Nonnatural Amino Acids for Site-Specific Protein Conjugation

Abstract

Over the years, several chemical reactions have been developed that enable the covalent conjugation of synthetic molecules to natural proteins. The resulting bioconjugates have become important tools in the study of natural proteins. Furthermore, they form a new class of protein-based pharmaceuticals and biomaterials. However, classical bioconjugation reactions to natural amino acids suffer from poor site-specificity. To overcome this problem, a variety of uniquely reactive non-natural amino acids have recently been designed. These can be incorporated into proteins by specifically engineered bacterial strains. Such reactive non-natural amino acids create new possibilities for bio-orthogonal conjugation to proteins. This review first gives an overview of the various methods for site-specific introduction of non-natural amino acids into proteins. Both semisynthetic and entirely recombinant methods are addressed. Then, a detailed description is given of the reactive non-natural amino acids that have already been recombinantly introduced into proteins. The bio-orthogonal reactions that can be used for conjugation to these reactive non-natural amino acids are also discussed. These include the alkyne/azide 'click' reaction, carbonyl condensations, Michael-type additions, and Mizoroki-Heck substitutions.

Staudinger Ligation as a Method for Bioconjugation

“…The azide is preferred as the chemical handle because of the high degree of oxidation of the phosphine moiety in aqueous solutions. The azide can be introduced into a protein chemically, that is, by a diazotransfer reaction [39] or biochemically, either by using genetic engineering for the incorporation of azide-containing amino acids into proteins [40] or by post-translational modifications. The latter concept was used in a combined effort by the research groups of Tirrell and Bertozzi.…”

Vinyl Sulfone: A Multi-Purpose Function in Proteomics

“…This classical residue-specific modification chemistry, however, is rarely sufficiently selective to distinguish one residue within a sea of chemical functionality and for this reason more intricate approaches have been developed in recent times to introduce a unique chemical handle in the target protein that is orthogonal to the remainder of the proteome (Hackenberger & Schwarzer, 2008). Direct incorporation of non-canonical amino acids into proteins via the subversion of the biosynthetic machinery is an attractive means of introducing selectively new functionality by either a site-specific or residue-specific manner (Beatty & Tirrell, 2009;de Graaf et al, 2009;Johnson et al, 2010;Liu & Schultz, 2010;Voloshchuk & Montclare, 2010;Young & Schultz, 2010) that in combination with recent and notorious advances in bioorthogonal reactions (nucleophilic addition to carbonyl, 1,3-dipolar cycloaddition reactions, Diels-Alder reactions, olefin cross-metathesis reactions and palladium-catalyzed cross-coupling reactions) has allowed an exquisite level of selectivity in the covalent modification of proteins (Wiltschi & Budisa, 2008;Sletten & Bertozzi, 2009;Lim & Lin, 2010;Tiefenbrunn & Dawson, 2010). In spite that major technical challenges have been overcome, a prodigious amount of lab work and the concurrently optimization of a larger set of parameters is normally required for those advanced and selective methodologies in comparison with conventional organic reaction development.…”

Selective chemical protein modification

“…Potential transformations, if they are to be relevant, are moulded by the need for biologically ambient conditions (that is, o37°C, pH 6-8, aqueous solvent) so as not to disrupt protein architecture and/or function. Ideally, this should proceed with near total conversion to generate homogenous constructs [2][3][4] . The applications of modified proteins are many; they are as varied as the in vivo tracking of protein-fluorophore conjugates 5 to the polyethylene glycol (PEG)ylation of therapeutic proteins to reduce immunogenicity 6 , from the production of materials with novel properties 7 to probing the mechanism of pathological enzymes 8 .…”