Profiling sulfur(VI) fluorides as reactive functionalities for chemical biology tools and expansion of the ligandable proteome

Here, we report a comprehensive profiling study of sulfur(VI) fluorides (SVI-F) in the context of multiple chemical biology applications and illustrate that these motifs present an exciting opportunity to develop tools for a wide scope of protein targets. SVI-Fs are reactive functionalities that offer utility for targeting almost any protein, as they can modify multiple residues including Lys, Tyr, His and Ser. A panel of SVI-F functionalities were studied with respect to hydrolytic stability and reactivity with nucleophilic amino acids. Subsequently, the reactivity of SVI-Fs with CAII and kinase proteins was investigated, in the context of both fragment binders and optimized probes. Finally, the performance of the SVI-F panel in chemoproteomic workflows was analyzed. The studies provided an in-depth understanding of the hydrolytic stability, protein reactivity and chemoproteomic utility of SVI-F functionalities that are suitable for direct incorporation into chemical tools. Such insights offer a valuable guide for the prospective design of SVI-F-containing ligands for various chemical biology workflows and demonstrate the wide range of proteins that SVI-Fs can capture, thus highlighting the opportunity for SVI-Fs to expand the liganded proteome

Efficient ligand discovery using sulfur(VI) fluoride reactive fragments

Sulfur(VI) fluorides (SFs) have emerged as valuable electrophiles for the design of "beyond-cysteine" covalent inhibitors and offer potential for expansion of the liganded proteome. Since SFs target a broad range of nucleophilic amino acids, they deliver an approach for the covalent modification of proteins without requirement for a proximal cysteine residue. Further to this, libraries of reactive fragments present an innovative approach for the discovery of ligands and tools for proteins of interest by leveraging a breadth of mass spectrometry analytical approaches. Herein, we report a screening approach that exploits the unique properties of SFs for this purpose. Libraries of SF-containing reactive fragments were synthesized, and a direct-to-biology workflow was taken to efficiently identify hit compounds for CAII and BCL6. The most promising hits were further characterized to establish the site(s) of covalent modification, modification kinetics, and target engagement in cells. Crystallography was used to gain a detailed molecular understanding of how these reactive fragments bind to their target. It is anticipated that this screening protocol can be used for the accelerated discovery of "beyond-cysteine" covalent inhibitors

Cross-Dehydrogenative Couplings between Indoles and β-Keto Esters : Ligand-Assisted Ligand Tautomerization and Dehydrogenation via a Proton-Assisted Electron Transfer to Pd(II)

Cross-dehydrogenative coupling reactions between -ketoesters and electron-rich arenes, such as indoles, proceed with high regiochemical fidelity with a range of -ketoesters and indoles. The mechanism of the reaction between a prototypical -ketoester, ethyl 2-oxocyclopentanonecarboxylate and N-methylindole, has been studied experimentally by monitoring the temporal course of the reaction by 1H NMR, kinetic isotope effect studies, and control experiments. DFT calculations have been carried out using a dispersion-corrected range-separated hybrid functional (B97X-D) to explore the basic elementary steps of the catalytic cycle. The experimental results indicate that the reaction proceeds via two catalytic cycles. Cycle A, the dehydrogenation cycle, produces an enone intermediate. The dehydrogenation is assisted by N-methylindole, which acts as a ligand for Pd(II). The compu-tational studies agree with this conclusion, and identify the turnover-limiting step of the dehydrogenation step, which involves a change in the coordination mode of the -keto ester ligand from an O,O’-chelate to an C-bound Pd enolate. This ligand tautom-erization event is assisted by the -bound indole ligand. Subsequent scission of the ’-C–H bond takes place via a proton-assisted electron transfer mechanism, where Pd(II) acts as an electron sink and the trifluoroacetate ligand acts as a proton acceptor, to pro-duce the Pd(0) complex of the enone intermediate. The coupling is completed in cycle B, where the enone is coupled with indole. Pd(TFA)2 and TFA-catalyzed pathways were examined experimentally and computationally for this cycle, and both were found to be viable routes for the coupling step

