Fluorescent peptides

• US 6,054,557 A
• Filed: 07/10/1996
• Issued: 04/25/2000
• Est. Priority Date: 04/04/1995
• Status: Expired due to Term

• Current Assignee: Advanced Bioconcept Company
• Original Assignee: Advanced Bioconcept (1994) Ltd.
• Inventors: Faure, Marie-Pierre, Vincent, Jean-Pierre, Gaudriault, Georges, Desjardins, Clarissa, Beaudet, Alain
• Primary Examiner(s): Tsang, Cecilia J.
• Assistant Examiner(s): DELACROIX MUIRHEI, CYBILLE
• Application Number:
  US08/682,810
• Time in Patent Office: 1,385 Days
• Field of Search: 530/350, 530/302, 530/324, 435/7.1
• US Class Current: 530/350
• CPC Class Codes: C07K 14/575 Hormones derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin C07K14/665, e.g. corticotropin C07K14/695
• CPC Class Codes: C07K 14/57536 Endothelin, vasoactive intestinal contractor [VIC]
• CPC Class Codes: C07K 14/57563 Vasoactive intestinal peptide [VIP]; Related peptides
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C07K 7/14 Angiotensins: Related peptides
• CPC Class Codes: G01N 33/5094 for blood cell populations red blood cells G01N33/80
• CPC Class Codes: G01N 33/56966 Animal cells

Abstract

The invention provides biologically active compounds of the formula: ##STR1## where R₁ is a light-emitting moiety and R₇ is a peptide of between 2 and 200 amino acids. X is selected from the group including ═O, ═S, --OH, ═C.tbd.O, ═NH, --H, --OR, --NR, --R, --R₃ R₄, wherein each R, R₄ and R₃, independently, is H or a C1-C6 branched or unbranched, substituted or unsubstituted, alkyl. The compounds are both biologically active and optically detectable. The peptide is chemically attached to the light-emitting moiety at an amino acid position which is not involved in binding to the peptide'"'"'s receptor. In this way, the peptide'"'"'s affinity for its receptor is not significantly decreased, and the compound retains high biological activity and can be easily detected using standard optical means.

Claims

1. A biologically active and fluorescent compound of the formula:

##STR5## wherein R₁ is a light-emitting moiety;

R₂ is galanin, a galanin analog based on conservative amino acid substitutions, a galanin derivative, or a fragment thereof; and

wherein X is selected from the group consisting of ═

O, ═

S, --OH, ═

C═

O, ═

NH, --H, --OR, --NH, --R and R₃ R₄, where each R, R₃ and R₄ independently is H or a branched or unbranched, substituted or unsubstituted C1-C6 alkyl group.

2. A method for generating a biologically active compound of claim 1, comprising:

reacting R₁ and R₂ in an aqueous solution to form a compound mixture;

contacting said compound mixture with a receptor for R₂ ; and

isolating from said compound mixture a compound exhibiting biological activity in the presence of said R₂ receptor.

3. The method of claim 2, wherein said isolating step comprises

binding said compound to said R₂ receptor to form a binding complex;

releasing said compound from said binding complex; and

isolating said biologically active compound.

4. A method for generating a biologically active peptide compound of claim 1, comprising:

reacting R₁ and R₂ in an aqueous solution to form a compound mixture;

contacting said compound mixture with a receptor for R₂ ;

isolating from the compound mixture a compound exhibiting biological activity in the presence of said R₂ receptor;

determining the amino acid position at which said R₁ moiety is bound to said R₂ peptide; and

synthesizing said compound of claim 1 by chemically attaching said R₁ moiety to said amino acid of said R₂ peptide.

5. A method for imaging cell receptor sites comprising contacting candidate cell receptor sites with a compound of claim 1, and detecting said bound compounds as an indication of said cell receptor sites.

6. A method for cell sorting comprising contacting a population of candidate cells with a compound of claim 1, and isolating cells bound to said compound.

7. A method for flow cytometry comprising contacting a population of cells with a compound of claim 1 and detecting cells bearing receptors on their surfaces by detecting cells bound to said compound.

8. A compound of claim 1;

wherein said compound is formed by;

reacting R₁ and R₂ in an aqueous solution to form a compound mixture;

contacting the compound mixture with a receptor for R₂ ;

isolating from the compound mixture a compound exhibiting biological activity in the presence of the R₂ receptor;

determining biologically inactive regions of the isolated compound; and

synthesizing said compound by covalently attaching R₁ to an amino acid in said biologically inactive region to form a labeled amino acid, and by attaching other amino acids to said labeled amino acid to form said compound.

9. A compound of claim 1, wherein R₂ is galanin or a fragment thereof.

10. A compound of claim 1, wherein R₂ is a galanin analog based on conservative amino acid substitutions, or a fragment thereof.

11. A compound of claim 1, wherein C(X) is linked to R₂ through an alpha carbon atom of an amino acid residue.

12. A compound of claim 1, wherein C(X) is linked to a residue of R₂ which is not required for biological activity.

13. A compound of claim 1, wherein R₂ binds to a human receptor.

14. A compound of claim 1, wherein C(X) is linked to Lys, the fifth amino acid residue of galanin.

15. A compound of claim 14, wherein C(X) is linked to the epsilon-amino group of a Lys residue.

16. A compound of claim 1, wherein R₁ is selected from fluorescein, FTC, Texas Red, phycoerythrin, rhodamine, carboxytetramethylrhodamine, DAPI, indopyra dyes, Cascade blue coumarin, NBD, Lucifer Yellow, propidium iodide, a porphyrin, BODIPY, CY³, CY⁵, CY⁹, and derivatives and analogs thereof.

