Synthesis and use of acyl fluorides of cyanine dyes

• US 6,342,326 B1
• Filed: 05/10/2000
• Issued: 01/29/2002
• Est. Priority Date: 05/10/2000
• Status: Expired due to Fees

• Current Assignee: Beckman Coulter Incorporated (Danaher Corporation)
• Original Assignee: Beckman Coulter Incorporated (Danaher Corporation)
• Inventors: Milton, Raymond C.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US09/568,446
• Time in Patent Office: 629 Days
• Field of Search: 435/6, 430/93, 430/581, 436/519, 536/25-32, 548/427
• US Class Current: 430/93
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C09B 23/02 the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups

Abstract

This invention provides dyes, particularly cyanine and related dyes, with acyl fluoride activating groups, the dyes having the general formula

wherein:

each dotted line represents carbon atoms necessary to form a fused substituted or unsubstituted aromatic ring;

n is an integer selected from the group consisting of 1, 2 and 3;

X and Y are selected from the group consisting of S, O, N, CH₂ and C(CH₃)₂;

at least one of said R₁ and R₂ comprises a sulfonic acid or sulfonate group attached to the aromatic ring; and

R₃ and R₄ are independently selected from the group consisting of alkylcarboxylate, activated alkylcarboxylate and an inert group; wherein at least one of said R₃ and R₄ groups is alcylcarboxylate or activated alkylcarboxylate with the carboxyl group converted to an acyl fluoride. The inert group is a group that is inert towards acyl fluoride and has a sterical structure which allows aminoacylation of the acyl fluoride group.

Claims

1. An activated dye having a formula:

wherein;

each dotted line represents carbon atoms necessary to form a fused substituted or unsubstituted aromatic ring;

n is an integer selected from the group consisting of 1, 2 and 3;

X and Y are selected from the group consisting of S, O, N, CH₂ and C(CH₃)₂;

at least one of said R₁ and R₂ comprises a sulfonic acid or sulfonate group attached to the aromatic ring; and

R₃ and R₄ are independently selected from the group consisting of alkylcarboxylate, activated alkylcarboxylate and an inert group;

wherein at least one of said R₃ and R₄ groups is alcylcarboxylate or activated alkylcarboxylate with a carboxyl group converted to an acyl fluoride, and wherein said inert group is a group that is inert towards acyl fluoride and has a sterical structure which allows aminoacylation of the acyl fluoride group.

2. The activated dye of claim 1, wherein said aromatic ring is phenyl, naphthyl, or heterocyclic ring.

3. The activated dye of claim 1, wherein both R₃ and R₄ are acyl fluorides.

4. The activated dye of claim 1, wherein the inert group is selected from the group consisting of alkanes, alkenes, dienes, alkynes, arenes, ketones, amides, and ethers.

5. The activated dye of claim 1, wherein said alkyls possess 1 to 12 carbon atoms.

6. The activated dye of claim 1, wherein one or both R₃ and R₄ have a substituent group on the alkylcarbon adjacent to the acyl fluoride which is inert towards the acyl fluoride and has a sterical structure that permits aminoacylation of the acyl fluoride group.

7. The activated dye of claim 1, wherein said dye is a cyanine dye.

8. The activated dye of claim 7, wherein said cyanine dye is selected from the group consisting of Cy5, benzyl Cy5, dibenzyl Cy5, Cy7, benzyl Cy7 and dibenzyl Cy7.

9. The activated dye of claim 1, wherein said dye is selective towards nitrogen nucleophiles over competing species.

10. The activated dye of claim 9, wherein said competing species are oxygen nucleophiles.

11. The activated dye of claim 1, wherein said dye is soluble in aqueous solutions.

12. A method for activating a dye comprising the steps of:

(a) providing a dye having at least one carboxylic group;

(b) providing a fluorinating reagent capable of reacting with the carboxylic group of said dye to form acyl fluoride; and

(c) reacting said dye with said fluorinating agent under a condition sufficient to form a dye with at least one acyl fluoride group,

wherein said dye has a structure;

 

wherein;

each dotted line represents carbon atoms necessary to form a fused substituted or unsubstituted aromatic ring;

n is an integer selected from the group consisting of 1, 2 and 3;

X and Y are selected from the group consisting of S, O, N, CH₂ and C(CH₃)₂;

at least one of said R₁ and R₂ comprises a sulfonic acid or sulfonate group attached to the aromatic ring; and

R₃ and R₄ are independently selected from the group consisting of alkylcarboxylate, activated alkylcarboxylate and an inert group;

wherein at least one of said R₃ and

R₄ groups is alcylcarboxylate or activated alkylcarboxylate with a carboxyl group converted to an acyl fluoride and wherein said inert group is a group that is inert towards acyl fluoride and has a sterical structure which allows aminoacylation of the acyl fluoride group.

