A protein functionalization platform based on selective reactions at methionine residues

Abstract

Nature has a remarkable ability to carry out site-selective post-translational modification of proteins, therefore enabling a marked increase in their functional diversity¹. Inspired by this, chemical tools have been developed for the synthetic manipulation of protein structure and function, and have become essential to the continued advancement of chemical biology, molecular biology and medicine. However, the number of chemical transformations that are suitable for effective protein functionalization is limited, because the stringent demands inherent to biological systems preclude the applicability of many potential processes². These chemical transformations often need to be selective at a single site on a protein, proceed with very fast reaction rates, operate under biologically ambient conditions and should provide homogeneous products with near-perfect conversion^2-7. Although many bioconjugation methods exist at cysteine, lysine and tyrosine, a method targeting a less-explored amino acid would considerably expand the protein functionalization toolbox. Here we report the development of a multifaceted approach to protein functionalization based on chemoselective labelling at methionine residues. By exploiting the electrophilic reactivity of a bespoke hypervalent iodine reagent, the S-Me group in the side chain of methionine can be targeted. The bioconjugation reaction is fast, selective, operates at low-micromolar concentrations and is complementary to existing bioconjugation strategies. Moreover, it produces a protein conjugate that is itself a high-energy intermediate with reactive properties and can serve as a platform for the development of secondary, visible-light-mediated bioorthogonal protein functionalization processes. The merger of these approaches provides a versatile platform for the development of distinct transformations that deliver information-rich protein conjugates directly from the native biomacromolecules.

Figure 1. The evolution of a methionine-selective protein functionalization strategy

(a) Existing protein functionalization tactics, and the potential for methionine-selective bioconjugation; R = peptide or protein, R¹ = various organic groups, R² = aryl or ester group (b) Functionalized hypervalent iodine reagents enable methionine-selective protein modification, leading to methionine-based bioorthogonal protein diversification. X = leaving group; R³ = functional payload.

Figure 2. Evolution of a methionine-selective bioconjugation strategy.

(a) Initial results for functionalization at methionine with hypervalent iodine reagents. (b) Optimal process for the thiourea-accelerated methionine selective bioconjugation of exenatide.

Figure 3. Scope of the methionine-selective bioconjugation strategy.

(a) Range of polypeptide/protein substrates. (b) Functionalized iodonium reagents compatible with the bioconjugation. (c) A stimuli responsive strategy for reversing methionine bioconjugation.

Figure 4. Exploiting the multi-faceted reactivity of the protein-sulfonium conjugate.

(a) A photocatalytic design plan for secondary protein diversification. (b) A system for photo-redox mediated reduction of the sulfonium conjugate and examples of the substrate scope; in the case of 17e, further reduction of C–S bond in 13 was not observed³⁵. Interestingly, the reduction of 13 proceeds in light, without the Ir-photocatalyst, but the yield was greatly diminished. (c). Secondary protein functionalization via photoredox radical cross coupling between diazo sulfonium-protein conjugate and C-4 substituted Hantzsch ester.