Process for preparing cyanine dye labeling reagents

• US 7,241,897 B2
• Filed: 01/21/2004
• Issued: 07/10/2007
• Est. Priority Date: 03/12/2002
• Status: Expired due to Term

• Current Assignee: Enzo Life Sciences Incorporated (Enzo Biochem Incorporated)
• Original Assignee: Enzo Life Sciences Incorporated (Enzo Biochem Incorporated)
• Inventors: Stavrianopoulos, Jannis G., Rabbam, Elazar
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US10/761,906
• Publication Number:
  US 20040203038A1
• Time in Patent Office: 1,266 Days
• Field of Search: 548/100, 548/122, 548/400, 548/416, 548/453, 548/454, 548/455, 548/457, 548/458, 548/460, 436/800, 536/23.1, 536/26.6
• US Class Current: 548/100
• 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
• CPC Class Codes: C09B 47/00 Porphines; Azaporphines non-dyeing compounds C07D487/22
• CPC Class Codes: C09B 57/00 Other synthetic dyes of known constitution
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine

Abstract

This invention provides for labeling reagents, labeled targets and processes for preparing labeling reagents. The labeling reagents can take the form of cyanine dyes, xanthene dyes, porphyrin dyes, coumarin dyes or composite dyes. These labeling reagents are useful for labeling probes or targets, including nucleic acids and proteins. These reagents can be usefully applied to protein and nucleic acid probe based assays. They are also applicable to real-time detection processes.

Claims

1. A process for preparing a cyanine dye labeling reagent, said process comprising the steps of:

(a) providing;

(i) a first intermediate compound comprising;

wherein X₁ comprises carbon, oxygen, nitrogen or sulfur; and

(ii) a second intermediate compound comprising;

wherein X₂ comprises carbon, oxygen, nitrogen or sulfur;

wherein at least one of R₁ through R₁₀ comprises a reactive group capable of forming a carbon-carbon linkage with a target, and

(iii) linking reagents suitable for linking said first intermediate compound and said second intermediate compound;

(b) forming a reaction mixture comprising said first intermediate compound (i), said second intermediate compound (ii), and said linking reagents under conditions to link (i) and (ii) to form

wherein at least one of R₁ through R₁₀ comprises a reactive group capable of forming a carbon-carbon linkage with a target, wherein said reactive group comprises an alkene group, an alkyne group, or a metallo-organic compound, and wherein n is an integer of 1, 2 or 3, and wherein X₁ and X₂ independently comprise carbon, oxygen, nitrogen or sulfur.

2. The process of claim 1, wherein R1 through R10 independently comprise hydrogen, C₁-C₆ alkyl, a C₁-C₄ alkyl group having a hydrophilic substituent comprising sulfonate, carboxylate, hydroxyl, substituted amines and quaternary amines, aliphatic, alkenes, alkynes, charged or polar groups, or combinations of any of the foregoing.

3. The labeling reagent of claim 1, wherein said metallo-organic compound comprises mercury, zinc, copper or platinum.

4. The labeling reagent of claim 1, wherein said metallo-organic compound comprises an alkene group or an alkyne group.

5. The process of claim 1, wherein said reactive group attached to said compound formed in step b further comprises a backbone that comprises at least two consecutive peptide bonds.

6. The process of claim 5, wherein at least one of said two consecutive peptide bonds are separated by a single atom.

7. The process of claim 6, wherein said single atom comprises C, N, S, O or P.

8. The process of claim 5, wherein said backbone comprises one or more carbon atoms.

9. The process of claim 5, wherein said backbone comprises at least one non-carbon atom.

10. The process of claim 9, wherein said non-carbon atom comprises sulfur, oxygen or nitrogen.

11. The process of claim 5, wherein said backbone further comprises at least one additional moiety comprising peptide bonds, amino acids, aliphatic chains from C₁ through C₂₀, alkene groups, alkyne groups, saturated or unsaturated or partially saturated rings, heterocyclic rings and sugars.

12. The process of claim 5, wherein said backbone comprises a di-peptide or an oligo-peptide.

13. The process of claim 12, wherein said di-peptide or oligo-peptide comprises (glycine)₂ or (glycine)₄.