Side-Chain-Functionalized Polypeptides

Figure 1.6 The ionizable amino acids possess some of the most important side-chain functional groups for bioconjugate applications. The C- and N-terminal of each polypeptide chain also is included in this group.

Most of the other posttranslational modifications involving the N- or C-terminus (Table 1) as well as the side-chain functionalities (Table 2) of the polypeptide chains occur under the control of enzymes that also dictate the regioselectivity of such chemical transformations. This regioselectivity is difficult to attain by synthetic procedures. Sophisticated protection schemes are required when additional chemistry must be performed on preassembled peptides, unless enzymatic methods can be used to supplement the synthetic strategies. As a consequence, the use of suitably modified amino acids as synthons is generally the preferred approach as will be discussed in the following sections. 

The biological function of peptides and proteins depends on their native conformation. The side-chain functionalities of the a-amino acids that comprise peptides and proteins have profound effects on their properties. These functionalities reside in the 20 naturally occurring a-amino acids, which have different propensities for formation of the three major secondary structural conformations. 1 In addition to these naturally occurring a-amino acids whose primary structure enables the polypeptide to fold into a predictable secondary and tertiary structure, the incorporation of unnatural amino acids has opened important areas of research. 

In summary, protein molecules may contain up to nine amino acids that are readily derivatizable at their side chains aspartic acid, glutamic acid, lysine, arginine, cysteine, histidine, tyrosine, methionine, and tryptophan. These nine residues contain eight principal functional groups with sufficient reactivity for modification reactions primary amines, carboxylates, sulfhydryls (or disulfides), thioethers, imidazolyls, gua-nidinyl groups, and phenolic and indolyl rings. All of these side chain functional groups in addition to the N-terminal a-amino and the C-terminal a-carboxylate form the full complement of polypeptide reactivity within proteins (Fig. 12). 

Ogata et al. published several applications with graft copolymers of poly(7 benzyl-L-glutamate) onto suspension polymerized crosslinked polystyrene beads containing aminomethyl functions [39] (Scheme 6). 4- 7 7 3 benzyl ester group of the polypeptide side chain was converted either to benzyl amide by aminolysis or to free acid by hydrolysis and the effects... 

Chemical modification of amino acid side chain functionalities will also serve to cleave specific peptide bonds selectively. Chemical cleavage of a polypeptide chain exploits the unique reactivity of chemically modified side chains of particular amino acids in the labilization of adjacent peptide bonds by neighbouring group participation (68). The residues investigated so far for this purpose have been methionine, cysteine and the aromatic amino acids including tryptophan (438-440, 443). 

Much of protein engineering concerns attempts to explore the relationship between protein stmcture and function. Proteins are polymers of amino acids (qv), which have general stmcture +H3N—CHR—COO , where R, the amino acid side chain, determines the unique identity and hence the stmcture and reactivity of the amino acid (Fig. 1, Table 1). Formation of a polypeptide or protein from the constituent amino acids involves the condensation of the amino-nitrogen of one residue to the carboxylate-carbon of another residue to form an amide, also called peptide, bond and water. The linear order in which amino acids are linked in the protein is called the primary stmcture of the protein or, more commonly, the amino acid sequence. Only 20 amino acid stmctures are used commonly in the cellular biosynthesis of proteins (qv). 

Through combined effects of noncovalent forces, proteins fold into secondary stmctures, and hence a tertiary stmcture that defines the native state or conformation of a protein. The native state is then that three-dimensional arrangement of the polypeptide chain and amino acid side chains that best facihtates the biological activity of a protein, at the same time providing stmctural stabiUty. Through protein engineering subde adjustments in the stmcture of the protein can be made that can dramatically alter its function or stabiUty. 

To understand the function of a protein at the molecular level, it is important to know its three-dimensional stmcture. The diversity in protein stmcture, as in many other macromolecules, results from the flexibiUty of rotation about single bonds between atoms. Each peptide unit is planar, ie, oJ = 180°, and has two rotational degrees of freedom, specified by the torsion angles ( ) and /, along the polypeptide backbone. The number of torsion angles associated with the side chains, R, varies from residue to residue. The allowed conformations of a protein are those that avoid atomic coUisions between nonbonded atoms. 

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