Reactive cyanine compounds

• US 8,309,059 B2
• Filed: 08/31/2010
• Issued: 11/13/2012
• Est. Priority Date: 08/31/2009
• Status: Active Grant

• Current Assignee: Promega Corporation
• Original Assignee: Promega Corporation
• Inventors: Corona, Cesear, Klaubert, Dieter, McDougall, Mark
• Primary Examiner(s): Hartley, Michael G
• Assistant Examiner(s): Schlientz, Leah
• Application Number:
  US12/872,467
• Publication Number:
  US 20110053162A1
• Time in Patent Office: 805 Days
• Field of Search: 424/9.6, 548/455
• US Class Current: 424/9.6
• CPC Class Codes: A61K 49/0032 Methine dyes, e.g. cyanine dyes
• CPC Class Codes: A61K 49/0056 Peptides, proteins, polyamino acids
• CPC Class Codes: C09B 23/0008 substituted on the polymethine chain
• CPC Class Codes: C09B 23/0066 the polymethine chain being part of a carbocyclic ring,(e.g. benzene, naphtalene, cyclohexene, cyclobutenene-quadratic acid)
• CPC Class Codes: C09B 23/06 three >CH- groups, e.g. carbocyanines
• CPC Class Codes: C09B 23/083 five >CH- groups
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

The invention provides compounds and compositions of Formulas I-VII, and methods of using the compounds. The compounds can be used to prepare dye conjugates that are uniformly and substantially more fluorescent on proteins, nucleic acids or other biopolymers, than conjugates labeled with structurally similar known carbocyanine dyes. In addition to having more intense fluorescence emission than structurally similar dyes at virtually identical wavelengths, and decreased artifacts in their absorption spectra upon conjugation to biopolymers, the compounds can have greater photostability and/or higher absorbance (extinction coefficients) at the wavelength(s) of peak absorbance than such structurally similar dyes.

Claims

1. A compound of Formula I:

2. The compound of claim 1 wherein R^x is:

an activated ester of a carboxylic acid that includes a —

C(═

O)O—

group covalently bonded to a succinimidyl, a sulfosuccinimidyl, a maleimide, or a 1-oxybenzotriazolyl group;

—

NH₂;

—

OH;

—

SO₂Cl;

—

SO₂Br;

—

SH;

—

B(OH)₂;

—

B(OR)₂ wherein R is alkyl or aryl;

—

O—

P(N(alkyl)₂)(O-alkylene-CN);

—

N═

C═

O;

—

C(═

O)—

Cl;

—

C(═

O)—

Br;

—

C(═

O)—

I;

—

C(═

O)H;

—

N₃;

—

C(═

O)CN;

a maleimide group;

a diazirinyl group;

an azidoaryl group;

a psoralen derivative;

a benzophenone;

a 6′

-O-linked-4-cyanobenzothiazole;

a (C₁-C₇)alkyl-methylene chloride;

a (C₁-C₇)alkyl-methylene bromide;

a (C₁-C₇)alkyl-methylene iodide;

—

CO₂H;

a (C₁-C₇)alkyl-methylene iodide;

or SO₃H.

3. The compound of claim 1 wherein R¹, R², R³, R⁴, R⁵, and R⁶ are each independently H, F, or sulfo.

4. The compound of claim 1 wherein W₁ forms a 6-membered ring having a —

CH₂—

, —

O—

, —

S—

, or —

NH—

para to the N of the W₁ ring in Formula I or II.

5. The compound of claim 1 wherein W₂ forms a 6-membered ring having a —

CH₂—

, —

O—

, —

S—

, or —

NH—

para to the N of the W₂ ring in Formula I or II.

6. The compound of claim 1 wherein W₁, W₂, or both, are substituted on a carbon of the ring with —

(CH₂)_m—

OH, where m is 1 to about 12.

7. The compound of claim 1 wherein each Y is CR⁷R⁷, one R₇ of Y is methyl, and the other R⁷ of Y is -L-R^x.

8. The compound of claim 1 wherein at least one L is (C₁-C₁₂)alkyl or (C₁-C₁₂)alkyl-B—

(CH₂CH₂O)_n(CH₂)_m—

wherein n is 1-6, m is 1-8, and B is —

NHC(═

O)—

, —

C(═

O)NH—

, —

OC(═

O)—

, —

C(═

O)O—

, —

O—

, —

NH—

, or a direct bond.

