Squarate dyes and their use in fluorescent sequencing method

• US 6,140,494 A
• Filed: 05/12/1999
• Issued: 10/31/2000
• Est. Priority Date: 04/19/1996
• Status: Expired due to Fees

• Current Assignee: Amersham Pharmacia UK Limited, Amersham Pharmacia Biotech UK Ltd. (Danaher Corporation)
• Original Assignee: Amersham Pharmacia Biotech UK Ltd. (Danaher Corporation)
• Inventors: West, Richard Martin, Hamilton, Alan Lewis, Briggs, Mark Samuel Jonathan, Bruce, Ian Edward, Cummins, William Jonathan
• Primary Examiner(s): Houtteman, Scott W.
• Application Number:
  US09/171,303
• Time in Patent Office: 538 Days
• Field of Search: 536/26.6, 536/25.34, 568/839, 549/510
• US Class Current: 536/26.6
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C09B 57/007 Squaraine dyes

Abstract

Novel squarate dyes are described, and adducts of these dyes with biologically significant chemical species such as nucleosides or nucleotides. The adducts have useful properties for fluorescent nucleic acid sequencing methods.

Claims

1. A squarate dye of the formula (I) or (II) or (IIa) ##STR15## where each Z is independently O or S or CR¹₂,

n=1-3,

each of s and m is 0, 1 or 2,

R¹ is lower alkyl (1-4 carbon chain),

each R² is independently selected from electron donating and electron withdrawing groups or is a branched or straight chain of up to 30 carbon atoms incorporating one to five positively charged nitrogen atoms,

each R³ is independently selected from:

alkylene, alkenylene and alkynylene (1-20 carbon chain), or is a branched or straight chain of up to 30 carbon atoms incorporating one to five ether oxygen atoms or arylene rings or positively charged nitrogen atoms,

at least one X is a nucleophilic functional group, such as OH, SH or NH₂, or alternatively a grouping capable of reacting with a nucleophile,

and any other X present is independently selected from H and SO₃^- and the residue of a squarate dye of formula (I) or (II) or (IIa) and another fluorochrome,

provided that at least one R² is SO₃^- and/or at least one X is phosphoramidite.

3. A squarate dye according to claim 1 or claim 2, wherein at least one R² is SO₃^-.

4. A squarate dye according to claim 1 or claim 2, wherein n is 1 and Z is --C(CH₃)₂.

5. A squarate dye according to claim 1 or claim 2, wherein 1 to 5 SO₃^- groups are present.

6. A squarate dye according to claim 1 or claim 2, wherein at least one X is selected from CO₂ H, activated carboxyl, CO active ester, NCS, O phosphoramidite, NCOCH₂ l and ##STR17##

7. A squarate dye according to claim 1 or claim 2, wherein each R² is individually selected from halogen, alkoxy, primary secondary and tertiary amine, nitro, SO₃^- and --R³ X.

8. An adduct of a biological molecule with a squarate dye according to claim 1 or claim 2.

12. A fluorescent labelling complex comprising:

a first or donor fluorochrome having first absorption and emission spectra;

a second or acceptor fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of said first fluorochrome;

at least one linker for covalently attaching said first and second fluorochromes for transfer of resonance energy transfer between said first and second fluorochromes;

a target bonding group capable of forming a covalent bond with a target compound;

wherein at least one of the said first and second fluorochromes is a squarate dye according to claim 1 or claim 2.

2. A squarate dye of the formula (III) or (IV) or (IVa) ##STR16## where each Z is independently O or S or CR¹₂,

n=1-3,

each of s and m is 0, 1 or 2,

R¹ is lower alkyl (1-4 carbon chain),

each R² is independently selected from electron donating and electron withdrawing groups or is a branched or straight chain of up to 30 carbon atoms incorporating one to five positively charged nitrogen atoms,

each R³ is independently selected from:

alkylene, alkenylene and alkynylene (1-20 carbon chain), or is a branched or straight chain of up to 30 carbon atoms incorporating one to five ether oxygen atoms or arylene rings or positively charged nitrogen atoms,

at least one X is a nucleophilic functional group, such as OH, SH or NH₂, or alternatively a grouping capable of reacting with a nucleophile

and any other X present is independently selected from H and SO₃^- and the residue of a squarate dye of formula (III), (IV) or (IVa) and another fluorochrome,

A is O, NR⁴ or S,

R⁴ is alkyl, alkenyl, alkynyl or H, and

each Y is independently X or H,

provided that at least one R² is SO₃^- and/or at least one X is phosphoramidite.

9. An adduct of a nucleoside or nucleotide or analogue or oligonucleotide or nucleic acid with a squarate dye of the formula (I) or (II) or (IIa) or (III) or (IV) or (IVa) where each Z is independently O or S or CR¹₂,

n=1-3,

R¹ is lower alkyl (1-4 carbon chain),

each R² is independently selected from electron donating and electron withdrawing groups such as halogen, alkoxy, primary secondary and tertiary amino, nitro, SO₃^-, and --R³ --X, or is a branched or straight chain of up to 30 carbon atoms incorporating one to five positively charged nitrogen atoms,

each R³ is independently selected from:

alkylene, alkenylene and alkynylene (1-20 carbon chain), or is a branched or straight chain of up to 30 carbon atoms incorporating one to five ether oxygen atoms or arylene rings or positively charged nitrogen atoms,

at least one X is a nucleophilic functional group, such as OH, SH or NH₂, or alternatively a grouping capable of reacting with a nucleophile, in which case X is preferably selected from the following

CO₂ H, activated carboxyl such as acid halide or anhydride, CO active ester, NCS, O phosphoramidite, NC(O)CH₂ l and ##STR18## any other X present is independently selected from H and SO₃^- and the residue of a squarate dye (whereby dimers and oligomers of the dyes shown as monomers of formula (I), (II), (IIa), (III), (IV) and (IVa) are envisaged), or other fluorochrome,

each of s and m is 0, 1 or 2,

A is O, NR⁴ or S,

R⁴ is alkyl, alkenyl, alkynyl or H, and

each Y is independently X or H.

10. An adduct as claimed in claim 9, wherein the adduct has the formula Q--N--CO--Sq, where Q is a nucleotide or nucleotide analogue or oligonucleotide residue, and Sq is a residue of a squarate dye, the two being joined by an amide linkage formed between an amine group of Q and a carboxylate group of Sq.

11. An improved fluorescent sequencing method, which comprises using an adduct according to claim 9 or claim 10.