Protein site-selective modification

B. G. Davis, R. C. Lloyd, and J. Bryan Jones, Controlled site-selective glycosylation of proteins by a combined site-directed mutagenesis and chemical modification approach, J. Org. Chem., 63 (1998) 9614-9615. 

Relative to acylation strategies, modification via reductive alkylation preserves the overall charge state (and thus the solubility) of the protein. In general, this technique also suffers from poor site selectivity. 

By far the most widely used methods for site-selective protein modification target cysteine residues. In contrast to lysine, cysteine is one of the rarest amino acids (10), and it is unusual to find it in the reduced form on protein surfaces. Thus, it is often possible to introduce a uniquely reactive cysteine residue using genetic methods. Similar to lysine-modifying reagents, many cysteine-reactive small molecules are commercially available because of the success of this overall strategy. 

The unexpected specificity which can be achieved with functional group modification reagents is an apparent consequence of the native protein s ability to impose a unique chemical environment on a given amino acid under a given set of experimental conditions. It is important to emphasize that the site-specific modification of a protein is a kinetic phenomenon and selective modifications result from the ability of the protein to alter the reaction rate of a single residue under one clearly defined condition of pH, ionic strength and temperature. For example, it is entirely possible that if, at pH 7.0, one lysine residue is substantially more reactive than either free lysine or other lysine residues in the protein it may well be less reactive than these at pH 9.0. 

Drugs with simple or common pharmacophores cannot discriminate small differences among binding sites. Pharmacophores for ADME/Tox proteins are generally not complex [39], By contrast, drugs with complex or unusual pharmacophores have the potential to bind selectively to target proteins. Therefore chemical modification intended to increase the complexity of the pharmacophore is one way to avoid unfavorable effects arising from promiscuous interactions. 

Circular dichroism (CD) has played an important role in our studies on the modification of enzymes and hormones with Co(III). The objective of these studies has been to incorporate selectively substitution inert metal ions at specifically modified sites in proteins as probes of biological function. Significant information concerning the catalytic mechanism of carboxypeptidase A (CPA) (1) has been obtained from a site specific modification of tyrosine 248 with Co(III) (2). The method developed for CPA has been extended to other enzymes and hormones in order to devg op an improved method for incorporating stable radioisotopes t Co) into proteins. The substitution-inertness of Co(III) provides the necessary stability in these derivatives (3). 

Fig. 10.3-2 Common strategies for protein bioconjugation, targeting lysine, cysteine, aspartic acid, and glutamic acid residues. In most situations, only cysteine modification reactions are site selective.

Transition metal-mediated reactions provide an exceptionally powerful set of tools for site-selective protein modification. These strategies have had a striking impact on organic synthesis over the last three decades due to the ability of transition metals to activate otherwise unreactive functional groups with... 

The application of site-specific modification of enzymes and other proteins has become increasingly common, and a wide range of chemical classes is available from which to select or design a reagent for exploring a particular enzyme. Structural similarity to the natural ligand is always desirable to ensure target sped-... 

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