Re‐Engineering the Genetic Code: Combining Molecular Biology and Organic Chemistry (2003)

Imagine…︁ if the biomachinery of nature accepted every uncoded amino acid site-specifically as desired. Spectacular advances in breaking the genetic code to expand the set of amino acids available for protein biosynthesis are highlighted, and examples of the post-translational chemical modification of proteins containing these nonnatural amino acids are presented (see scheme). Major breakthroughs have been made in the biosynthetic incorporation of noncoded amino acids into proteins.1 The research groups of Schultz,2 Yokoyama,3 and Tirrell4 in particular have shown that the diversity of proteins may be expanded to include a far wider range of residues than the 20 proteogenic amino acids by using nature's own biosynthetic machinery. Examples are the inclusion in proteins of amino acids bearing functional groups that serve as photoactivatable cross-linkers, spin labels, fluorescence labels, or metal chelators, of glycosylated amino acids, and of a biotinylated amino acid for affinity labeling.5 Herein we will focus on amino acids labeled with biologically inert but chemically reactive (bioorthogonal) functional groups that allow the selective post-translational covalent modification of proteins (Scheme 1). The incorporation of such reactive amino acids into proteins brings together the worlds of molecular biology and organic chemistry to open up a fertile research area with many applications. The incorporation of nonproteogenic amino acids into proteins has been successfully carried out in a wide range of systems, both in vitro and in vivo (Figure 1). tRNA can be charged with an amino acid outside or inside the cell, either by stoichiometric chemical ligation or by catalytic means. Because of the different options available and requirements posed, a single method does not yet exist for the incorporation of any given amino acid. However, investigations are being directed toward self-perpetuating living-model systems. Wonderful examples have been set in studies involving the acetylcholine receptor in Xenopus oocytes6 and cultured human neurons.7 tRNAs can be chemically charged extracellularly with a noncoded amino acid of choice. The success of this method depends on the ability of the ribosome to handle the resulting AA-tRNA for translation. However, the absolute amounts of protein thus obtained are generally limited because any single tRNA has to be stoichiometrically ligated and delivered to the cells. AA-tRNAs produced catalytically are less limited in supply, but bring about other considerations. When the substrate tolerance of native aminoacyl-tRNA synthases (aaRSs) is exploited, intracellular acylation results in the proteome-wide expression of modified proteins. For example, Met-aaRS allows selenomethione as well as amino acids with less closely related side chains to be loaded.8 When the site-specific incorporation of a noncoded amino acid is intended, an aaRS suitable for the acylation of a specific tRNA that can not be loaded with coded amino acids by the target expression system is required. Together with a plasmid specifying the desired protein product, these orthogonal tRNA/aaRS pairs must then be transfected into the host system. Several such pairs have been described that mediate the incorporation of the desired amino acid at the specified position by acylating suppressor tRNAs, thus enabling read-through at amber codons (pathway A, Figure 1),9 or take advantage of specific Watson–Crick base pairing at the third (wobble) codon residue (pathway B, Figure 1).4 This latter method does, however, result in the incorporation of the nonnatural amino acid into other cellular proteins in which the redundant codon is used to code for the “expected” natural amino acid by nature itself. Alternatively, the use of four-base codons in E. coli has been exploited, whereby nonnatural amino acids are incorporated by the cognate four-base-anticodon AA-tRNAs within the context of a three-base coding frame (pathway C, Figure 1).10 Another possibility is the use of ribozymes to acylate tRNA.11 Currently this is done outside the cell, but in the future ribozymes may be found to tolerate even more unusual side chains than altered aaRSs do. The in vivo application of ribozymes will certainly be welcomed. Biochemical strategies for the incorporation of nonproteogenic amino acids into proteins. As expected, the less-complicated and better-documented translational machinery of prokaryotes has allowed more rapid progress to be made in bacteria than has been possible in eukaryotes. It was even shown that the enzymes required for a complete biosynthetic route to p-aminophenylalanine (pAF) could be added to E. coli, which had also been transfected with a tRNA/aaRS pair specific for this amino acid,12 to give a truly autonomous system with a repertoire of 21 amino acids to build up proteins. However, issues of post-translational modification or folding may require eukaryotic expression. To this end, cell lines may be supplemented with a prokaryotic orthogonal tRNA/aaRS pair. This has been demonstrated in cell-free systems13 and may prove especially useful when the toxicity of the amino acid or the delivery of chemically ligated AA-tRNA could preclude a living-cell approach. Recent progress includes the introduction of orthogonal tRNA/aaRS pairs into living eukaryotic cells, such as yeast1 and cultured-human-cell lines.3, 14 To date over 100 nonproteogenic amino acids have been incorporated into proteins biosynthetically.5 To emphasize the potential of proteins bearing chemically reactive nonnatural amino acids, the azidohomoalanine (1) should be highlighted (Scheme 1). The small, inert, and biocompatible azido group in 1 has shown its versatility in further post-translational bioorthogonal (or abiotic) chemical transformations. The research groups of Bertozzi and Tirrell substituted the eight methionines in the reporter protein murine dihydrofolate reductase (mDHFR) for 1 and selectively labeled this protein with antigenic FLAG peptides15 or fluorophoric coumarin dyes16 through a Staudinger ligation reaction in crude cell lysates (Scheme 2). Nonnatural amino acids bearing a bioorthogonal chemical handle. Covalent functionalization of an azide-labeled protein through bioorthogonal reactions. The copper(I)-catalyzed azide–alkyne [3+2] dipolar cycloaddition (a so-called click reaction17) in proteins is also noteworthy (Scheme 2). Link and Tirrell incorporated 1 into the outer-membrane protein C and subsequently allowed the azide groups to react with a biotinylated alkyne reagent, as could be monitored after staining with fluorescent avidin.18 Although not strictly bioorthogonal owing to the presence of many endogenous keto groups in living cells (e.g. in sugars), p-acetyl-L-phenylalanine (3) has also been used for in vivo chemical decoration with hydrazide-containing exogenous reagents.19 We would like to end this Highlight with some hints for future directions. Expanding the still very limited palette of truly bioorthogonal reactions is an important task for synthetic organic chemists. As an example, the transition-metal-mediated and mild Suzuki cross-coupling reaction between the pseudo proteogenic p-iodo-L-phenylalanine (4) and an exogenous aryl or alkyl boronic acid can be envisaged.20 The pseudo proteogenic homoallylglycine (5) and homopropargylglycine (6) might be used for further post-translational chemical modification by the very mild alkene-, alkyne-, or enyne-metathesis reactions.21 Intramolecular metathesis reactions within a protein by incorporation of multiple homoallylglycine residues (5) would lead to cross-links that could serve as highly stable disulfide-bridge isosteres. Finally, one could envisage such cross-links resulting from a [3+2]-cycloaddition reaction between the azide-containing amino acids 1 or 2 and the alkyne-containing amino acid 6. A slight preview of the enhanced stability of proteins containing non-natural amino acids has been given by incorporating trifluoroleucine instead of leucine into leucine zipper proteins, which led to higher temperature and denaturant tolerance.22 In conclusion, it is clear that exciting developments are taking place at the interface of molecular biology and organic chemistry. The recently discovered biotechnological tools that make it possible to include noncoded amino acids in proteins give access to tailor-made proteins for which numerous applications can be envisaged. The results obtained so far have whetted our appetite for the things to come, and we expect them to have a heavy impact on several fields, such as proteomics, (bio)materials, biomedical research, (combined homogeneous and bio)catalysis, and synthesis.