FLUOROGENIC PEPTIDES AND THEIR METHOD OF PRODUCTION

• US 20110039289A1
• Filed: 03/14/2008
• Published: 02/17/2011
• Est. Priority Date: 03/14/2008
• Status: Abandoned Application

• Current Assignee: Assaymetrics Limited
• Original Assignee: Assaymetrics Limited
• Inventors: Smith, John A., Graves, Pierre
• Application Number:
  US12/922,649
• Publication Number:
  US 20110039289A1
• Time in Patent Office: Days
• US Class Current: 435/23
• CPC Class Codes: C07K 1/13 Labelling of peptides
• CPC Class Codes: C07K 5/101 the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
• CPC Class Codes: C07K 5/1016 and aromatic or cycloaliphatic
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase

Abstract

A method of making a fluorogenic peptide with an acridone fluorophor is provided, the method comprising the steps of: (a) providing an acridone derivative, said acridone derivative having first and second reactive groups (or precursors thereof), (b) providing a solid phase support provided with reactive species for reacting with the first reactive group of the acridone derivative (c) causing the solid phase support and acridone derivative to react so that the acridone derivative is attached to the solid phase support (d) providing the peptide or reagents for the formation of the peptide, and (e) subsequent to step (c), causing the reaction of the second acridone reactive group with the peptide or one or more of the reagents for the formation of the peptide.

Claims

1. A method of making a fluorogenic peptide with an acridone fluorophor, the method comprising the steps of:

(a) providing an acridone derivative, said acridone derivative having first and second reactive groups (or precursors thereof),

(b) providing a solid phase support provided with reactive species for reacting with the first reactive group of the acridone derivative

(c) causing the solid phase support and acridone derivative to react so that the acridone derivative is attached to the solid phase support

(d) providing the peptide or reagents for the formation of the peptide, and

(e) subsequent to step (c), causing the reaction of the second acridone reactive group with the peptide or one or more of the reagents for the formation of the peptide.

2. A method according to claim 1 wherein acridone derivative provided in step (a) includes or comprises a precursor of the first or second reactive group, and wherein the method further comprises treating the acridone derivative provided in step (a) such that the first or second reactive group is formed from the precursor.

3. A method according to claim 1 or claim 2 wherein the first reactive group comprises a carboxyl group.

4. A method according to claim 1 wherein the second reactive group comprises an amino group.

5. A method according to claim 2 wherein the precursor comprises a protecting moiety.

7. A method according to claim 1 wherein one or both of the first and second reactive groups (or precursors thereof) are linked to the acridone ring by a spacer group.

8. A method according to claim 1 wherein the second reactive group (or precursor thereof) is attached to the “

2”

or “

4”

position of the acridone, and the first reactive group (or precursor thereof) is attached to the “

5”

position of the acridone ring, the substituent positions being given below in Formula (1);

9. A method according to claim 8 wherein the first reactive group comprises a carboxyl group and the second reactive group comprises an amino group.

11. A method according to claim 1 wherein the one or more reagents for the formation of a peptide comprises at least one amino acid residue and an amino acid protecting group.

12. A method according to claim 11 comprising causing the at least one amino acid residue to react with the second reactive group.

14. A method according to claim 1 comprising the step of cleaving the peptide from the support.

15. A method according to claim 1 wherein the peptide comprises from 1 to 10 residues.

16. A method according to claim 1 wherein the peptide comprises at least one residue of one or more of tyrosine, nitrotyrosine, nitrophenylalanine and tryptophan.

6. (canceled)

10. (canceled)

13. A method according to claim 13 further comprising removing the amino acid protecting group and reacting a further amino acid residue with the deprotected amino acid residue.

17. A solid support to which a fluorogenic reporter is attached, the fluorogenic reporter being an acridone derivative.

18. A solid support to which a fluorogenic reporter is attached as claimed in claim 17 wherein the acridone is linked to the solid support via a linking group.

19. (canceled)

20. (canceled)

21. A kit for the synthesis of peptides, said kit comprising a solid support suitable for use in solid phase peptide synthesis, an acridone derivative optionally attached to the solid support or alternatively for reaction with the solid support to form an acridone derivative attached to the solid support, and compounds for the synthesis of peptides.

22. The use of 2-aminoacridone or derivative provided with an amino group attached to the “

2”

position (referring to Formula I) to form a fluorogenic peptide, the linkage between the 2-aminoacridone or derivative and the peptide being between said amino group and the peptide

23. (canceled)

24. A fluorogenic peptide having the structure:

25. A method of determining the proteolytic activity of an enzyme suspected of being a proteolytic enzyme, the method comprising:

(a) providing a sample comprising a substrate to which is attached an acridone moiety, the acridone moiety being attached via a cleavage site for a proteolytic enzyme to a quenching moiety capable of quenching fluorescent radiation emitted by the acridone moiety

(b) providing the enzyme suspected of being a proteolytic enzyme in admixture with the cleavage site and

(c) illuminating the acridone moiety with radiation so as to cause the acridone moiety to fluoresce; and

(d) monitoring the properties of the light emitted from the sample.

26. (canceled)

27. (canceled)

28. (canceled)