β‑Fluorovinylsulfonamide as a Highly Reactivity- and Structure-Tunable Electrophile for Covalent Targeting of Proteins

Covalent targeting of proteins with small molecule probes
is a
powerful approach for analyzing and manipulating biological functions.
Recent advancements in the design of target selective covalent ligands
further expand the scope of accessible proteins in this approach.
Here we report β-fluorovinylsulfonamide (FVS) as a versatile
electrophile for the design of covalent ligands. FVS irreversibly
reacts with cysteine residues in proteins to afford stable vinyl sulfide
adducts. The reactivity of FVS is maintained with the introduction
of various substituents at the β-position, allowing for fine-tuning
of the electrophilic reactivity and flexible molecular design of FVS-based
covalent ligands. These reaction properties were leveraged in the
development of highly selective covalent ligands for RSK2 and BTK.
FVS compounds are also able to react with lysine residues in proteins
to form enamine-type reversible covalent adducts, which are susceptible
to hydrolysis under neutral aqueous conditions. This lysine reactivity
of FVS led to the discovery of FVS compound 53 as a reversible
covalent ligand for PFKL, which selectively reacts with K677 of PFKL
and allosterically upregulates PFKL activity in living cells.

利用小分子探针实现蛋白质的共价靶向，是分析与调控生物学功能的有力手段。近年来靶标选择性共价配体的设计进展进一步拓展了该策略可靶向的蛋白质范围。本文报道β-氟乙烯磺酰胺（β-fluorovinylsulfonamide, FVS）作为一类通用亲电基团用于共价配体的设计。FVS可与蛋白质中的半胱氨酸残基发生不可逆反应，生成稳定的乙烯硫醚加合物。在β位引入各类取代基后，FVS的反应活性得以保留，这使得我们能够精准调控其亲电反应活性，并灵活设计基于FVS的共价配体。我们利用该反应特性开发了针对RSK2与BTK的高选择性共价配体。此外，FVS类化合物还可与蛋白质中的赖氨酸残基反应，生成烯胺型可逆共价加合物，该类加合物在中性水溶液条件下易发生水解。基于FVS的赖氨酸反应活性，我们发现化合物53可作为PFKL的可逆共价配体，其能特异性靶向PFKL的K677位点，并在活细胞中变构上调PFKL的活性。