Dye Compounds and the Use of their Labelled Conjugates

• US 20100009353A1
• Filed: 05/16/2007
• Published: 01/14/2010
• Est. Priority Date: 05/18/2006
• Status: Active Grant

• Current Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Inventors: Romanov, Nikolai Nikolaevich, Barnes, Colin
• Granted Patent: US 8,178,360 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61K 49/0041 Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal in vivo G01N
• CPC Class Codes: A61K 49/0052 Small organic molecules oligomers, polymers, dendrimers A61K49/0054
• CPC Class Codes: C09B 11/24 Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine

Abstract

Novel rhodamine dye compounds, labelled conjugates comprising the dyes are described, together with methods for their use. The dyes and labelled conjugates are useful as molecular probes in a variety of applications, such as in assays involving staining of cells, protein binding, and analysis of nucleic acids, such as hybridization assays and nucleic acid sequencing.

Claims

1. A compound of the formula s:

wherein X is SO₃H or SO₃⁻

,

M⁺ is a common counter ion,

k, n, m are independently integers of from 1 to 6,

R₁ is H or an alkyl or substituted alkyl group, R₂ is H, alkyl or substituted alkyl group, halogen, carboxy, carboxamide, hydroxy- or alkoxy group, or R₂ together with R₁ or R₅ is a carbon or heterosubstituted chain forming a ring, R₃ is H, alkyl or substituted alkyl group, halogen, carboxy, carboxamide, hydroxy- or alkoxy group or R₃ together with R₄ or R₆ is a carbon or heterosubstituted chain forming a ring, R₄ is H, or an alkyl or substituted alkyl group,

R₅ and R₆ are H, alkyl or substituted alkyl group, halogen, hydroxy- or alkoxy group,

R₈ is H, halogen, hydroxy- or alkoxy group, alkyl or substituted alkyl group or together with R₁ is a carbon or heterosubstituted carbon chain forming a ring,

R₉ is H, halogen, hydroxy- or alkoxy group, alkyl or substituted alkyl group or together with R₄ is a carbon or heterosubstituted carbon chain forming a ring,

R₇ is OH, OR₁₀ where R₁₀ is alkyl or substituted alkyl, or NR₁₁R₁₂ where R₁₁ and R₁₂ are independently H, alkyl or a substituted alkyl, or is joined to form aliphatic or aromatic cyclic or substituted aliphatic-, aromatic- or heterocyclic rings,

Z is halogen, amino or substituted amino, hydroxyl-, alkoxy-, carboxy-, carboxamido- or a substituted carboxamido group, SO₃H or SO₃⁻

and a is 0 to 4, wherein if a is 0, R₇ is NR₁₁R₁₂ where R₁₁ and R₁₂ are independently H, alkyl or a substituted alkyl.

2. The compound of claim 1 wherein X is SO₃⁻

, n is 3, R₁, R₄, R₅, R₆, R₈ and R₉ are all H, R₂ and R₃ are H or CH₃, Z is carboxy-, carboxamido- or a substituted carboxamido group, a is 1 and R₇ is OH.

3. A compound of claim 1 wherein R₁ is linked to R₂ or R₈ via a chain of CH₂ groups to form a ring, and R₄ is linked to R₃ or R₉ via a chain of CH₂ groups to form a ring.

5. The compound of claim 3, wherein X is SO₃⁻

, n is 3, R₁ and R₂ form a six membered ring R₃ and R₄ form a six membered ring, R₅, R₆, R₈ and R₉ are H, Z is carboxy-, carboxamido- or a substituted carboxamido group, a is 1 and R₇ is OH.

6. A compound of claim 1 wherein R1 and R4 are (CH₂)_mSO₃⁻

.

7. The compound of claim 6 wherein X is SO₃⁻

, n=3, m=3 and R₂, R₃, R₅, R₆, R₈ and R₉ are all H, a is 0 and R₇ is OH.

9. A nucleotide or oligonucleotide labelled with a compound according to claim 1.

13. A labelled nucleotide or oligonucleotide according to claim 9 wherein the label is attached to the C5 position of a pyrimidine base or the C7 position of a 7-deaza purine base through a linker moiety.

14. A labelled nucleotide or oligonucleotide according to claim 13 wherein said linker moiety comprises a moiety selected from the group consisting of:

wherein X is selected from the group comprising O, S, NH and NQ;

wherein Q is a C1-10 substituted or unsubstituted alkyl group, Y is selected from the group comprising O, S, NH and N(allyl), T is hydrogen or a C1-10 substituted or unsubstituted alkyl group.

16. A labelled nucleotide according to claim 13, further comprising a 3′

OH blocking group covalently attached to the ribose or deoxyribose sugar of the nucleotide.

20. A kit comprising two or more nucleotides wherein at least one nucleotide is a labelled nucleotide according to claim 9.

23. A kit according to claim 20 wherein each nucleotide can be cleaved from an attached compound by chemical or enzymatic treatment.

26. A kit according to claim 23 wherein said chemical treatment comprises treating with a phosphine reagent.

28. A kit according to claim 20 wherein two of the labelled nucleotides are excited using a single laser.

33. A kit according to claim 20 wherein a first of four nucleotides is the a labelled nucleotide and the second, third, and fourth nucleotides are each labelled with a different compound, wherein each compound has a distinct absorbance maximum and each of the compounds is distinguishable from the other three compounds.

34. A kit according to claim 33 wherein the second and third labelled nucleotides have a distinct absorbance maximum above 600 nm.

37. A kit according to claim 33 wherein the first of four nucleotides is the labelled nucleotide and a second and third nucleotide are each labelled with a different compound, wherein each of the compounds of the second and third nucleotides can be excited using a single laser with a wavelength between 630-700 nm.

39. A kit according to claim 33 wherein the first of four nucleotides is the labelled nucleotide and a second, third, and fourth nucleotide are each labelled with a different compound, wherein the compound of the second nucleotide can be excited using a laser with a wavelength at 532 nm and each of the compounds of the third and fourth nucleotides can be excited using a single laser with a wavelength at 660 nm.

44. A kit according to claim 20 further comprising a DNA polymerase.

55. A method of synthesising a compound according to claim 1 utilising the following starting materials in a condensation with substituted or unsubstituted phthalic anhydride derivatives:

4. (canceled)

8. (canceled)

10-12. -12. (canceled)

15. (canceled)

17-19. -19. (canceled)

21-22. -22. (canceled)

24-25. -25. (canceled)

27. (canceled)

29-32. -32. (canceled)

35-36. -36. (canceled)

38. (canceled)

40-43. -43. (canceled)

45-54. -54. (canceled)

56-57. -57. (canceled)