Squarate dyes and their use in fluorescent sequencing method
First Claim
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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.
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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.
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Citations
12 Claims
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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. - View Dependent Claims (3, 4, 5, 6, 7, 8, 12)
- 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,
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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.
- 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,
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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. - View Dependent Claims (10, 11)
- 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,
Specification