Proteins enantioseparation

The ability of proteins to form enantioselective interactions with a large variety of drugs is used in chiral affinity chromatography. Protein CSPs that are most frequently used for the enantioseparation of pharmaceuticals include bovine serum albumin (BSA), human serum albumin... 

From the method development and robustness point of view, the temperature is a parameter that controls equilibria such as pK and enantiomer—chiral selector complexation, or induces structural changes in, e.g., proteins.For chiral separations, generally a lower temperature results in better enantioseparation, but even the opposite has been observed. Sometimes a raise in temperature does not so much affect the enantiomeric separation, but increases the resolution between an enantiomer and a matrix component. ... 

The use of polysaccharide-based CSPs instead of protein-based CSPs often increases the peak efficiency and facilifafes faster separafions. Papini ef al. [159] recently developed a method for the enantioseparation of lorazepam and on a Chiralpak OD-R column and an enzymatic hydrolysis was used to determine the amount of the glucoronide metabolite of lorazepam present. The separation was performed in 7 min with an LOQ of 1 and 10 ng/mL for lorazepam in plasma and urine, respectively. Another relatively fast separation for chiral analysis was published by Lausecker and Eischer [188]. They developed a method for determination of the drug candidate R483 within... 

Olsson and Blomberg [141] enantioseparated omeprazole and its metabolite 5-hydroxyomeprazole using open tubular capillary electrochromatography with immobilized avidin as chiral selector. The separation was performed with open tubular capillary electrochromatography. The protein avidin was used as the chiral selector. Avidin was immobilized by a Schiffs base type of reaction where the protein was via glutral-dehyde covalently bonded to the amino-modified wall of a fused-silica capillary, 50 /an i.d. Both racemates were baseline resolved. Resolution... 

Haginaka, J. (2001) Protein-based chiral stationary phases for high-performance liquid chromatography enantioseparations, J. Chromatogr. A 906, 253-273. 

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

Another marked disadvantage should be mentioned. As a consequence of the macromolecular nature of the selector, its molar loading on the support material is quite low. Since the number of binding sites per protein molecule is also limited (in contrast to polysaccharide and other polymeric CSPs consisting of the same repetitive sub-selector units), these CSPs have a low loadability [203]. This greatly restricts their use for preparative enantioseparations. 

The use of a convective macroporous polymer as an alternative support material instead of silica for the preparation of protein-based CSPs has successfully been demonstrated by Hofstetter et al. [221]. Enantioseparation was performed using a polymeric flow-through-type chromatographic support (POROS-EP, 20 pm polymer particles with epoxy functionalities) and covalently bound BSA as chiral SO. Using flow rates of up to 10 ml/min, rapid enantiomer separation of acidic compounds, including a variety of amino acid derivatives and drugs, could be achieved within a few minutes at medium efficiencies, typical for protein chiral stationary phases (Fig. 9.13). 

Table 13.2 Enantioseparation of N-protected proteinous amino acids on carbamate-type cinchona CSPs°.

Taken together, these results indicate that new enantioseparation proteins can be designed by using a computational chemical method. The feasibility of this approach was supported by the results of the quantitative analysis of enzyme reactions. 

Chromatographic enantioseparations may be performed using two methods called direct and indirect. In this former method, chiral stationary phases (polymers of natural origin, protein phases, synthetic polymers with build-in chiral selectors, etc.) are used or appropriate chiral selectors (cyclodextrins, crown ethers, etc.) are added to the mobile phase [7]. 

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