POLYMETHINE COMPOUNDS WITH LONG STOKES SHIFTS AND THEIR USE AS FLUORESCENT LABELS

Abstract

The present disclosure relates to new compounds and their use as fluorescent labels. The compounds may be used as fluorescent labels for nucleotides in nucleic acid sequencing applications. The labels are advantageous due to their long Stokes shifts.

##STR00001##

Claims

1. A compound of formula (I) or mesomeric forms thereof: ##STR00034## wherein mCat+ or mAn is an organic or inorganic positively/negatively charged counterion and m is an integer 0-3; x is an integer 0-2; Ra.sub.1 is H, SO.sub.3.sup., sulfonamide, halogen, hydroxy, alkoxy, amino or a further ring fused to an adjacent carbon atom where the ring may contain SO.sub.3.sup., sulfonamide, halogen substituents; Rb.sub.1 is SO.sub.3.sup., sulphonamide, halogen, hydroxy, alkoxy, amino, COOH or an amide or ester thereof and n is 0-3; each of Rc.sub.1 and Rc.sub.2 is independently alkyl or substituted alkyl; each of Rd.sub.1 and Rd.sub.2 is independently H, alkyl, aryl, substituted alkyl, or substituted aryl; and Re.sub.1 is alkyl, substituted alkyl, aryl or substituted aryl; wherein either Rc.sub.1, Rb.sub.1 or Re.sub.1 contains a COOH or COO.sup. or an amide or ester thereof.

2. A compound according to claim 1 of formula (I) or mesomeric forms thereof: ##STR00035## wherein mCat+ or mAn is an organic or inorganic positively/negatively charged counterion and m is an integer 0-3; Ra.sub.1 is H, SO.sub.3.sup., sulfonamide, halogen, hydroxy, alkoxy, amino or a further ring fused to an adjacent carbon atom where the ring may contain SO.sub.3.sup., sulfonamide, halogen substituents; Rb.sub.1 is SO.sub.3.sup., sulphonamide, halogen, hydroxy, alkoxy, amino, COOH or an amide or ester thereof and n is 0-3; each of Rc.sub.1 and Rc.sub.2 is independently alkyl or substituted alkyl; each of Rd.sub.1 and Rd.sub.2 is independently H, alkyl, aryl, substituted alkyl, or substituted aryl; and Re.sub.1 is alkyl, substituted alkyl, aryl or substituted aryl; wherein either Rc.sub.1, Rb.sub.1 or Re.sub.1 contains a COOH or COO.sup. or an amide or ester thereof.

3. A compound according to claim 1 or claim 2 wherein Re.sub.1 is alkyl and Rb.sub.1 is COOH or COO.sup. or an amide or ester thereof.

4. A compound according to claim 1 or claim 2 wherein Re.sub.1 is an alkyl group substituted with COOH or COO.sup. or an amide or ester thereof.

5. A compound according to claim 1 or claim 2 wherein Re.sub.1 is an aryl group substituted with COOH or COO.sup. or an amide or ester thereof.

6. A compound according to claim 1 or claim 2 wherein Re.sub.1 is an aryl group substituted with COOH or COO.sup. or an amide or ester thereof wherein there is an alkyl group between the aryl ring and the COOH or COO.sup. or an amide or ester thereof.

7. A compound according to claim 1 or claim 2 wherein Re.sub.1 is an aryl group substituted with CH.sub.2COOH or CH.sub.2COO.sup. or an amide or ester thereof.

8. A compound according to any one of claims 4 to 7 wherein n is 0.

9. A compound according to any one of claims 4 to 7 wherein n is 1 and Rb.sub.1 is SO.sub.3.sup. or sulphonamide.

10. A compound according to any one of claims 1 to 9 wherein each of Rc.sub.1 and Rc.sub.2 is methyl.

11. A compound according to any one of claims 1 to 9 wherein Rc.sub.1 or Rc.sub.2 is methyl, ethyl, propyl or (CH.sub.2).sub.qSO.sub.3.sup. where q is 1-6.

12. A compound according to claim 11 wherein either Rc.sub.1 or Rc.sub.2 is (CH.sub.2).sub.4SO.sub.3.sup..

13. A compound according to any one preceding claim wherein Ra.sub.1 is H, SO.sub.2NH.sub.2 or SO.sub.3.sup..

14. A compound according to any one of claims 1 to 12 wherein Ra.sub.1 is a ring fused to an adjacent carbon atom.

15. A compound according to claim 14 wherein the ring contains one or more SO.sub.3.sup. or sulphonamide substituents.

16. A compound according to any one preceding claim wherein each of Rd.sub.1 and Rd.sub.2 is independently H or methyl.

17. A compound according to claim 1 which is represented by formula (II): ##STR00036## or a salt thereof wherein mCat+ or mAn is an organic or inorganic positively/negatively charged counterion and m is an integer 0-3; each n is independently 0-6; Ra.sub.1 is H, SO.sub.3.sup., sulfonamide, halogen, or a further ring fused to an adjacent carbon atom where the ring may contain SO.sub.3.sup., sulfonamide, halogen substituents; Rb.sub.1 is SO.sub.3.sup., sulphonamide, halogen, COOH or an amide or ester thereof and n is 0-3; and X is OH, O.sup. or an ester or amide.

