Enantiomers proteins

The inherent chirality of proteins makes them very good candidates for the separation of enantiomers. Proteins which can tolerate organic solvents, as well as high temperatures, and which can function over a wide range of pH are useful as chiral stationary phases. Table 22-2 shows some of the characteristics of these proteins [138]. 

Care should be exercised when attempting to interpret in vivo pharmacological data in terms of specific chemical—biological interactions for a series of asymmetric compounds, particularly when this interaction is the only parameter considered in the analysis (10). It is important to recognize that the observed difference in activity between optical antipodes is not simply a result of the association of the compound with an enzyme or receptor target. Enantiomers differ in absorption rates across membranes, especially where active transport mechanisms are involved (11). They bind with different affinities to plasma proteins (12) and undergo alternative metaboHc and detoxification processes (13). This ultimately leads to one enantiomer being more available to produce a therapeutic effect. 

Although most anesthetics are achiral or are adininistered as racemic mixture, the anesthetic actions are stereoselective. This property can define a specific, rather than a nonspecific, site of action. Stereoselectivity is observed for such barbiturates as thiopental, pentobarbital, and secobarbital. The (3)-enantiomer is modestly more potent (56,57). Additionally, the volatile anesthetic isoflurane also shows stereoselectivity. The (3)-enantiomer is the more active (58). Further evidence that proteins might serve as appropriate targets for general anesthetics come from observations that anesthetics inhibit the activity of the enzyme luciferase. The potencies parallel the anesthetic activities closely (59,60). 

Tocainide is rapidly and well absorbed from the GI tract and undergoes very fitde hepatic first-pass metabolism. Unlike lidocaine which is - 30% bioavailable, tocainide s availability approaches 100% of the administered dose. Eood delays absorption and decreases plasma levels but does not affect bio availability. Less than 10% of the dmg is bound to plasma proteins. Therapeutic plasma concentrations are 3—9 jig/mL. Toxic plasma levels are >10 fig/mL. Peak plasma concentrations are achieved in 0.5—2 h. About 30—40% of tocainide is metabolized in the fiver by deamination and glucuronidation to inactive metabolites. The metabolism is stereoselective and the steady-state plasma concentration of the (3)-(—) enantiomer is about four times that of the (R)-(+) enantiomer. About 50% of the tocainide dose is efirninated by the kidneys unchanged, and the rest is efirninated as metabolites. The elimination half-life of tocainide is about 15 h, and is prolonged in patients with renal disease (1,2,23). 

Chiral Chromatography. Chiral chromatography is used for the analysis of enantiomers, most useful for separations of pharmaceuticals and biochemical compounds (see Biopolymers, analytical techniques). There are several types of chiral stationary phases those that use attractive interactions, metal ligands, inclusion complexes, and protein complexes. The separation of optical isomers has important ramifications, especially in biochemistry and pharmaceutical chemistry, where one form of a compound may be bioactive and the other inactive, inhibitory, or toxic. 

Separation of enantiomers by physical or chemical methods requires the use of a chiral material, reagent, or catalyst. Both natural materials, such as polysaccharides and proteins, and solids that have been synthetically modified to incorporate chiral structures have been developed for use in separation of enantiomers by HPLC. The use of a chiral stationary phase makes the interactions between the two enantiomers with the adsorbent nonidentical and thus establishes a different rate of elution through the column. The interactions typically include hydrogen bonding, dipolar interactions, and n-n interactions. These attractive interactions may be disturbed by steric repulsions, and frequently the basis of enantioselectivity is a better steric fit for one of the two enantiomers. ... 

The predominance of L-amino acids in biological systems is one of life s most intriguing features. Prebiotic syntheses of amino acids would be expected to produce equal amounts of L- and D-enantiomers. Some kind of enantiomeric selection process must have intervened to select L-amino acids over their D-connterparts as the constituents of proteins. Was it random chance that chose L- over D-isomers ... 

A. Walhagen and F.-E. Edholm, Coupled-column cliromatography of immobilized protein phases for direct separation and determination of dmg enantiomers in plasma , 7. Chromatogr. 473 371-379 (1989). 

