Work on biomimetic ligands, as defined by Clonis, was initiated in 1984 and is exemplified by two development projects.29 The first biomimetic dye ligand was developed for trypsin by linking benzamidine to the reactive chlorotriazine through
a diaminomethylbenzene group.29 The ligand was designed on the basis of the unusual cationic substrate preference of trypsin-like enzymes. In a second example,
Reactive Blue 2 was specifically redesigned to confer specificity for calf intestinal alkaline phosphatase.30
From there, it was a short step to generate well-defined dye adsorbents in order to develop a series of adsorbents for which
the ligand structures and the ligand density were known and could be controlled.31 However, there are many more variables in the performance of affinity chromatography. Greater degrees of sophistication
are required, both in the design of the ligand and the ligand-adsorbent conjugate and in the execution of the technique. In
a second step toward designed ligands (but still restricted to the concept of "designer dyes"), the interaction between the
"parent" dye (Cibacron Blue) and analogs with horse liver alcohol dehydrogenase was established using X-ray crystallography.32 A new set of terminal-ring (opposite to the anthraquinone) analogs were synthesized with favorable affinity for ADH. These
ligands belong to a second generation of adsorbents, the results of rational molecular design techniques.
Lowe et al. reviewed the advent of computer-aided ligand design, which has subsequently developed to include design based
on X-ray crystallographic data, NMR protein structures, and homology data from suitable databases.33 This has been made possible by concomitant software developments.33 , design may start from peptide ligand models as mimics of protein-protein binding interactions. Peptides themselves can
be used as ligands, but present an expensive and less stable choice for bioprocessing. Instead, the peptide template can be
used to model a synthetic alternative.
There are several different approaches to ligand design and synthesis that form the basis of the technology used today for
commercial development of third-generation adsorbents, including:
- optimization of existing ligands (analog synthesis);
- rational design (computer modeling of ligand structures);
- systematic screening of ligand arrays;
- rational design combined with library construction and screening.
Often, insufficient structural information is available for a target protein, and, in some cases, even the target may be unknown.
If a biological ligand is known or a peptide ligand has already been developed, those models may be used. It is not surprising,
therefore, that most commercially developed ligands and adsorbents have been derived from targeted construction and screening
of solid phase libraries. Lowe recently reviewed combinatorial approaches to affinity chromatography.34 Although virtual libraries can be constructed from, for example, the Available Chemicals Directory's 2.5 million entities,
and these libraries can be reduced to real and manageable sub-libraries, this route has not been commercially successful for
bioprocessing.35 It is important to note that earlier screening of solution phase libraries has also failed to produce any commercial adsorbents,
since the orientation of the ligand to its target in solution and as an immobilized entity may present different aspects to
the protein. In this article, only solid phase libraries and screening will be discussed.
Figure 2. Basic Chemistry of Ligand Synthesis from Dichlorotriazine-Agarose
It is possible to identify two major steps in the development of "customized" ligands: 1) construction and screening of suitable
libraries, known as intelligent combinatorial chemistry and 2) development of the ligand adsorbent conjugate.