Assessing Squarates as Amine-Reactive Probes

Area of Science:

• Chemical Biology
• Organic Chemistry
• Biochemistry

Background:

• Covalent probes are vital for identifying ligand-binding sites on proteins.
• Lysine residues are abundant and attractive targets for covalent probes.
• Achieving amine-specific and regioselective reactions with binding site lysines is a significant challenge.

Purpose of the Study:

• To investigate the potential of squarates and squaramides as selective covalent labeling agents for lysine residues.
• To compare the reactivity and selectivity of squarates/squaramides with other amine-reactive electrophiles.
• To assess the utility of squarates in affinity-based chemoproteomic applications.

Main Methods:

• Kinetic analysis of squarate and monosquaramide reactions with amines.
• Comparison of squarate reactivity with other bioconjugation handles like N-hydroxysuccinimidyl esters.
• Evaluation of squarate selectivity using the enzyme GlfT2 from Mycobacterium tuberculosis, which has multiple surface-exposed lysines.

Main Results:

• Monosquaramides exhibit mild electrophilicity, favoring longer residence times for selective lysine labeling.
• Squarate reactivity can be modulated by substituents; electron-withdrawing groups and dithionosquarates enhance reactivity.
• N-hydroxysuccinimidyl esters react significantly faster, indicating lower selectivity.
• Squarates demonstrated high selectivity in labeling GlfT2, primarily modifying a single lysine near a binding site, leading to covalent inhibition.

Conclusions:

• Squaric esters and squaramides are effective and selective covalent labeling agents for lysine residues.
• Their tunable reactivity and selectivity make them valuable tools for affinity-based chemoproteomics.
• These findings pave the way for improved methods to identify and study protein-ligand interactions.