Abstract
Site‐directed (gene) mutagenesis has been the most useful method available for the conversion of one amino acid residue of a given protein into another. Until relatively recently, this strategy was limited to the twenty standard amino acids. The ongoing maturation of stop codon suppression and related technologies for unnatural amino acid incorporation has greatly expanded access to nonstandard amino acids by expanding the scope of the translational apparatus. However, the necessity for translation of genetic changes restricts the diversity of residues that may be incorporated. Herein we highlight an alternative approach, termed post‐expression mutagenesis, which operates at the level of the very functional biomolecules themselves. Using the lens of retrosynthesis, we highlight prospects for new strategies in protein modification, alteration, and construction which will enable protein science to move beyond the constraints of the “translational filter” and lead to a true synthetic biology.
Highlights
Retrosynthetic Analysis of Protein ModificationTo overcome the limitations in generality of early strategies for chemical approaches to mutagenesis (lack of generality in the side chains which can be accessed and sometimes harsh reaction conditions), the application of retrosynthetic logic, as a convenient tool for allowing one to escape occasional subjective narrowness of design,[13] helps to identify further disconnections and thence synthons
Site-directed mutagenesis has been the most useful method available for the conversion of one amino acid residue of a given protein into another
Through the suppression of certain codons[3] with tRNAs loaded with unscience to move beyond the constraints of the “translational filter” and lead to a true synthetic biology
Summary
To overcome the limitations in generality of early strategies for chemical approaches to mutagenesis (lack of generality in the side chains which can be accessed and sometimes harsh reaction conditions), the application of retrosynthetic logic, as a convenient tool for allowing one to escape occasional subjective narrowness of design,[13] helps to identify further disconnections and thence synthons. For example, whilst other Cb electrophile tag-andmodify reactions have been explored more broadly in proteins (e.g., Cb-Seg: new nucleophile[15] or Dha: as a Cb electrophile for Sg,[16] and Seg[17]) the potential for a Cb nucleophile to react with a heteroatom electrophile (an inverted polarity disconnection) has not yet been described. In this Minireview, we seek to explore the potential generality of future disconnections (and putative yet unrealized reactions) further. By examining a generic polypeptide backbone two broadly defined disconnections are immediately apparent (Scheme 1): amide backbone (disconnections F–H) and side-chain disconnections (disconnections A–E and I)
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