Proteins, chiral recognition

From the atomic to the macroscopic level chirality is a characteristic feature of biological systems and plays an important role in the interplay of structure and function. Originating from small chiral precursors complex macromolecules such as proteins or DNA have developed during evolution. On a supramolecular level chirality is expressed in molecular organization, e.g. in the secondary and tertiary structure of proteins, in membranes, cells or tissues. On a macroscopic level, it appears in the chirality of our hands or in the asymmetric arrangement of our organs, or in the helicity of snail shells. Nature usually displays a preference for one sense of chirality over the other. This leads to specific interactions called chiral recognition. 

Noncovalent interactions play a key role in biodisciplines. A celebrated example is the secondary structure of proteins. The 20 natural amino acids are each characterized by different structures with more or less acidic or basic, hydrophilic or hydrophobic functionalities and thus capable of different intermolecular interactions. Due to the formation of hydrogen bonds between nearby C=0 and N-H groups, protein polypeptide backbones can be twisted into a-helixes, even in the gas phase in the absence of any solvent." A protein function is determined more directly by its three-dimensional structure and dynamics than by its sequence of amino acids. Three-dimensional structures are strongly influenced by weak non-covalent interactions between side functionalities, but the central importance of these weak interactions is by no means limited to structural effects. Life relies on biological specificity, which arises from the fact that individual biomolecules communicate through non-covalent interactions." " Molecular and chiral recognition rely on... 

Different classifications for the chiral CSPs have been described. They are based on the chemical structure of the chiral selectors and on the chiral recognition mechanism involved. In this chapter we will use a classification based mainly on the chemical structure of the selectors. The selectors are classified in three groups (i) CSPs with low-molecular-weight selectors, such as Pirkle type CSPs, ionic and ligand exchange CSPs, (ii) CSPs with macrocyclic selectors, such as CDs, crown-ethers and macrocyclic antibiotics, and (iii) CSPs with macromolecular selectors, such as polysaccharides, synthetic polymers, molecular imprinted polymers and proteins. These different types of CSPs, frequently used for the analysis of chiral pharmaceuticals, are discussed in more detail later. 

Kempe M, Mosbach K. Chiral recognition of N alpha-protected amino acids and derivatives in non-covalently molecularly imprinted polymers. Int J Peptide Protein Res 1994 44 603 -606. 

In most cases, the protein is immobilized onto y-aminopropyl silica and covalently attached using a cross-linking reagent such as A/.Af-carbonyldiiinida/.ole. The tertiary structure or three dimensional organization of proteins arc thought to be important for their activity and chiral recognition. Therefore, mobile phase conditions that cause protein denal-uralion" or loss of tertiary structure must he avoided. 

Owing to their size and the feasibility of controlling the dendrimer structure and globular morphology via their synthesis, dendrimers with chiral branching scaffold are of interest as potential protein mimics. In addition, the introduction of chiral branching units or spacers into the molecular scaffold should lead to the development of non-symmetrical macromolecular conformations and provide chiral cavities for asymmetrical catalysis or chiral recognition processes. 

A liquid chromatographic method is utilized for the determination of clopidogrel bisulfate in samples of the bulk drug substance. The method uses a column (L57 column size 15 cm x 4.6 mm) packed with ovomucoid (a chiral-recognition protein) that is chemically bonded to silica particles of 5 /im diameter and a pore size of 120 A. Both the reference standard and the sample to be analyzed are dissolved in methanol, and then diluted with mobile phase. The mobile phase is 75 25 0.01 M phosphate buffer /acetonitrile, and the flow rate is adjusted to 1.0 ml/min. Observation is made on the basis of the UV absorbance at 220 nm, and the clopidogrel peak has a relative retention time about 1.0 min. 

The chiral recognition mechanisms in NLC and NCE devices are similar to conventional liquid chromatography and capillary electrophoresis with chiral mobile phase additives. It is important to note here that, to date, no chiral stationary phase has been developed in microfluidic devices. As discussed above polysaccharides, cyclodextrins, macrocyclic glycopeptide antibiotics, proteins, crown ethers, ligand exchangers, and Pirkle s type molecules are the most commonly used chiral selectors. These compounds... 

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