Affinity-Guided Oxime Chemistry for Selective Protein Acylation in Live Tissue Systems

Abstract

Catalyst-mediated protein modification is a powerful approach for the imaging and engineering of natural proteins. We have previously developed affinity-guided 4-dimethylaminopyridine (AGD) chemistry as an efficient protein modification method using a catalytic acyl transfer reaction. However, because of the high electrophilicity of the thioester acyl donor molecule, AGD chemistry suffers from nonspecific reactions to proteins other than the target protein in crude biological environments, such as cell lysates, live cells, and tissue samples. To overcome this shortcoming, we here report a new acyl donor/organocatalyst system that allows more specific and efficient protein modification. In this method, a highly nucleophilic pyridinium oxime (PyOx) catalyst is conjugated to a ligand specific to the target protein. The ligand-tethered PyOx selectively binds to the target protein and facilitates the acyl transfer reaction of a mild electrophilic N-acyl-N-alkylsulfonamide acyl donor on the protein surface. We demonstrated that the new catalytic system, called AGOX (affinity-guided oxime) chemistry, can modify target proteins, both in test tubes and cell lysates, more selectively and efficiently than AGD chemistry. Low-background fluorescence labeling of the endogenous cell-membrane proteins, carbonic anhydrase XII and the folate receptor, in live cells allowed for the precise quantification of diffusion coefficients in the protein's native environment. Furthermore, the excellent biocompatibility and bioorthogonality of AGOX chemistry were demonstrated by the selective labeling of an endogenous neurotransmitter receptor in mouse brain slices, which are highly complicated tissue samples.

Photocatalytic proximity labelling of MCL-1 by a BH3 ligand

“…Hamachi and co-workers have developed a wide range of LDL methods capable of selectively modifying native proteins in a 'traceless' manner, in which the ligand leaves its binding site after the labelling reaction and the protein is able to perform its native function 14 . These methods include exchange/cleavage approaches based on electrophilic phenylsulfonate esters [15][16][17] , acyl imidazoles 18,19 , N-sulfonyl pyridines 20 , or N-acyl-N-alkyl sulfonamides 21 and catalyst tethering approaches based on N,N-dimethylaminopyridine (DMAP) [22][23][24] or oxime reagents 25 . In addition, several groups have developed LDL reagents incorporating transition metal catalysts.…”

Considerations on Probe Design for Affinity‐Guided Protein Conjugation

“…The nucleophilic catalyst is commonly modified with the ligands of the proteins of interest to bring the catalyst to the protein surface. In this approach, acyl donors of relatively poor electrophilicity are activated for conjugation by the catalyst …”

“…The optimized oxime–acyl donor strategy was used for labeling in complex mixtures such as cell lysates, where it showed superior protein selectivity compared to the DMAP–thiophenol thioester strategy. Furthermore, the delicate conjugation reaction of the oxime–acyl donor pair displayed very high selectivity for labeling of a membrane receptor in brain tissue slices, which was not possible using the DMAP–thiophenol thioester strategy …”

“…The approach of ligand‐guided conjugation enables selective labeling of target protein in buffers and more complex solutions such as cell lysates, but this is also, to our knowledge, the only complexation moiety class that allows for selectively guided covalent protein labeling in living systems. By pairing with a suitable reactive moiety, it has been possible to covalently modify proteins of interest on or in live cells, in brain tissue slices, and even in mice . In most cases, the probes have been used for labeling of receptors or other membrane bound proteins, thereby, avoiding the issue of traversing the cell membrane.…”

Photocatalytic Proximity Labelling of MCL-1 by a BH3 Ligand

“…14 These methods include exchange/cleavage approaches based on electrophilic phenylsulfonate esters, 15-17 acyl imidazoles, 18,19 N-sulfonyl pyridines, 20 or N-acyl-N-alkyl sulfonamides 21 and catalyst tethering approaches based on N,N-dimethylaminopyridine (DMAP) [22][23][24] or oxime reagents. 25 In addition, several groups have developed LDL reagents incorporating transition metal catalysts. Ball and co-workers used rhodium(II) metallopeptides to selectively modify side-chains on protein surfaces with functionalised diazo compounds.…”