Protein-type CSPs

The observation of enantioselective binding of chiral molecules to certain classes 

CSPs incorporating BSA have been successfully employed to separate the enantiomers of acidic and neutral compounds [161-163], such as N-acylated amino acids, aromatic amino acids and sulfoxides. Retention and enantioselectivity can be optimized by adjusting pH, ionic strength and organic modifier content of the mobile phase. 

HSA bears structural and functional resemblance to BSA, and HSA-type CSPs [164] also show similar enantioselective binding preferences for acidic and neutral drug molecules, such as 2-aryloxypropanoic acids [165], warfarin [166] and benzodiazepines [167]. The chiral recognition mechanism of HSA has been the subject of a number of investigations [168], which revealed that enantioselective binding occurs primarily at two well-defined hydrophobic sites. Acidic drugs have been shown to bind preferentially to the so-called warfarin-azapropazone (site I) and neutral drugs to the indol-benzodiazepine site (site II). 

The investigation of chiral recognition of drug molecules by proteins may re- 

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The usefulness of protein-type CSPs has already been shown in particular Chiral-AOP and Ultron ES-OVM (see Table 2) have a very broad range of enantioselectivity. It is not, however, possible to systematically predict the resolution on such CSPs, but the overall retention, selectivity and efficiency can be modified to a certain extent by altering several key variables ... 

Besides the conventional factors causing peak broadening in LC. another source for lower efficiency in LC with chiral stationary phases may be the existence of at least two different adsorption sites (the stereoselective and non-stereoselective ones) that may considerably differ in their adsorption kinetics (heterogeneous mass transfer kinetics) and thus cause peak broadening and tailing. These factors have been investigated and modelled by Fomstedt et al.. e.g. for protein type CSPs [75,91-9.3] and their contribution was determined for different analytes and different type of CSPs (bovine serum albumin, cellobiohydrolase 1, tris(4-methylbenzoyl) cellulose) [94j. In a recent study, these authors reported on the adsorption isotherms as well as selective and nonselective contributions of propranolol enantiomers on a cellobiohydrolase 1 CSP in dependence of the mobile phase pH [95[. 

Protein type CSPs — representinf> a clci.ss of polymeric type CSPs which can he u.sed with aqueous mobile phases... 

Parallel to the n-donor-acceptor CSPs related to Pirkle s pioneering work for the understanding of chiral recognition phenomena and for gaining insight into SO-SA complexation principles on the molecular level, the protein type CSPs can claim a major credit for the rapid development of chiral technology in pharmaceutical and life sciences. 

One popular strategy to isolate and identify the binding domain of a protein type CSP is to compare the retention and enantioselectivity behaviour of CSPs prepared with whole proteins and with isolated protein domains. Such a study has been performed by Pinkerton et al. [204) with turkey ovomucoid. Columns made from whole-turkey ovomucoid displayed chiral activity toward many racemates, whereas the fused first and second domain resolved only a selected number of aromatic weak bases. The first and second domains independently expressed no appreciable chiral recognition activity. The third domain, however, exhibited enantioselective protein binding for fused-ring aromatic weak acids, and glycosylation of this domain did not affect chiral recognition. 

ENANTIOSEPARATION OF PHARMACEUTICALLY RELEVANT CHIRAL COMPOUNDS USING PROTEIN TYPE CSPs... 

Over the years, a wide variety of diverse proteins have been tested for their potential to serve as the SOs of novel CSPs with different application profiles. For example, flavoprotein 216], avidin and conalbumin [217], as well as other proteins like egg-yolk riboflavin binding protein [218], have been immobilized onto silica. However, neither their enantioselectivity profiles nor their performance was significantly better than those of commercially available protein type CSPs. 

Recently, a mixed AGP-HSA protein type CSP has been prepared which showed a wider range of applications than the individual HSA and AGP CSPs. For two compounds, the enantioselectivity on the mixed CSP was higher than on the HSA CSP [219 [. Similarly,... 

Protein type CSPs have to be considered as important and widely used tools for enantiomer separations (Table 9.9) and for analyzing stereoselective phenomena of biological significance, in particular for stereoselective drug-protein binding studies. The latter application, however, is beyond the scope of this report and the reader is referred to recent articles on this topic [4,223-232]. 

In addition, due to the intermediate size of the chiral SO units, the loadability may not compete with CSPs based on low-molecular SOs however, it is much higher than for protein type CSPs [278]. 

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