The 1970s academic focus on protein purification is particularly evident in a 1979 review listing over 100 different proteins
and enzymes purified using immobilized triazine dyes.17 This also heralds a migration from a dye descriptor to a chemical identification of the core triazine. Additionally, it
is notable that the dyes under investigation were triazine, substituted at two positions and coupled to the matrix through
the third.
At this time it became evident that it was necessary to screen dye-ligand columns to find the most selective adsorbent.18,19
Atkinson et al. noted that "this dye (C.I. Reactive Blue 2), however, is only one of a large family of triazine dyes, most
of which bind proteins," indicating the need for a systematic approach to selection.20
It was also believed at this time that these ligand adsorbents would be highly suited to downstream processing.21
Stellwagen (1990) noted that "since the color, and hence the structure, of each reactive dye is different, each reactive dye
will have a somewhat different affinity for a bifunctional site on a given protein. Unfortunately, the affinity of a particular
reactive dye for a bifunctional site cannot be predicted with any confidence, necessitating an empirical screening procedure
to optimize chromatography."22 Only two years later, Lowe et al. wrote, "A fundamental advantage of affinity techniques is their predictive and rational
character, since the ligand selected is designed to interact specifically with the protein to be purified."23
At this pivotal point, dye affinity technology began to shift to de novo synthesis and rational ligand design.9 However, because some triazinyl derivatives are colored — although most are not — the concept of dye ligand chromatography,
in which the ligand is chosen from a random battery of commercial dyestuffs, has lived on past its expiration date. Two reviews
have summarized the field in the context of bioprocessing applications.24, 25
GENESIS OF SYNTHETIC LIGAND ALTERNATIVES
The shortcomings of dyes as affinity ligands has been recognized for some time. They are rarely single chemical entities and
— produced as bulk chemicals for a consumer product industry — they do not fulfill the rigorous requirements of biopharmaceutical
development and manufacturing. It had been noted that textile dyes contain isomers of the main product, stabilizing and diluent
agents, as well as anti-dusting agents such as dodecylbenzene. Of course, these impurities and contaminants must be removed.26 In one study of Cibacron Blue F3GA, up to 15 different colored components were identified. Furthermore, different preparations
did not necessarily contain the same components.27 In 1988, Burton et al. reported, for the first time, the synthesis of single isomer variants of C.I. Reactive Blue 2, thus
marking the link between dye structure and protein binding.28
Additionally, the interactions between target proteins and dye structures present many different alternatives for highly specific
binding of proteins at their active sites and less specific interactions at other sites. These reactions may include a complex
combination of electrostatic, hydrophobic, hydrogen bonding, and charge transfer reactions. Thus, ligand engineering or design
is a way of targeting specific binding.
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