Dye intermediate and method

Dyes have applications in many technologies, both new and well established, which count amongst them textile dyeing, optical data storage media and various detection methods, for both chemical and biological use. Within these applications various properties of the dyes are exploited in their actual detection which may ultimately be traced back to absorption, reflectance and luminescence (chemiluminescence, fluorescence and phosphorescence). However, a recurring theme of many applications is the need to form a relatively permanent link, in the form of a covalent bond, between the dye and the substrate of choice. The substrate may be inter alia: a relatively inert surface or probe possibly of insoluble polymeric origin; a macromolecule of biological origin e.g. a protein, antibody or oligonucleotide; or a small molecule of either biological or synthetic origin e.g. a hapten for use in directed or molecular recognition based applications; or a molecule which may modify the properties of the dye e.g. a water solubilising group such as a carbohydrate or other polar residues; or a species which protects the dyes from or decreases the rate of photofading of the dye, such as hindered amines and various transition metal complexes; or possibly another dye conjugate system thus enabling the possibility of energy transfer or fluorescence quenching based applications. The requirement for a covalent linkage means that many commercial products, spanning a large range of dye classes and possessing the desired spectral properties, cannot be used. This therefore necessitates either the modification of an existing product or the complete synthesis from appropriate materials such that a suitable grouping is available for linking the dye and substrate. There are many different sorts of linkage known, the main requirements being that the linkage is relatively stable and that it is easily introduced in high yield or efficiency. One of the most versatile groupings is that of the carboxylic acid which is readily converted to a range of reactive species or used to form a link to another substance containing the reactive linking species of choice. Various classes of dyes are provided having acid, ester or amide groups for covalent linking to biomolecules. The dyes may be prepared by use of a compound of formula (I) where Rcomprises a linker and a carboxy including acid, salt, ester including N-hydroxysuccinimide, activated ester or amide group; R, R, Rand Rare H, C-Calkyl or aralkyl