Modified carbocyanine dyes and their conjugates

• US 7,927,830 B2
• Filed: 05/29/2008
• Issued: 04/19/2011
• Est. Priority Date: 09/29/2000
• Status: Expired due to Fees

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Leung, Wai-Yee, Cheung, Ching-Ying, Yue, Stephen
• Primary Examiner(s): CHU, YONG LIANG
• Application Number:
  US12/129,671
• Publication Number:
  US 20090035810A1
• Time in Patent Office: 1,055 Days
• Field of Search: 435/40.5, 436/86, 436/94
• US Class Current: 435/40.5
• CPC Class Codes: A61K 41/0057 Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
• CPC Class Codes: C07C 66/02 Anthraquinone carboxylic acids
• CPC Class Codes: C07D 209/08 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, directly attached to carbon atoms of the hetero ring
• CPC Class Codes: C07D 209/12 Radicals substituted by oxygen atoms
• CPC Class Codes: C07D 209/14 Radicals substituted by nitrogen atoms, not forming part of a nitro radical
• CPC Class Codes: C07D 209/16 Tryptamines
• CPC Class Codes: C07D 209/18 Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
• CPC Class Codes: C07D 209/24 with an alkyl or cycloalkyl radical attached to the ring nitrogen atom
• CPC Class Codes: C07D 209/54 Spiro-condensed
• CPC Class Codes: C07D 209/60 Naphtho [b] pyrroles; Hydrogenated naphtho [b] pyrroles
• CPC Class Codes: C07D 209/96 Spiro-condensed ring systems
• CPC Class Codes: C07D 401/04 directly linked by a ring-member-to-ring-member bond
• CPC Class Codes: C07D 403/06 linked by a carbon chain containing only aliphatic carbon atoms
• CPC Class Codes: C07D 403/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 403/14 containing three or more hetero rings
• CPC Class Codes: C07D 417/06 linked by a carbon chain containing only aliphatic carbon atoms
• CPC Class Codes: C07D 417/14 containing three or more hetero rings
• CPC Class Codes: C07D 471/04 Ortho-condensed systems
• CPC Class Codes: C07D 498/04 Ortho-condensed systems
• CPC Class Codes: C07K 1/13 Labelling of peptides
• CPC Class Codes: C09B 23/02 : the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
• CPC Class Codes: C09B 23/06 : three >CH- groups, e.g. carbocyanines
• CPC Class Codes: C09B 23/083 : five >CH- groups
• CPC Class Codes: C09B 23/107 : four >CH- groups
• CPC Class Codes: C12Q 1/6825 : Nucleic acid detection involving sensors
• CPC Class Codes: C40B 70/00 : Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes
• CPC Class Codes: G01N 1/30 : Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• CPC Class Codes: G01N 2001/302 : Stain compositions
• CPC Class Codes: G01N 31/22 : using chemical indicators G01N31/02 takes precedence
• CPC Class Codes: G01N 33/533 : with fluorescent label
• CPC Class Codes: G01N 33/535 : with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
• CPC Class Codes: G01N 33/58 : involving labelled substances G01N33/53 takes precedence
• CPC Class Codes: G01N 33/581 : with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
• CPC Class Codes: G01N 33/582 : with fluorescent label
• CPC Class Codes: Y10T 436/143333 : Saccharide [e.g., DNA, etc.]

Abstract

Chemically reactive carbocyanine dyes incorporating an indolium ring moiety that is substituted at the 3-position by a reactive group or by a conjugated substance, and their uses, are described. Conjugation through this position results in spectral properties that are uniformly superior to those of conjugates of spectrally similar dyes wherein attachment is at a different position. The invention includes derivative compounds having one or more benzo nitrogens.

Claims

1. A method of staining a biological sample, comprising:

combining a dye solution comprising a compound having the formula;

2. The method of claim 1, wherein R² is sulfoalkyl.

3. The method of claim 1, wherein R¹² is alkyl.

4. The method of claim 1, wherein R¹² is ethyl.

5. The method of claim 1, wherein R⁴ is methyl.

6. The method of claim 1, wherein R¹⁴ is methyl.

7. The method of claim 1, wherein each of the sulfoalkyl groups are sulfopropyl groups.

8. The method of claim 1, wherein R⁷ and R¹⁷ are sulfo.

9. The method of claim 1, wherein n is 1.

10. The method of claim 1, wherein n is 2.

11. The method of claim 1, wherein R³ is —

(CH₂)_d(CONH(CH₂)_e)_z′

—

R_x, wherein R_x is a succinimidyl ester of a carboxylic acid.

12. The method of claim 10, wherein;

d is 3 or 5 and z′

is 0.

13. The method of claim 1, wherein R³ is —

(CH₂)_d(CONH(CH₂)_e)_z′

—

R_x, wherein R_x is a maleimide.

14. The method of claim 12, wherein d is 3 or 5;

e is 2; and

z′

is 1.

15. The method of claim 1, wherein R¹⁶ is alkyl.

16. The method of claim 1, wherein the salt is sodium.

17. The method according to claim 1, wherein the compound has the formula:

18. The method according to claim 1, wherein the compound has the formula:

19. The method according to claim 1, wherein the sample comprises cells, proteins, or nucleic acid polymers.

20. The method according to claim 1, wherein the sample is contained in or on a solid or semi-solid matrix that is a membrane, an electrophoretic gel, silicon chip, a glass slide, a microwell plate or a microfluidic chip.

21. A method for forming a dye-conjugate of a protein, peptide, or a nucleic acid polymer, said method comprising:

combining a dye solution comprising a compound of the formula;

23. The kit of claim 21, further comprising instructions for fluorescent labeling a biological sample.

22. A kit for fluorescent labeling of a biological sample, comprising:

a dye solution comprising a compound having the formula;

24. A composition comprising a compound having the formula: