Protein modification via alkyne hydrosilylation using a substoichiometric amount of ruthenium(ii) catalyst

Abstract

Transition metal catalysis has emerged as a powerful strategy to expand synthetic flexibility of protein modification. Herein, we report a cationic Ru(ii) system that enables the first example of alkyne hydrosilylation between dimethylarylsilanes and O-propargyl-functionalized proteins using a substoichiometric amount or low-loading of Ru(ii) catalyst to achieve the first C-Si bond formation on full-length substrates. The reaction proceeds under physiological conditions at a rate comparable to other widely used bioorthogonal reactions. Moreover, the resultant gem-disubstituted vinylsilane linkage can be further elaborated through thiol-ene coupling or fluoride-induced protodesilylation, demonstrating its utility in further rounds of targeted modifications.

Fig. 1. (a) General metal-mediated protein modification protocols using excess of metal catalysts and (b) our approach via Ru(ii)-catalyzed alkyne hydrosilylation.

Scheme 1. Hydrosilylation of peptide 27 with biotinylated hydrosilane 29. ^aConversion (%) determined by HPLC.

Scheme 2. (a) Tandem hydrosilylation and hydrazine condensation reaction. (b) gem-Disubstituted vinylsilane reactivity under thiol–ene coupling conditions and fluoride-induced protodesilylation, giving thioether 34 and O-allyl 35 in 81% and 85% isolated yields, respectively. ^aConversion (%) determined by HPLC.

Fig. 2. Selective labeling of O-propargyl (OP) modified protein substrates via hydrosilylation. (a) The structures of unnatural amino acids 37 and 38 and other reagents used in this study. (b) Modification of solvent-exposed lysine residues on lysozyme (Lyz) with 36 and subsequent labeling with 29. (c) Selective labeling of OP-Lyz via hydrosilylation with 29 and 1. Lyz (–) and OP-Lyz (+) (125 μM) was independently incubated with 29 (250 μM) and 1 (10 mol%) for 2 h at 37 °C and the presence of biotinylated protein was detected by Western blot using α-biotin-HRP conjugated antibody. (d) Genetic encoding and fluorescent labeling of 37 via hydrosilylation. (e) In-gel fluorescence demonstrating specific labeling of sfGFP-37 ₁₅₀ with 39. In (c) and (e), equal protein loading was verified by Coomassie staining.

Scheme 3. (a) Site-specific incorporation of 40 into C2Am via C2Am-Dha and (b) subsequent hydrosilylation with 8. Found masses corresponding to OP-C2Am (16 394 Da), Oxidized-OP-C2Am (16 435 Da), and VS-C2Am (16 704 Da).