Profiling Sulfur(VI) Fluorides as Reactive Functionalities for Chemical Biology Tools and Expansion of the Ligandable Proteome

Chemical probes are valuable tools to explore the function of proteins. Incorporation of electrophiles into small molecules enables covalent capture of protein interactions and provides access to powerful technologies including chemoproteomic profiling and reactive fragment screening. Current approaches have been largely limited to protein pockets containing cysteine, so establishing strategies to target other amino acid residues is essential to expanding the applicability across the proteome. Here, we profiled sulfur(VI) fluorides (SVI-F) as reactive functionalities that can modify multiple residues including Lys, Tyr, His and Ser, thus offering utility for targeting almost any protein. These studies provided an in-depth understanding of SVI-F functionalities, including hydrolytic stability, protein reactivity and utility in chemoproteomics. Such insights offer a valuable guide for the prospective design of SVI-F-containing ligands for various chemical biology workflows and illustrate the wide range of proteins that SVI-Fs can capture, thus highlighting the opportunity for SVI-Fs to expand the liganded proteome

Cross-Dehydrogenative Couplings between Indoles and β‑Keto Esters: Ligand-Assisted Ligand Tautomerization and Dehydrogenation via a Proton-Assisted Electron Transfer to Pd(II)

Cross-dehydrogenative coupling reactions between β-ketoesters and electron-rich arenes, such as indoles, proceed with high regiochemical fidelity with a range of β-ketoesters and indoles. The mechanism of the reaction between a prototypical β-ketoester, ethyl 2-oxocyclopentanonecarboxylate, and <i>N</i>-methylindole has been studied experimentally by monitoring the temporal course of the reaction by ¹H NMR, kinetic isotope effect studies, and control experiments. DFT calculations have been carried out using a dispersion-corrected range-separated hybrid functional (ωB97X-D) to explore the basic elementary steps of the catalytic cycle. The experimental results indicate that the reaction proceeds via two catalytic cycles. Cycle A, the dehydrogenation cycle, produces an enone intermediate. The dehydrogenation is assisted by <i>N</i>-methylindole, which acts as a ligand for Pd­(II). The computational studies agree with this conclusion, and identify the turnover-limiting step of the dehydrogenation step, which involves a change in the coordination mode of the β-keto ester ligand from an <i>O</i>,<i>O</i>′-chelate to an α-C-bound Pd enolate. This ligand tautomerization event is assisted by the π-bound indole ligand. Subsequent scission of the β′-C–H bond takes place via a proton-assisted electron transfer mechanism, where Pd­(II) acts as an electron sink and the trifluoroacetate ligand acts as a proton acceptor, to produce the Pd(0) complex of the enone intermediate. The coupling is completed in cycle B, where the enone is coupled with indole. Pd­(TFA)₂ and TFA-catalyzed pathways were examined experimentally and computationally for this cycle, and both were found to be viable routes for the coupling step

Profiling Sulfur(VI) Fluorides as Reactive Functionalities for Chemical Biology Tools and Expansion of the Ligandable Proteome

Here, we report a comprehensive profiling of sulfur(VI) fluorides (SVI-Fs) as reactive groups for chemical biology applications. SVI-Fs are reactive functionalities that modify lysine, tyrosine, histidine, and serine sidechains. A panel of SVI-Fs were studied with respect to hydrolytic stability and reactivity with nucleophilic amino acid sidechains. The use of SVI-Fs to covalently modify carbonic anhydrase II (CAII) and a range of kinases was then investigated. Finally, the SVI-F panel was used in live cell chemoproteomic workflows, identifying novel protein targets based on the type of SVI-F used. This work highlights how SVI-F reactivity can be used as a tool to expand the liganded proteome