18. A compound according to any one preceding claim wherein the compound is attached to a nucleotide or oligonucleotide.

19. A nucleotide or oligonucleotide labelled with a compound according to claims 1 to 17.

20. A labelled nucleotide or oligonucleotide according to claim 18 wherein the label is attached via an amide linkage formed from the COOH moiety.

21. A labelled nucleotide or oligonucleotide according to claims 18 or 19 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.

22. A labelled nucleotide or oligonucleotide according to claims 19 to 21, further comprising a 3 OH blocking group covalently attached to the ribose or deoxyribose sugar of the nucleotide.

23. A kit comprising two or more nucleotides wherein at least one nucleotide is a labelled nucleotide according to claims 19 to 22.

24. A kit according to claim 22 wherein two of the labelled nucleotides are measured by detection at the same wavelength.

25. A kit according to claim 24 comprising four nucleotides wherein a first nucleotide is a labelled nucleotide according to claims 18 to 21, a second nucleotide is labelled with a label that emits at the same wavelength as the first labelled nucleotide, a third nucleotide is labelled with a mixture of labels and the fourth is unlabelled such that each of the four labelled nucleotides are distinguishable from each other.

26. A kit according to claim 22 to 25 wherein the first labelled nucleotide has a Stokes shift of greater than 100 nm and the second labelled nucleotide has a Stokes shift of less than 50 nm.

27. Use of a nucleotide according to any one of claims 19 to 22, an oligonucleotide according to claims 19 to 22 or a kit according to any one of claims 23 to 26 in sequencing, expression analysis, hybridisation analysis, genetic analysis, RNA analysis or protein binding assays.

28. Use according to claim 27 on an automated sequencing instrument wherein said automated sequencing instrument comprises two lasers operating at different wavelengths and a detection system having a single detection channel set to a fixed emission wavelength.

29. A method of synthesising a compound according to claims 1 to 17 utilising the following starting material: ##STR00037## or a salt thereof wherein x is 0-2; Ra.sub.1 is H, SO.sub.3.sup., sulfonamide, halogen, or a further ring fused to an adjacent carbon atom where the ring may contain SO.sub.3.sup., sulfonamide, halogen substituents; Rb.sub.1 is SO.sub.3.sup., sulphonamide, halogen or COOH and n is 0-3; each of Rc.sub.1 and Rc.sub.2 is independently alkyl or substituted alkyl; and each of Rd.sub.1 and Rd.sub.2 is independently H, alkyl, aryl, substituted alkyl, or substituted aryl.

Description

DESCRIPTION OF FIGURES

[0118] FIG. 1 illustrates fluorescence intensities of nucleotide labelled with a new dye (NR5201s), an example of the type disclosed herein, and a structural analogue (NR550S0) when their solutions excited at 540 nm. NR550S0 (structure shown below) is a polymethine dye having an indole at both ends of the polymethine chain, as is typical of fluorescent dyes having a stokes shift in the region of 40-50 nm. The dye having the stokes shift of 40-50 nm shows a higher fluorescence signal than the long stokes shift dye.

[0119] FIG. 2 illustrates fluorescence intensities of FFN's based on new dye (NR5201s) and its commercial structural analogue (NR550S0) when their solutions excited at 460 nm. Unlike at 540 nm, the long stokes shift dye is brighter when excited at 460 nm. Thus the two labels can be differentiated when measured at 590 nm emission based in the excitation wavelength.

EXPERIMENTAL DETAILS

SM2

[0120] ##STR00022##

[0121] These starting materials were prepared from pyrilium salts (SM1) using ethyl orthoformmate or its derivatives

##STR00023##

Example 1

SM2-1

[0122] ##STR00024##

[0123] 2,4,6-Trimethylpyrilium tetrafluoroborate (1 ekv), triethylortoformate (1.5 ekv) and N,N-diphenylformamidine (1.1. ekv) in acetic acid were heated for 6 h at 80 C. Reaction mixture was left overnight at room temperature and the product was filtered off as yellow crystals. Yield 87%.

Indopyrilocyanines (X).

[0124] ##STR00025##

[0125] These starting materials were prepared from pyrilium salts derivatives (SM2) using N-substituted indolium salts

Example 2

(X)-1

[0126] ##STR00026##

[0127] Equimolar amounts of SM2-1 and N-Phenyl-2,3,3-trimethylindolium salt in mixture of acetic acid, acetic anhydride and pyridine (1:1:0.5) were stirred for 3 h at 80 C.

[0128] The product was precipitated with diethyl ether and column purified. Yield 48%.

Indopyridocyanines

[0129] These dyes were prepared from pyrilium derivatives (X) using substituted amines or their salts

##STR00027##

Example 3-1 (NR5201s)

[0130] ##STR00028##

[0131] Starting materials were stirred for 5 min in ethanol then N-ethyl-N,N-diisopropylamine added and stirring was continued for 0.5 h. After solvent evaporation the dye was collected, washed with water. Yield 95%.