17. A compound of claim 1 wherein C(X) comprises a linking moiety selected from C═

O and --C═

S.

18. A biologically active and fluorescent compound of the formula:

##STR6## wherein R₁ is a light-emitting moiety;

R₂ is neuropeptide Y(NPY), peptide YY(PYY) or a derivative or analog based on conservative amino acid substitutions thereof, or a fragment thereof; and

wherein X is selected from the group consisting of ═

O, ═

S, --OH, ═

C═

O, ═

NH, --H, --OR, --NH, --R and R₃ R₄, where each R, R₃ and R₄ independently is H or a branched or unbranched, substituted or unsubstituted C1-C6 alkyl group.

19. A compound of claim 18, wherein C(X) is linked to R₂ through a lysine residue.

20. A biologically active and fluorescent compound of the formula:

space="preserve" listing-type="equation">R.sub.2 --(CH.sub.2).sub.5 --NH--C(Z)--R.sub.1

wherein R₁ is a light-emitting moiety;

R₂ is enkephalin, an enkephalin derivative, or an enkephalin analog based on conservative substitutions, or a fragment thereof, wherein R₂ has no more than five amino acid residues; and

C(Z) is (C═

O), (C═

S), (C═

NH), (CH₂), or CH(OR), where R is H or a C_1-6 branched or unbranched, substituted or unsubstituted, alkyl.

21. A compound of claim 20, wherein R₂ is enkephalin or a fragment thereof.

22. A method for generating a biologically active compound of claim 21 comprising:

reacting R₁ and R₂ in an aqueous solution to form a compound mixture;

contacting said compound mixture with a receptor for R₂ ; and

isolating from said compound mixture a compound exhibiting biological activity in the presence of said R₂ receptor.

23. The method of claim 22, wherein said isolating step comprises binding said compound to said R₂ receptor to form a binding complex;

releasing said compound from said binding complex and isolating said biologically active compound.

24. A compound of claim 20, wherein R₂ is an enkephalin analog based on conservative amino acid substitutions, or a fragment thereof.

25. A compound of claim 20, wherein C(Z) is linked to R₂ through an alpha carbon of an amino acid residue.

26. A compound of claim 20, wherein C(Z) is linked to a residue of R₂ which is not required for biological activity.

27. A compound of claim 20, wherein R₁ is selected from fluorescein, FTC, Texas Red, phycoerythrin, rhodamine, carboxytetramethylrhodamine, DAPI, indopyra dyes, Cascade blue coumarin, NBD, Lucifer Yellow, propidium iodide, a porphyrin, BODIPY, CY³, CY⁵, CY⁹, and derivatives and analogs thereof.

28. A compound of claim 20, wherein R₂ binds to a human receptor.

29. A method for generating a biologically active peptide compound of claim 20 comprising:

reacting R₁ and R₂ in an aqueous solution to form a compound mixture;

contacting said compound mixture with a receptor for R₂ ;

isolating from the compound mixture a compound exhibiting biological activity in the presence of said R₂ receptor;

determining the amino acid position at which said R₁ moiety is bound to said R₂ peptide; and

synthesizing said compound of claim 20 by chemically attaching said R₁ moiety to said amino acid of said R₂ peptide.

30. A method for imaging cell receptor sites comprising contacting candidate cell receptor sites with a compound of claim 20, and detecting receptor bound compounds as an indication of said cell receptor sites.

31. A method for cell sorting comprising contacting a population of candidate cells with a compound of claim 20, and isolating cells bound to said compound.

32. A method for flow cytometry comprising contacting a population of cells with a compound of claim 20 and detecting cells bearing receptors on their surfaces by detecting cells bound to said compound.

33. A compound of claim 20 wherein said compound is formed by:

reacting R₁ and R₂ in an aqueous solution to form a compound mixture;

contacting the compound mixture with a receptor for R₂ ;

isolating from the compound mixture a compound exhibiting biological activity in the presence of the R₂ receptor;

determining biologically inactive regions of the isolated compound; and

synthesizing said compound by covalently attaching R₁ to an amino acid in said biologically inactive region to form a labeled amino acid, and by attaching other amino acids to said labeled amino acid to form said compound.

34. A biologically active and fluorescent compound of the formula:

##STR7## wherein R₁ is a light-emitting moiety;

R₂ is an opioid peptide that binds to a mu or delta opioid receptor which is not neurotensin;

R₇ is a C1-C6 branched or unbranched, substituted or unsubstituted, alkyl; and

X is selected from the group consisting of ═

O, ═

S, --OH, ═

C═

O, --NH, --H, --OR, --NR, --R, and R₃ R₄, wherein each R, R₃, and R₄, independently, is H or a C1-C6 branched or unbranched, substituted or unsubstituted, alkyl.

35. The compound of claim 34, wherein R₇ is --NH--(CH₂)₅ --NH--.