Modified carbocyanine dyes and their conjugates

• US 6,974,873 B2
• Filed: 10/01/2001
• Issued: 12/13/2005
• Est. Priority Date: 09/29/2000
• Status: Expired due to Term

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Leung, Wai-Yee, Cheung, Ching-Ying, Yue, Stephen
• Primary Examiner(s): SEAMAN, D MARGARET M
• Application Number:
  US09/969,853
• Publication Number:
  US 20020064794A1
• Time in Patent Office: 1,534 Days
• Field of Search: 548/455
• US Class Current: 548/455
• 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

or its salts, wherein

R² is alkyl or sulfoalkyl;

R¹² is alkyl or sulfoalkyl;

R³ is carboxyalkyl;

R⁴ is alkyl;

R¹³ is alkyl;

R¹⁴ is alkyl;

R⁶ is hydrogen or sulfo;

R⁷ is hydrogen or sulfo;

R⁸ is hydrogen or sulfo;

R⁹ is hydrogen or sulfo;

R¹⁶ is hydrogen or sulfo;

R¹⁷ is hydrogen or sulfo;

R¹⁸ is hydrogen or sulfo;

R¹⁹ is hydrogen or sulfo;

wherein sulfo is sulfonic acid or sulfonate; and

,

n is 1, 2 or 3;

with a biological sample in a concentration sufficient to yield a detectable optical response under desired conditions.

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

combining a dye solution comprising a compound having the formula

or its salts, wherein

R² is alkyl or sulfoalkyl;

R¹² is alkyl or sulfoalkyl;

R⁴ is alkyl;

R¹³ is alkyl;

R¹⁴ is alkyl;

R⁶ is hydrogen or sulfo;

R⁷ is hydrogen or sulfo;

R⁸ is hydrogen or sulfo;

R⁹ is hydrogen or sulfo;

R¹⁶ is hydrogen or sulfo;

R¹⁷ is hydrogen or sulfo;

R¹⁸ is hydrogen or sulfo;

R¹⁹ is hydrogen or sulfo;

wherein sulfo is sulfonic acid or sulfonate; and

,

n is 1, 2 or 3;

with a biological sample in a concentration sufficient to yield a detectable optical response under desired conditions.

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

combining a dye solution comprising a compound having the formula

or its salts, wherein;

R² is alkyl or sulfoalkyl;

R¹² is alkyl or sulfoalkyl;

R⁴ is alkyl;

R¹⁹ is alkyl;

R¹⁴ is alkyl;

R⁶ is hydrogen or sulfo;

R⁷ is hydrogen or sulfo;

R⁸ is hydrogen or sulfo;

R⁹ is hydrogen or sulfo;

R¹⁶ is hydrogen or sulfo;

R¹⁷ is hydrogen or sulfo;

R¹⁸ is hydrogen or sulfo;

R¹⁹ is hydrogen or sulfo;

wherein sulfo is sulfonic acid or sulfonate and,

n is 1, 2 or 3;

with a biological sample in a concentration sufficient to yield a detectable optical response under desired conditions.

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

combining a dye solution comprising a compound having the formula

or its salts, wherein;

R² is alkyl or sulfoalkyl;

R¹² is alkyl or sulfoalkyl;

wherein sulfo is sulfonic acid or sulfonate; and

,

n is 1, 2 or 3;

with a biological sample in a concentration sufficient to yield a detectable optical response under desired conditions.

5. 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

or its salts, wherein;

R² is alkyl or sulfoalkyl;

R¹² is alkyl or sulfoalkyl;

R⁴ is alkyl;

R¹³ is alkyl;

R¹⁴ is alkyl;

R⁶ is hydrogen or sulfo;

R⁷ is hydrogen or sulfo;

R⁸ is hydrogen or sulfo;

R⁹ is hydrogen or sulfo;

R¹⁶ is hydrogen or sulfo;

R¹⁷ is hydrogen or sulfo;

R¹⁸ is hydrogen or sulfo;

R¹⁹ is hydrogen or sulfo;

wherein sulfo is sulfonic acid or sulfonate and,

n is 1, 2 or 3;

with a sample comprising a protein, peptide or a nucleic acid polymer whereby a dye conjugate is formed.

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

combining a dye solution comprising a compound having the formula

or its salts, wherein

R² is alkyl or sulfoalkyl;

R¹² is alkyl or sulfoalkyl;

R³ is a protein, peptide or a nucleic acid polymer;

R⁴ is alkyl;

R¹³ is alkyl;

R¹⁴ is alkyl;

R⁶ is hydrogen or sulfo;

R⁷ is hydrogen or sulfo;

R⁸ is hydrogen or sulfo;

R⁹ is hydrogen or sulfo;

R¹⁶ is hydrogen or sulfo;

R¹⁷ is hydrogen or sulfo;

R¹⁸ is hydrogen or sulfo;

R¹⁹ is hydrogen or sulfo;

wherein sulfo is sulfonic acid or sulfonate; and

,

n is 1, 2 or 3;

with a biological sample in a concentration sufficient to yield a detectable optical response under desired conditions.