Example 3-2

[0132] ##STR00029##

[0133] Starting materials were stirred for 5 min in ethanol then N-ethyl-N,N-diisopropylamine added. Stirring was continued for 0.5 h. After solvent evaporation the dye was collected, washed with water. Yield 95%.

Example 3-3

[0134] ##STR00030##

[0135] Starting materials were mixed in ethanol then N-ethyl-N,N-diisopropylamine added. Reaction mixture was stirred for 0.5 h. After solvent evaporation the dye was collected, washed with water. Yield 95%.

Synthesis of Dye Labelled Nucleotide Triphosphates

[0136] Dye Conjugate pppT-NR5201s

##STR00031##

Preparation:

[0137] Anhydrous DMa (5 mL) and Hunig's Base (0.06 mL) were added to the dried sample of the dye (3-1) (80 mg). A solution of TSTU, (0.25 g) in 5 mL of dry DMA was then added to this. The reaction mixture was stirred at room temperature for 1 h. After activation was completed (TLC: 15% H.sub.2O in CH.sub.3CN) this solution was added to the solution of pppT-LN3 (0.23 g) in water (7 mL). The reaction mixture was stirred at room temperature under nitrogen atmosphere for 3 h. The reaction mixture was cooled down to 4 C. with an ice-bath, then a solution of 0.1 M TEAB (5 mL) in water was added and the mixture was stirred at room temperature for 10 min. The reaction mixture was applied to column with 75 g of DEAE Sephadex resin suspension in 0.05 M TEAB solution in water and washed with TEAB (concentration gradient from 0.10 M up to 0.75 M). Red coloured fractions were collected, the solvent evaporated and then the residue co-evaporated again with water to remove more TEAB and vac down to dryness. The dye was then re-dissolved in TEAB 0.1 M. This solution was filtered through a syringe filter 0.2 nm pore size and the product was purified by HPLC using C18 reverse phase column with acetonitrile-0.1 M TEAB. Yield 78%.

Dye Conjugate pppA-NR5201s

##STR00032##

Preparation:

[0138] Anhydrous DMA (5 mL) and Hunig's Base (0.06 mL) were added to the dried sample of the dye (3-1) (80 mg). A solution of TSTU, (0.25 g) in 5 mL of dry DMA was then added to this. The reaction mixture was stirred at room temperature for 2 h. After activation was completed (TLC: 15% H.sub.2O in CH.sub.3CN) this solution was added to the solution of pppA-LN3 (0.25 g) in water (7 mL). The reaction mixture was stirred at room temperature under nitrogen atmosphere for 24 h. The reaction mixture was cooled down to 4 C. with an ice-bath, then a solution of 0.1 M TEAB (5 mL) in water was added and the mixture was stirred at room temperature for 10 min. The reaction mixture was applied to column with 75 g of DEAE Sephadex resin suspension in 0.05 M TEAB solution in water and washed with TEAB (concentration gradient from 0.10 M up to 0.75 M). Red coloured fractions were collected, the solvent evaporated and then the residue co-evaporated again with water to remove more TEAB and vac down to dryness. The dye was then re-dissolved in TEAB 0.1 M. This solution was filtered through a syringe filter 0.2 nm pore size and the product was purified by HPLC using C18 reverse phase column with acetonitrile-0.1 M TEAB. Yield 75%.

Fluorescence Properties

[0139] FIG. 1 illustrates fluorescence intensities of nucleotide labelled with a new dye (NR5201s), an example of the type disclosed herein, and a structural analogue (NR550S0) when their solutions excited at 540 nm. NR550S0 (structure shown below) is a polymethine dye having an indole at both ends of the polymethine chain, as is typical of fluorescent dyes having a stokes shift in the region of 40-50 nm. The dye having the stokes shift of 40-50 nm shows a higher fluorescence signal than the long stokes shift dye.

##STR00033##

[0140] FIG. 2 illustrates fluorescence intensities of FFN's based on new dye (NR5201s) and its commercial structural analogue (NR550S0) when their solutions excited at 460 nm. Unlike at 540 nm, the long stokes shift dye is brighter when excited at 460 nm. Thus the two labels can be differentiated when measured at 590 nm emission based in the excitation wavelength.

[0141] From the comparison of ratios of fluorescence intensities of a new dye NR5201s with long Stokes shift and its structural analogue with a normal stokes shift, one can see the advantage of using new dyes due to a variance in signal intensities when solutions are excited at different wavelengths.

[0142] In the charts above, for dye NR550S0 the ratio of fluorescence intensities at 540 nm (193.0) and at 460 nm (10.1) is 19.3 as the dye does not absorb efficiently at 460 nm. In the same conditions for the new dye NR5201s, the ratio of fluorescence intensities at 540 nm (93.0) and 460 nm (40.1) is only about 2 as the longer stokes shift means the dye has a much higher level or absorbance at 460 nm. Due to these unique properties, the new dyes as disclosed herein allow more efficient data analysis as the signal to noise is improved, and allow sequencing platforms to operate using fewer than the conventional four detection channels.