13. The method of claim 12, wherein said aromatic ring is phenyl, naphthyl, or heterocyclic ring.

14. The method of claim 12, wherein both R₃ and R₄ are alkylcarboxylates.

15. The method of claim 12, wherein the inert group is selected from the group consisting of alkanes, alkenes, dienes, alkynes, arenes, ketones, amides, and ethers.

16. The method of claim 12, wherein one or both R₃ and R₄ have a substituent group on the alkylcarbon adjacent to the acyl fluoride which is inert towards and has a sterical structure that permits aminoacylation of the acyl fluoride group.

17. The method of claim 12, wherein said alkyls possess 1 to 12 carbon atoms.

18. The method of claim 12, wherein said dye is a cyanine dye.

19. The method of claim 18, wherein said cyanine dye is selected from the group consisting of Cy5, benzyl Cy5, dibenzyl Cy5, Cy7, benzyl Cy7 and dibenzyl Cy7.

20. A method for activating a dye comprising the steps of:

(a) providing a dye having at least one carboxylic group;

(b) providing a fluorinating reagent selected from the group consisting of diethylaminosulfur trifluoride (DAST), cyanuric fluoride and tetramethylfluoro-formadinium hexafluorophosphate (TFFH); and

(c) reacting said dye with said fluorinating agent under a condition sufficient to form a dye with at least one acyl fluoride group.

21. The method of claim 20, wherein said fluorinating agent is DAST.

22. The method of claim 21, wherein said amount of DAST combined with said cyanine dye is greater than about 0.75 molar equivalents.

23. The method of claim 22, wherein said amount of DAST combined with said cyanine dye is greater than about 1.5 molar equivalents.

24. The method of claim 20, wherein said dye is dissolved in a solvent prior to reacting with the fluorinating agent.

25. The method of claim 24, wherein the solvent is acetonitrile, DMF, or a mixture thereof.

26. The method of claim 20, further comprising an additional step of precipitating the activated dye from the mixture.

27. The method of claim 26, wherein the activated dye is precipitated by adding diethyl ether.

28. A method of labeling biological and non-biological materials comprising the steps of:

(a) providing an activated dye with at least one acyl fluoride group;

(b) providing a material containing a nitrogen nucleophile; and

(c) reacting said dye with said material under a condition sufficient to couple the dye to the material,

wherein said dye has a structure;

 

wherein;

each dotted line represents carbon atoms necessary to form a fused substituted or unsubstituted aromatic ring;

n is an integer selected from the group consisting of 1, 2 and 3;

X and Y are selected from the group consisting of S, O, N, CH₂ and C(CH₃)₂;

at least one of said R₁ and R₂ comprises a sulfonic acid or sulfonate group attached to the aromatic ring; and

R₃ and R₄ are independently selected from the group consisting of alkylcarboxylate, activated alkylcarboxylate and an inert group;

wherein at least one of said R₃ and

R₄ groups is alcylcarboxylate or activated alkylcarboxylate with carboxyl group converted to an acyl fluoride and wherein said inert group is a group that is inert towards acyl fluoride and has a sterical structure which allows aminoacylation of the acyl fluoride group.

29. The method of claim 28, wherein said biological material is selected from the group consisting of proteins, cells, amino-modified nucleic acids and combinations thereof.

31. The method of claim 28, wherein said non-biological materials are polymers.

32. The method of claim 28, wherein said aromatic ring is phenyl, naphthyl, or heterocyclic ring.

33. The method of claim 28, wherein both R₃ and R₄ are acyl fluorides.

34. The method of claim 28, wherein the inert group is selected from the group consisting of alkanes, alkenes, dienes, alkynes, arenes, ketones, amides, ethers.

35. The method of claim 28, wherein said alkyls possess 1 to 12 carbon atoms.

36. The method of claim 28, wherein one or both R₃ and R₄ have a substituent group on the alkylcarbon adjacent to the acyl fluoride which is inert towards the acyl fluoride and has a sterical structure that permits aminoacylation of the acyl fluoride group.

37. The method of claim 28, wherein said dye is a cyanine dye.

38. The method of claim 37, wherein said cyanine dye is selected from the group consisting of Cy5, benzyl Cy5, dibenzyl Cy5, Cy7, benzyl Cy7 and dibenzyl Cy7.

30. A method of labeling amino-modified oligonucleotides comprising the steps of:

(a) providing an activated dye with at least one acyl fluoride group;

(b) providing an amino-modified oligonucleotide; and

(c) reacting said dye with said amino-modified oligonucleotide under a condition sufficient to couple the dye to the amino-modified oligonucleotide.

39. The method of claim 30, wherein said activated dye is added in a molar excess as compared to the amino-modified oligonucleotide.