A study of the reactivity of S^(VI)–F containing warheads with nucleophilic amino-acid side chains under physiological conditions

Abstract

Sulfonyl fluorides (SFs) have recently emerged as a promising warhead for the targeted covalent modification of proteins. Despite numerous examples of the successful deployment of SFs as covalent probe compounds, a detailed exploration of the factors influencing the stability and reactivity of SFs has not yet appeared. In this work we present an extensive study on the influence of steric and electronic factors on the reactivity and stability of the SF and related S-F groups. While SFs react rapidly with N-acetylcysteine, the resulting adducts were found to be unstable, rendering SFs inappropriate for the durable covalent inhibition of cysteine residues. In contrast, SFs afforded stable adducts with both N-acetyltyrosine and N-acetyllysine; furthermore, we show that the reactivity of arylsulfonyl fluorides towards these nucleophilic amino acids can be predictably modulated by adjusting the electronic properties of the warhead. These trends were largely conserved when the covalent reaction occurred within a protein binding pocket. We have also obtained a crystal structure depicting covalent modification of the catalytic lysine of a tyrosine kinase (FGFR1) by the ATP analog 5'-O-3-((fluorosulfonyl)benzoyl)adenosine (m-FSBA). Highly reactive warheads were demonstrated to be unstable with respect to hydrolysis in buffered aqueous solutions, indicating that warhead reactivity must be carefully tuned to provide optimal rates of protein modification. Our results demonstrate that the reactivity of SFs complements that of more commonly studied acrylamides, and we hope that this work spurs the rational design of novel SF-containing covalent probe compounds and inhibitors, particularly in cases where a suitably positioned cysteine residue is not present.

The Application of Fluorine‐Containing Reagents in Structural Proteomics

“…The PMSF, however, was unstable in aqueous solutions. Grimster and co‐workers evaluated the reactivity of aryl SFs by using protected amino acids, and they demonstrated no reaction besides SF hydrolysis of the N‐acetylated serine. SFs are most reactive to N ‐acetylcysteine (NAC), but the product is susceptible to hydrolysis.…”

Fluorierte Reagenzien in der Strukturproteomik

“…Das PMSF war jedoch unter wässrigen Bedingungen instabil. Grimster und Mitarbeiter untersuchten die Reaktivität von Aryl‐SFs, indem sie geschützte Aminosäuren verwendeten, und zeigten, dass keine Reaktion außer der SF‐Hydrolyse von N‐acetyliertem Serin stattfand …”

“…Grimster und Mitarbeiter untersuchten die Reaktivitätv on Aryl-SFs,i ndem sie geschützte Aminosäuren verwendeten, und zeigten, dass keine Reaktion außer der SF-Hydrolyse von N-acetyliertem Serin stattfand. [38] SFs sind am reaktivsten gegen N-Acetylcystein (NAC), jedoch geht das entstehende Produkt eine Hydrolysereaktion ein. Die Reaktivitäten von SFs in Bezug auf N-Acetyltyrosin und N-a-Acetyllysin sind niedriger als die gegen NAC, generieren aber in beiden Fällen stabile Produkte.…”

Covalent Small Molecules as Enabling Platforms for Drug Discovery

“…Notably, lysine has been pursued intensively with multiple publications detailing lysine‐reactive warheads such as activated esters, sulfur(VI)‐based reactive centres (sulfonyl fluorides and fluorosulfates) and α,β‐unsaturated systems . Some of these warheads can exhibit surprising aqueous stability, supporting their use in biological systems . Additionally, these warheads can result in improved selectivity relative to structurally related reversible inhibitors .…”