Proteins (BSA or ovomucoid, OVM) have also been successful in the preparative resolution of enantiomers by liquid-liquid extraction, either between aqueous and lipophilic phases [181] or in aqueous two-phase systems (ATPS) [123, 180]. The resolution of d,l-kynurenine [180] and ofloxacin and carvediol [123] were performed using a countercurrent extraction process with eight separatory funnels. The significant number of stages needed for these complete resolutions in the mentioned references and others [123, 180, 189], can be overcome with more efficient techniques. Thus, the resolution of d,l-kynurenine performed by Sellergren et al. in 1988 by extraction experiments was improved with CCC technologies 10 years later [128]. 

S. G. Allenmark, Separation of enantiomers by protein-based chiral phases in A practical approach to chiral separations by liquid chromatogra.phy, G. Subramanian, VCH, Weinheim (1994) Chapter 7. 

Proteins. A chiral stationary phase with immobilized a -acid glycoprotein on silica beads was introduced by Hermansson in 1983 [18, 19]. Several other proteins such as chicken egg albumin (ovalbumin), human serum albumin, and cellohy-drolase were also used later for the preparation of commercial CSPs. Their selectivity is believed to occur as a result of excess of dispersive forces acting on the more retained enantiomer [17]. These separation media often exhibit only modest loading capacity. 

One of the most useful applications of chiral derivatization chromatography is the quantification of free amino acid enantiomers. Using this indirect method, it is possible to quantify very small amounts of enantiomeric amino acids in parallel and in highly complex natural matrices. While direct determination of free amino acids is in itself not trivial, direct methods often fail completely when the enantiomeric ratio of amino acid from protein hydrolysis must be monitored in complex matrices. 

The method described above is applicable to a wide range of samples for the determination of amino acids in different matrices. For example, the amino acid composition and distribution of single enantiomers has been determined in protein hydrolysates, orange juice (Fig. 7-11), yogurt and seawater [23]. 

Problem 9.3. Alanine, an amino acid found in proteins, is chiral. Draw the two enantiomers of ala- nine using the standard convention of solid, wedged, and dashed lines. 

The synthesis of an a-amino acid from an achiral precursor by any of the methods described in the previous section yields a racemic mixture, with equal amounts of S and R enantiomers. To use an amino acid in the laboratory synthesis of a naturally occurring protein, however, the pure S enantiomer must be obtained. 

Figure 8.41 Separation of the enantiomers of the common protein amino acids (N-perfluoropropionylamide isopropyl esters) on a 20 m X 0.25 mm I.D. open tubular column coated with Chirasil-Val. (Reproduced with permission from ref. 764. Copyright Elsevier Scientific Publishing Co.)...

Although anosmias to these compounds occur at similar levels, some communicative value may arise from the persistence of signal emissions which are not enantiomerically pure (Carman, 1993 Wysocki et al., 1999). In secreted mixtures, the alternate versions of such compounds are produced in a constant ratio since they have identical volatility and hence provide stable informational content to the receiver. Support for this idea comes from the results of the NMR mapping of the BT binding site within the MUP1 carrier (Zidek et al., 1999). Here the protein-ligand complex does exist in the expected ratio, and for both enantiomers, although the orientation of the bound thiazole was interpreted as opposite to that indicated by previous X-ray analyses. 

There is a wide variety of commercially available chiral stationary phases and mobile phase additives.32 34 Preparative scale separations have been performed on the gram scale.32 Many stationary phases are based on chiral polymers such as cellulose or methacrylate, proteins such as human serum albumin or acid glycoprotein, Pirkle-type phases (often based on amino acids), or cyclodextrins. A typical application of a Pirkle phase column was the use of a N-(3,5-dinitrobenzyl)-a-amino phosphonate to synthesize several functionalized chiral stationary phases to separate enantiomers of... 

Capillary electrophoresis employing chiral selectors has been shown to be a useful analytical method to separate enantiomers. Conventionally, instrumental chiral separations have been achieved by gas chromatography and by high performance liquid chromatography.127 In recent years, there has been considerable activity in the separation and characterization of racemic pharmaceuticals by high performance capillary electrophoresis, with particular interest paid to using this technique in modem pharmaceutical analytical laboratories.128 130 The most frequently used chiral selectors in CE are cyclodextrins, crown ethers, chiral surfactants, bile acids, and protein-filled... 

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