Research Article

Posttranslational mutagenesis: A chemical strategy for exploring protein side-chain diversity

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Science  04 Nov 2016:
Vol. 354, Issue 6312, aag1465
DOI: 10.1126/science.aag1465

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Radicals push proteins beyond genes

Chemically modifying proteins after their translation can expand their structural and functional roles (see the Perspective by Hofmann and Bode). Two related methods describe how to exploit free radical chemistry to form carbon-carbon bonds between amino acid residues and a selected functional group. Wright et al. added a wide range of functional groups to proteins containing dehydroalanine precursors, with borohydride mediating the radical chemistry. Yang et al. employed a similar approach, using zinc in combination with copper ions. Together, these results will be useful for introducing functionalities and labels to a wide range of proteins.

Science, this issue pp. 597 and 623; see also p. 553

Structured Abstract

INTRODUCTION

Natural posttranslational modifications (PTMs) to proteins expand the chemical groups available to proteins. The ability to expand posttranslational functional group diversity in an unbounded manner could, in principle, allow exploration and understanding of how these groups modulate biological function. Natural PTMs feature bonds to heteroatoms (non-carbon) made at the γ (Cys Sγ, Thr Oγ, Ser Oγ) or ω (Lys Nω, Tyr Oω) positions of side chains. However, one of the central features of biomolecules is C (sp3)–C (sp3) bond formation. Because all amino acid side chains contain this C–C bond, mastering its construction on proteins could allow free-ranging structural alteration of residues in proteins (both natural and unnatural).

RATIONALE

In principle, C (sp3)–C (sp3) disconnections at the β,γ C–C bond would allow the chemical installation of a wide range of amino acid functionalities. Traditional two-electron chemistry (using nucleophiles and electrophiles) requires reagents that are often incompatible with biological substrates and/or water. Free radicals can be tolerant of aqueous conditions and unreactive (and thereby compatible) with the majority of functionality present in biomolecules. We therefore reasoned that mild, carbon-centered free radical chemistry would be enabled by matching free-radical reactivity with a suitable, uniquely reactive functional group partner that possesses a chemical affinity for such singly occupied molecular orbitals. The amino acid residue dehydroalanine (Dha) can be readily introduced in a site-selective manner genetically, biosynthetically, or chemically; upon reaction with a suitable radical, Dha would favorably generate a stabilized Cα radical 1. Suitable “quenching” of the central Cα radical intermediate 1 generated after formation of the critical C–C bond would thus allow “chemical mutation” of the side chain.

RESULTS

A range of precursor halides (R-Hal, Hal = I or Br) allowed the creation of radicals R•. These radicals reacted selectively with Dha in peptides and proteins with excellent site selectivity and regioselectivity (>98% β,γ) and typically with a diastereoselectivity of ~1:1. Combined use of R-Hal with NaBH4 under low-oxygen conditions suppressed competing oxidation and disubstitution side reactions of intermediates 1. This allowed for rapid reactions (typically 30 min) with improved efficiency across a range of representative protein types and scaffolds (all α, α/β folds, all β, receptor, enzyme, antibody). The reactivity of primary, secondary, and tertiary alkyl halides allowed installation of natural, simple hydrophobic residue side chains. Charged or polar protic (e.g., OH, NH) functionality in amino acid side chains was also possible. Even the use of side-chain reagents in unprotected form proved possible, thus highlighting not only exquisite chemoselectivity but also compatibility with common biological functional groups. These transformations enabled the creation of a wide diversity of natural, unnatural, posttranslationally modified (methylated, glycosylated, phosphorylated, hydroxylated) and labeled (fluorinated, isotopically labeled) side chains, as well as difficult-to-access but important residues in proteins (e.g., methyl-Arg, citrulline, ornithine, methyl-Gln, phospho-Ser).

CONCLUSION

This approach to chemical editing of amino acid residues, outside of the rigid constraints of the ribosome and enzymatic processing, may prove to be a general technology for accessing diverse, previously unattainable proteins.

Posttranslational chemical mutagenesis through C (sp3)–C (sp3) bond-forming radical reactions.

Modification in a protein after translation using C–C bond formation allows construction of many side chains, not just the modification of existing natural amino acid residues. t-Leu, tert-leucine; Orn, ornithine; Cit, citrulline.

Abstract

Posttranslational modification of proteins expands their structural and functional capabilities beyond those directly specified by the genetic code. However, the vast diversity of chemically plausible (including unnatural but functionally relevant) side chains is not readily accessible. We describe C (sp3)–C (sp3) bond-forming reactions on proteins under biocompatible conditions, which exploit unusual carbon free-radical chemistry, and use them to form Cβ–Cγ bonds with altered side chains. We demonstrate how these transformations enable a wide diversity of natural, unnatural, posttranslationally modified (methylated, glycosylated, phosphorylated, hydroxylated), and labeled (fluorinated, isotopically labeled) side chains to be added to a common, readily accessible dehydroalanine precursor in a range of representative protein types and scaffolds. This approach, outside of the rigid constraints of the ribosome and enzymatic processing, may be modified more generally for access to diverse proteins.

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