Carrier molecule, covalent attachment

Cell membranes are lipophilic and designed to be barriers against large anionic molecules, although there is a natural mechanism for intercellular transport of anionic oligonucleotides. In order to enhance membrane transport, antisense oligonucleotides are frequentiy modified by covalent attachment of carrier molecules or lipophilic groups. 

Although small molecules do not usually stimulate antibody formation, antibodies against them can usually be raised if the small molecule, or hapten, is covalently attached to a large immunologically active carrier protein. 

Phosphopantetheine tethering is a posttranslational modification that takes place on the active site serine of carrier proteins - acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs), also termed thiolation (T) domains - during the biosynthesis of fatty acids (FAs) (use ACPs) (Scheme 23), polyketides (PKs) (use ACPs) (Scheme 24), and nonribosomal peptides (NRPs) (use T domain) (Scheme 25). It is only after the covalent attachment of the 20-A Ppant arm, required for facile transfer of the various building block constituents of the molecules to be formed, that the carrier proteins can interact with the other components of the different multi-modular assembly lines (fatty acid synthases (FASs), polyketide synthases (PKSs), and nonribosomal peptide synthetases (NRPSs)) on which the compounds of interest are assembled. The structural organizations of FASs, PKSs, and NRPSs are analogous and can be divided into three broad classes the types I, II, and III systems. Even though the role of the carrier proteins is the same in all systems, their mode of action differs from one system to another. In the type I systems the carrier proteins usually only interact in cis with domains to which they are physically attached, with the exception of the PPTases and external type II thioesterase (TEII) domains that act in trans. In the type II systems the carrier proteins selectively interact... 

FIGURE 21-4 Acyl carrier protein (ACP). The prosthetic group is 4 -phosphopantetheine, which is covalently attached to the hydroxyl group of a Ser residue in ACP. Phosphopantetheine contains the B vitamin pantothenic acid, also found in the coenzyme A molecule. Its —SH group is the site of entry of malonyl groups during fatty acid synthesis. 

The covalent attachment of enzymes to water-insoluble carriers is usually the preferred immobilization method for sensor manufacturing. Obviously, the selected procedure should avoid the loss of enzymatic activity and keep the accessibility of the binding site to the substrate molecules. Unfortunately, this is usually not the case and due to the severe conditions of many of these procedures, major activity losses and/or changes on the substrate selectivity are produced during immobilization. Some authors have pointed out that the enzyme activity decreases approximately one fifth per formed bond [66]. 

The immune system does not recognize relatively small molecules, so we must use a trick to prepare antibodies with binding sites that are specific for a particular drug. As shown in Figure 1 IF-Id, we attach the drug covalently to an antigenic carrier molecule such as bovine serum albumin (BSA), a protein that is obtained from the blood of cattle. 

Antibodies binding to proteins, viruses, or cells with high specificity and affinity can be readily prepared by immunization, which simply consists of injecting the antigen of interest into an experimental animal. Antibodies binding to small organic molecules, called haptens, are also accessible by covalently attaching haptens to a carrier protein, usually KLH (keyhole limpet... 

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