Advances in chemical protein modification.
Chemical protein modification has emerged as an invaluable tool for the development of modified proteins. The complementary use of both genetic and chemical methods has provided a large toolbox that allows the preparation of almost unlimited protein constructs with either natural or synthetically modified residues. Such a protein chemodiversity, usually achieved after translation and commonly referred to as post-translational protein modifications (PTMs), is often responsible for the vast biodiversity found in nature. These modifications include acylation, methylation, phosphorylation, sulfation, farnesylation, ubiquitination, and glycosylation, among others, and play a pivotal role in important cellular processes including trafficking, differentiation, migration, and signaling. Consequently, reproducing in a highly efficient and controlled way such natural modifications of proteins (by introducing natural PTMs) would provide an invaluable tool to study their precise function. Additionally, the possibility offered by the introduction and (bio)orthogonal modification of unnatural moieties/amino acids (usually improving the properties of natural PTMs during isolation, analysis, and processing) makes site-selective modification of proteins a key tool for interrogating and intervening biological systems both in vitro and in vivo. Given the range of chemical modification methods available, it is now possible to decide which residue to target and which modification to attach in order to confer the desired property/function (affinity probes, fluorophores, reactive tags, etc.). For example, increasing the circulation half-life of a therapeutic protein may be achieved by the addition of polyethylene glycol (PEG). On the other hand, the use of a spectroscopic label to monitor biomolecule distribution in vivo enables the construction of highly selective imaging agents. Despite the vast progress in the field of bioconjugation chemistry, scientists still face many challenges, not only synthetically but also from a processing, manufacturing, safety, and stability perspective. A number of methods have been developed and applied for the modification of particular proteins and therefore may not be applicable to any protein of interest. Thus, there remains a need for the development of complementary reactions for the site-selective chemical modification of proteins that are mild, efficient, and robust. Several reviews covering different aspects of the chemical synthesis of proteins, from general native chemical ligation strategies and the modification of endogenous amino acids to more specialized topics such as click modification protocols, the introduction of particular PTMs including glycosylation, PEGylation, and polymerization of protein-based initiators, and the challenging labeling of a specific protein of interest in a complex cellular mixture using the so-called “bioorthogonal” reactions, have been published during the past decade. While the later publications describe different protein syntheses/modifications in detail, the aim of the present review is not to be an exhaustive survey of all available bioconjugation methodologies but to discuss recent chemical strategies for the site-selective modification of proteins such as fast sulfur exchange or stable thioether formation, photo and metal-free cycloadditions, and other particularly challenging metal-mediated protocols. This review will be divided into two sections: transition metal-free and transition metal-mediated approaches. For clarity, we will use the following terminology throughout this manuscript: residue/amino acid/site-selective (or simply site-selective) reactions are those transformations that preferentially modify one amino acid residue over the others (e.g., cysteine versus lysine) and, thus, can be considered examples of chemoselective reactions; on the other hand transformations described as regioselective preferentially modify only one of a set of the same amino acid, in particular when more than one is present in the same molecule (e.g., solvent-exposed lysine versus internal lysine).