Squarate dyes and their use in fluorescent sequencing method

A squarate dye of the formula (III) or (IV) or (IVa) ##STR16## where each Z is independently O or S or CR.sup.1.sub.2,

• n=1-3,
• each of s and m is 0, 1 or 2,
• R.sup.1 is lower alkyl (1-4 carbon chain),
• each R.sup.2 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 R.sup.3 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 NH.sub.2, or alternatively a grouping capable of reacting with a nucleophile
• and any other X present is independently selected from H and SO.sub.3.sup.- and the residue of a squarate dye of formula (III), (IV) or (IVa) and another fluorochrome,
• A is O, NR.sup.4 or S,
• R.sup.4 is alkyl, alkenyl, alkynyl or H, and
• each Y is independently X or H,
• provided that at least one R.sup.2 is SO.sub.3.sup.- and/or at least one X is phosphoramidite.

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 CR.sup.1.sub.2,

• n=1-3,
• R.sup.1 is lower alkyl (1-4 carbon chain),
• each R.sup.2 is independently selected from electron donating and electron withdrawing groups such as halogen, alkoxy, primary secondary and tertiary amino, nitro, SO.sub.3.sup.-, and --R.sup.3 --X, or is a branched or straight chain of up to 30 carbon atoms incorporating one to five positively charged nitrogen atoms,
• each R.sup.3 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 NH.sub.2, or alternatively a grouping capable of reacting with a nucleophile, in which case X is preferably selected from the following
• CO.sub.2 H, activated carboxyl such as acid halide or anhydride, CO active ester, NCS, O phosphoramidite, NC(O)CH.sub.2 l and ##STR18## any other X present is independently selected from H and SO.sub.3.sup.- 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, NR.sup.4 or S,
• R.sup.4 is alkyl, alkenyl, alkynyl or H, and
• each Y is independently X or H.