9. The compound of claim 8 wherein at least one L is (C₁-C₁₀)alkyl or (C₁-C₁₂)alkyl-C(═

O)NH—

(CH₂CH₂O)n(CH₂)m- wherein n is 1-6 and m is 1-8.

10. The compound of claim 1 wherein R⁹ of Formula II is —

OPh-NH—

(C₁-C₁₂)alkyl-R^x, or —

OPh-C(═

O)NH—

(CH₂CH₂O)_n(CH₂)_m—

R^x, wherein n is 1-6, m is 1-8.

11. The compound of claim 1 wherein m of Formula II is 1.

12. The compound of claim 1 wherein n of Formula I is 1 or 2.

13. The compound of claim 1 wherein two, three, or four -L-R^x group are present.

14. The compound of claim 1 wherein the inorganic anion is a halo anion.

16. A method to detect a selected molecule in a sample, comprising:

a) contacting a sample suspected of having a selected molecule with a composition comprising a conjugate comprising a compound of claim 1 and a ligand for the selected molecule so as to yield a mixture; and

b) detecting the presence or amount of the compound in the mixture.

17. The method of claim 16 wherein the conjugate comprises a compound of claim 1 wherein -L-R^x is -linker-A-X, wherein the linker is a multiatom straight or branched carbon chain that optionally includes one or more N, S, or O atoms;

wherein -A-X is a substrate for a dehalogenase, and X is a halogen.

18. The method of claim 16 wherein the ligand is an oligonucleotide.

19. The method of claim 16 wherein the ligand is a cyanobenzothiazole moiety.

20. The method of claim 16 wherein the cyanobenzothiazole moiety is a cyanobenzothiazole moiety of formula (VIII):

21. A kit comprising a conjugate comprising a dye conjugate used to label a molecule in a sample comprising a compound of claim 1 and a ligand for the molecule.

15. A compound selected from the group consisting of

5-((1E,3E,5E)-5-(1,1-dimethyl-5,6-dihydro-1H-pyrrolo[3,2,1-ij]quinolin-2(4H)-ylidene)penta-1,3-dienyl)-6-(6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl)-6-methyl-1,2,3,6-tetrahydropyrrolo[3,2,1-ij]quinolinium trifluoroacetate (3525);

6-(6-((2-Cyanoethoxy)(diisopropylamino)phosphinooxy)hexyl)-5-((1E,3E,5E)-5-(1,1-dimethyl-5,6-dihydro-1H-pyrrolo[3,2,1-ij]quinolin-2(4H)-ylidene)penta-1,3-dienyl)-6-methyl-1,2,3,6-tetrahydropyrrolo[3,2,1-ij]quinolinium chloride (3742);

(E)-2-((2E,4E)-5-(6-(6-(2,5-Dioxopyrrolidin-1-yloxy)-6-oxohexyl)-6-methyl-7-sulfo-1,2,3,6-tetrahydropyrrolo[3,2,1-ij]quinolinium-5-yl)penta-2,4-dienylidene)-1,1-dimethyl-2,4,5,6-tetrahydro-1H-pyrrolo[3,2,1-ij]quinoline-9-sulfonate (3526);

6-(25-chloro-6-oxo-10,13,16,19-tetraoxa-7-azapentacosyl)-5-((1E,3E,5E)-5-(1,1-dimethyl-9-sulfo-5,6-dihydro-1H-pyrrolo[3,2,1-ij]quinolin-2(4H)-ylidene)penta-1,3-dienyl)-6-methyl-1,2,3,6-tetrahydropyrrolo[3,2,1-ij]quinolinium-7-sulfonate (3665);

5-((1E,3E)-3-((E)-2-(1,1-Dimethyl-5,6-dihydro-1H-pyrrolo[3,2,1-ij]quinolin-2(4H)-ylidene)ethylidene)-6-hydroxyhex-1-enyl)-6,6-dimethyl-1,2,3,6-tetrahydropyrrolo[3,2,1-ij]quinolinium chloride (3688);

(E)-2-((E)-3-((E)-2-(6,6-Dimethyl-7-sulfo-1,2,3,6-tetrahydropyrrolo[3,2,1-ij]quinolinium-5-yl)vinyl)-6-hydroxyhex-2-enylidene)-1,1-dimethyl-2,4,5,6-tetrahydro-1H-pyrrolo[3,2,1-ij]quinoline-9-sulfonate (3786);

