Modified Carbocyanine Dyes and Their Conjugates

• US 20090035809A1
• Filed: 05/29/2008
• Published: 02/05/2009
• Est. Priority Date: 09/29/2000
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Leung, Wai-Yee, Cheung, Ching-Ying, Yue, Stephen
• Granted Patent: US 8,614,302 B2
• Time in Patent Office: Days
• US Class Current: 435/40.500
• 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 compound having the formula:

2. The compound according to claim 1, wherein R³ comprises a protein, a peptide, an amino acid, a dextran, a hormone, a lectin, a lipopolysaccharide, a biological cell, a non-biological microparticle, a nucleotide, an oligonucleotide, a small-molecule drug, avidin or streptavidin, biotin, or tyramide.

3. The compound according to claim 1, wherein R³ comprises an acrylamide, an activated ester of a carboxylic acid, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an amine, an anhydride, an aniline, an aryl halide, an azide, an aziridine, a boronate, a carboxylic acid, a diazoalkane, a haloacetamide, a halotriazine, a hydrazide, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a reactive platinum complex, a sulfonyl halide, a thiol group, or a photoactivatable group.

4. The compound according to claim 1, wherein R³ comprises an antibody or fragment thereof, a blood component protein, a blood vessel proliferation inhibition factor, an enzyme, an enzyme inhibitor, a hormone, an IgG-binding protein, a fluorescent protein, a growth factor, a metal-binding protein, a microorganism or portion thereof, a neuropeptide, a peptide toxin, a phospholipid-binding protein, or a structural protein.

5. The compound according to claim 1, wherein the atoms of W are —

CH, —

C₁—

CR^1′

, and —

N(R¹²)_β

′

, where β

′

is 1, but no more than one of said atoms in W is —

N(R¹²)_β

′

, and each R^1′

is amino, sulfo, trifluoromethyl, halogen, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ alkylamino, or C₂-C₁₂ dialkylamino, wherein each alkyl portion of which is optionally substituted by substituents selected from the group consisting of carboxy, sulfo, amino, and hydroxy;

6. A compound having the formula:

7. The compound according to claim 6, wherein R³ comprises a carboxylic acid, a succinimidyl ester of a carboxylic acid, hydrazide, a maleimide, an antibody or fragment thereof, a fluorescent protein, a lectin, a nucleotide, an oligonucleotide, a peptide, protein, a small-molecule drug, or a tyramide.

8. The compound according to claim 6, wherein

R⁴, R¹³ and R¹⁴ are each methyl;

R⁶, R⁸, R⁹, R¹⁶, R¹⁷ and R¹⁸ are each H;

R⁷ is sulfo;

R² and R¹² are independently methyl or sulfopropyl; and

n is 2.

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

combining a dye solution comprising a compound having the formula;

10. The method according to claim 9, wherein R³ comprises a protein, a peptide, an amino acid, a dextran, a hormone, a lectin, a lipopolysaccharide, a biological cell, a non-biological microparticle, a nucleotide, an oligonucleotide, a small-molecule drug, avidin or streptavidin, biotin, or tyramide.

11. The method according to claim 9, wherein R³ comprises an acrylamide, an activated ester of a carboxylic acid, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an amine, an anhydride, an aniline, an aryl halide, an azide, an aziridine, a boronate, a carboxylic acid, a diazoalkane, a haloacetamide, a halotriazine, a hydrazide, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a reactive platinum complex, a sulfonyl halide, a thiol group, or a photoactivatable group.

12. The method according to claim 9, wherein R³ comprises an antibody or fragment thereof, a blood component protein, a blood vessel proliferation inhibition factor, an enzyme, an enzyme inhibitor, a hormone, an IgG-binding protein, a fluorescent protein, a growth factor, a metal-binding protein, a microorganism or portion thereof, a neuropeptide, a peptide toxin, a phospholipid-binding protein, or a structural protein.

13. The method according to claim 9, wherein the sample comprises cells, proteins, or nucleic acid polymers.

14. The method according to claim 9, wherein the sample is contain in or on a solid or semi-solid matrix this a membrane an electrophoretic gel, silicon chip, a glass slide, a microwell plate or a microfluidic chip.

15. 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

16. The method according to claim 15, wherein R³ comprises a carboxylic acid, an amine, an activated ester of a carboxylic acid, a halotriazine, a hydrazine, a maleimide, a reactive platinum complex.

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

a dye solution comprising a compound having the formula;