glu, Asp, Ser, Thr; A.sub.3 =Gly or Glyc; A.sub.4 =Arg or Lys; R.sub.2 =4-nitroaniline with the proviso that A.sub.3 is Gly when A.sub.4 is Lys and that A.sub.3 is Glyc when A.sub.4 is Arg. The invention also discloses the process for the preparation of the peptide derivatives, the method for the determination of the bacterial endotoxins by the use of the invented peptide derivatives and the use of these derivatives for determination of bacterial endotoxins.

## Claims

### Claim 1

Peptide derivatives having the general formula:
R.sub.1 -A.sub.1 -A.sub.2 -A.sub.3 -A.sub.4 -R.sub.2 or its salt
wherein
R.sub.1 =H or a protective group
A.sub.1 =H, Ile, Leu or Val
A.sub.2 =Glu, Asp, Ser, or Thr
A.sub.3 =Gly or Glyc
A.sub.4 =Arg or Lys
R.sub.2 =is a group that upon enzymatic hydrolysis gives para-nitroaniline (pNA),
with the proviso that A.sub.3 is Gly when A.sub.4 is Lys and Glyc when A.sub.4 is Arg.

### Claim 2 (depends on 1)

Peptide derivatives according to claim 1 wherein A.sub.1 =Ile.

### Claim 3 (depends on 1)

Peptide derivatives according to claim 1 having the formula selected from the group consisting of:
.alpha.-Ac-Ile-Glu-Gly-Lys-pNA,
.alpha.-Ac-Ile-Ser-Gly-Lys-pNA,
.alpha.-Ac-Ile-Thr-Gly-Lys-pNA, and
.alpha.-Ac-Ile-Glu-Glyc-Arg-pNA.

### Claim 4

A method for quantitatively determining bacterial endotoxins in a sample which comprises incubating the sample with a Limulus clotting enzyme and a substrate for said enzyme, wherein said substrate is a peptide derivative having the following formula:
R.sub.1 -A.sub.1 -A.sub.2 -A.sub.3 -A.sub.4 -R.sub.2 or its salt
where
R.sub.1 =H or a protective group
R.sub.1 =H, Ile, Leu or Val
A.sub.2 =Glu, Asp, Ser, or Thr
A.sub.3 =Gly or Glyc
A.sub.4 =Arg or Lys
R.sub.2 =an aromatic or heterocyclic group which by enzymatic hydrolysis gives a compound R.sub.2 -NH.sub.2 which can be determined quantitatively,
with the proviso that A.sub.3 is Gly when A.sub.4 is Lys and Glyc when A.sub.4 is Arg.

### Claim 5 (depends on 4)

The method of claim 4 when A is Ile.

### Claim 6 (depends on 5)

The method of claim 5 wherein said peptide derivative is selected from the group consisting of:
.alpha.-Ac-Ile-Glu-Gly-Lys-pNA,
.alpha.-Ac-Ile-Ser-Gly-Lys-pNA,
.alpha.-Ac-Ile-Thr-Gly-Lys-pNA, and
.alpha.-Ac-Ile-Glu-Glyc-Arg-pNA.

### Claim 7 (depends on 4)

The method of claim 4 wherein said clotting enzyme is Limulus Amebocyte Lysate.

### Claim 8 (depends on 4)

The method of claim 4 wherein R.sub.2 -NH.sub.2 is para-nitroaniline.