5-((1E,3E)-3-(1,1-Dimethyl-5,6-dihydro-1H-pyrrolo[3,2,1-ij]quinolin-2(4H)-ylidene)prop-1-enyl)-6-(6-hydroxyhexyl)-6-methyl-1,2,3,6-tetrahydropyrrolo[3,2,1-ij]quinolinium chloride (3785);

Sodium 2-((1E,3Z)-3-(1-(5-carboxypentyl)-1-methyl-9-sulfonato-5,6-dihydro-1H-pyrrolo[3,2,1-ij]quinolin-2(4H)-ylidene)prop-1-enyl)-1-methyl-1-(4-sulfonatobutyl)-1,4,5,6-tetrahydropyrrolo[3,2,1-ij]quinolinium-9-sulfonate (3845);

Sodium 2-((1E,3Z)-3-(1-(6-(2-(2-(6-chlorohexyloxy)ethoxy)ethylamino)-6-oxohexyl)-1-methyl-9-sulfonato-5,6-dihydro-1H-pyrrolo[3,2,1-ij]quinolin-2(4H)-ylidene)prop-1-enyl)-1-methyl-1-(4-sulfonatobutyl)-1,4,5,6-tetrahydropyrrolo[3,2,1-ij]quinolinium-9-sulfonate (3838);

6-((Z)-5-((2E,4E)-5-(6,6-Dimethyl-1,2,3,6-tetrahydrobenzo[f]pyrrolo[3,2,1-ij]quinolinium-5-yl)penta-2,4-dienylidene)-6-methyl-2,3,5,6-tetrahydro-1H-benzo[f]pyrrolo[3,2,1-ij]quinolin-6-yl)hexanoate (3846);

Sodium 2-((1E,3E,5Z)-5-(1-(6-(2-(2-(6-chlorohexyloxy)ethoxy)ethylamino)-6-oxohexyl)-1-methyl-9-sulfonato-5,6-dihydro-1H-pyrrolo[3,2,1-ij]quinolin-2(4H)-ylidene)penta-1,3-dienyl)-1-methyl-1-(4-sulfonatobutyl)-1,4,5,6-tetrahydropyrrolo[3,2,1-ij]quinolinium-9-sulfonate (3847);

Sodium 5-((1E,3E,5Z)-5-(1-(5-carboxypentyl)-1-methyl-9-sulfonato-5,6-dihydro-1H-pyrrolo[3,2,1-ij]quinolin-2(4H)-ylidene)penta-1,3-dienyl)-6-methyl-6-(4-sulfonatobutyl)-1,2,3,6-tetrahydropyrrolo[3,2,1-ij]quinolinium-7-sulfonate (3848);

Sodium 5-((E)-2-((E)-2-(4-carboxyphenoxy)-3-((E)-2-(1-methyl-9-sulfonato-1-(4-sulfonatobutyl)-5,6-dihydro-1H-pyrrolo[3,2,1-ij]quinolin-2(4H)-ylidene)ethylidene)cyclohex-1-enyl)vinyl)-6-methyl-6-(4-sulfonatobutyl)-1,2,3,6-tetrahydropyrrolo[3,2,1-ij]quinolinium-7-sulfonate (3855);

Sodium 2-((E)-2-((E)-2-(4-(2-(2-(6-chlorohexyloxy)ethoxy)ethylcarbamoyl)phenoxy)-3-((E)-2-(1-methyl-9-sulfonato-1-(4-sulfonatobutyl)-5,6-dihydro-1H-pyrrolo[3,2,1-ij]quinolin-2(4H)-ylidene)ethylidene)cyclohex-1-enyl)vinyl)-1-methyl-1-(4-sulfonatobutyl)-1,4,5,6-tetrahydropyrrolo[3,2,1-ij]quinolinium-9-sulfonate (3856);

or

5-((E)-2-((E)-3-((Z)-2-(1-(25-Chloro-6-oxo-10,13,16,19-tetraoxa-7-azapentacosyl)-1-methyl-9-sulfo-5,6-dihydro-1H-pyrrolo[3,2,1-ij]quinolin-2(4H)-ylidene)ethylidene)-2-(4-sulfophenoxy)cyclohex-1-enyl)vinyl)-6-methyl-6-(4-sulfobutyl)-1,2,3,6-tetrahydropyrrolo[3,2,1-ij]quinolinium-7-sulfonate (3921).